Siting, design and operational controls for snow disposal sites.

The Municipality of Anchorage (MOA), at 61 degrees north latitude, ploughs and hauls snow from urban streets throughout the winter, incorporating grit and chloride applied to street surfaces for traffic safety. Hauled snow is stored at snow disposal facilities, where it melts at ambient spring temperatures. MOA studies performed from 1998 through 2001 show that disposal site melt processes can be manipulated, through site design and operation practices, to control chloride and turbidity in meltwater. An experimental passive "V-swale" pad configuration tested by MOA investigators reduced site meltwater turbidity by an order of magnitude (to about 50 NTU from the 500 NTU typical of more conventional planar pad geometry). The MOA has developed new siting, design and operational criteria for snow disposal facilities to conform to the tested V-swale pad configuration.

Structure of Na+,K+-ATPase at 11-A resolution: comparison with Ca2+-ATPase in E1 and E2 states.

Na+,K+-ATPase is a heterodimer of alpha and beta subunits and a member of the P-type ATPase family of ion pumps. Here we present an 11-A structure of the heterodimer determined from electron micrographs of unstained frozen-hydrated tubular crystals. For this reconstruction, the enzyme was isolated from supraorbital glands of salt-adapted ducks and was crystallized within the native membranes. Crystallization conditions fixed Na+,K+-ATPase in the vanadate-inhibited E2 conformation, and the crystals had p1 symmetry. A large number of helical symmetries were observed, so a three-dimensional structure was calculated by averaging both Fourier-Bessel coefficients and real-space structures of data from the different symmetries. The resulting structure clearly reveals cytoplasmic, transmembrane, and extracellular regions of the molecule with densities separately attributable to alpha and beta subunits. The overall shape bears a remarkable resemblance to the E2 structure of rabbit sarcoplasmic reticulum Ca2+-ATPase. After aligning these two structures, atomic coordinates for Ca2+-ATPase were fit to Na+,K+-ATPase, and several flexible surface loops, which fit the map poorly, were associated with sequences that differ in the two pumps. Nevertheless, cytoplasmic domains were very similarly arranged, suggesting that the E2-to-E1 conformational change postulated for Ca2+-ATPase probably applies to Na+,K+-ATPase as well as other P-type ATPases.

Structure-function relationships in the Ca(2+)-binding and translocation domain of SERCA1: physiological correlates in Brody disease.

Alanine-scanning mutagenesis of all amino acids in transmembrane helices M4, M5, M6 and M8, which contain known Ca2+ binding residues in the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum, revealed patches of mutation-sensitivity in M4, M5 and M6, but in M8. A six-residue motif, (E/D)GLPA(T/V), in M4 and M6 and its counterpart in M5 were highlighted by mutagenesis. Site-directed disulfide mapping of helices M4 and M6 demonstrated that these transmembrane helices associate as a right-handed coiled-coil. This structural information, combined with the earlier analysis of the association of each Ca2+ binding residue with either Ca2+ binding site I or site II, permitted the development of a "side-by-side" model for the two Ca2+ binding sites in the Ca(2+)-ATPase. In about half of Brody disease families, mutations create stop codons which delete all or part of the Ca2+ binding and translocation domain, resulting in loss of SERCA1 function and muscle disease.

Factorial structure of the Wisconsin Card Sorting Test.

This study examined the factorial structure of the Wisconsin Card Sorting Test (WCST) in normal university students (N = 135) and a mixed clinical sample (N = 139). Two highly stable orthogonal factors were observed accounting for 70 and 21 per cent of the variance, respectively. Factor I was interpreted as reflecting undifferentiated executive function while Factor II may measure cognitive abilities associated with attentional function. This work can serve as the basis for further examination of the construct validity of the WCST and has implications for its use.

Site-directed disulfide mapping of helices M4 and M6 in the Ca2+ binding domain of SERCA1a, the Ca2+ ATPase of fast twitch skeletal muscle sarcoplasmic reticulum.

In an attempt to define the spatial relationships among SERCA1a transmembrane helices M4, M5, M6, and M8, involved in Ca2+ binding, all six cysteine residues were removed from predicted transmembrane sequences by substitution with Ser or Ala. The cysteine-depleted protein retained 44% of wild type Ca2+ transport activity. Pairs of cysteine residues were then reintroduced to determine whether their juxtaposition would result in the formation of disulfide cross-links between transmembrane helices. In initial studies designed to map the juxtaposition of Ca2+ binding residues, Cys was substituted for Glu309 or Gly310 in transmembrane sequence M4, in combination with the substitution of Cys for Glu771 in M5; for Asn796, Thr799, or Asp800 in M6; or for Glu908 in M8. These double mutants all retained the capacity to form a phosphoenzyme intermediate from Pi (but not from ATP in the presence of Ca2+), and in all but mutants E309C/N796C and G310C/N796C, phosphoenzyme formation was insensitive to 100 microM Ca2+. These results support the view that both Glu309 and Asn796 contribute to Ca2+ binding site II, which is not required for conversion of E2, the substrate for Pi phosphorylation, to E1. Cross-linking in mutants E309C/N796C and G310C/D800C established reference points for the orientation of M4 and M6 relative to each other and provided the basis for the prediction of potential additional cross-links. Strong links were formed with the pairs T317C/A804C and T317C/L807C near the cytoplasmic ends of the two helices and with A305C/L792C and A305C/L793C near the lumenal ends. These combined results support the conclusion that M4 and M6 form a right-handed coiled-coil structure that forms part of the pathway of Ca2+ translocation. In addition to providing a possible explanation for the mutation sensitivity of several pairs of residues in these helices, the proposed association of M4 and M6 supports a new model for the orientation of the two Ca2+ binding sites among transmembrane helices M4, M5, and M6.

Scanning mutagenesis reveals a similar pattern of mutation sensitivity in transmembrane sequences M4, M5, and M6, but not in M8, of the Ca2+-ATPase of sarcoplasmic reticulum (SERCA1a).

Scanning mutagenesis was performed on all amino acids in transmembrane sequences M5, M6, and M8, which, together with M4, make up the Ca2+ binding domain of the Ca2+-ATPase of sarcoplasmic reticulum (SERCA1a). When these transmembrane sequences were displayed on a helical net, examination of the effects of 101 novel point mutations and 95 prior mutations carried out on 92 transmembrane amino acids revealed "patches" of sensitivity to mutation in M4, M5, and M6 but not in M8. The patches of mutation-sensitive residues spanned 6 of the 7 tiers of the helical net and covered about 240 degrees at their widest point in tiers 3 or 4 and 140 degrees in tiers 2 and 5. A contiguous column of mutation-insensitive hydrophobic amino acids was found in M4 and M6 and in tiers 4 to 7 of M5. A six-residue motif, (E/D)GLPA(T/V) in tiers 3 and 4 of M4 and M6 with Ca2+-binding residues Glu309 and Asp800 as the first residue, was highlighted by mutation sensitivity. Elements of the motif could also be discerned in M5, but reading in the C-terminal to N-terminal direction. Mutation sensitivity in tier 5 of M4 mirrored mutation sensitivity of tier 5 in M6, although the amino acid sequences were not similar. The motif or its counterpart was found in a region in M4, M5, and M6 that is made up of tiny or small amino acids but is bounded by tiers with a larger percentage of bulky amino acids. Tiers 3, 4, and 5 of M4, M5, and M6 contain Ca2+ binding and affinity mutations, E1P to E2P block mutations and E2P dephosphorylation mutations, indicating an important role for these central tiers in Ca2+ binding and in the conformational changes that accompany Ca2+ translocation. Analysis of M8 revealed only a single mutation-sensitive residue, the Ca2+-binding amino acid, Glu908. This residue and a mutation-insensitive residue, Ala912, were the only vestiges of the motif that was found in M4 and M6. Additional mutations to Glu908 provided further evidence for its role in Ca2+ binding. Since mutation of M8 failed to identify residues involved in blocking conformational changes or altering Ca2+ affinity, it is apparent that M8 plays a peripheral role in Ca2+ binding and translocation in comparison with M4, M5, and M6.

Functional consequences of alterations to hydrophobic amino acids located in the M4 transmembrane sector of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Those hydrophobic residues between Ile298 and Ile315 in transmembrane segment M4 of the Ca(2+)-ATPase of sarcoplasmic reticulum, not previously mutated, were mutated systematically in ways that would alter their size or polarity, and functional consequences were measured. Fourteen residues in this sequence are organized as juxtapositions of large, hydrophobic (Val, Leu, Ile) and small (Ala, Gly) residues, and these were altered so that large residues were substituted for small and vice versa. Several mutants exhibited diminished Ca2+ transport, but mutants A305V and A306V lost all Ca2+ transport function. In both cases, the mutants were phosphorylated with ATP in the presence of Ca2+ and with inorganic phosphate only in the absence of Ca2+, indicating that the Ca(2+)-binding sites were intact. Reduced Ca2+ affinity, as measured by Ca2+ dependence of phosphorylation from ATP, was observed for mutant A305V. In both mutants, the ADP-insensitive phosphoenzyme intermediate (E2P) decayed slowly relative to the wild-type enzyme, suggesting that the E2P to E2 conformational transition was impaired, slowing the rate of the phosphatase reaction. Double mutants which reversed the order of Val304 and Ala305 and Ala306 and Ile307, resulted in the same phenotype as the single Ala mutations. These results, combined with our previous demonstration that Glu309 is a Ca2+ binding residue, that Pro312 is involved in E1P to E2P conformational changes, and that Gly310 is involved in E2P to E2 conformational changes, support the hypothesis that transmembrane segment M4 plays a key role in the Ca2+ transport function of the Ca(2+)-ATPase through its involvement in both the binding of Ca2+ and the subsequent conformational changes which bring about the translocation of Ca2+ to the lumen of the membrane.

Expandable intraluminal graft: a preliminary study. Work in progress.

To overcome the problem of recurrence of stenosis after vascular balloon dilatations, we developed an expandable, intraluminal graft that allows dilatation of the lesion and simultaneous placement of a supportive endoprosthesis to prevent recoil of the arterial wall. The graft is made of continuous, woven, stainless steel wire. The resulting tubular mesh has a wall thickness of 200-450 micron and 80% open surface. The grafts, mounted on angioplasty catheters, are introduced through 8-12-F Teflon sheaths. Eleven grafts of 6, 8, and 10 mm in diameter by 20 mm long were placed in the aorta, common carotid, superior mesenteric, iliac, and renal arteries of dogs. Six grafts showed no stenosis in follow-up studies of up to 8 weeks. Two grafts had moderate stenosis as a result of neointimal hyperplasia. Two partial and one complete graft thrombosis occurred in nonheparinized animals in which the graft outflow was restricted. Anticoagulant was not used on a long-term basis. Light and electron microscopy studies showed complete covering of the graft's inner surface by endothelium at 3 weeks.

Linear discriminant function analysis in neuropsychological research: some uses and abuses.

The present paper addressed the continued misinterpretation and misapplication of linear discriminant function analysis in neuropsychological research. Methodological problems concerning the influence of shrinkage and stepwise selection procedures on LDFA are virtually ignored and affect both the classification and inferential application of LDFA. Throughout the paper examples of potential abuses of LDFA were cited and data from a familiar research problem was employed to demonstrate procedures which enable more accurate interpretation of LDFA results. Linear discriminant function analysis and its multivariate equivalents are powerful and flexible tools for exploring group differences provided appropriate applications and interpretations of results are made.