Identification of PKCalpha isoform-specific effects in cardiac myocytes using antisense phosphorothioate oligonucleotides

Members of the mammalian protein kinase C (PKC) superfamily play key regulatory roles in multiple cellular processes. In the heart, PKC signaling is involved in hypertrophic agonist-induced gene expression and hypertrophic growth. To investigate the specific function of PKC signaling in regulating cardiomyocyte growth, we used antisense oligonucleotides to inhibit PKC alpha, the major isozyme present in the neonatal heart. Transfection of cultured neonatal cardiomyocytes with antisense PKCalpha oligonucleotides resulted in a marked reduction in both PKCalpha mRNA and protein levels. PKCalpha antisense treatment also reduced phenylephrine (PE)-induced PKC activity and perinuclear translocation of PKCalpha. Antisense inhibition of PKCalpha led to reduction of PE-induced increase in skeletal alpha-actin mRNA levels and atrial natriuretic peptide (ANP) secretion but had no significant effects on PE-induced beta-myosin heavy chain, ANP, or B-type natriuretic peptide (BNP) gene expression. On the other hand, antisense PKCalpha treatment attenuated endothelin-1-induced increase in ANP and BNP peptide secretion, whereas endothelin-1-induced gene expression of ANP and BNP remained unchanged. The hypertrophic agonist-induced growth of cardiomyocytes, characterized by increased [(3)H]leucine incorporation, was not affected with antisense PKCalpha treatment. Furthermore, we found that PE-induced increase in extracellular signal-regulated kinase (ERK) activity was partially inhibited by antisense PKCalpha treatment, implicating ERK as a downstream mediator for PKCalpha signaling. These results indicate that PKCalpha isozyme is involved in hypertrophic signaling in cardiomyocytes and provide novel strategies for future studies to identify other cellular targets controlled selectively by PKCalpha or other PKC isozymes.     

Genetic risk determines the emergence of diabetes-associated autoantibodies in young children

Timing of onset of autoimmunity is a prerequisite for unmasking triggers and pathogenesis of type 1 diabetes. We followed 4,590 consecutive newborns with 8 or 3% HLA-DQB1 conferred risk for type 1 diabetes at 3-, 6-, or 12-month intervals up to 5.5 years of age. Islet cell autoantibodies (ICAs) and, in the 137 children with ICAs, insulin autoantibodies (IAAs), GAD65 autoantibodies (GADAs), and IA-2 protein autoantibodies (IA-2As) were measured. Children with high genetic risk developed ICAs more often than those with moderate risk (log-rank P = 0.0015); 85 and 91% remained ICA negative by 5 years of age, respectively. The time of appearance of biochemical autoantibodies was then compared with the appearance of ICAs. IAAs and GADAs emerged usually before ICAs (means -1.8 and -1.5 months, respectively) and IA-2As after ICAs (mean 2.0 months). Ninety-five percent of all IAAs, GADAs, and IA-2As seroconversions occurred in a cluster (-12 to 8 months) around the ICA seroconversion. We conclude that diabetes-associated autoantibodies emerged in children with predisposing HLA-DQB1 alleles after 3 months of age at a constant tempo, determined by the genetic risk level, usually in the order of IAA, GADA, ICA, and IA-2A. Seroconversion to multiple autoantibody positivity usually occurred tightly clustered in time.     

Initial rightward orienting bias in clinical tasks: normal subjects and right hemispheric stroke patients with and without neglect

In order to develop the diagnosis of hemi-inattention in patients with right hemispheric cerebrovascular accident (RCVA), the initial starting point of cancellation performance was studied in seven commonly used visual cancellation tasks, an Object Finding task, and a blindfold Tactuo-motor search task. The subject groups consisted of 34 patients with RCVA and 31 healthy subjects. Patients were divided into groups of contralateral neglect and no neglect. One additional case of ipsilesional neglect and one of nonlateralised attentional disorder in relation to early orienting bias, are reported. Patients with contralateral neglect showed a strong tendency to start their cancellation performance on the right. Also, half of the nonneglect patient group demonstrated right bias in initiating their cancellation performance, suggesting the presence of a subclinical hemi-inattention group. Contrary to expectations, also a small proportion of normal subjects were right-biased. Task dependent differences in the assessment of early rightward orienting bias were found.     

Brain catecholamine metabolism in catechol-O-methyltransferase (COMT)-deficient mice

Catechol-O-methyltransferase (COMT) catalyses the O-methylation of compounds having a catechol structure and its main function involves the elimination of biologically active or toxic catechols and their metabolites. By means of homologous recombination in embryonic stem cells, a strain of mice has been produced in which the gene encoding the COMT enzyme is disrupted. We report here the levels of catecholamines and their metabolites in striatal extracellular fluid in these mice as well as in homogenates from different parts of the brain, under normal conditions and after acute levodopa administration. In immunoblotting studies, COMT-knockout mice had no COMT protein in brain or kidney tissues but the amounts of catecholamine synthesizing and other metabolizing enzyme proteins were normal. Under normal conditions, COMT deficiency does not appear to affect significantly brain dopamine and noradrenaline levels in spite of relevant changes in their metabolites. This finding is consistent with previous pharmacological studies with COMT inhibitors and confirms the pivotal role of synaptic reuptake processes and monoamine oxidase-dependent metabolism in terminating the actions of catecholamines at nerve terminals. In contrast, when COMT-deficient mice are challenged with l-dihydroxyphenylalanine, they show an extensive accumulation of 3,4-dihydroxyphenylacetic acid and dihydroxyphenylglycol and even dopamine, revealing an important role for COMT under such situations. Notably, in some cases these changes appear to be Comt gene dosage-dependent, brain-region specific and sexually dimorphic. Our results may have implications for improving the treatment of Parkinson's disease and for understanding the contribution of the natural variation in COMT activity to psychiatric phenotypes.     

Is the learning curve for laparoscopic fundoplication determined by the teacher or the pupil?

BACKGROUND: For all surgical procedures, a surgeons' learning curve can be anticipated during which complication rates are increased. The aims of this study were to evaluate individual learning curves for a group of surgeons performing laparoscopic fundoplication and to evaluate if the Procedicus MIST-simulator (Mentice Inc., G?teborg, Sweden) accurately predicts surgical performance. METHODS: Twelve Nordic centers participated, each contributing with a "master" and a "pupil" surgeon. The pupils were tested in the simulator and thereafter performed their first 20 supervised operations. All procedures were videotaped and evaluated by 3 independent reviewers. RESULTS: A significant decrease in operative time (P <0.001) and a trend (P = 0.12) toward improved score were seen during the series. The master significantly affected the pupil's score (P =0.0137). The simulator-test showed no correlation with the operative score. CONCLUSIONS: Individual learning curves varied, and the teacher was shown to be the most important factor influencing the pupil's performance score. The correlation between assessed performance and patient outcome will be further investigated.     

Anterior knee pain after intramedullary nailing of a tibial shaft fracture: an ultrasound study of the patellar tendons of 36 patients

OBJECTIVES: Chronic anterior knee pain is a common complication following intramedullary nailing of a tibial shaft fracture. The etiology of pain is often not known. This study sonographically examined the patellar tendons of patients with a nailed tibial shaft fracture. DESIGN: Prospective study. SETTING: University hospital. PATIENTS: Fifty consecutive patients with a nailed tibial shaft fracture were initially included in the study. Thirty-six of them could be measured at an average of 2.5 +/- 0.5 years after nail insertion (1.0 +/- 0.3 years after nail extraction). INTERVENTION: Reamed intramedullary nailing with 2 interlocking bolts at both ends of the nail (Grosse-Kempf-nail, Howmedica). MAIN OUTCOME MEASUREMENTS: The ultrasound investigation of the patellar tendons of the 36 patients. RESULTS: Twelve (33%) patients were painless and 24 (67%) patients had anterior knee pain at follow-up. With the reference to the mean difference in the thickness of the distal part of the patellar tendon in the operated limb versus nonoperated limb, the result was 1.4 +/- 1.1 mm in the chronic pain group and 2.6 +/- 2.5 mm in the painless group (P = 0.135, [95% confidence interval for the group difference = -0.4-2.8]). The corresponding values for the proximal part of the patellar tendon was 1.4 +/- 1.3 mm in the chronic pain group and 2.3 +/- 2.3 mm in the painless group (P = 0.251, [95% confidence interval for the group difference = -0.7-2.4]). There were no statistically significantly differences between study groups in the blood circulation of the patellar tendon or at the entry point, calcification of the patellar tendon, granulation tissue at the entry point, or occurrence of low echo areas in the patellar tendon. CONCLUSION: After intramedullary nailing of a tibial shaft fracture, patients with or without anterior knee pain show similar changes in the ultrasound investigation of their patellar tendons. Based on those findings, it does not appear to make any difference as to the approach used (paratendinous or transtendinous) for intramedullary nailing of the tibia.     

Sodium transport in airway epithelium correlates with lung compliance in healthy newborn infants

To study the relation between sodium transport in airway epithelium and postnatal pulmonary adaptation, we measured nasal potential difference at 1 to 4 hours and lung compliance at 21 to 48 hours after birth in 20 healthy infants. Sodium transport correlated with lung compliance ( r 2 = 0.40, P < .003). Airway sodium transport plays a role in postnatal pulmonary adaptation.     

Carotid plaque mast cells associate with atherogenic serum lipids, high grade carotid stenosis and symptomatic carotid artery disease. Results from the helsinki carotid endarterectomy study

OBJECTIVE: Increased numbers of mast cells (MCs) are present in ruptured coronary plaques, suggesting to play a role in acute coronary syndromes. We evaluated the distribution densities of MCs, macrophages and T cells in carotid plaques and correlated these findings to stroke risk factors as well as history of stroke or TIA. METHODS AND RESULTS: Seventy-eight carotid samples from 75 patients (16 plaques from asymptomatic patients and 62 from patients with recent ischemic symptoms) undergoing carotid endarterectomy with an internal carotid stenosis >70% that were immunostained and quantified for MCs, macrophages and T cells. The MC distribution density showed positive correlation with the degree of carotid stenosis (p = 0.012), serum levels of total cholesterol (p = 0.021), LDL cholesterol (p = 0.013) and triglycerides (p = 0.005), and an inverse correlation with serum HDL cholesterol levels (p = 0.001). The average MC density (p = 0.023), but not the macrophage (p = 0.58) or T cell (p = 0.74) density, was higher in the symptomatic than in the asymptomatic patients. In a comparison of plaques ipsilateral and contralateral to the thromboembolic event, the densities of the three types of inflammatory cells were similar. CONCLUSIONS: Increased MC distribution density is associated with an atherogenic serum lipid profile, high-grade carotid artery stenosis and symptomatic carotid artery disease. These findings suggest a potential involvement of MCs in the pathophysiology of carotid artery stenosis.     

A rare truncating mutation in ADH1C (G78Stop) shows significant association with Parkinson disease in a large international sample

BACKGROUND: Alcohol dehydrogenases (ADHs) may be involved in the pathogenesis of neurodegenerative disorders because of their multiple roles in detoxification pathways and retinoic acid synthesis. In a previous study, significant association of an ADH class IV allele with Parkinson disease (PD) was found in a Swedish sample. PATIENTS: The previously associated single-nucleotide polymorphism plus 12 further polymorphisms in the ADH cluster on human chromosome 4q23 were screened for association in an extension of the original sample that now included 123 Swedish PD patients and 127 geographically matched control subjects. A rare nonsense single-nucleotide polymorphism in ADH1C (G78stop, rs283413) was identified in 3 of these patients but in no controls. To obtain sufficient power to detect a possible association of this rare variant with disease, we screened a large international sample of 1076 PD patients of European ancestry and 940 matched controls. RESULTS: The previously identified association with an ADH class IV allele remained significant (P<.02) in the extended Swedish study. Furthermore, in the international collaboration, the G78stop mutation in ADH1C was found in 22 (2.0%) of the PD patients but only in 6 controls (0.6%). This association was statistically significant (chi(2)(1) = 7.5; 2-sided P = .007; odds ratio, 3.25 [95% confidence interval, 1.31-8.05]). In addition, the G78stop mutation was identified in 4 (10.0%) of 40 Caucasian index cases with PD with mainly hereditary forms of the disorder. CONCLUSION: Findings presented herein provide further evidence for mutations in genes encoding ADHs as genetic risk factors for PD.