Crystal structure of imidazole glycerol phosphate synthase: a tunnel through a (beta/alpha)8 barrel joins two active sites.

BACKGROUND: Imidazole glycerol phosphate synthase catalyzes a two-step reaction of histidine biosynthesis at the bifurcation point with the purine de novo pathway. The enzyme is a new example of intermediate channeling by glutamine amidotransferases in which ammonia generated by hydrolysis of glutamine is channeled to a second active site where it acts as a nucleophile. In this case, ammonia reacts in a cyclase domain to produce imidazole glycerol phosphate and an intermediate of purine biosynthesis. The enzyme is also a potential target for drug and herbicide development since the histidine pathway does not occur in mammals. RESULTS: The 2.1 A crystal structure of imidazole glycerol phosphate synthase from yeast reveals extensive interaction of the glutaminase and cyclase catalytic domains. At the domain interface, the glutaminase active site points into the bottom of the (beta/alpha)(8) barrel of the cyclase domain. An ammonia tunnel through the (beta/alpha)(8) barrel connects the glutaminase docking site at the bottom to the cyclase active site at the top. A conserved "gate" of four charged residues controls access to the tunnel. CONCLUSIONS: This is the first structure in which all the components of the ubiquitous (beta/alpha)(8) barrel fold, top, bottom, and interior, take part in enzymatic function. Intimate contacts between the barrel domain and the glutaminase active site appear to be poised for crosstalk between catalytic centers in response to substrate binding at the cyclase active site. The structure provides a number of potential sites for inhibitor development in the active sites and in a conserved interdomain cavity.

Nuclease activity is essential for RecBCD recombination in Escherichia coli.

RecBCD has two conflicting roles in Escherichia coli. (i) As ExoV, it is a potent double-stranded (ds)DNA exonuclease that destroys linear DNA produced by restriction of foreign DNA. (ii) As a recombinase, it promotes repair of dsDNA breaks and genetic recombination in the vicinity of chi recombination hot-spots. These paradoxical roles are accommodated by chi-dependent attenuation of RecBCD exonuclease activity and concomitant conversion of the enzyme to a recombinase. To challenge the proposal that chi converts RecBCD from a destructive exonuclease to a recombinogenic helicase, we mutated the nuclease catalytic centre of RecB and tested the resulting mutants for genetic recombination and DNA repair in vivo. We predicted that, if nuclease activity inhibits recombination and helicase activity is sufficient for recombination, the mutants would be constitutive recombinases, as has been seen in recD null mutants. Conversely, if nuclease activity is required, the mutants would be recombination deficient. Our results indicate that 5' --> 3' exonuclease activity is essential for recombination by RecBCD at chi recombination hot-spots and at dsDNA ends in recD mutants. In the absence of RecB-dependent nuclease function, recombination becomes entirely dependent on the 5' --> 3' single-stranded (ss)DNA exonuclease activity of RecJ and the helicase activity of RecBC(D).

Relation of useful field of view and other screening tests to on-road driving performance.

The purpose of this study was to examine the value of a clinical driving assessment battery in predicting performance on an on-road driving test. 43 participants referred to the Bryn Mawr Rehab Adapted Driving Program for evaluation of driving ability underwent an evaluation consisting of a predriver screening and an on road driving test. The predriver screening included a vision screening, a reaction rime task, a split-attention task, the Hooper Visual Organization Test, verbal and symbolic sign recognition, and assessment of Useful Field of View. Logistic regression analyses were applied to identify which predriver screening variables could be used to predict outcome on the on road driving test (pass/fail); UFOV was that best single predictor. The addition of screening tests beyond UFOV alone did not increase predictive validity. These findings suggest that UFOV may serve as an indicator of the need for further driving assessment.

A general high-performance liquid chromatography-based assay for the hydrolysis of N-acyl glutamates.

A rapid and simple HPLC assay has been developed to separate and quantify N-acyl glutamates and the corresponding carboxylic acids of the acyl moiety. This method was specifically developed to assay hydrolytic activity for glutamate carboxypeptidases. Although established assays for specific substrates of such enzymes exist, they may not be amenable for examining the hydrolytic activity of new substrate probes. This assay was developed to accommodate such probes.

The hotspot conversion paradox and the evolution of meiotic recombination.

Studies of meiotic recombination have revealed an evolutionary paradox. Molecular and genetic analysis has shown that crossing over initiates at specific sites called hotspots, by a recombinational-repair mechanism in which the initiating hotspot is replaced by a copy of its homolog. We have used computer simulations of large populations to show that this mechanism causes active hotspot alleles to be rapidly replaced by inactive alleles, which arise by rare mutation and increase by recombination-associated conversion. Additional simulations solidified the paradox by showing that the known benefits of recombination appear inadequate to maintain its mechanism. Neither the benefits of accurate segregation nor those of recombining flanking genes were sufficient to preserve active alleles in the face of conversion. A partial resolution to this paradox was obtained by introducing into the model an additional, nonmeiotic function for the sites that initiate recombination, consistent with the observed association of hotspots with functional sites in chromatin. Provided selection for this function was sufficiently strong, active hotspots were able to persist in spite of frequent conversion to inactive alleles. However, this explanation is unsatisfactory for two reasons. First, it is unlikely to apply to obligately sexual species, because observed crossover frequencies imply maintenance of many hotspots per genome, and the viability selection needed to preserve these would drive the species to extinction. Second, it fails to explain why such a genetically costly mechanism of recombination has been maintained over evolutionary time. Thus the paradox persists and is likely to be resolved only by significant changes to the commonly accepted mechanism of crossing over.

Perceived benefits of and barriers to exercise and stage of exercise adoption in young adults.

College students (N = 432) completed questionnaires assessing current exercise participation, intention to become more active, perceived benefits of exercise, and perceived barriers to exercise. Confirmatory factor analyses of the benefit and barrier items suggested that a model containing 4 benefit factors (social, psychological, body image, and health) and 4 barrier factors (time-effort, social, physical effects, and specific obstacles) fit better than other models with fewer factors. Subscales derived from these factors revealed specific differences across participants who were categorized into 1 of 4 stages of exercise adoption. These findings confirm the multidimensional nature of perceived benefits of and barriers to exercise. Implications for the promotion and maintenance of exercise are discussed.

Chi recombination activity in phage lambda decays as a function of genetic distance.

In Escherichia coli, chi is a recombination hotspot that stimulates RecBCD-dependent exchange at and to one side of itself. chi activity is highest at chi and decreases with distance from chi. The decrease in chi activity may be a simple property of the physical distance over which chi can stimulate recombination. Alternatively, the decay in chi activity with distance may reflect the high likelihood that chi-stimulated recombination occurs in a single chi-proximal act, to the exclusion of additional chi-stimulated exchanges more distal to chi. To test the models, we determined if chi activity decreases as a function of physical distance (i.e., DNA base pairs) or genetic distance (homologous DNA base pairs). Our results indicate that chi activity decays as a function of genetic distance. In addition, we found that the sbcB gene product (exonuclease I, a 3'-->5' ssDNA exonuclease) modulates the distance over which chi can act. In contrast, the recJ gene product (a 5'-->3' ssDNA exonuclease) does not alter the decay of chi activity.

The recombination hot spot chi activates RecBCD recombination by converting Escherichia coli to a recD mutant phenocopy.

The products of the recB and recC genes are necessary for conjugal recombination and for repair of chromosomal double-chain breaks in Escherichia coli. The recD gene product combines with the RecB and RecC proteins to comprise RecBCD enzyme but is required for neither recombination nor repair. On the contrary, RecBCD enzyme is an exonuclease that inhibits recombination by destroying linear DNA. The RecD ejection model proposes that RecBCD enzyme enters a DNA duplex at a double-chain end and travels destructively until it encounters the recombination hot spot sequence chi. Chi then alters the RecBCD enzyme by weakening the affinity of the RecD subunit for the RecBC heterodimer. With the loss of the RecD subunit, the resulting protein, RecBC(D-), becomes deficient for exonuclease activity and proficient as a recombinagenic helicase. To test the model, genetic crosses between lambda phage were conducted in cells containing chi on a nonhomologous plasmid. Upon delivering a double-chain break to the plasmid, lambda recombined as if the cells had become recD mutants. The ability of chi to alter lambda recombination in trans was reversed by overproducing the RecD subunit. These results indicate that chi can influence a recombination act without directly participating in it.

Chi and the RecBC D enzyme of Escherichia coli.

The products of genes recB and recC are responsible for conjugal recombination and for the repair of chromosomal double chain breaks in Escherichia coli. The product of the recD gene, which combines with the RecB and RecC proteins to comprise the RecBCD enzyme, is not required for either recombination or repair. On the contrary, RecBCD enzyme is a potent exonuclease which inhibits recombination by destroying linear DNA. The RecD Ejection model supposes that RecBCD enzyme enters DNA at a double-chain end and travels destructively along the DNA until (typically) it encounters the recombination hotspot sequence chi. Chi then alters the RecBCD enzyme by weakening the affinity of the RecD subunit for the RecBC heterodimer. With the loss of the RecD subunit from the enzyme, the resulting protein, RecBC(D-), becomes deficient for exonuclease activity and proficient as a recombinagenic helicase. Thus, the RecD Ejection model proposes that chi participates in recombination by acting as a toggle to convert RecBCD (a powerful exonuclease) to RecBC(D-) (a recombinase). We review the properties of RecBCD and its cognate site chi, including recent results that support both the RecD Ejection model and the view that chi plays only an indirect role in recombination.

The role of violence in decisions about hospitalization from the psychiatric emergency room.

OBJECTIVE: The authors evaluated the relationship between violent behavior and decision making about hospitalization from the psychiatric emergency room. METHOD: The medical charts of 321 patients evaluated in an urban psychiatric emergency room during a 4-week period were reviewed retrospectively. Violent behavior was defined as physical attacks on persons or fear-inducing behavior before or during the evaluation in the emergency room; and its value in predicting hospitalization decisions was assessed with logistic regression analyses that also included 12 demographic, clinical, and contextual variables. RESULTS: A model predicting hospitalization decisions was developed and cross-validated. Although violent patients were more likely to be hospitalized than nonviolent patients, clinical variables such as diagnosis and overall severity of psychiatric impairment were more important than violent behavior in predicting hospitalization decisions. CONCLUSIONS: Despite legal pressures to focus on overt behaviors such as violence as a basis for liability prevention and civil commitment, clinicians in this study did not allocate inpatient resources to preventively detain persons unlikely to benefit from treatment. Rather, they hospitalized the most severely disturbed patients, with diagnoses such as schizophrenic and manic disorders for which a widely accepted therapeutic armamentarium exists. The results are consistent with clinical recommendations that in the evaluation of the violent patient, attention needs to be given to the underlying disorder, since violent behavior itself can result from diverse causes only some of which require inpatient psychiatric treatment.

Dissecting the molecular mechanism of ion-solute cotransport: substrate specificity mutations in the putP gene affect the kinetics of proline transport.

Rare mutations that alter the substrate specificity of proline permease cluster in discrete regions of the putP gene, suggesting that they may replace amino acids at the active site of the enzyme. If putP substrate specificity mutations directly after the active site of proline permease, the mutants should show specific defects in the kinetics of proline transport. In order to test this prediction, we examined the kinetics of three putP substrate specificity mutants. One class of mutation increases the Km over 120 fold but only decreases the Vmax fourfold. Such Km mutants may be specifically defective in substrate recognition, thus identifying an amino acid critical for substrate binding. Another class of mutation decreases the Vmax 80-fold without changing the Km. Vmax mutants appear to alter the rate of substrate translocation without affecting the substrate binding site. The last class of mutation alters both the Km and Vmax of proline transport. These results indicate that substrate specificity mutations alter amino acids critical for Na+/proline symport.

Mutations of putP that alter the lithium sensitivity of Salmonella typhimurium.

The putP gene encodes the major proline permease in Salmonella typhimurium that couples transport of proline to the sodium electrochemical gradient. To identify residues involved in the cation binding site, we have isolated putP mutants that confer resistance to lithium during growth on proline. Wild-type S. typhimurium can grow well on proline as the sole carbon source in media supplemented with NaCl, but grows poorly when LiCl is substituted for NaCl. In contrast to the growth phenotype, proline permease is capable of transporting proline via Na+/proline or Li+/proline symport. Therefore, we selected mutants that grow well on media containing proline as the sole carbon source in the presence of lithium ions. All of the mutants assayed exhibit decreased rates of Li+/proline and Na+/proline cotransport relative to wild type. The location of each mutation was determined by deletion mapping: the mutations cluster in two small deletion intervals at the 5' and 3' termini of the putP gene. The map positions of these lithium resistance mutations are different from the locations of the previously isolated substrate specificity mutations. These results suggest that Lir mutations may define domains of the protein that fold to form the cation binding site of proline permease.

Regulation of proline utilization in Salmonella typhimurium: molecular characterization of the put operon, and DNA sequence of the put control region.

The two genes required for proline utilization (put) in Salmonella typhimurium form a divergent operon. Extensive genetic evidence suggests that transcription of the put operon is autoregulated by the putA gene product, a membrane-associated dehydrogenase. In order to understand the mechanism of regulation, we characterized plasmid clones of the put operon. A 7.5 kb clone contains both of the put structural genes and regulatory sites. This clone only expressed two unique proteins corresponding to the putA and putP gene products. By comparing the physical and genetic maps of the put operon, the position of the put regulatory region was defined and the DNA sequence of this region was determined. Analysis of the DNA sequence indicated several potential regulatory sites for the put genes. Based on genetic and physical mapping studies, the most likely regulatory sites are two convergent promoters approximately 30 bp apart. A 27 bp palindrome located between the two promoters may be the operator for autoregulation by the PutA protein. The putA translational start site is 40 bp downstream of its putative mRNA start site. The putP promoter and its translational start site are separated by a 400 bp untranslated region.

Do infants see emotional expressions in static faces?

To determine whether young infants discriminate photographs of different emotions on an affect-relevant basis or on the basis of isolated features unrelated to emotion, groups of 17-, 23-, and 29-week-olds were habituated to slides of 8 women posing either Toothy Angry, Nontoothy Angry, or Nontoothy Smiling facial expressions and were then shown 2 new women in the familiarized expression and in a novel Toothy Smiling expression. At all 3 ages, recovery to the novel Toothy Smiling faces occurred only after habituation to Nontoothy faces (whether smiling or angry), not after habituation to Toothy Angry faces, indicating that infants had been responsive to nonspecific features of the photographs (presence or absence of bared teeth) rather than to affectively relevant configurations of features. In a second experiment, 2 older age groups (35 and 41 weeks) also proved to be insensitive to affect-related aspects of still faces, though more so for angry than for happy expressions. It is suggested that the young infant's difficulty in extracting emotional information from static stimuli may be attributable to the absence of the critical invariants (dynamic, multimodally specified) that characterize naturalistic expressions of emotion.

Abstraction of invariant face expressions in infancy.

To determine whether infants can abstract invariant face expressions across different persons (i.e., can form face expression categories), groups of 18-, 24-, and 30-week-old infants (18 boys and 18 girls per group) were habituated by the infant control procedure to photographs of 4 different female faces all wearing an identical expression (happy or surprise). In an immediately following test phase, categorization was inferred from greater generalization of habituation (less recovery of fixation) to 2 new female faces in the familiarized expression than to the same new faces in the altered (novel) expression. To rule out the possibility that generalization at test might be due to failure to discriminate the new persons, control groups of 18 boys and 18 girls at each age saw the same test faces following repeated presentations of only 1 of the 4 habituation faces. The results indicated that not until 30 weeks could infants differentiate happy and surprise expressions on a categorical basis. At 24 weeks they could distinguish a surprise expression following habituation to happy faces, but could not do the reverse. At 18 weeks they could do neither. Overall, the performance of girls was superior to that of boys. The findings are consistent with recent evidence suggesting that the ability to extract invariant configural information relative to the human face does not emerge until about 7 months of age.