Evidence for phosphorylation-dependent conformational changes in methylesterase CheB.

Enhancement of methylesterase activity of the response regulator CheB is dependent upon phosphorylation of the N-terminal regulatory domain of the enzyme. This domain plays a dual role in the regulation of methylesterase activity with an inhibitory effect in the unphosphorylated state and a stimulatory effect in the phosphorylated state. Structural studies of the unphosphorylated state have indicated that the basis for the regulatory domain's inhibitory effect is partial blockage of access of substrate to the active site suggesting that the activation upon phosphorylation involves a repositioning of the two domains with respect to each other. We report in this study evidence for phosphorylation-dependent conformational changes in CheB. Differences in rates of proteolytic cleavage by trypsin between the phosphorylated and unphosphorylated states have been observed at three sites in the protein with one site, 113, within the regulatory domain and two sites, 134 and 148, lying within the interdomain linker. These results support the hypothesis for the mechanism for the activation of CheB wherein phosphorylation of a specific aspartate residue within the N-terminal domain results in a propagated conformational change within the regulatory domain leading to a repositioning of its two domains. Presumably, structural changes in the regulatory domain of CheB facilitate a repositioning of the N- and C-terminal domains, leading to stimulation of methylesterase activity.

Social problem solving in children with acquired brain injuries.

OBJECTIVES: To assess the performance of children with acquired brain injuries (ABIs) on a measure of social problem solving and to examine the relationships between participant characteristics and performance on the Social Knowledge Interview (SKI) and between parent-reported child behavior and performance on the SKI. DESIGN: Between-group comparisons using correlational analyses, matched pairs t-tests, and analysis of covariance (ANCOVA). PARTICIPANTS: Thirty-one children 6-12 years old with ABI and 31 control participants, matched on age and sex. MAIN OUTCOME MEASURE: The SKI, a measure of social problem-solving skills. RESULTS: Using matched pairs t-tests and ANCOVA, groups were compared on several SKI measures, including the number of unique responses generated for each problem scenario, the quality of those responses, and the ability to select the best response from a set of alternatives. When equated for socioeconomic status (SES), ABI and control participants performed similarly on the SKI; however, a trend for children with ABI to generate more assertive responses was observed. Performance on the SKI was positively correlated with IQ and related to parent-reported adaptive behavior. In children with ABI, performance was also related to primary lesion location and treatment regimen. CONCLUSIONS: Children with ABI are as capable of judging the appropriateness of behavior and generating response options on an analog measure of social problem solving as were their typically developing peers. However, those individual children with ABI who are more likely to have social problems may be identified by the qualitative aspects of their responses on analog tasks. These findings have implications for the identification of children with social skills deficits following ABI and for the development of effective rehabilitation strategies.

Identification of viral mutants by mass spectrometry.

A method to identify mutations of virus proteins by using protein mass mapping is described. Comparative mass mapping was applied to a structural protein of the human rhinovirus Cys1199 --> Tyr mutant and to genetically engineered mutants of tobacco mosaic virus. The information generated from this approach can rapidly identify the peptide or protein containing the mutation and, in cases when nucleic acid sequencing is required, significantly narrows the region of the genome that must be sequenced. High-resolution matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and tandem mass spectrometry were used to identify amino acid substitutions. This method provides valuable information for those analyzing viral variants and, in some cases, offers a rapid and accurate alternative to nucleotide sequencing.

Antiviral agent blocks breathing of the common cold virus.

A dynamic capsid is critical to the events that shape the viral life cycle; events such as cell attachment, cell entry, and nucleic acid release demand a highly mobile viral surface. Protein mass mapping of the common cold virus, human rhinovirus 14 (HRV14), revealed both viral structural dynamics and the inhibition of such dynamics with an antiviral agent, WIN 52084. Viral capsid digestion fragments resulting from proteolytic time-course experiments provided structural information in good agreement with the HRV14 three-dimensional crystal structure. As expected, initial digestion fragments included peptides from the capsid protein VP1. This observation was expected because VP1 is the most external viral protein. Initial digestion fragments also included peptides belonging to VP4, the most internal capsid protein. The mass spectral results together with x-ray crystallography data provide information consistent with a "breathing" model of the viral capsid. Whereas the crystal structure of HRV14 shows VP4 to be the most internal capsid protein, mass spectral results show VP4 fragments to be among the first digestion fragments observed. Taken together this information demonstrates that VP4 is transiently exposed to the viral surface via viral breathing. Comparative digests of HRV14 in the presence and absence of WIN 52084 revealed a dramatic inhibition of digestion. These results indicate that the binding of the antiviral agent not only causes local conformational changes in the drug binding pocket but actually stabilizes the entire viral capsid against enzymatic degradation. Viral capsid mass mapping provides a fast and sensitive method for probing viral structural dynamics as well as providing a means for investigating antiviral drug efficacy.

Mass spectrometric methods for evaluating point mutations.

Two methods for internally calibrating spectra resulting from the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis of partially digested proteins are described. Partial digestion of proteins results in a large number of ion signals present in the MALDI-TOF mass spectrum, which in turn represent a significant over-sampling of each amino acid present in the analyte. This over-sampling allows ion signals of undisputed origin to be used as internal calibrants for the evaluation of fragments suspected to contain point mutations. Correlated with the correct amino acid sequence, the mass values of all ion signals (calibrants and analytes) are observed to fall into a single low-error data set. Conversely, empirically derived data applied to an incorrect sequence split the data into subsets of different errors. The methods take advantage of the self-consistent nature of data generated during the enzymatic mass mapping of proteins using MALDI-TOF, and they aid in the rapid, sensitive and accurate evaluation of point mutations present in proteins.

Applications of mass spectrometry in combinatorial chemistry.

This article describes the use of two mass spectrometric techniques, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry, toward a variety of challenging problems in drug discovery and identification. Quantitative ESI was used to screen for inhibitor activity of two different enzymatic glycosylation reactions resulting in the identification of the most effective inhibitors and the determination of their IC50 (inhibitor concentration at 50% inhibition). Also described is a combinatorial extraction method used with automated MALDI mass spectrometry to improve upon the clinical analysis of the immunosuppressant drug cyclosporin A (CsA). Optimization was performed by generating an array of solvent systems which were screened (by MALDI-MS) for the most efficient extraction of CsA from whole blood. Ultimately a 70/30 hexane:CHCl3 mixture was identified as the most efficient binary solvent system for such extractions. In addition it was demonstrated that peptides and carbohydrates, covalently linked to a polymeric support (through a photolabile linker), can be directly analyzed by MALDI in a single step which requires no pretreatment of the sample to induce cleavage from the support. The UV laser light in the MALDI experiment was used to simultaneously promote the analyte's photolytic cleavage from the solid support and its gas phase ionization for subsequent mass spectral analysis. Overall, the strength of mass spectrometry lies in its versatility, making it a powerful analytical technique with which to characterize the diversity of compounds found in combinatorial libraries.

Studies on reactogenicity and immunogenicity of attenuated bivalent cold recombinant influenza type A (CRA) and inactivated trivalent influenza virus (TI) vaccines in infants and young children.

Fifty-two infants seronegative to or without prior infection with influenza type A viruses were enrolled in a study to evaluate reactogenicity and immunogenicity of three bivalent cold recombinant type A (CRA) and two trivalent inactivated influenza (TI) vaccines. Controls consisted of infants receiving normal saline by nose drops (Pli.n.) or intramuscularly (Pli.m.). CRA and TI vaccines were monitored for local and systemic reactions after vaccination. Serum specimens obtained prior to and 6 weeks postvaccination were analysed for neutralizing antibody to influenza H1N1 and H3N2 viruses. CRA vaccines and Pli.n. recipients had similar numbers of acute respiratory infections and comparable rates of illnesses during the trial. Significantly fewer CRA vaccinees without an intercurrent viral infection had fever (0/16 versus 4/10, p = 0.04) and cough (4/16 versus 9/10, p = 0.002) than CRA vaccinees with a confirmed intercurrent viral infection. Recipients of TI vaccine and Pli.m. did not develop reactions at the injection site. For each of the CRA vaccines tested, a dominant CRA virus was identified. The dominant CRA viruses were isolated from a greater number of infants or for a longer duration than the non-dominant CRA viruses. All 14 non-dominant CRA viruses were recovered from infants within the first week after vaccination; 24 of 77 dominant CRA viruses were recovered more than 7 days after vaccination. The immunogenicity of CRA vaccines was not affected by a confirmed intercurrent viral infection or low titres of influenza-specific antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

Outcome of pediatric resuscitation.

A one-year prospective study of 105 resuscitations in 74 children was done at Milwaukee Children's Hospital. Resuscitation outcome was correlated to location of arrest, level of monitoring at time of arrest, and type of arrest. Type of arrest was the only analyzed variable that influenced outcome. This study indicates that outcome for children requiring resuscitation for respiratory arrest without cardiac arrest is reasonably good (25% mortality). However, the outcome for children requiring resuscitation for cardiac or cardiorespiratory arrest is poor (87% to 89% mortality or severe morbidity). Children who suffered a respiratory arrest without cardiac arrest had a better outcome than did adults. However, children who had a cardiac or cardiopulmonary arrest had the same poor outcome as did adults.

Use of 6-hydroxydopamine to deplete brain catecholamines in the rhesus monkey: effects on urinary catecholamine metabolites and behavior.

The purpose of this study was to determine: 1) whether 6-hydroxydopamine (6-OHDA), previously shown to deplete brain catecholamines (CA) in rodents, depletes brain CA in rhesus monkeys; 2) whether depletion of brain CA produces changes in behavior; and, 3) whether urinary output of 3-methoxy-4-hydroxyphenylglycol (MHPG) reflects brain norepinephrine (NE) depletions. Repeated intracerebroventricular (ICV) injection of 6-OHDA (N = 20; 15.5-73.3 mg/subject) produced chronic changes in social behavior and, at higher dosages, reduced output of urinary MHPG. However, 4 weeks after the last ICV 6-OHDA injection, urinary MHPG excretion returned to baseline values and whole brain CA content was not reliably different from control. A single treatment with 6-OHDA microinjected into the substantia nigra (SN) (N = 12; 120-240 microgram/subject) produced chronic whole brain depletions of brain CA without depleting serotonin. Reductions in brain CA were associated with a specific set of motor behaviors, aphagia, and adipsia. SN 6-OHDA produced greater brain NE depletions than ICV 6-OHDA, but urinary MHPG output was not reduced. SN 6-OHDA treated subjects showed chronic changes in social behavior and were more sensitive to the operant response rate decreasing effects of alpha-methyl-para-tyrosine (AMPT) than control subjects. Subjects with the largest depletions of brain dopamine (DA) (greater than 90%) were hypokinetic, rigid, and had a distal limb tremor. These results show that SN but not ICV injection of 6-OHDA can deplete brain CA in the rhesus monkey. The most prominent behavioral changes were characterized by disturbances in motor function. Urinary MPHG output does not reflect depletions of brain NE in this species.

Effects of imipramine treatment of separation-induced social disorders in rhesus monkeys.

Two groups of young rhesus monkeys were subjected to repetitive peer separations, a procedure that has been shown to produce depressivelike reactions in infant monkeys. Midway through the procedure one group was treated with the antidepressant imipramine hydrochloride, the other with a saline placebo. In comparison with placebo treatment, the imipramine treatment yielded significant behavioral improvement in a form and with a time course similar to that seen when the drug is given clinically to human depressives. We discuss the implications of the findings.

The effect of electrically induced convulsions on the behavior of normal and abnormal rhesus monkeys.

The social behavior of six juvenile rhesus monkeys was studied in a playroom before, during, and after a series of electrically induced convulsive (EIC) treatments. Three subjects had been reared in a normal environment, and showed the usual levels of social activity for monkeys of this age. Three were socially deprived early in life and showed higher levels of self-directed behaviors and lower levels of social behaviors. At different times all animals received both EIC ad sham EIC, three times per week for 4 weeks, and were observed in a playroom. In general the experimental subjects showed an increase in environmental activity as a consequence of EIC treatments while the control subjects showed decreases in environmental activity and in several social behaviors. The control subjects showed clear increases in self-disturbance behaviors while the experimental animals tended to show a decrease. The patterns of these changes thus showed clearly different characteristics of response to EIC and sham EIC as a function of early rearing condition. The results are discussed in terms of possible models for further study of the effects of EIC on physiological and social variables in a controlled laboratory setting.

Thyrotropin releasing hormone: antagonism of pentobarbital narcosis in the monkey.

The effect of TRH on pentobarbital narcosis in 21 rhesus monkeys was examined. Vital signs monitored included respiration rate, heart rate, temperature, sleeping time,and time of reappearance of certain reflexes. Blood samples were obtained for pentobarbital assay. Two dose schedules for TRH administration were used. One group of 6 animals received a single dose of 20 mg/kg 30 min after barbiturate administration, while the other group were received 3 injections of 20 mg/kg spaced at 30, 40 and 50 min after injection of pentobarbital. Both groups were sex balanced. TRH administration resulted in dramatically increased respiration and heart rates and arrested the progress of barbiturate induced hypothermia. The extended dose schedule prolonged increased respiration rate and a differential effect of TRH on pentobarbital induced hypothermia across sexes was observed. All animals regained reflexes sooner and sleeping time was reduced by 22%. No differences in pentobarbital blood levels with TRH were observed. These results extend earlier work in rodents to primates and suggest a possible use of TRH in cases of acute barbiturate intoxication.

Mother-infant separation in rhesus monkeys as a model of human depression. A reconsideration.

Nineteen rhesus monkeys between the ages of 5.9 and 8.5 months were separated from their mothers in five different studies. While in two of the studies, data indicated behavioral responses roughly parallel to Bowlby's protest-despair response to maternal separations, data across all five studies were sufficiently variable to bring this technique into serious question as a reliable and predictable animal model for neurobiologic and rehabilitative studies.

Kinetics and mechanisms of drug action on microorganisms XXII: Effects of aminosidine with and without organism pretreatment with bacteriostatic agents.

Escherichia coli generation in the logarithmic growth phase was inhibited in peptone broth USP at pH 7.0 without kill below 3.0 mug/ml of aminosidine. Above this value, the logarithms of the number of viables of the drug-treated culture ultimately decreased linearly with time and the slopes of these plots were independent of concentration. A concentration-dependent lag in the time of attainment of the cidal action was observed, and the extent of this lag was related to the ease of emergence of resistant organisms. The minimal concentration for cidal action increased with increasing concentrations of nutrients and with decreasing pH. Pretreatment of the cultures with novobiocin and tetracycline lessened the minimum bactericidal concentration of aminosidine whereas chloramphenicol pretreatment increased it. Tetracycline pretreatment inhibited the emergence of aminosidine-resistant organisms.

Abortifacient effects of prostaglandin E2 in the pigtailed macaque (Macaca nemestrina).

15(S)15-methyl-prostaglandin E2 was evaluated for abortifacient effects at various stages of pregnancy in five Macaca nemestrina. Despite its reported effectiveness in inducing abortions in human beings, it was concluded that this compound is not appropriate for collecting fetal material of known gestational age for morphologic and histologic examinations.