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.

Single-kernel analysis of fumonisins and other fungal metabolites in maize from South African subsistence farmers.

Fumonisins are important Fusarium mycotoxins mainly found in maize and derived products. This study analysed maize from five subsistence farmers in the former Transkei region of South Africa. Farmers had sorted kernels into good and mouldy quality. A total of 400 kernels from 10 batches were analysed; of these 100 were visually characterised as uninfected and 300 as infected. Of the 400 kernels, 15% were contaminated with 1.84-1428 mg kg(-1) fumonisins, and 4% (n=15) had a fumonisin content above 100 mg kg(-1). None of the visually uninfected maize had detectable amounts of fumonisins. The total fumonisin concentration was 0.28-1.1 mg kg(-1) for good-quality batches and 0.03-6.2 mg kg(-1) for mouldy-quality batches. The high fumonisin content in the batches was apparently caused by a small number (4%) of highly contaminated kernels, and removal of these reduced the average fumonisin content by 71%. Of the 400 kernels, 80 were screened for 186 microbial metabolites by liquid chromatography-tandem mass spectrometry, detecting 17 other fungal metabolites, including fusaric acid, equisetin, fusaproliferin, beauvericin, cyclosporins, agroclavine, chanoclavine, rugulosin and emodin. Fusaric acid in samples without fumonisins indicated the possibility of using non-toxinogenic Fusaria as biocontrol agents to reduce fumonisin exposure, as done for Aspergillus flavus. This is the first report of mycotoxin profiling in single naturally infected maize kernels.

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.

Aspergillus acidus from Puerh tea and black tea does not produce ochratoxin A and fumonisin B2.

Puerh tea is a unique Chinese fermented tea. Unlike other teas it is stored for a long period of time. Aspergillus niger is claimed to be the dominant microorganism in the Puerh tea manufacturing process and also to be common on tea in general. A. niger sensu stricto is known to produce the mycotoxins ochratoxin A, fumonisins B(2) and B(4). With this in mind, we performed a preliminary study to determine if production of these mycotoxins by black Aspergilli isolated from Puerh and black tea can occur. An examination of 47 isolates from Puerh tea and black tea showed that none of these was A. niger. A part of the calmodulin gene in 17 isolates were sequenced, and these 17 isolates were all identified as Aspergillus acidus (=A. foetidus var. acidus). The rest of the 47 isolates were also identified as A. acidus from their metabolite profile. Neither production of ochratoxin A nor fumonisins B(2) and B(4) by any of the 47 isolates were observed.

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.

Prolonged residual paralysis after a single intubating dose of rocuronium.

It is often argued that neuromuscular monitoring is unnecessary when only one dose of an intermediate-acting neuromuscular blocking agent is given. This case report documents that it may take more than 3.5 h before it is possible to antagonize a block caused by a normal dose of rocuronium (0.6 mg kg(-1)). Possible causes of the extremely prolonged duration of action are discussed, as is the importance of quantitative neuromuscular monitoring.

Good clinical research practice in pharmacodynamic studies of neuromuscular blocking agents II: the Stockholm revision.

The set of guidelines for good clinical research practice (GCRP) in pharmacodynamic studies of neuromuscular blocking agents, which was developed following an international consensus conference in Copenhagen, has been revised and updated following the second consensus conference in Stockholm in 2005. It is hoped that these guidelines will continue to help researchers in the field and assist the pharmaceutical industry and equipment manufacturers in enhancing the standards of the studies they sponsor.

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.

Myocardial late gadolinium enhancement cardiovascular magnetic resonance in hypertrophic cardiomyopathy caused by mutations in troponin I.

OBJECTIVE: To examine the influence of genotype on late gadolinium enhancement (LGE) and the potential of cardiovascular magnetic resonance (CMR) to detect preclinical hypertrophic cardiomyopathy. DESIGN: Prospective, blinded cohort study of myocardial LGE in a genetically homogeneous population. PATIENTS: 30 patients with disease causing mutations in the recognised hypertrophic cardiomyopathy gene for cardiac troponin I (TNNI3): 15 with echocardiographically determined left ventricular hypertrophy (LVH+) and 15 without (LVH-). MAIN OUTCOME MEASURES: CMR measures of regional left ventricular function, wall thickness, and mass, and the extent and distribution of LGE. RESULTS: LGE was found in 12 (80%) LVH+ patients but with variable extent (mean 15%, range 3-48%). LGE was also found in two (13%) LVH- patients but the extent was limited (3.6%) and both patients were found to have an abnormal ECG and regional hypertrophy by cine CMR. The extent of LGE was positively associated with clinical markers of sudden death risk (21% with > or = 2 risk factors v 7% with < or = 1 risk factor, p = 0.02) and left ventricular mass (r = 0.56, p < 0.001) and was inversely associated with ejection fraction (r = -0.58, p < 0.001). Segmental analysis showed that as regional wall thickness increased, LGE was more prevalent (p < 0.0001) and more extensive (r = 0.98, p = 0.001). CONCLUSION: In patients with disease causing mutations in TNNI3, focal fibrosis was not detected by LGE CMR before LVH and ECG abnormalities were present. Once LVH is present, LGE is common and the extent correlates with adverse clinical parameters. This suggests that focal fibrosis is closely linked to disease development.

Prevalence and clinical significance of systolic impairment in hypertrophic cardiomyopathy.

OBJECTIVES: To determine the frequency of systolic impairment (SI) and its impact on the natural history of hypertrophic cardiomyopathy (HCM). METHODS: 1080 patients (mean (SD) age 43 (15) years, 660 men) with HCM were evaluated. Initial assessment included history, examination, 48 hour Holter monitoring, cardiopulmonary exercise testing, and echocardiography; SI was defined as a fractional shortening (FS) < or = 25%. Survival data were collected at clinic visits or by direct communication with patients and their general practitioners. The results of serial echocardiography in 462 patients with normal FS at presentation are also reported. RESULTS: 26 (2.4%) patients (49 (14) years, 18 men) had SI at the initial visit. During follow up (58 (49) months), nine (34.6%) died or underwent cardiac transplantation compared with 108 (10.2%) patients with normal FS (p = 0.01). Five year survival from death (any cause) or transplantation was 90.1% (95% confidence interval (CI) 87.8 to 92.4) in patients with normal systolic function versus 52.4% (95% CI 25.2 to 79.6, p < 0.0001) in patients with SI. In patients who underwent serial echocardiography, 22 (4.8%, aged 41 (15) years) developed SI over 66 (40) months; the annual incidence of SI was 0.87% (95% CI 0.54 to 1.31). On initial evaluation patients who developed SI had a higher frequency of syncope (67 (15.2%) v 10 (45.5%) of those who did not develop SI, p = 0.001), non-sustained ventricular tachycardia (91 (20.6%) v 11 (50%), p = 0.002), and an abnormal blood pressure response on exercise (131 (29.7%) v 15 (68.2%), p = 0.001). Patients with SI had greater wall thinning (p = 0.001), left ventricular cavity enlargement (p < 0.0005), and deterioration in New York Heart Association functional class (p = 0.001) during follow up. Thirteen (59.1%) patients who progressed to SI died or underwent transplantation compared with 38 (8.6%) patients who maintained normal systolic function. CONCLUSIONS: SI is an infrequent complication of HCM but, when present, is associated with a poor prognosis.

Scandinavian research in anaesthesiology 1981-2000: visibility and impact in EU and world context.

BACKGROUND: We wished to assess the development in number and impact of publications in anaesthesiology and intensive care medicine from 1981 to 2000 in the four Scandinavian countries: Sweden, Norway, Finland, and Denmark. For comparison, we also analyzed data from the UK and the Netherlands. METHODS: Publication and citation data from 1981 to 2000 were gathered from National Science Indicators (2001), covering 33 journals indexed in Current Contents. Data were analyzed in running 5-year periods. The following informetric indicators were used: absolute number of publications; absolute number of citations; absolute citation impact (average number of citations per publication per 5-year period); citation impact relative to the European Union and the world; and the percentage of cited papers from each country. RESULTS: The annual number of publications from Denmark was stable over the 20-year period. Sweden increased its production by 35%, while the remaining four countries showed increases from 100% to 146%. Thus, Sweden and Denmark lost visibility within the European Union (EU) and in world context. The EU and world citation shares of Finland and Norway increased slightly, whereas those of Sweden, Denmark, the UK, and the Netherlands all declined significantly. The absolute citation impact (ACI) increased for all the four Scandinavian countries. The ACI of the Netherlands did not change and was surpassed by all the Scandinavian countries by 1994-98, while the UK finished below the other five countries. CONCLUSIONS: (1) The annual number of publications from Sweden, Norway, Finland, the UK, and the Netherlands increased after the late eighties, whereas the net publication output from Denmark was stagnant over the 20-year period investigated; (2) the international publication and citation visibility of Finland and Norway increased slightly, as opposed to the significant decrease seen by the other four countries; (3) judging from the increase in absolute and relative citation impact and in the percentage of cited papers, the recognition of publications from the four Scandinavian countries increased over the past 20 years.

Remifentanil vs. alfentanil infusion in non-paralysed patients: a randomized, double-blind study.

BACKGROUND AND OBJECTIVE: Remifentanil has a short duration of action and constant elimination, which allow administration of high doses, without prolonging recovery. Remifentanil has been compared to alfentanil, as part of a total intravenous anaesthetic technique, where remifentanil provided better anaesthetic conditions than alfentanil, without adverse effect on recovery. However, these results were obtained during anaesthesia involving neuromuscular blockade, which may mask both signs of insufficient anaesthesia and side-effects such as muscle rigidity. The aim of this study was to compare remifentanil with alfentanil for anaesthesia without neuromuscular blockade. METHODS: We performed a prospective, randomized, double-blind, four-centre study to compare remifentanil infusion 15 microg kg(-1) h(-1) and alfentanil infusion 60 microg kg(-1) h(-1), using a total intravenous technique for non-paralysed patients, and the laryngeal mask airway for airway management. We enrolled 192 patients, 18-65 yr of age with ASA I-II, undergoing minor surgery. The primary endpoint was the number of patients having pre-defined responses to surgical stimulation. A number of secondary criteria was evaluated to assess undesirable properties of the procedures. RESULTS: In the alfentanil group, 85% of patients responded to surgical stimulation, vs. 35% in the remifentanil group (P < 0.0001). No difference was found in recovery data, or in any other parameter than those related to insufficient anaesthesia. CONCLUSIONS: The remifentanil-based technique provided significantly better anaesthetic conditions than the alfentanil-based technique in the setting of this study, without causing any significant adverse effects.

Pharmacokinetics and pharmacodynamics of mivacurium in patients phenotypically heterozygous for the usual and atypical plasma cholinesterase variants (UA).

BACKGROUND: Mivacurium is hydrolyzed by plasma cholinesterase (pChe). The purpose of this study was to evaluate the pharmacodynamics and the pharmacokinetics of the three isomers of mivacurium in patients phenotypically heterozygous for the usual and the atypical pChe variant (UA). METHODS: Thirty-two patients were included in a dose-response study, in which the patients received one of four doses of mivacurium. An additional bolus dose of mivacurium, to a total of 0.1 mg kg-1, was given followed by a continuous infusion adjusted to maintain 91-99% neuromuscular block. The times to different levels of recovery following the infusion were measured using mechanomyography and train-of-four (TOF) nerve stimulation. Twelve of the patients with an estimated duration of anaesthesia of more than 90 min were (randomly) selected for the pharmacokinetic part of the study. Venous samples were taken for determination of the three isomers of mivacurium. These results were compared with results from a previous study in phenotypically normal patients (UU). RESULTS: The estimated ED50 and ED95 were 24 and 69 microg kg-1, respectively. The median (range) infusion rate was 3.7 microg kg-1 min-1 (1.2-2.9) and the time to a TOF ratio of 0.7 was 29.8 min (16.1-44.8). The median clearances of the cis-cis, cis-trans and trans-trans isomers were 3.7, 29 and 28 ml kg-1 min-1, respectively. The elimination half-lives of the isomers were 45, 6.7 and 6.3 min, respectively. CONCLUSION: In patients heterozygous for the usual and the atypical variant (UA), the potency of mivacurium is higher, the infusion requirements lower and the rate of spontaneous recovery prolonged, compared with phenotypically normal patients. The clearances of the active isomers are significantly lower and the elimination half-lives longer in heterozygous patients than in phenotypically normal patients (UU). The pharmacokinetics of the inactive cis-cis isomer was not affected.