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Attention-deficit/hyperactivity disorder and obesity in US males and females, age 8-15 years: National Health and Nutrition Examination Survey 2001-2004

Tue, 06/16/2015 - 10:20am

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: Youth with ADHD may be at increased risk for obesity. Medications used to treat ADHD can affect weight. Few studies have investigated possible gender differences in associations between ADHD and obesity.

WHAT THIS STUDY ADDS: Nationally representative of US youth aged 8-15 years. Height and weight were measured, and ADHD assessed by structured diagnostic interview and parent report. Associations between ADHD and obesity are reported for males and females to enable gender comparisons.

OBJECTIVE: To investigate how associations between attention-deficit/hyperactivity disorder (ADHD) and obesity differ by gender and medication use in a nationally representative sample of US youth in which height and weight were measured.

METHODS: Youth age 8-15 (n = 3050) studied in the National Health and Nutrition Examination Survey 2001-2004. Obesity was defined as > /=95th percentile of US body mass index-for-age reference. ADHD was determined by asking parents if child had been diagnosed and using the Diagnostic Interview Schedule for Children IV. Gender-stratified multivariable logistic regression was used to estimate odds of obesity for youth with ADHD (medicated and unmedicated) relative to youth without ADHD.

RESULTS: Males with ADHD who were medicated had lower odds of obesity compared to males without ADHD (adjusted odds ratio [OR] = 0.42, 95% confidence interval [CI] = 0.23-0.78). Unmedicated males with ADHD were as likely as males without ADHD to be obese (adjusted OR = 1.02, 95% CI = 0.43-2.42). The odds of obesity for females taking medication for ADHD did not differ statistically from those of females without ADHD (adjusted OR = 1.21, 95% CI = 0.52-2.81). Females with ADHD not taking medication had odds of obesity 1.54 times those of females without ADHD; however, the 95% CI (0.79-2.98) was wide and not statistically significant at alpha = 0.05.

CONCLUSIONS: Associations between ADHD and obesity are influenced by treatment of ADHD with medication and may differ by gender. Youth with ADHD who are not treated with medication are as or more likely than youth without ADHD to be obese. the Study of Obesity.

Educating first responders to provide emergency services to individuals with disabilities

Tue, 06/16/2015 - 10:20am

OBJECTIVE: Individuals with disabilities experience more negative outcomes due to natural and manmade disasters and emergencies than do people without disabilities. This vulnerability appears to be due in part to knowledge gaps among public health and safety emergency planning and response personnel (responders). We assessed the effectiveness of an online program to increase emergency responder knowledge about emergency planning and response for individuals with disabilities.

METHODS: Researchers developed an online course designed to teach public health, emergency planning and management, and other first response personnel about appropriate, efficient, and equitable emergency planning, response, interaction, and communication with children and adults with disabilities before, during, and after disasters or emergencies. Course features included an ongoing storyline, exercises embedded in the form of real-life scenarios, and game-like features such as points and timed segments.

RESULTS: Evaluation measures indicated significant pre- to post-test gains in learner knowledge and simulated applied skills.

CONCLUSION: An online program using scenarios and simulations is an effective way to make disability-related training available to a wide variety of emergency responders across geographically disparate areas.

Disruption of sonic hedgehog signaling in Ellis-van Creveld dwarfism confers protection against bipolar affective disorder

Tue, 06/16/2015 - 10:20am

Ellis-van Creveld syndrome, an autosomal recessively inherited chondrodysplastic dwarfism, is frequent among Old Order Amish of Pennsylvania. Decades of longitudinal research on bipolar affective disorder (BPAD) revealed cosegregation of high numbers of EvC and Bipolar I (BPI) cases in several large Amish families descending from the same pioneer. Despite the high prevalence of both disorders in these families, no EvC individual has ever been reported with BPI. The proximity of the EVC gene to our previously reported chromosome 4p16 BPAD locus with protective alleles, coupled with detailed clinical observations that EvC and BPI do not occur in the same individuals, led us to hypothesize that the genetic defect causing EvC in the Amish confers protection from BPI. This hypothesis is supported by a significant negative association of these two disorders when contrasted with absence of disease (P=0.029, Fisher's exact test, two-sided, verified by permutation to estimate the null distribution of the test statistic). As homozygous Amish EVC mutations causing EvC dwarfism do so by disrupting sonic hedgehog (Shh) signaling, our data implicate Shh signaling in the underlying pathophysiology of BPAD. Understanding how disrupted Shh signaling protects against BPI could uncover variants in the Shh pathway that cause or increase risk for this and related mood disorders.

Generalized Identity Matching to Sample after Multiple-Exemplar Training in Capuchin Monkeys

Tue, 06/16/2015 - 10:20am

A multiple-exemplar identity matching-to-sample baseline was established to encourage development of generalized IDMTS performances in three adult male capuchins. Mask (blank comparison) or Shuffled S- procedures were used to promote select (sample-S+) control in baseline relations and to assess stimulus control relations in generalized IDMTS tests. The IDMTS baseline comprised eight 3-stimulus sets or four 4-stimulus sets. Probe trials with new stimulus sets were substituted for baseline sets in successive testing sessions and subsequently converted to new baseline relations. All monkeys exhibited high accuracy on generalized IDMTS tests. A monkey who was given the Mask procedure in training and tests showed generalized IDMTS with select relations predominating. Two monkeys who were given training and testing with the Shuffled S- procedure performed somewhat better on Shuffled S- IDMTS test trials than on test trials that contained non-shuffled test IDMTS trials thus suggesting that exclusion of familiar nonmatching comparison stimuli from baseline in Shuffled S-test trials contributed to the higher accuracy scores with the former procedures. Development of select relations appeared to be a positive predictor of development of generalized IDMTS.

Blank-comparison matching-to-sample reveals a false positive symmetry test in a capuchin monkey

Tue, 06/16/2015 - 10:20am

A positive symmetry test result was obtained with a capuchin monkey that had previously exhibited virtually errorless AB and BA arbitrary matching-to-sample (MTS) with different stimuli. The symmetry test (BA) followed the acquisition of a new AB relation. It seemed possible, however, that the positive result could have occurred through the exclusion of previously defined comparison stimuli and not because the new AB and BA relations had the property of symmetry. To assess this possibility, a blank-comparison MTS procedure was implemented that permitted the separate assessment of select and reject (i.e., exclusion) control with both baseline and BA matching relations. In this assessment, the monkey did not exhibit reliable BA matching when exclusion was not possible, thus showing that the symmetry result was a false positive. However, the study demonstrated the feasibility of using a blank comparison MTS procedure with capuchins. The present results may set the stage for more successful methodology for establishing desired forms of relational stimulus control in capuchins and ultimately improving the assessment of relational learning capacity in that species, other nonhuman species, and nonverbal humans.

Emergency preparedness of families of children with developmental disabilities: what public health and safety emergency planners need to know

Tue, 06/16/2015 - 10:20am

OBJECTIVE: To assess the emergency preparedness knowledge, behaviors, and training needs of families of children with developmental disabilities (DD).

DESIGN: An online survey.

PARTICIPANTS: A sample of 314 self-selecting US parents/guardians of children with DD, aged birth-21 years. MAIN

OUTCOME MEASURES: 1) Preparedness self-assessment; 2) self-report regarding the extent to which families followed 11 specific preparedness action steps derived from publicly available preparedness guides; and 3) parent training and support needs.

RESULTS: Although most participants assessed themselves to be somewhat to moderately well prepared, even those who reported being "very well prepared" had taken fewer than half of 11 recommended action steps. Most participants expressed a need for preparedness support; virtually all the respondents felt that training was either important or very important.

CONCLUSIONS: Children with disabilities are known to be particularly vulnerable to negative disaster impacts. Overall, parents in this study appeared under-prepared to meet family disaster needs, although they recognized its importance. The results suggest opportunities and methods for public health and safety planning, education and outreach to parents of children with DD who would benefit from targeted training such as information and skill building to develop effective family preparedness plans and connections to local emergency management and responders.

Interacting with the National Database for Autism Research (NDAR) via the LONI Pipeline workflow environment

Tue, 06/16/2015 - 10:20am

Under the umbrella of the National Database for Clinical Trials (NDCT) related to mental illnesses, the National Database for Autism Research (NDAR) seeks to gather, curate, and make openly available neuroimaging data from NIH-funded studies of autism spectrum disorder (ASD). NDAR has recently made its database accessible through the LONI Pipeline workflow design and execution environment to enable large-scale analyses of cortical architecture and function via local, cluster, or "cloud"-based computing resources. This presents a unique opportunity to overcome many of the customary limitations to fostering biomedical neuroimaging as a science of discovery. Providing open access to primary neuroimaging data, workflow methods, and high-performance computing will increase uniformity in data collection protocols, encourage greater reliability of published data, results replication, and broaden the range of researchers now able to perform larger studies than ever before. To illustrate the use of NDAR and LONI Pipeline for performing several commonly performed neuroimaging processing steps and analyses, this paper presents example workflows useful for ASD neuroimaging researchers seeking to begin using this valuable combination of online data and computational resources. We discuss the utility of such database and workflow processing interactivity as a motivation for the sharing of additional primary data in ASD research and elsewhere.

The Three NITRCs: A Guide to Neuroimaging Neuroinformatics Resources

Tue, 06/16/2015 - 10:20am

Information management is critical as the landscape of neuroscience related shared resources (data, software, computation, etc.) expands. Since 2006, the Neuroimaging Informatics Tools and Resources Clearinghouse (NITRC: RRID:nif-0000-00202) has provided a comprehensive support infrastructure for resources in the neuroimaging domain. Funded by the NIH Blueprint for Neuroscience Research as well as four NIH institutes , NITRC’s mission is to facilitate finding and comparing neuroimaging resources for neuroimaging analyses. Over the years the scope of these resources has expanded to support scientific domains from MR to PET, SPECT, CT, MEG/EEG, optical imaging, genetic imaging, clinical neuroinformatics and computational neuroscience. A broad set of initiatives have been developed to support these research areas.

Hippocampal volume reduction in female but not male recent abstinent methamphetamine users

Tue, 06/16/2015 - 10:20am

Growing evidence suggests abnormalities in brain morphology including hippocampal structure in patients with methamphetamine (MA) dependence. This study was performed to examine hippocampal volume in abstinent MA users, and to further explore its relationship with cognitive function. 30 abstinent MA users (20 males and 10 females) with average 5.52 months of duration of abstinence and 29 healthy controls (19 males and 10 females) age 18-45 years old were recruited for clinical assessment and imaging scan. FreeSurfer was used to segment the hippocampus bilaterally, and hippocampal volumes were extracted for group and gender comparisons. Cognitive function was measured using the CogState Battery Chinese language version (CSB-C). Analysis of covariance (ANCOVA) controlling for education showed a significant group by gender interaction for the right hippocampal relative volume adjusted for total brain size (p=0.020); there was a significant difference between male controls and female controls (p < 0.001), but such a difference did not exist between male patients and female patients (p=0.203). No significant correlations were found between hippocampal volume and cognitive measures. There seems to be a gender difference in how MA affects hippocampal volume in abstinent MA users. Hippocampus might be an important treatment target for cognitive improvement and functional recovery in this patient population, especially in females.

Quantitative Measures of Craniofacial Dysmorphology in a Family Study of Schizophrenia and Bipolar Illness

Tue, 06/16/2015 - 10:20am

Several laboratories, including ours, have reported an overrepresentation of craniofacial (CF) anomalies in schizophrenia (SZ). How might this dysmorphology arise in a brain-based disorder? Because the brain and face derive from shared embryologic primordia and morphogenetic forces, maldevelopmental processes may result in both CF and brain dysmorphology. Our approach is 2-pronged. First, we have employed, for the first time in the study of psychiatric disorders, objective measures of CF morphology that utilize an extensive normative database, permitting computation of standardized scores for each subject. Second, we have rendered these findings biologically interpretable by adopting principles of embryology in the analysis of dysmorphology. Dependent measures in this investigation focused on derivatives of specific embryonic primordia and were contrasted among probands with psychotic disorders, their first-degree relatives, and normal controls (NC). Subject groups included patients with a diagnosis of SZ (N = 39) or bipolar (BP) disorder with psychotic features (N = 32), their clinically unaffected relatives (N = 82 and N = 41, respectively), and NC (N = 95) subjects. Anomalies involving derivatives of frontonasal and mandibular embryonic primordia showed a clear association with psychotic illness, as well as familial aggregation in relatives in both diagnostic groups. In contrast, one class of CF anomalies emerged only among SZ probands and their first-degree relatives: dysmorphology arising along the junction of the frontonasal and maxillary prominence derivatives, manifested as marked asymmetries. This class was not overrepresented among the BP patients nor among their relatives, indicating that this dysmorphology appears to be specific to SZ and not a generalized feature of psychosis. We discuss these findings in light of embryologic models that relate brain regions to specific CF areas.

Physical Activity Enjoyment, Perceived Barriers, and Beliefs Among Adolescents With and Without Intellectual Disabilities

Tue, 06/16/2015 - 10:20am

BACKGROUND: Youth with intellectual disabilities (ID) exhibit low levels of physical activity, but the underlying contributors to behavior are unclear. We compared physical activity enjoyment, perceived barriers, beliefs, and self-efficacy among adolescents with ID and typically developing (TD) adolescents.

METHODS: A questionnaire was administered to 38 adolescents with ID (mean age 16.8 years) and 60 TD adolescents (mean age 15.3 years). Of the original 33 questionnaire items, 23 met the test-retest reliability criteria and were included in the group comparisons.

RESULTS: Fewer adolescents with ID reported that they have someone to do physical activity with (64% vs. 93%, p < 0.001), and a greater proportion of adolescents with ID perceived that physical activities were too hard to learn (41% vs. 0%, p < 0.001). Fewer adolescents with ID believed that physical activity is good for their health (92% vs. 100%, p=0.05). More adolescents with ID reported a dislike of individual physical activities (p=0.02). A large proportion of adolescents with ID (84%) responded that they were good at doing physical activities, but the difference between groups was only of borderline significance. (95% of TD adolescents, p=0.06).

CONCLUSIONS: Adolescents shared many of the same perceptions about physical activity, but some important differences between groups were identified.

Barriers to Physical Activity in Children With Autism Spectrum Disorders: Relationship to Physical Activity and Screen Time

Tue, 06/16/2015 - 10:20am

BACKGROUND: Individual, social, and community barriers to physical activity (PA) experienced by children with autism spectrum disorder (ASD) make PA participation more difficult and may contribute to increased screen time. METHODS: We compared the prevalence of parent-reported barriers to PA among 58 typically developing (TD) children and 53 children with an ASD, 3-11 years, and assessed the association between barriers and PA participation and screen time among children with ASD. RESULTS: Parents of children with ASD reported significantly more barriers than parents of TD children. Based on parent-report, 60% of children with ASD required too much supervision compared to no TD children (p<0.001). Parents of children with ASD were more likely to report that adults lack skills needed to include their child (58%), that their child has few friends (45%), and that other children exclude their child (23%). The number of parent-reported barriers to PA was inversely correlated with the hours spent in PA per year (r=-0.27, p=0.05) and positively related to total screen time (r=0.32, p < 0.03). CONCLUSIONS: These findings underscore the need for community-based PA programs designed to meet the special requirements of this population and policies that compel schools and other government-supported organizations for inclusion and/or targeted programming.

Relationship among Glutamine, gamma-Aminobutyric Acid, and Social Cognition in Autism Spectrum Disorders

Tue, 06/16/2015 - 10:20am

OBJECTIVE: An imbalance of excitatory and inhibitory neurotransmission in autism spectrum disorder (ASD) has been proposed. We compared glutamate (Glu), glutamine (Gln), and gamma-aminobutyric acid (GABA) levels in the anterior cingulate cortex (ACC) of 13 males with ASD and 14 typically developing (TD) males (ages 13-17), and correlated these levels with intelligence quotient (IQ) and measures of social cognition.

METHODS: Social cognition was evaluated by administration of the Social Responsiveness Scale (SRS) and the Reading the Mind in the Eyes Test (RMET). We acquired proton magnetic resonance spectroscopy ((1)H-MRS) data from the bilateral ACC using the single voxel point resolved spectroscopy sequence (PRESS) to quantify Glu and Gln, and Mescher-Garwood point-resolved spectroscopy sequence (MEGA-PRESS) to quantify GABA levels referenced to creatine (Cr).

RESULTS: There were higher Gln levels (p=0.04), and lower GABA/Cre levels (p=0.09) in the ASD group than in the TD group. There was no difference in Glu levels between groups. Gln was negatively correlated with RMET score (rho=-0.62, p=0.001) and IQ (rho=-0.56, p=0.003), and positively correlated with SRS scores (rho=0.53, p=0.007). GABA/Cre levels were positively correlated with RMET score (rho=0.34, p=0.09) and IQ (rho=0.36, p=0.07), and negatively correlated with SRS score (rho=-0.34, p=0.09).

CONCLUSIONS: These data suggest an imbalance between glutamatergic neurotransmission and GABA-ergic neurotransmission in ASD. Higher Gln levels and lower GABA/Cre levels were associated with lower IQ and greater impairments in social cognition across groups.

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Mon, 06/15/2015 - 1:01pm

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

Broad spectrum aminoglycoside phosphotransferase type III from Enterococcus: overexpression, purification, and substrate specificity

Mon, 06/15/2015 - 12:02pm

The aminoglycoside phosphotransferases (APHs) are responsible for the bacterial inactivation of many clinically useful aminoglycoside antibiotics. We report the characterization of an enterococcal enzyme, APH(3')-IIIa, which inactivates a broad spectrum of aminoglycosides by ATP-dependent O-phosphorylation. Overproduction of APH(3')-IIIa has permitted the isolation of 30-40 mg of pure protein/(L of cell culture). Purified APH(3')-IIIa is a mixture of monomer and dimer which is slowly converted to dimer only over time. Dimer could be dissociated into monomer by incubation with 2-mercaptoethanol, suggesting that dimerization is mediated by formation of disulfide bond(s). Both monomer and dimer show Km values in the low micromolar range for good substrates such as kanamycin and neomycin, and kcat values of 1-4 s-1. All aminoglycosides show substrate inhibition except amikacin and kanamycin B. Determination of minimum inhibitory concentrations indicates a positive correlation between antibiotic activity and kcat/Km, but not with Km or kcat. NMR analysis of phosphorylated kanamycin A has directly demonstrated regiospecific phosphoryl transfer to the 3'-hydroxyl of the 6-aminohexose ring of the antibiotic. Analysis of structure-activity relationships with a variety of aminoglycosides has revealed that the deoxystreptamine aminocyclitol ring plays a critical role in substrate binding. This information will form the basis for future design of inhibitors of APH(3')-IIIa.

Regiospecificity of aminoglycoside phosphotransferase from Enterococci and Staphylococci (APH(3')-IIIa)

Mon, 06/15/2015 - 12:02pm

The broad-spectrum aminoglycoside phosphotransferase, APH(3')-IIIa, confers resistance to several aminoglycoside antibiotics in opportunistic pathogens of the genera Staphylococcus and Enterococcus. The profile of the drug resistance phenotype suggested that the enzyme would transfer a phosphate group from ATP to the 3'-hydroxyl of aminoglycosides. In addition, resistance to the 3'-deoxyaminoglycoside antibiotic, lividomycin A, suggested possible transfer to the 5"-hydroxyl of the ribose [Trieu-Cuot, P., and Courvalin, P. (1983) Gene 23, 331-341]. Using purified overexpressed enzyme, we have prepared and purified the products of APH(3')-IIIa-dependent phosphorylation of several of aminoglycoside antibiotics. Mass spectral analysis revealed that 4,6-disubstituted aminocyclitol antibiotics such as amikacin and kanamycin are monophosphorylated, while 4,5-disubstituted aminoglycosides such as butirosin A, ribostamycin, and neomycin B are both mono- and diphosphorylated by APH(3')-IIIa. Using a series of one- and two-dimensional 1H, 13C, and 31P NMR experiments, we have unambiguously assigned the regiospecificity of phosphoryl transfer to several antibiotics. The 4,6-disubstituted aminocyclitol antibiotics are exclusively phosphorylated at the 3'-OH hydroxyl, and the 4,5-disubstituted aminocyclitol antibiotics can be phosphorylated at both the 3'- and 5"-hydroxyls. The first phosphorylation can occur on either the 3'- or 5"-hydroxyl group of neomycin B or butirosin A. Initial phosphotransfer to the 3'-position predominates for butirosin while the 5"-OH is favored for neomycin. These results open the potential for the rational design of aminoglycoside kinase inhibitors based on functionalization of either the 6-aminohexose or the pentose rings of aminoglycoside antibiotics.

Mechanism of aminoglycoside 3'-phosphotransferase type IIIa: His188 is not a phosphate-accepting residue

Mon, 06/15/2015 - 12:02pm

BACKGROUND: The enzyme aminoglycoside 3'-phosphotransferase Type IIIa (APH(3')-IIIa), confers resistance to many aminoglycoside antibiotics by regiospecific phosphorylation of their hydroxyl groups. The chemical mechanism of phosphoryl transfer is unknown. Based on sequence homology, it has been suggested that a conserved His residue, His188, could be phosphorylated by ATP, and this phospho-His would transfer the phosphate to the incoming aminoglycoside. We have used chemical modification, site-directed mutagenesis and positional isotope exchange methods to probe the mechanism of phosphoryl transfer by APH(3')-IIIa.

RESULTS: Chemical modification by diethylpyrocarbonate implicated His in aminoglycoside phosphorylation by APH(3')-IIIa. We prepared His to Ala mutants of all four His residues in APH(3')-IIIa and found minimal effects of the mutations on the steady-state phosphorylation of several aminoglycosides. One of these mutants, His188Ala, was largely insoluble when compared to the wild-type enzyme. Positional isotope exchange experiments using gamma-[18O]-ATP did not support a double-displacement mechanism.

CONCLUSIONS: His residues are not required for aminoglycoside phosphorylation by APH(3')-IIIa. The conserved His 188 is thus not a phosphate accepting residue but does seem to be important for proper enzyme folding. Positional isotope exchange experiments are consistent with direct attack of the aminoglycoside hydroxyl group on the gamma-phosphate of ATP.

Structure of an enzyme required for aminoglycoside antibiotic resistance reveals homology to eukaryotic protein kinases

Mon, 06/15/2015 - 12:02pm

Bacterial resistance to aminoglycoside antibiotics is almost exclusively accomplished through either phosphorylation, adenylylation, or acetylation of the antibacterial agent. The aminoglycoside kinase, APH(3')-IIIa, catalyzes the phosphorylation of a broad spectrum of aminoglycoside antibiotics. The crystal structure of this enzyme complexed with ADP was determined at 2.2 A. resolution. The three-dimensional fold of APH(3')-IIIa reveals a striking similarity to eukaryotic protein kinases despite a virtually complete lack of sequence homology. Nearly half of the APH(3')-IIIa sequence adopts a conformation identical to that seen in these kinases. Substantial differences are found in the location and conformation of residues presumably responsible for second-substrate specificity. These results indicate that APH(3') enzymes and eukaryotic-type protein kinases share a common ancestor.

Spectinomycin kinase from Legionella pneumophila. Characterization of substrate specificity and identification of catalytically important residues

Mon, 06/15/2015 - 12:02pm

The bacterium Legionella pneumophila is the responsible agent for Legionnaires' disease and has recently been shown to harbor a gene encoding a kinase that confers resistance to the aminoglycoside antibiotic spectinomycin (Suter, T. M., Viswanathan, V. K., and Cianciotto, N. P. (1997) Antimicrob. Agents Chemother. 41, 1385-1388). We report the overproduction, purification, and characterization of this spectinomycin kinase from an expressing system in Escherichia coli. The purified protein shows stringent substrate specificity for spectinomycin with Km = 21.5 microM and kcat = 24.2 s-1 and does not bind other aminoglycosides including kanamycin, amikacin, neomycin, butirosin, streptomycin, or apramycin. Purification of spectinomycin phosphate followed by characterization by mass spectrometry and 1H, 13C, and 31P NMR established the site of phosphorylation to be at the hydroxyl group at position 9. Thus this enzyme is designated APH(9)-Ia (where APH is aminoglycoside kinase). The enzyme was inactivated by the electrophilic ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine, consistent with a nucleophilic residue such as Lys lining the nucleotide binding pocket. Site-directed mutagenesis of Lys-52 and Asp-212 to Ala confirmed that these residues were important for catalysis, with Lys-52 playing a potential role in ATP binding and Asp-212 in phosphoryl transfer. Thio and solvent isotope effect experiments in the presence of either Mg2+ or Mn2+ were consistent with a kinetic mechanism in which phosphate transfer does not contribute significantly to the rate-limiting step. These results establish that APH(9)-Ia is a highly specific antibiotic resistance kinase and provides the requisite mechanistic information for future structural studies.

Aminoglycoside phosphotransferases: proteins, structure, and mechanism

Mon, 06/15/2015 - 12:02pm

Aminoglycoside antibiotics constitute an important class of clinically useful drugs which are imperiled by the emergence of resistant organisms. Aminoglycoside resistance in the clinics is primarily due to the presence of modifying enzymes which N-acetylate, O-adenylate or O-phosphorylate the antibiotics. The latter family of enzymes are termed the aminoglycoside phosphotransferases or kinases and are the subject of this review. There are seven classes of aminoglycoside phosphotransferases (APH(3'), APH(2''), APH(3'off'), APH(6), APH(9), APH(4), APH(7'')) and many isozymes in each class, and although there is very little overall general sequence homology among these enzymes, certain signature residues and sequences are common. The recent determination of the three-dimensional structure of the broad spectrum aminoglycoside kinase APH(3')-IIIa complexed with the product ADP, in addition to mechanistic and mutagenic studies on this and related enzymes, has added a great deal to our understanding of this class of antibiotic resistance enzyme. In particular, the revelation of structural and mechanistic similarities between APHs and Ser/Thr and Tyr kinases has set the stage for future inhibition studies which could prove important in reversing aminoglycoside resistance.