Three-dimensional magnetic resonance microscopy of materials.

Several aspects of magnetic resonance microscopy are examined employing three-dimensional (3D) back-projection reconstruction techniques in combination with either simple Bloch-decay methods or MREV-8 multiple-pulse line narrowing techniques in the presence of static field gradients. Applications to the areas of ceramic processing, catalyst porosity measurements and the characterization of polymeric materials are presented. The focus of the discussion centers on issues of sensitivity and resolution using this approach compared with other methods. Advantages and limitations of 3D microscopy over more commonly employed slice selection protocols are discussed, as well as potential remedies to some of the inherent limitations of the technique.

Herpes thymidine kinase mutants with altered catalytic efficiencies obtained by random sequence selection.

We have obtained 190 active Herpes simplex virus type 1 thymidine kinase mutants by substituting a 33 nucleotide sequence with 20% degeneracy for a portion of the nucleotide sequence that encodes the putative thymidine binding site [K.M. Munir, D.C. French, D.K. Dube and L.A. Loeb (1992) J. Biol. Chem., 167, 6584-6589]. In order to classify these mutants with respect to thymidine kinase activity we determined the ability of Escherichia coli harboring these mutants to form colonies in the presence of varying concentrations of thymidine. Escherichia coli harboring one of the mutant enzymes was able to form colonies at a concentration of thymidine lower than did the wild type. It was able to phosphorylate thymidine more rapidly than the wild type both in vivo and in vitro. The increased thymidine kinase activity was manifested by (i) a 42% enhanced uptake of [methyl-3H]thymidine into E. coli, (ii) a 2.4 times higher rate of [methyl-3H]thymidine incorporation into acid-insoluble material and (iii) a 5-fold increase in the kcat of the purified enzyme compared to the wild type. Herpes thymidine kinase purified from other mutants that formed colonies at higher thymidine concentrations than that of the wild type exhibited a decrease in kcat. The kcat of one of these mutant thymidine kinases was 10(-4) of that of the wild type enzyme. This study demonstrates that a spectrum of mutant enzymes with different catalytic properties can be obtained by selection from a plasmid with random sequence substitutions and this can be done in the absence of rational protein design.

Thymidine kinase mutants obtained by random sequence selection.

Knowledge of the catalytic properties and structural information regarding the amino acid residues that comprise the active site of an enzyme allows one, in principle, to use site-specific mutagenesis to construct genes that encode enzymes with altered functions. However, such information about most enzymes is not known and the effects of specific amino acid substitutions are not generally predictable. An alternative approach is to substitute random nucleotides for key codons in a gene and to use genetic selection to identify new and interesting enzyme variants. We describe here the construction, selection, and characterization of herpes simplex virus type 1 thymidine kinase mutants either with different catalytic properties or with enhanced thermostability. From a library containing 2 x 10(6) plasmid-encoded herpes thymidine kinase genes, each with a different nucleotide sequence at the putative nucleoside binding site, we obtained 1540 active mutants. Using this library and one previously constructed, we identified by secondary selection Escherichia coli harboring thymidine kinase mutant clones that were unable to grow in the presence of concentrations of 3'-azido-3'-deoxythymidine (AZT) that permits colony formation by E. coli harboring the wild-type plasmid. Two of the mutant enzymes exhibited a reduced Km for AZT, one of which displayed a higher catalytic efficiency for AZT over thymidine relative to that of the wild type. We also identified one mutant with enhanced thermostability. These mutants may have clinical potential as the promise of gene therapy is increasingly becoming a reality.

Conflict and friendship relations in middle childhood: behavior in a closed-field situation.

Disagreements between school-aged children were examined as a function of friendship status. 66 same-sex dyads were selected, including equal numbers of "best friends" and nonfriends, who were then observed while playing a board game (a closed-field situation). Conflicts occurred more frequently among friends than among nonfriends and lasted longer. Friends did not talk more during their conflicts than nonfriends, but assertions were used selectively according to friendship and sex: With friends, girls used assertions accompanied by rationales more frequently than boys whereas boys used assertions without rationales more frequently than girls. These sex differences were not evident during conflicts between nonfriends. Results are discussed in relation to the social constraints intrinsic to closed-field competitive conditions as they apply to friendship relations in middle childhood.

Permissible amino acid substitutions within the putative nucleoside binding site of herpes simplex virus type 1 encoded thymidine kinase established by random sequence mutagenesis [corrected].

We determined the essentiality of all amino acid replacements within an 11-codon sequence in the putative nucleoside-binding site of thymidine kinase encoded by herpes simplex virus type 1. This involved partial randomization of 11 codons in the gene to create a degenerate library, followed by genetic complementation using a tk- Escherichia coli strain and selection of unnatural active enzymes. We produced and tested 53,000 variants; of which 190 were found to be biologically active. Sequence analyses of functional variants revealed a high degree of flexibility in accommodating different types of amino acid substitutions in this region. However, no replacement was tolerated at proline-173, whereas tyrosine-172 could be replaced by only phenylalanine. To further define permissible substitutions at specified positions, we constructed a library with randomization at only four test codons. We produced and tested 600,000 variants; of which only 5 were active. Again proline-173 was conserved, and only tyrosine and phenylalanine were found at position 172. The identification of these conserved amino acids should provide important insights into the understanding of the structural basis of catalysis by this enzyme.

Artificial mutants generated by the insertion of random oligonucleotides into the putative nucleoside binding site of the HSV-1 thymidine kinase gene.

We have obtained 42 active artificial mutants of HSV-1 thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) by replacing codons 166 and 167 with random nucleotide sequences. Codons 166 and 167 are within the putative nucleoside binding site in the HSV-1 tk gene. The spectrum of active mutations indicates that neither Ile166 nor Ala167 is absolutely required for thymidine kinase activity. Each of these amino acids can be replaced by some but not all of the 19 other amino acids. The active mutants can be classified as high activity or low activity on two bases: (1) growth of Escherichia coli KY895 (a strain lacking thymidine kinase activity) in the presence of thymidine and (2) uptake of thymidine by this strain, when harboring plasmids with the random insertions. E. coli KY895 harboring high-activity plasmids or wild-type plasmids can grow in the presence of low amounts of thymidine (less than 1 microgram/mL), but are unable to grow in the presence of high amounts of thymidine. On the other hand, E. coli KY895 harboring low-activity plasmids can grow at a high concentration of thymidine (greater than 50 microgram/mL) in the media. The high-activity plasmids also have an enhanced [3H]dT uptake. The amounts of thymidine kinase activity in vitro in unfractionated extracts do not correlate with either growth at low thymidine concentration or the rate of thymidine uptake. Heat inactivation studies indicate that the mutant enzymes are without exception more temperature-sensitive than the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneity of peer-rejected boys: aggressive and nonaggressive subtypes.

Cluster analyses were employed in 2 studies to explore the possibility that discernible subtypes exist within the population of peer-rejected boys. In Study 1, 41 rejected 9- and 10-year-old boys were identified using nomination sociometrics. 8 teacher rating, behavior observation, and social problem solving interview measures were entered into the analysis. In Study 2, 48 9-, 10-, and 11-year-old rejected boys were identified using rating sociometrics. 9 teacher rating and peer rating measures were entered into the cluster analysis. 2 large clusters emerged in each of the 2 samples. Consistent patterns were seen across both studies when children within each cluster were compared with each other and with those in a popular comparison group. Boys in one cluster exhibited high aggression, low self-control, behavior problems, and withdrawn behavior. Boys in the other cluster exhibited withdrawal but did not obtain elevated scores on measures of aggression, behavior problems, or self-control. Findings of rejected-child heterogeneity have significant implications for the design of treatment programs and further research on peer relationship difficulties of children.

Behavior problems of peer-neglected and peer-rejected elementary-age children: parent and teacher perspectives.

Rejected, neglected, popular, and average-status children were selected on the basis of positive and negative sociometric measures from a total sample of 870 8- and 11-year-old children. Teachers completed the School Behavior Checklist and parents completed the Child Behavior Checklist for selected children. No age or sex differences were found. On both scales, rejected children were found to exhibit more behavior problems than neglected, popular, or average children. Neglected children did not exhibit more behavior problems than children of average status.