Chromosome 11p15 paternal isodisomy in focal forms of neonatal hyperinsulinism.

CONTEXT: Focal forms of congenital hyperinsulinism are due to a constitutional heterozygous mutation of paternal origin in the ABCC8 gene, more often than the KCNJ11 gene, located in the 11p15.1 region. This mutation is associated with the loss of the maternally inherited 11p15.1 to 11p15.5 region in the lesion. We investigated the possible occurrence of a compensatory duplication of the paternal 11p15.1-11p15.5 region. MATERIALS AND METHODS: A combined immunohistochemistry and fluorescent in situ hybridization study on beta-cell interphase nuclei with probes covering two genes located in this region (ABCC8 and CDKN1C genes) was performed in four cases of focal forms of hyperinsulinism. RESULTS: beta-Cells in the lesions of four cases of focal congenital hyperinsulinism were diploid for chromosomes 11 and 13. The 11p15.1 to 11p15.2 and 11p15.4 to 11p15.5 regions containing ABCC8 and CDKN1C genes, respectively, were present with two copies. Loss of the maternal allele was confirmed in these focal lesions with microsatellite markers flanking the ABCC8 and CDKN1C genes, whereas a heterozygous mutation in the ABCC8 gene was inherited from the father. CONCLUSIONS: There is a duplication of the paternal allele on chromosome 11 in the focal forms of hyperinsulinism lesion. The paternal isodisomy observed rendered the beta-cells homozygous for ABCC8 mutation and harbored a K-channel defect in the lesion similar to that observed in diffuse forms of congenital hyperinsulinism.

NMR and fluorescence spectroscopy approaches to secondary and primary active multidrug efflux pumps.

Multidrug efflux pumps are found in all major transporter families. Along with a lack of three-dimensional structure information, the mechanism of drug recognition, energy coupling with drug translocation and the catalytic cycle are so far not understood. In the present study, we present first data of a fluorescence-based assay to study the pH-gradient-mediated activity of the multidrug antiporter EmrE, by co-reconstitution with the light-driven proton pump bacteriorhodopsin. In addition to biochemical approaches, the emerging technique, solid-state NMR, can be used for the investigation of these transporters. A number of experiments based on MAS (magic angle sample spinning) NMR are available to provide data on protein structure and dynamics, drug binding and protein-lipid interactions. However, these experiments dictate a number of constraints with respect to sample preparation that will be discussed for proteins from the SMR (small multidrug resistance transporter) family. In addition, 2H-NMR is used to probe protein mobility of Lactococcus lactis ABC transporter, LmrA.

A CGH study of 27 patients with CHARGE association.

CHARGE association is a non-random occurrence of congenital malformations including coloboma, heart disease, choanal atresia, retarded growth and/or retarded development, genital hypoplasia, ear anomalies and/or deafness. The cause of this association remains unknown. Various genetic mechanisms have been proposed, including a contiguous gene syndrome but, so far, no recurrent locus has been identified. To address this question, we decided to perform a comparative genomic hybridization (CGH) study on a cohort of 27 patients with CHARGE association and a normal standard karyotype. We found two chromosomal anomalies: a der(9)t(9;13) derived from a paternal translocation and a der(6)t(4;6) of unknown origin. This suggests that chromosome imbalances may well mimic CHARGE association. Therefore patients with CHARGE association must be carefully tested with classical and molecular cytogenetic techniques to detect a potential chromosome imbalance. It is expected that more stringent diagnostic criteria of CHARGE association could define a more homogeneous group of patients where a single genetic cause might be identified.

In vitro studies of membrane protein folding.

The study of membrane protein folding is a new and challenging research field. Consequently, there are few direct studies on the in vitro folding of membrane proteins. This review covers work aimed at understanding folding mechanisms and the intermolecular forces that drive the folding of integral membrane proteins. We discuss the kinetic and thermodynamic studies that have been undertaken. Our review also draws on closely related research, mainly from purification studies of functional membrane proteins, and gives an overview of some of the successful methods. A brief survey is also given of the large body of mutagenesis and fragment work on membrane proteins, as this too has relevance to the folding problem. It is noticeable that the choice of solubilizing detergents and lipids can determine the success of the method, and indeed it appears that particular lipid properties can be used to control the rate and efficiency of folding. This has important ramifications for much in vitro folding work in that it aids our understanding of how to obtain and handle folded, functional protein. With this in mind, we also cover some relevant properties of model, lipid-bilayer systems.

Progressive stabilization of intermediate and transition states in protein folding reactions by introducing surface hydrophobic residues.

It can be argued from the principle of solvent exclusion that the introduction of hydrophobic residues onto the surface of a protein will not destabilize the folded state because the nonpolar side chain will be at least as exposed in the unfolded state as it is when the protein chain is folded. A comparison of the folding pathway of wild type and 11 site-directed mutants of CD2.d1 shows this to be true. In fact, owing to partial burial of nonpolar groups as folding proceeds, we find that the rapidly formed intermediate state and, to a greater extent, the transition state are generally stabilized by hydrophobic surface mutations. This effect is slightly moderated in the folded state presumably by the perturbation of van der Waals' contacts and/or local electrostatic interactions that have a greater influence in this fully compact structure. The fact that in all but one case we find that stabilization of the rapidly collapsed intermediate is accompanied by a faster acquisition of the folded state refutes the argument that I states are generally "off pathway" conformations or ensembles that lead to the inhibition of otherwise more rapid folding trajectories.

Effects of mutations on the thermodynamics of a protein folding reaction: implications for the mechanism of formation of the intermediate and transition states.

We have measured changes in heat capacity, entropy, and enthalpy for each step in the folding reaction of CD2.d1 and evaluated the effects of core mutations on these properties. All wild-type and mutant forms fold through a rapidly formed intermediate state that precedes the rate-limiting transition state. Mutations have a pronounced effect on the enthalpy of both the intermediate and folded states, but in all cases a compensatory change in entropy results in a small net free-energy change. While the enthalpy change in the folded state can be attributed to a loss of van der Waals interactions, it has already been shown that changes in the stability of the intermediate are dominated by changes in secondary structure propensity [Lorch et al. (1999) Biochemistry 38, 1377-1385]. It follows that the thermodynamic basis of beta-propensity is enthalpic in origin. The effects of mutations on the enthalpy and entropy of the transition state are smaller than on the ground states. This relative insensitivity to mutation is discussed in the light of theories concerning the nature of the rate-limiting barrier in folding reactions.

Effects of core mutations on the folding of a beta-sheet protein: implications for backbone organization in the I-state.

A series of core mutations were introduced into beta-strand segments of an immunoglobulin fold (the isolated first domain of CD2, CD2.d1) to examine their influence on the rapidly formed intermediate state (I-state) which transiently accumulates in the folding reaction [Parker, M. J., and Clarke, A. R. (1997) Biochemistry 36, 5786-5794]. The residue changes were chemically conservative, each representing the removal of one or two methylene groups from aliphatic side chains. Predictably, the mutations destabilize the folded state with respect to the unfolded state by about 1.1 +/- 0.7 kcal mol-1 per methylene group removed. However, when the folding reaction is dissected by transient kinetic analysis into its component steps, six out of the nine mutations lead to a stabilization of the I-state. The direction and magnitude of these effects on the global stability of the transient intermediate are well correlated with changes in secondary structure propensity occasioned by the substitutions. The results show that, although side chain interactions are extremely weak in this early phase of folding, the beta-strand conformation of the polypeptide chain is established. In the next phase of the reaction, the rate-limiting transition state is attained by the formation of a tightly localized hydrophobic nucleus which includes residues V30, I18, and V78. Interestingly, in almost all immunoglobulin domains of extracellular proteins, the latter pair are cysteine residues which form a disulfide bridge.

Thermodynamic properties of transient intermediates and transition states in the folding of two contrasting protein structures.

The N-terminal domain of phosphoglycerate kinase (N-PGK) and domain 1 of the T-cell adhesion protein CD2 (CD2.d1) fold through rapidly formed and transiently populated intermediate states in reactions which have no kinetic complications arising from proline isomerization or disulfide bonding. We have evaluated the thermodynamic parameters (DeltaCp, change in heat capacity; DeltaS, entropy change; DeltaH, enthalpy change) for each experimentally accessible step in these folding reactions. Despite their different topologies and amino acid compositions, the individual steps [U-I (unfolded to intermediate state), I-t (intermediate to major transition state), and t-F (transition state to the fully folded state)] have closely similar qualitative properties in the two proteins. For both, the heat capacity changes are proportional to m-value changes (Deltam) for every step in the reaction, but the ratio DeltaCp/Deltam is lower for N-PGK, presumably owing to a much larger compliment of aromatic amino acids in the core. According to measurements of DeltaCp and Deltam, the I-states are highly condensed (65-70% for N-PGK and 40-45% dehydrated for CD2.d1), yet the changes in entropy in the U-to-I transition are small, showing that the entropy gained from desolvation must be balanced by that lost in ordering the chain. The high degree of conformational order in the I-state, implied by these measurements, is mirrored by the extensive, native secondary structure revealed by amide exchange measurements [Hosszu, L. L. P., et al. (1997) Nat. Struct. Biol. 4, 801-804; Parker, M. J., et al. (1997) Biochemistry 36, 13396-13405]. At 25 degreesC the transition state barrier has an entirely enthalpic origin, the entropic contribution being favorable. The latter observation implies that, during the consolidation of structure occurring in the I-to-F step, further dehydration (positive DeltaS) precedes side-chain locking (negative DeltaS). Only after the transition state is surmounted do we see a net entropic penalty arising from the widespread ordering of side chains.

Meta-analysis of botulinum toxin treatment of spasmodic dysphonia: a review of 22 studies.

This meta-analysis focuses on the treatment effects of the use of botulinum toxin in laryngeal dystonia. The vocal symptoms are characterized by spasms of the laryngeal muscles thus the use of the term spasmodic dysphonia (SD). The objective of the intervention, i.e. botulinum toxin treatment, is to reduce or eliminate the vocal spasms and thus improve the acoustic regularity of the voice. The purpose of the present study was to assess the efficacy of botulinum toxin in the treatment of SD by engaging in a 'best synthesis' systematic summary of existing research. The average treated SD patient in the 22 studies subjected to meta-analysis obtained 97% improvement as a result of treatment with botulinum toxin.

Acquisition of native beta-strand topology during the rapid collapse phase of protein folding.

The 98 residue C-terminal domain of the cell-surface receptor protein CD2 (CD2.D1) has a beta-sandwich fold belonging to the immunoglobulin superfamily but lacking the usual disulfide bridges. Kinetic studies on the folding/unfolding of CD2.D1 reveal that folding proceeds through a rapidly formed intermediate state [Parker, M. J., & Clarke, A. R. (1997) Biochemistry 36, 5786-5794]. To characterize the structural properties of this intermediate we have performed a series of amide hydrogen exchange studies using the pH competition method, in which folding and exchange are initiated simultaneously. The complex beta-sheet topology of this molecule makes it an ideal object for examining the acquisition of backbone hydrogen bonds made between sequence-local and sequence-distant segments of the chain during folding. The pattern of protected amides in the intermediate reveal that the essential features of the beta-sheet topology of CD2.D1 are defined early in the folding pathway, before the development of intimate side chain interactions characteristic of the native state. The results are discussed in light of current issues concerning the mechanistic relevance of kinetic protein folding intermediates.

Structure of a kinetic protein folding intermediate by equilibrium amide exchange.

A combination of equilibrium amide exchange and kinetic folding data show that the essential features of the complex topology of the N-terminal domain of a thermophilic phosphoglycerate kinase are established on a millisecond or faster timescale, before the rate-limiting step in the folding pathway commences.

The utility of meta-analysis in the determination of efficacy of treatment in aphasia: a reply to Robey (1994).

In response to Robey (1994) we argue that his judgment of our study and conclusions (Whurr, Lorch, & Nye, 1992) are inaccurate. We point out that our study was in fact an analysis of the effects of treatment for aphasic patients. Further, the results obtained in Robey's analysis, though obtained via a different strategy of analysis, yielded essentially the same degree of overall treatment outcome effect. Thus, we conclude that while Robey does provide a different model of effect size data organization and measurement, the results are no different and do not in fact contradict our data.

The use of botulinum toxin in the treatment of adductor spasmodic dysphonia.

Botulinum toxin injections have been used to treat 31 patients with adductor spasmodic dysphonia. Injections of 3.00-3.75 units of botulinum toxin were performed bilaterally into the thyroarytenoid muscle. This treatment significantly decreased the standard deviation of the fundamental frequency of the speech sample, indicating a reduction in the variability of pitch amongst patients. A total of 96% of patients' subjective diary reports showed an improvement with a median of 7 days to peak effect and a 5 week duration of peak effect.

Treatment of cervical dystonia hand spasms and laryngeal dystonia with botulinum toxin.

One hundred and twenty-six patients with different forms of focal dystonia (89 with cervical dystonia, 12 with hand cramps and 25 with laryngeal dystonia) were treated with localised injections of botulinum toxin. Mean doses per muscle were 200 mouse units (m.u.) for treating cervical dystonia, 40-120 m.u. for forearm muscles in writers' cramp and 3.7 m.u. for the thyroarytenoid muscle in laryngeal dystonia. Responder rates have been above 80% in all patient groups and beneficial effects could be reproduced over follow-up periods of up to 4 years. The commonest side-effects were dysphagia after treatment of spasmodic torticollis, weakness of neighbouring muscles after injections for hand cramps and breathiness and hypophonia following laryngeal injections. All these were transient and generally well tolerated. It is concluded that botulinum toxin injections are a safe and effective treatment in all three types of focal dystonia.

A meta-analysis of studies carried out between 1946 and 1988 concerned with the efficacy of speech and language therapy treatment for aphasic patients.

An examination of the empirical evidence for the efficacy of speech and language therapy treatment for adult aphasic patients is undertaken with the aid of meta-analysis which affords a statistical method of systematic data summary and synthesis. Patient characteristics and treatment outcomes are correlated to identify factors that contribute to the demonstration of a treatment effect. One of the most striking results of this retrospective study was the identification of the overwhelming failure to report data or include, in experimental controls, variables that might crucially affect outcome.

Verb finding in aphasia.

Word finding for nouns and verbs was examined in a heterogeneous group of aphasics (N = 9) by comparing the ability to generate synonyms and sentences for the same set of 20 nouns and 20 verbs. Synonym Generation performance resembled that of an age-matched group of normal control subjects (n = 9): In both groups, some subjects produced comparable numbers of synonyms for nouns and verbs while other subjects produced significantly fewer synonyms for verbs. Essentially the same two patterns were displayed on Sentence Generation using the frequency of "empty" nouns (e.g., 'it', 'man') and "empty" verbs (e.g., 'is', 'do') as an index of word-finding difficulty: In both groups, some subjects produced comparable numbers of empty nouns and verbs, while other subjects produced significantly more empty verbs. However, the Sentence Generation performance of one aphasic subject stood out overall by her tendency to avoid empty verbs and produce incomplete sentences. This pattern of performance was interpreted as a breakdown in an early stage of sentence planning that may be directly related to her diagnosis of transcortical motor aphasia.

Emotional and non-emotional facial behaviour in patients with unilateral brain damage.

Aspects of emotional facial expression (responsivity, appropriateness, intensity) were examined in brain-damaged adults with right or left hemisphere cerebrovascular lesions and in normal controls. Subjects were videotaped during experimental procedures designed to elicit emotional facial expression and non-emotional facial movement (paralysis, mobility, praxis). On tasks of emotional facial expression, patients with right hemisphere pathology were less responsive and less appropriate than patients with left hemisphere pathology or normal controls. These results corroborate other research findings that the right cerebral hemisphere is dominant for the expression of facial emotion. Both brain-damaged groups had substantial facial paralysis and impairment in muscular mobility on the hemiface contralateral to site of lesion, and the left brain-damaged group had bucco-facial apraxia. Performance measures of emotional expression and non-emotional movement were uncorrelated, suggesting a dissociation between these two systems of facial behaviour.

Effect of emotional context on bucco-facial apraxia.

Patients with left- and right-hemisphere cerebrovascular pathology and normal adult controls were videotaped while executing tasks of bucco-facial praxis in emotional and nonemotional conditions. Each practic movement was assessed for accuracy and motor execution. Left-brain-damaged patients were significantly impaired on these tasks relative to right-damaged patients and controls. When emotional context was provided, apractic performance improved significantly.

The expression and perception of facial emotion in brain-damaged patients.

This study examined the expression and perception of facial emotion in patients with unilateral cerebrovascular pathology. Subjects were 12 right brain-damaged (RBD), 15 left brain-damaged (LBD) aphasic, and 16 normal control (NC) right-handed males. Expressions were elicited during posed and spontaneous conditions. Both positive and negative emotions were studied. RBDs were significantly impaired, relative to LBDs and NCs, in expressing and perceiving facial emotion. There were no group differences as a function of condition, but there were differences as a function of emotional valence. Qualitative performance differences also were observed. There was no evidence that the ability to produce a particular emotion was related to the ability to identify the same emotion. Overall, these findings support the notion that the right cerebral hemisphere is dominant for expressing and perceiving facial emotion.