[Knowledge used in duration judgments of two moving objects].

Forty-nine undergraduates observed two cars traveling in the same direction on a CRT display for various duration. They then chose the car that they believed had run longer, and gave the reason for their choice. There were three types of tasks. Correct judgment was possible for the first type, by logically applying either of two pieces of knowledge about duration: "duration equals temporal end point minus temporal start point" (Knowledge alpha) or "duration equals distance divided by speed" (Knowledge beta). Knowledge alpha alone was useful for the second type, while only Knowledge beta led to correct judgment for the third. Main results were as follows: (1) Undergraduates were more likely to use Knowledge alpha than beta regardless of the types. (2) None of repeatedly making judgment, thinking about reasons for judgment, or receiving failure feedback was very helpful making participants become aware of necessity of using Knowledge beta for the third type. (3) Only some 20% of undergraduates were able to use proper knowledge specifically required for each type.

Binding pockets of the beta(1)- and beta(2)-adrenergic receptors for subtype-selective agonists.

We examined the subtype-selective binding site of the beta-adrenergic receptors (betaARs). The beta(1)/beta(2)-chimeric receptors showed the importance of the second and seventh transmembrane domains (TM2 and TM7) of the beta(2)AR for the binding of the beta(2)-selective agonists such as formoterol and procaterol. Alanine-substituted mutants of TM7 of the beta(2)AR showed that Tyr(308,) located at the top of TM7, mainly contributed to beta(2) selectivity. However, Tyr(308) interacted with formoterol and procaterol in two different ways. The results of Ala- and Phe-substituted mutants indicated that the phenyl group of Tyr(308) interacted with the phenyl group in the N-substituent of formoterol (hydrophobic interaction), and the hydroxyl group of Tyr(308) interacted with the protonated amine of procaterol (hydrophilic interaction). In contrast to beta(2)AR, TM2 is a major determinant that beta(1)-selective agonists such as denopamine and T-0509 bound the beta(1)AR with high affinity. Three amino acids (Leu(110), Thr(117), and Val(120)) in TM2 of the beta(1)AR were identified as major determinants for beta(1)-selective binding of these agonists. Three-dimensional models built on the basis of the predicted structure of rhodopsin showed that Tyr(308) of the beta(2)AR covered the binding pocket formed by TM2 and TM7 from the upper side, and Thr(117) of the beta(1)AR located in the middle of the binding pocket to provide a hydrogen bonding for the beta(1)-selective agonists. These data indicate that TM2 and TM7 of the betaAR formed the binding pocket that binds the betaAR subtype-selective agonists with high affinity.

Contribution of stimulus attributes to errors in duration and distance judgments--a developmental study.

In Matsuda's 1996 study, 4- to 11-yr.-old children (N = 133) watched two cars running on two parallel tracks on a CRT display and judged whether their durations and distances were equal and, if not, which was larger. In the present paper, the relative contributions of the four critical stimulus attributes (whether temporal starting points, temporal stopping points, spatial starting points, and spatial stopping points were the same or different between two cars) to the production of errors were quantitatively estimated based on the data for rates of errors obtained by Matsuda. The present analyses made it possible not only to understand numerically the findings about qualitative characteristics of the critical attributes described by Matsuda, but also to add more detailed findings about them.

Evolving catalytic antibodies in a phage-displayed combinatorial library.

In vitro affinity maturation for evolving catalytic antibodies has been demonstrated by generating a diverse repertoire of the appropriate complementarity-determining regions on a phage surface. Phage display is followed by a selection based on binding to an altered antigen that was not used at the time of immunization, and provides variants with new catalytic activity and substrate specificity. This library format reduces the time needed to isolate the desired catalytic antibody fragments to under 2 weeks.

Solution structure of ferredoxin from the thermophilic cyanobacterium Synechococcus elongatus and its thermostability.

The three-dimensional structure of ferredoxin, purified from the thermophilic cyanobacterium Synechococcus elongatus, was determined in aqueous solution by two-dimensional proton nuclear magnetic resonance. In addition to the 946 distance constraints from nuclear Overhauser effect connectivities, we added 241 distance constraints derived from the crystal structure of Spirulina platensis ferredoxin to the 19 residues close to the [2Fe-2S] iron-sulfur center, where crosspeaks disappeared due to paramagnetic effects. The atomic root-mean-square difference of the ten converged structures from the mean structure was 0.61(+/-0.12) A for backbone atoms (N, C(alpha), C'). The main-chain structure was almost the same as the crystal structures of other mesophile ferredoxins, but comparison of the side-chain structures revealed an extension of the hydrophobic core, a unique hydrophobic patch on the surface of the large beta-sheet, and two unique charge networks in this thermostable ferredoxin structure, some of which might contribute to thermostability.

Site-directed mutagenesis of active site contact residues in a hydrolytic abzyme: evidence for an essential histidine involved in transition state stabilization.

Specific molecular interactions involved in catalysis by antibody 6D9 were investigated by site-directed mutagenesis. The catalytic antibody 6D9, which was generated against a transition state analog (III), hydrolyzes a non-bioactive chloramphenicol monoester derivative (I) to produce chloramphenicol (II). Construction of a three-dimensional molecular model of 6D9 and sequence comparison within a panel of related antibodies suggested candidates for catalytic residues, His (L27d), Tyr (L32), Tyr (H58) and Arg (H100b); these were targeted for the site-directed mutagenesis study. The Y-H58-F and R-H100b-A mutants possessed catalytic activities comparable to that of the wild-type, and the Y-H58-H and Y-L32-F mutant displayed an approximately fivefold decrease in k(cat)/Km. In the transition state analysis, the plots of logK(TSA) versus log(k(cat)/Km) for the mutants are linear, with a slope of approximately 1.0, indicating that the entire hapten-binding energy in the mutants is also utilized to bind the transition state and to accelerate the catalysis. In addition, a dramatic change in the catalytic activity was observed when the histidine residue (27d) in the CDR1 light chain was replaced with alanine. The H-L27d-A mutant had no detectable catalytic activity. This mutation led to a large, 40-fold reduction in transition state binding, with no change in substrate binding. Coupled with the previous kinetic studies and chemical modifications of the intact 6D9 antibody, this mutagenesis study has demonstrated that His L27d plays an essential role in stabilization of the transition state, the mechanism of catalysis by the 6D9 antibody.

Interaction between the amino-terminal SH3 domain of CRK and its natural target proteins.

CRK is a human homolog of chichen v-Crk, which is an adaptor protein. The SH2 domain of CRK binds to several tyrosine-phosphorylated proteins, including the epidermal growth factor receptor, p130(Cas), Shc, and paxillin. The SH3 domain, in turn, binds to cytosolic proteins of 135-145, 160, 180, and 220 kDa. We screened expression libraries by Far Western blotting, using CRK SH3 as a probe, and identified partial cDNA sequences of four distinct proteins, including C3G, DOCK180, EPS15, and clone ST12. The consensus sequence of the CRK SH3 binding sites as deduced from their amino acid sequences was Pro+3-Pro+2-X+1-Leu0-Pro-1-X-2-Lys-3. The interaction of the CRK SH3 domain with the DOCK180 peptide was examined with an optical biosensor, based on the principles of surface plasmon resonance. A low dissociation constant of the order of 10(-7) resulted from a high association rate constant (kassoc = 3 x 10(4)) and low dissociation rate constant (kdiss = 3 x 10(-3)). All CRK-binding proteins except clone ST12 also bound to another adaptor protein, Grb2. Mutational analysis revealed that glycine at position +1 of ST12 inhibited the binding to Grb2 while retaining the high affinity binding to CRK SH3. The result suggests that the amino acid at position +1 also contributes to the high affinity binding of the peptides to the SH3 domain of Grb2, but not to that of CRK.

Developmental characteristics of topographic EEG in school-age children using an autoregressive model.

Developmental characteristics of the resting EEG were investigated in 47 school-age children using statistical analysis. Total and component EEG power were statistically analyzed between the subject groups from 7 to 14 year-old using our autoregressive pattern discrimination system. In early school-age children, significant topographic differences were seen in theta waves, while in late school-age children, the differences were found in alpha waves in the frontal and occipital regions, and in beta waves in the frontal region.

Determinants of protein side-chain packing.

The problem of protein side-chain packing for a given backbone trace is investigated using 3 different prediction models. The first requires an exhaustive search of all possible combinations of side-chain conformers, using the dead-end elimination theorem. The second considers only side-chain-backbone interactions, whereas the third neglects side-chain-backbone interactions and instead keeps side-chain-side-chain interactions. Predictions of side-chain conformations for 11 proteins using all 3 models show that removal of side-chain-side-chain interactions does not cause a large decrease in the prediction accuracy, whereas the model having only side-chain-side-chain interactions still retains a significant level of accuracy. These results suggest that the 2 classes of interactions, side-chain-backbone and side-chain-side-chain, are consistent with each other and work concurrently to stabilize the native conformations. This is confirmed by analyses of energy spectra of the side-chain conformations derived from the fourth prediction model, the Independent model, which gives almost the same quality of the prediction as the dead-end elimination. The analyses indicate that the 2 classes of interactions simultaneously increase the energy difference between the native and nonnative conformations.

A common ancestry for multiple catalytic antibodies generated against a single transition-state analog.

Immunization with a single haptenic transition-state analog generates a few catalytic antibodies among the dozens of antibodies capable of binding the hapten. The diversity of the immune response has raised some fundamental issues, such as How do catalytic and noncatalytic antibodies differ on a structural basis? To address this issue, the variable region primary sequences of 11 antibodies (including 6 catalytic and 5 noncatalytic antibodies) elicited against a single haptenic transition-state analog were deduced from cDNA sequences. Cluster analyses using phylogenetic trees constructed by the neighbor-joining method have revealed that the amino acid sequences of noncatalytic antibodies bear no relationship to one another, while the catalytic antibodies share significant structural identity. Furthermore, no catalytic antibodies possessing amino acid sequences with high homology to those of noncatalytic antibodies were detected. Five catalytic antibodies examined showed 89-95% and 74-84% sequence homologies in the complete light- and heavy-chain variable regions, respectively. Thus, it seems likely that the catalytic antibodies elicited against a single hapten use the canonical set of variable region genes. Interestingly, one catalytic antibody showed only limited sequence similarity to the other catalytic antibodies and was found to exhibit a distinctly different substrate specificity. From the broad range of their binding constants to the hapten, it is unlikely that highly homologous catalytic antibodies are generated as a result of simple high-affinity choices. These results emphasize the utility of rationally designed transition-state analogs for the induction of antibody molecules with catalytic activity.

Extrarenal Wilms' tumour.

Extrarenal Wilms' tumour is rare and its imaging has received scant mention in the literature. We describe a 2-year-old boy with a firm mass in the right flank. CT, MRI and ultrasonography showed an inhomogeneous solid mass located in the retroperitoneum, which was separate from the right kidney. Angiography showed an enlarged right gonadal artery and irregularly tortuous vessels in the tumour similar to intrarenal Wilms' tumour ("spider leg" or "creeping vine" appearance). Histopathological examination confirmed an extrarenal Wilms' tumour.