Mutant small heat shock protein B3 causes motor neuropathy: utility of a candidate gene approach.

OBJECTIVE: Idiopathic peripheral neuropathy is common and likely due to genetic factors that are not detectable using standard linkage analysis. We initiated a candidate gene approach to study the genetic influence of the small heat shock protein (sHSP) gene family on an axonal motor and motor/sensory neuropathy patient population. METHODS: The promoter region and all exonic and intronic sequences of the 10 sHSP genes (HSPB1-HSPB10) were screened in a cohort of presumed nonacquired, axonal motor and motor/sensory neuropathy patients seen at the Ohio State University Neuromuscular Clinic. RESULTS: A missense mutation in the gene encoding small heat shock protein B3 (HSPB3, also called HSP27, protein 3) was discovered in 2 siblings with an asymmetric axonal motor neuropathy. Electrophysiologic studies revealed an axonal, predominantly motor, length-dependent neuropathy. The mutation, HSPB3(R7S), is located in the N-terminal domain and involves the loss of a conserved arginine. CONCLUSIONS: The discovery of an HSPB3 mutation associated with an axonal motor neuropathy using a candidate gene approach supports the notion that the small heat shock protein gene family coordinately plays an important role in motor neuron viability.

SMN mRNA and protein levels in peripheral blood: biomarkers for SMA clinical trials.

BACKGROUND: Clinical trials of drugs that increase SMN protein levels in vitro are currently under way in patients with spinal muscular atrophy. OBJECTIVE: To develop and validate measures of SMN mRNA and protein in peripheral blood and to establish baseline SMN levels in a cohort of controls, carriers, and patients of known genotype, which could be used to follow response to treatment. METHODS: SMN1 and SMN2 gene copy numbers were determined in blood samples collected from 86 subjects. Quantitative reverse transcription PCR was used to measure blood levels of SMN mRNA with and without exon 7. A cell immunoassay was used to measure blood levels of SMN protein. RESULTS: Blood levels of SMN mRNA and protein were measured with high reliability. There was little variation in SMN levels in individual subjects over a 5-week period. Levels of exon 7-containing SMN mRNA and SMN protein correlated with SMN1 and SMN2 gene copy number. With the exception of type I SMA, there was no correlation between SMN levels and disease severity. CONCLUSION: SMN mRNA and protein levels can be reliably measured in the peripheral blood and used during clinical trials in spinal muscular atrophy, but these levels do not necessarily predict disease severity.

Light scattering and transmission electron microscopy studies reveal a mechanism for calcium/calmodulin-dependent protein kinase II self-association.

Calmodulin (CaM)-kinase II holoenzymes composed of either alpha or beta subunits were analyzed using light scattering to determine a mechanism for self-association. Under identical reaction conditions, only alphaCaM-kinase II holoenzymes self-associated. Self-association was detected at a remarkably low enzyme concentration (0.14 microM or 7 microg/mL). Light scattering revealed two phases of self-association: a rapid rise that peaked, followed by a slower decrease that stabilized after 2-3 min. Electron microscopy identified that the rapid rise in scattering was due to the formation of loosely packed clusters of holoenzymes that undergo further association into large complexes of several microns in diameter over time. Self-association required activation by Ca(2+)/CaM and was strongly dependent on pH. Self-association was not detected at pH 7.5, however, the extent of this process increased as reaction pH decreased below 7.0. A peptide substrate (autocamtide-2) and inhibitor (AIP) designed from the autoregulatory domain of CaM-kinase II potently prevented self-association, whereas the peptide substrate syntide-2 did not. Thus, CaM-kinase II self-association is isoform specific, regulated by the conditions of activation, and is inhibited by peptides that bind to the catalytic domain likely via their autoregulatory-like sequence. A model for CaM-kinase II self-association is presented whereby catalytic domains in one holoenzyme interact with the regulatory domains in neighboring holoenzymes. These intersubunit-interholoenzyme autoinhibitory interactions could contribute to both the translocation and inactivation of CaM-kinase II previously reported in models of ischemia.

Psychometric evaluation of an inpatient psychiatric care consumer satisfaction survey.

This study assessed the psychometric properties of a questionnaire designed to measure consumer satisfaction with inpatient psychiatric care. To this end, 37 inpatient psychiatric units from across the United States agreed to participate. The questionnaire was completed by 1,351 individuals, or a responsible party, for an average response rate of 53%. The factor analysis identified six scales: Nonclinical Services, Psychiatric Care, Staff, Medical Outcome, Patient Education, and Program Components/Activities. The internal reliability of the scales was high to moderate (.88 to .74). Results of a stepwise regression model showed good criterion-related validity, explaining 58% of the variance in overall quality ratings. Little shrinkage in this variance occurred when the model was cross-validated. Also, differences in satisfaction levels were noted for select facility and consumer characteristics. Results are interpreted as providing support for the reliability and validity of a newly developed consumer satisfaction survey for use in evaluating inpatient psychiatric care.

Identification of domains essential for the assembly of calcium/calmodulin-dependent protein kinase II holoenzymes.

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), as isolated from brain, is a multimeric complex composed predominantly of two subunits, alpha and beta, products of unique genes. Little is known about how subunit composition influences holoenzyme structure or how the domain(s) of each subunit interact to form holoenzymes. We show here that holoenzymes composed of only alpha or only beta subunits exhibit different biophysical properties. The S values of alpha and beta are 17.2 and 14.5 S while the Stokes's radii are 85 and 111 A, respectively, indicating their structures are different. C-terminal truncations of the alpha subunit show that amino acids 382-478 are necessary for holoenzyme formation and that amino acids 427-478 contribute to holoenzyme stability. Additionally, the C-terminal domains of both the alpha subunit, alpha315-478, and beta subunit, beta314-542, formed oligomers indicating the sufficiency of the C-terminal domain for multimer formation. Using the yeast two-hybrid system we show, in vivo, that full-length subunits, alpha1-478 and beta1-542, interact with themselves or each other interchangeably. Additionally, the C-terminal domains of the alpha subunit, alpha315-478 and beta subunit, beta314-542 associated with themselves in a manner indistinguishable from their association with full-length alpha or beta subunits. Further studies revealed that the C-terminal domains of the alpha and beta subunits contain information necessary for interaction with beta but not alpha. These data are summarized into a model describing the assembly of CaM kinase II holoenzymes.

Inactivation and self-association of Ca2+/calmodulin-dependent protein kinase II during autophosphorylation.

The time-dependent loss in enzyme activity associated with the autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase) was altered by both pH and ATP concentration. These parameters also influenced the extent to which soluble CaM-kinase undergoes self-association to form large aggregates of sedimentable enzyme. Specifically, autophosphorylation of CaM-kinase in 0.01 mM ATP at pH 6.5 resulted in the formation of sedimentable enzyme and a 70% loss of enzyme activity. Under similar conditions at pH 7.5, the enzyme lost only 30% of its activity, and no sedimentable enzyme was detected. In contrast to 0.01 mM ATP, autophosphorylation of CaM-kinase at pH 6.5 in 1 mM ATP did not result in a loss of activity or the production of sedimentable enzyme, even though the stoichiometry of autophosphorylation was comparable. Electron microscopy studies of CaM-kinase autophosphorylated at pH 6.5 in 0.01 mM ATP revealed particles 100-300 nm in diameter that clustered into branched complexes. Inactivation and self-association of CaM-kinase were influenced by the conditions of autophosphorylation in vitro, suggesting that both the catalytic and physical properties of the enzyme may be sensitive to fluctuations in ATP concentration and pH in vivo.

Ca2+/calmodulin kinase II translocates in a hippocampal slice model of ischemia.

Rat hippocampal slices were exposed to conditions that simulate an ischemic insult, and the subcellular distribution and the enzymatic activity of Ca2+/calmodulin-dependent protein kinase II (CaM kinase) were monitored. Semiquantitative western blots using a monoclonal antibody to the 50-kDa alpha subunit showed that there was a significant redistribution of the enzyme from a supernatant to a pellet fraction after 10 min of an anoxic/aglycemic insult. No significant change in the total amount of CaM kinase enzyme was detected in the homogenates for up to 20 min of exposure to the insult. Ca2+/CaM-dependent enzyme activity did not significantly change in the pellet during the 20-min insult. Supernatant activity decreased throughout the insult. The persistence of Ca2+/CaM-dependent CaM kinase activity in the pellet fraction and the detected movement of enzyme from the supernatant to the pellet indicate that redistribution may be an important mechanism in regulating the cellular location of CaM kinase activity.

Channel permeant cations compete selectively with noncompetitive inhibitors of the nicotinic acetylcholine receptor.

Previous work suggests that noncompetitive inhibitor (NCI) ligands and channel permeant cations bind to sites within the nicotinic acetylcholine receptor ion channel. We have used ethidium as a fluorescent probe of the NCI site to investigate interactions between NCI ligands and channel permeant cations. We found that ethidium can be completely displaced from the receptor by a variety of inorganic monovalent and divalent cations. The rank order of monovalent cation affinities was found to be Tl+ greater than Rb+ greater than or equal to K+ greater than Cs+ greater than Na+ greater than Li+. The monovalent cation Kd values vary markedly over a 40-fold range, from 3 to 121 mM. The Kd values and rank order correspond to values determined previously from electrophysiological data. Hill plots of the back titrations yield slopes of 1.0 for all monovalent cations, indicating a single class of independent sites, as shown previously for NCI ligands. Scatchard analysis of ethidium binding in the presence of Tl+ reveals a reduction in affinity and no changes in the maximal number of sites. In the presence of agonist the kinetics of ethidium dissociation induced by the addition of phencyclidine or cations alone or the simultaneous addition of both are nearly identical. The ethidium dissociation rate induced by either phencyclidine or cations is regulated by the occupation of the agonist sites in a similar manner. These results indicate that the effect of cations on NCI ligand binding occurs by mutually exclusive competition. We suggest that NCIs can regulate cation binding at a physiological cation recognition site that is likely part of the cation permeation path through the receptor channel.