Protein expression studies of desmoplakin mutations in cardiomyopathy patients reveal different molecular disease mechanisms.

Mutations in the gene for desmoplakin (DSP) may cause arrhythmogenic right ventricular cardiomyopathy (ARVC) and Carvajal syndrome (CS). Desmoplakin is part of all desmosomes, which are abundantly expressed in both myocardial and epidermal tissue and serve as intercellular mechanical junctions. This study aimed to investigate protein expression in myocardial and epidermal tissue of ARVC and CS patients carrying DSP mutations in order to elucidate potential molecular disease mechanisms. Genetic investigations identified three ARVC patients carrying different heterozygous DSP mutations in addition to a homozygous DSP mutation in a CS patient. The protein expression of DSP in mutation carriers was evaluated in biopsies from myocardial and epidermal tissue by immunohistochemistry. Keratinocyte cultures were established from skin biopsies of mutation carriers and characterized by reverse transcriptase polymerase chain reaction, western blotting, and protein mass spectrometry. The results showed that the mutation carriers had abnormal DSP expression in both myocardial and epidermal tissue. The investigations revealed that the disease mechanisms varied accordingly to the specific types of DSP mutation identified and included haploinsufficiency, dominant-negative effects, or a combination hereof. Furthermore, the results suggest that the keratinocytes cultured from patients are a valuable and easily accessible resource to elucidate the effects of desmosomal gene mutations in humans.

Arm-to-arm variation when evaluating neuromuscular block: an analysis of the precision and the bias and agreement between arms when using mechanomyography or acceleromyography.

BACKGROUND: Studies comparing acceleromyography and mechanomyography indicate that the two methods cannot be used interchangeably. However, it is uncertain to what extent differences in precision between the methods and the naturally occurring arm-to-arm variation have influenced the results of these studies. Accordingly, the purpose of this study was to examine the precision and the arm-to-arm variation, when the same method is used on both of the arms. METHODS: In the first part (n=20), mechanomyography was applied bilaterally and in the second part acceleromyography (n=20). Anaesthesia was induced with propofol and opioid, and neuromuscular block with rocuronium 0.6 mg kg(-1). The precision of the two methods and the bias and limits of agreement between the arms were evaluated using train-of-four (TOF) stimulation, without and with referral to the initial baseline value, that is, normalization. RESULTS: Both methods were found to be precise (<5% variation) without any difference between the dominant and non-dominant arms. There were no significant biases between the arms, except for the onset time obtained with acceleromyography, which was 10% longer for the dominant arm. However, the individual differences (limits of agreement) were wide (0.20-0.25 at TOF 0.90). Normalization during recovery did not change bias or limits of agreement between the arms. CONCLUSIONS: In the research setting, acceleromyography and mechanomyography are both precise methods without difference between the arms. Although there is no mean bias between the arms, both methods show wide individual differences (limits of agreement), which might to a large extend explain the differences often found when two different methods are compared on the contralateral arms. ClinicalTrial.gov identifier: NCT00472121; URL: http://clinicaltrials.gov/ct2/show/study/NCT00472121.

Acceleromyography and mechanomyography for establishing potency of neuromuscular blocking agents: a randomized-controlled trial.

BACKGROUND: Acceleromyography (AMG) is increasingly being used in neuromuscular research, including in studies establishing the potency of neuromuscular blocking and reversal agents. However, AMG is insufficiently validated for use interchangeably with the gold standard, mechanomyography (MMG) for this purpose. The aim of this study was to compare AMG and MMG for establishing dose-response relationship and potency, using rocuronium as an example. METHODS: We included 40 adult patients in this randomized-controlled single-dose response study. Anaesthesia was induced and maintained with propofol and opioid. Neuromuscular blockade was induced with rocuronium 100, 150, 200 or 250 microg/kg. Neuromuscular monitoring was performed with AMG (TOF-Watch SX) with pre-load (Hand Adapter) at one arm and MMG (modified TOF-Watch SX) on the other, using 0.1 Hz single twitch stimulation. Dose-response relationships were determined for both recording methods using log (dose) against probit (maximum block). The obtained slopes of the regression lines, ED(50), ED(95) and the maximum block were compared. RESULTS: The ED(50) and ED(95) [95% confidence interval (CI)] for AMG were 185 microg/kg(167-205 microg/kg) and 368 microg/kg(288-470 microg/kg), compared with 174 microg/kg(159-191 microg/kg) and 338 microg/kg(273-418 microg/kg) for MMG. There were no statistically significant biases in maximum block, ED(50), ED(95) or slopes obtained with the two methods. CONCLUSION: Our results indicate that any possible difference between AMG and MMG is so small that it justifies AMG to be used for establishing the potency of neuromuscular blocking agents. However, the wide CIs show that we cannot rule out a 13% higher ED(50) and a 26% higher ED(95) for AMG.

The undesirable effects of neuromuscular blocking drugs.

Neuromuscular blocking drugs are designed to bind to the nicotinic receptor at the neuromuscular junction. However, they also interact with other acetylcholine receptors in the body. Binding to these receptors causes adverse effects that vary with the specificity for the cholinergic receptor in question. Moreover, all neuromuscular blocking drugs may cause hypersensitivity reactions. Often the symptoms are mild and self-limiting but massive histamine release can cause systematic reactions with circulatory and respiratory symptoms and signs. At the end of anaesthesia, no residual effect of a neuromuscular blocking drug should be present. However, the huge variability in response to neuromuscular blocking drugs makes it impossible to predict which patient will suffer postoperative residual curarization. This article discusses the undesirable effects of the currently available neuromuscular blocking drugs including the definitions, diagnosis and causes of hypersensitivity reactions and postoperative residual curarisation.

Alpha-cardiac actin is a novel disease gene in familial hypertrophic cardiomyopathy.

We identified the alpha-cardiac actin gene (ACTC) as a novel disease gene in a pedigree suffering from familial hypertrophic cardiomyopathy (FHC). Linkage analyses excluded all the previously reported FHC loci as possible disease loci in the family studied, with lod scores varying between -2.5 and -6.0. Further linkage analyses of plausible candidate genes highly expressed in the adult human heart identified ACTC as the most likely disease gene, showing a maximal lod score of 3.6. Mutation analysis of ACTC revealed an Ala295Ser mutation in exon 5 close to 2 missense mutations recently described to cause the inherited form of idiopathic dilated cardiomyopathy (IDC). ACTC is the first sarcomeric gene described in which mutations are responsible for 2 different cardiomyopathies. We hypothesize that ACTC mutations affecting sarcomere contraction lead to FHC and that mutations affecting force transmission from the sarcomere to the surrounding syncytium lead to IDC.

Using micellar mole fractions to assess membrane protein stability in mixed micelles.

The increased focus on the structural and physical properties of membrane proteins has made it critical to develop methods that provide a reliable estimate of membrane protein stability. A simple approach is to monitor the protein's conformational changes in mixed detergent systems, typically consisting of an anionic (denaturing) and non-ionic (non-denaturing) component. Linear correlations between, e.g., the melting temperature and the bulk mole fraction of the anionic component have been observed. However, a potential complication is that the bulk mole fraction is not identical to the mole fraction in the mixed micelle, which is the local environment experienced by the membrane protein. Here, we present an extensive analysis of the thermal stability of the membrane-integrated domain of the outer membrane protein AIDA in the presence of different mixed micelles. In the micelle system SDS-octyl-polyoxyethylene, the melting temperature in the absence of SDS extrapolates to 113 degrees C using bulk mole fractions. However, for mixed micelles involving short-chain detergents or phospholipids, the melting temperature calculated using bulk mole fractions reaches values up to several hundred degrees higher than 113 degrees C and can only be obtained by extrapolation over a narrow mole fraction interval. Furthermore, there is a non-linear relationship between the melting temperature and bulk mole fractions for mixed micelle systems involving cationic detergents (also denaturing). We show that if we instead use the micellar mole fraction as a parameter for denaturing detergent strength, we obtain linear correlations which extrapolate to more or less the same value of the melting temperature. There remains some scatter in the extrapolated values of the melting temperature in different binary systems, which suggest that additional micellar interactions may play a role. Nevertheless, in general terms, the mixed micellar composition is a good parameter to describe the membrane protein's microenvironment. Note, however, that for the mixed micelle system involving SDS and dodecyl maltoside, which has been used by several research groups to determine membrane protein stability, the estimate provided by bulk mole fraction leads to similar values as that of micellar mole fractions.

The prefrontal "cortex" in the pigeon catecholamine histofluorescence.

The prefrontal cortex of mammals is densely innervated with dopaminergic fibers. We report a comparable, dense network of catecholamine (probably dopamine)-containing fluorescent fibers in the posterodorsolateral neostriatum of the pigeon. This region is clearly separable from paleostriatum augmentatum, lobus parolfactorius, posterior archistriatum, posteromedial corticoid and septum, all of which also show strong catecholamine fluorescence. Parallel biochemical, anatomical and neurobehavioral data support the suggestion that posterodorsolateral neostriatum in the pigeon may be comparable to the mammalian prefrontal cortex. Thus the telencephalic tissue represented as the prefrontal cortex in mammals and the posterodorsolateral neostriatum in the pigeon, may turn out to be a phylogenetically ancient neural device.

On the projections from the neostriatum to the cerebral cortex: the "displaced" neurons.

The entire dorsal and lateral cortex of one cerebral hemisphere of rats was infiltrated with different fluorescent tracers and the neostriatum was examined for labelled perikarya. In spite of the extensive infiltration of the cortex, such neurons were seen only sporadically in the ipsilateral neostriatum and almost only in the vicinity of the globus pallidus and the subcortical white matter. The size and shape of these neurons, and particularly their proximity to some cell groups which surround the neostriatum and project to the cerebral cortex, suggest that these neurons belong to the neighbouring structures such as the magnocellular nuclei of the basal forebrain, the claustrum and the VIb cortical layer.

Interactions of bambuterol with human serum cholinesterase of the genotypes EuEu (normal), EaEa (atypical) and EuEa.

Bambuterol, a carbamate ester prodrug of the bronchodilator terbutaline, was tested as inhibitor and substrate of human serum cholinesterases of the genotypes EuEu (the normal enzyme), EaEa (the atypical enzyme) and EuEa. The IC50 for the normal enzyme was 11 +/- 2.2 nM (mean, SD, N = 10) and for the atypical enzyme 140 +/- 6 nM (N = 13), indicating a much higher affinity of bambuterol to the normal enzyme. The heterozygotes showed a mixed behaviour; the major activity was inhibited like the normal enzyme (IC50 = 9.3 +/- 1.9 nM, N = 9), while a residual activity (10-15%) was inhibited by bambuterol like the atypical enzyme. At a bambuterol concentration of 100 nM each of the three cholinesterase genotypes responded uniquely to bambuterol; the normal enzyme was inhibited to 2.2 +/- 0.9%, the atypical enzyme to 58 +/- 4.6%, and the heterozygote to 10 +/- 1.2% of the basal activity. Bambuterol may therefore be added to the list of inhibitors useful in the genotyping of cholinesterases. Bambuterol was much less efficiently hydrolysed in serum containing the atypical cholinesterase than in serum containing the normal enzyme. The results of the hydrolysis experiments once again illustrate the difference in affinity of bambuterol to the genetic forms of cholinesterase, and also strengthen the evidence that cholinesterase is the major serum enzyme catalysing the hydrolysis of bambuterol.

A multipurpose vertical holeboard with automated recording of spatial and temporal response patterns for rodents.

A method is presented for manual or automated recording of rats' spontaneous nose-poking ('visit') behaviors to a vertical holeboard with a matrix of 45 or 54 holes. Several behavior parameters are presented: visit frequency, visit duration, temporal visit pattern, spatial visit pattern, stereotype of visits, diversity of visits and variability of visit patterns. The paper describes the development of the apparatus and some methods of analyzing and presenting the multi-parametric data. The use of the apparatus is illustrated with a one-trial appetitive conditioning task. After 5 min in a single 10-min session, a food pellet is presented, only once in a given hole, to provide reinforcement of a spontaneous visit to that hole. The behavior parameters are compared before and after reinforcement. When the one-trial conditioning effect was challenged with d-amphetamine, the behavior parameters changed in a graded manner depending upon the dose (0.25-6.0 mg/kg). The apparatus has also proven useful for studies of exploratory behavior without using food reinforcement following lesion or drug interventions.

Influences of the rearing conditions on functional properties of the rat's prefrontal system.

For a period of 42 days, starting on their 22nd day of life, rats were housed either in an 'enriched' maze-like environment in which acquisition of food and water was occasionally conditioned upon solution of spatial problems, or under 'impoverished' conditions with minimal stimulation and food and water always available. The animals were subsequently tested for initial acquisition and retention of spatial delayed alternation and a visual pattern discrimination. Finally, the effect of ablation of the medial prefrontal cortical area on the performance of the two tasks was investigated. The results showed an interaction between the rearing conditions and the order of the task presentation: the enriched environment clearly affected performance in the first task only. Thus, the group which started with delayed alternation was better in this task than any other group: the group which started with the visual discrimination performed worse in that task than any other group. The rearing conditions seemed to have no effects on the degree of impairment produced by a prefrontal cortical lesion. It is suggested that the presently used enriched environment initially biases the rats towards application of spatial hypotheses during problem solving behaviour and against the use of a visual hypothesis.

The prefrontal cortex and variants of sequential behaviour indications of functional differentiation between subdivisions of the rat's prefrontal cortex.

In two separate experiments we addressed the involvement of the rat's prefrontal cortex in mediation of the sequential ordering of the "components of behaviour". In both experiments the animals were required to operate two different and spatially distant manipulanda sequentially. While in the first experiment a light cue signalled which response would at any moment be appropriate no sensory cues offered procedural guidance during the second experiment. Both experiments focused on postoperative retention performance. In the first experiment we studied the consequences of ablation of the dorsal anteromedial cortex, the total anteromedial cortex and the suprarhinal cortex as compared to a sham operated control group. In the second experiment we compared a sham operated group to a group subjected to ablation of the total anteromedial cortex. While the proficiency of task performance was evaluated on the basis of the number of reinforcements obtained and the percentage of bar presses to be reinforced, additional analysis of behaviour included registration of the individual behavioural components and the sequential orders in which these were observed. The major findings were: (1) lesions within the anteromedial, prefrontal cortex (especially if including the ventral part of this region) are associated with significantly impaired performance of both the presently investigated tasks. (2) The demonstrated association between the anteromedial cortex and the mediation of the sequential arrangement of behavioural components does not seem to be secondary to changes in neither quality nor frequency of any individual behavioural component. (3) In the first experiment--where a cue light signalled which response would at any point in time be adequate--lesions within all parts of the prefrontal cortex (the suprarhinal as well as the anteromedial cortex) were associated with overproduction of errors, which in the behavioural sequence analysis had been characterized as "type B". We tentatively interpret such errors as reflecting a failure to utilize the visual cue offered by the signal lamp. Since lesions within all parts of the prefrontal cortex seem to be associated with "cue utilization" failures while only lesions involving the anteromedial cortex are reflected in impaired sequential arrangement of behavioural components the present studies seem to reflect a certain degree of functional specialization within the prefrontal cortex of the rat.

Behavioural effects of ablation of the pigeon-equivalent of the mammalian prefrontal cortex.

Six pigeons were trained to perform delayed alternation and brightness discrimination. Three of them underwent ablation of the posterodorsolateral neostriatum (PDLNS) which is believed to correspond to the mammalian prefrontal cortex. In the other three pigeons hyperstriatal lesions were induced by local injections of ibotenic acid. Ablation of PDLNS impaired performance of delayed alternation much more than did the hyperstriatal lesion. In brightness discrimination, a mild impairment occurred only on the first postoperative session and only in the PDLNS group. We conclude that the ablation of PDLNS in pigeons and of the prefrontal cortex in mammals induce similar impairments. Thus, the prefrontal cortex appears not to be a privilege of mammals, but may appear in different architectonic variants in all "higher" vertebrates.

Retention and relearning of spatial delayed alternation in rats after ablation of the prefrontal or total non-prefrontal isocortex.

In the present study we addressed the question whether, within the isocortex, the prefrontal area of the rat is uniquely involved in mediation of delayed alternation. In one group of rats the dorsolateral isocortex, from the dorsomedial shoulder to the dorsal lip of the rhinal sulcus was removed bilaterally in a single surgical session. In these rats delayed alternation in a T-maze was significantly less impaired than in rats with one stage bilateral removal of the medial prefrontal cortex. The prefrontal cortex seems not to depend crucially on isocortical input for its medication of delayed alternation.

Synaptic proteins in frontal and control brain regions of rats after exposure to spatial problems.

Synaptic proteins D1, D2, D3, synaptin and 14-3-2, as well as the glial protein glutamine synthetase, were measured by crossed immunoelectrophoresis in the anteromedial (prefrontal) cortex, occipital (visual) cortex and the anterior and posterior parts of the neostriatum of rats. The 3 experimental groups consisted of rats trained to criterion in a spatial delayed alternation, those run as yoked controls and, finally, rats kept in individual cages and not subjected to any training. Statistical analysis showed that two variables: behavioral procedures and brain regions, had a significant effect. Their interaction was also significant. Further analysis revealed that only in the prefrontal cortex of the yoked control animals was there a significant decrease of the synaptic membrane proteins D1, D2 and D3. Thus, particular behavioral treatment seems capable of affecting synapses in a specific 'association' cortical area. The change is more easily related to the amount of 'work' than to formation of 'memory trace' within the critical area.

Long-term retrograde labelling of neurons.

The intensity of labelling of neuronal perikarya with Fluoro-gold or rhodamine microspheres appeared unchanged in rats surviving one year after surgery. These tracers may be used for sequential labelling with long intervals and to study brain connections in precious specimens.

Strain differences in catecholamine content of pigeon brains.

Concentrations of dopamine, noradrenaline and DOPAC were measured in 6 regions of telencephalon and in the cerebellar cortex of adult white Carneaux, and mixed breed pigeons of either sex. In both groups the regional differences in the amount of dopamine replicated the pattern we published earlier. The absolute values, however, differed in these groups: in all telencephalic regions the amount of dopamine was lower in the Carneaux strain than in the mixed breed specimens; in 4 of these regions the difference was statistically significant. A similar tendency was observed in the noradrenaline concentration but the difference was significant only in two telencephalic samples and the cerebellum. The concentration of DOPAC was significantly smaller in two telencephalic regions of the Carneaux pigeons.

The prefrontal 'cortex' in the pigeon. Biochemical evidence.

Concentrations of dopamine and noradrenaline were determined in 6 regions of the telencephalon and in the cerebellum of the pigeon. Noradrenaline was rather evenly distributed. A significant variation was found of the dopamine-noradrenaline ratio (DA:NA), a measure which makes it possible to distinguish dopamine found in dopaminergic fibers from dopamine which is precursor of noradrenaline. The highest ratio was found in the anteroventromedial region (containing the presumed homologue of the mammalian neostriatum), and the next highest in the posteroventrolateral region (containing the archistriatum). Like in mammals, the lowest concentration of the non-precursor dopamine in the pigeon brain seems to be contained in the cerebellum. Among the regions which show physiological and anatomical similarities with the mammalian cerebral cortex, the DA:NA ratio was significantly higher in the posterodorsolateral, than in the posterodorsomedial and anterodorsomedial regions. The two dorsomedial regions contain the equivalents of the hippocampus and sensory cortical areas of mammals. The strong dopamine innervation of the posterodorsolateral region is comparable to that of the mammalian prefrontal cortex.

The N-CAM D2-protein as marker for synaptic remodelling in the red nucleus.

We have followed the time-course of changes in the concentration of 3 neuronal and one glial antigen in the red nucleus in rats after unilateral lesion of the cerebellorubral connections. The neuronal markers were the neuronal cell adhesion molecule (N-CAM) D2-protein which is prevalent in newly formed neuronal membranes, and the D1- and D3-proteins, which are found mainly in mature neuronal membranes. The glial marker was S-100, a cytoplasmic protein. Six days after the lesion no changes in the concentration of the markers were found in the partially deafferentiated red nucleus. However, 10 days after the lesion the D2-protein concentration was significantly increased, in contrast to the D1-protein concentration which was decreased. After a further 3 days the D2-protein concentration began to decrease, approaching the still significantly decreased D1-protein concentration. Twenty-one days after the lesion the marker protein concentrations were not significantly changed from normal. However, whereas the concentrations of neuronal membrane markers were lower, the glial S-100 concentration showed a tendency to increase. Furthermore, although the changes in D3-protein concentration were unable to reach statistical significance alone they always followed the direction of D1-protein and were significantly in variance with the changes in D2-protein and S-100 concentrations. Our results support the notion of the N-CAM D2-protein as a useful marker for synaptic turnover in adult brain.

Focal cortical seizures prevent HRP and HRP-WGA labeling only in neurons bidirectionally connected to the cortex.

Intracortical implants of polyacrylamide gel containing horseradish peroxidase labeled cortical efferents and perikarya in some cortical areas and a number of subcortical formations. When epileptogenic penicillin was added to the gel, no labeling was seen in the efferents and cell bodies of the cortex, thalamus, or claustrum, whereas the magnocellular nuclei of the basal forebrain, raphe nuclei and locus coeruleus did contain the label.