Regulation of HERG potassium channel activation by protein kinase C independent of direct phosphorylation of the channel protein

OBJECTIVE: Patients with HERG-associated long QT syndrome typically develop tachyarrhythmias during physical or emotional stress. Previous studies have revealed that activation of the beta-adrenergic system and consecutive elevation of the intracellular cAMP concentration regulate HERG channels via protein kinase A-mediated phosphorylation of the channel protein and via direct interaction with the cAMP binding site of HERG. In contrast, the influence of the alpha-adrenergic signal transduction cascade on HERG currents as suggested by recent reports is less well understood. The aim of the present study was to elucidate the biochemical pathways of the protein kinase C (PKC)-dependent regulation of HERG currents. METHODS: HERG channels were heterologously expressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage clamp technique. RESULTS: Application of the phorbol ester PMA, an unspecific protein kinase activator, shifted the voltage dependence of HERG activation towards more positive potentials. This effect could be mimicked by activation of conventional PKC isoforms with thymeleatoxin. Coexpression of HERG with the beta-subunits minK or hMiRP1 did not alter the effect of PMA. Specific inhibition of PKC abolished the PMA-induced activation shift, suggesting that PKC is required within the regulatory mechanism. The PMA-induced effect could still be observed when the PKC-dependent phosphorylation sites in HERG were deleted by mutagenesis. Cytoskeletal proteins such as actin filaments or microtubules did not affect the HERG activation shift. CONCLUSION: In addition to the known effects of PKA and cAMP, HERG channels are also modulated by PKC. The molecular mechanisms of this PKC-dependent process are not completely understood but do not depend on direct PKC-dependent phosphorylation of the channel.     

C-reactive protein as a predictor for incident diabetes mellitus among middle-aged men: results from the MONICA Augsburg cohort study, 1984-1998

BACKGROUND: Previous studies have suggested that low-grade systemic inflammation is involved in the pathogenesis of type 2 diabetes mellitus. OBJECTIVE: To investigate the association between C-reactive protein (CRP), the classic acute-phase protein, and incident type 2 diabetes mellitus among middle-aged men. METHODS: A total of 2052 initially nondiabetic men aged 45 to 74 years who participated in 1 of the 3 MONICA (Monitoring of Trends and Determinants in Cardiovascular Disease) Augsburg surveys between 1984 and 1995 were followed up for an average of 7.2 years. Incidence of diabetes was assessed by questionnaire mailed to participants in 1998. High-sensitive CRP was measured by an immunoradiometric assay. RESULTS: A total of 101 cases of incident diabetes occurred during the follow-up period. The age-standardized incidence rate was 6.9 per 1000 person-years. Men with CRP levels in the highest quartile (CRP > or = 2.91 mg/L) had a 2.7 times higher risk of developing diabetes (95% confidence interval, 1.4-5.2) compared with men in the lowest quartile (CRP < or = 0.67 mg/L) in a Cox proportional hazards model adjusted for age and survey. After further adjustment for body mass index, smoking, and systolic blood pressure, the observed association was significantly reduced and became nonsignificant. CONCLUSIONS: Low-grade systemic inflammation is associated with an increased risk of type 2 diabetes mellitus in middle-aged men. Inflammation could be one mechanism by which known risk factors for diabetes mellitus, such as obesity, smoking, and hypertension, promote the development of diabetes mellitus.     

Hydrolytically activated etoposide prodrugs inhibit MDR-1 function and eradicate established MDR-1 multidrug-resistant T-cell leukemia

Effective therapy of high-risk leukemia with established cytotoxic drugs may be limited by poor antitumor efficacy, systemic toxicity, and the induction of drug resistance. Here, we provide the first evidence that hydrolytically activated prodrugs may overcome these problems. For this purpose, VP16 was functionally blocked by hydrolytically cleavable carbonate linkers with unique characteristics to generate 2 novel prodrugs of VP16. First, we established a more than 3-log higher efficacy of the 2 prodrugs compared with VP16 on a panel of naturally drug-resistant tumor cell lines. Second, the prodrugs did overcome VP16-induced multidrug resistance-1 gene (MDR-1)-mediated multidrug resistance in vitro in a newly established VP16-resistant T-cell leukemia cell line MOVP-3 by functionally blocking MDR-1-mediated efflux. Third, in vivo studies showed a maximum tolerated dose of ProVP16-II (> 45mg/kg), which was at least 3-fold higher than that of VP16 (15 mg/kg). Finally, tests of ProVP16-II in a multidrug-resistant xenograft model of T-cell leukemia expressing MDR-1 indicated that only the mice treated with this prodrug revealed a complete and long-lasting regression of established, drug-resistant leukemia. In summary, the hydrolytically activated etoposide prodrugs proved effective against multidrug-resistant T-cell leukemia in vitro and in vivo and provide proof of concept for a highly promising new strategy for the treatment of MDR-1 drug-resistant malignancies.     

Phase-dependent expression of matrix metalloproteinases and their inhibitors in demyelinating canine distemper encephalitis

Matrix metalloproteinases (MMPs) are zinc- and calcium-dependent enzymes that cleave molecules of the extracellular matrix, and thus are able to open the blood-brain-barrier and affect myelin. Their inhibitors (TIMPs) are important candidates for the therapy of demyelinating diseases. To establish an immunohistochemical profile of MMP and TIMP expression in plaque variants in dogs with spontaneous demyelinating distemper encephalitis, paraffin-embedded cerebella were studied employing the avidin-biotin-peroxidase complex method with a panel of nine polyclonal (anti-MMP-1, -3, -7, -9, -12, -13, -14, -TIMP-1, and -2) and two monoclonal antibodies (anti-latent MMP-2, and -MMP-11). All MMPs and TIMPs were prominently up-regulated in acute and subacute non-inflammatory lesions, and double-labeling techniques showed that they were mainly expressed by astrocytes and brain macrophages/microglia. In subacute inflammatory and chronic plaques, a moderate to strong decrease of MMP and TIMP expression compared to acute lesions was observed. In these phases MMP-11, -12, and -13 were still moderately present. In addition to astro- and microglia, invading perivascular mononuclear cells were positive for MMPs and TIMPs. In summary, there seems to be a phase-dependent expression of MMPs and TIMPs in demyelinating canine distemper encephalitis, and an MMP-TIMP imbalance might account for the lesion progression in this disease.     

Human ependymomas reveal frequent deletions on chromosomes 6 and 9

Ependymomas are glial tumors of the brain and spinal cord. Genetic aberrations associated with the development of these tumors have not been fully identified yet. In previous cytogenetic and comparative genomic hybridization studies, multiple genomic imbalances in ependymomas were found, including partial or whole chromosome losses (1p, 4q, 6q, 9, 10, 11, 13, 16, 17, 19q, 20q and 22q). The aim of this study was to map particularly the commonly affected regions in ependymomas. Thirty-three pairs of matched normal and tumor specimens from ependymoma patients were genotyped using 34 polymorphic microsatellite markers distributed over 15 chromosomes. Loss of heterozygosity (LOH) was found in 26 of 33 tumors (78.8%). The most frequent LOHs were found on the long arms of chromosomes 6 (30.3%) and 9 (27.3%). LOH was also detected on 3p14 (13.3%), 10q23 (10.3%) and 11q (18.2%). Because of the high percentage of LOH on chromosome 6 and 9, we conclude that important tumor suppressor genes are situated on these two chromosomes.Dedicated to Prof. Dr. H.K. Mueller-Hermelink's 60th birthday.     

Neural correlates of episodic encoding and recognition of words in unmedicated patients during an acute episode of schizophrenia: a functional MRI study

OBJECTIVE: Memory impairment has been well documented in schizophrenia. In a previous study, the authors investigated patterns of brain activity during episodic encoding and recognition of words in remitted, stable schizophrenia outpatients being treated with novel antipsychotics. The same procedure was used in this study to investigate unmedicated patients during an acute episode of schizophrenia. METHOD: Functional magnetic resonance imaging was used to study regional brain activation in 10 unmedicated patients experiencing an acute episode of schizophrenia and 10 healthy comparison subjects during performance of a modified version of the words subtest of Warrington's Recognition Memory Test. RESULTS: Despite intact recognition performance, patients with schizophrenia showed reduced activation of anterior prefrontal, posterior cingulate, and retrosplenial areas relative to comparison subjects during word encoding. During word recognition, reduced activation was found in the patients' dorsolateral prefrontal and limbic/paralimbic regions. On the other hand, higher metabolism in bilateral anterior prefrontal cortices was observed. CONCLUSIONS: The results suggest that different neural pathways are engaged during episodic encoding and recognition of words in patients experiencing an acute episode of schizophrenia relative to healthy comparison subjects. Furthermore, acute psychosis may prevent practice effects, reflected in a failure to engage brain regions associated with successful episodic memory retrieval in healthy subjects.     

Bioelectrical behaviour of hypoxic human neocortical tissue under the influence of nimodipine and dimethyl sulfoxide

Nimodipine and dimethyl sulfoxide (DMSO) have been shown to affect electrophysiological responses in rodent brain tissue in an vitro model of hypoxia. In the present study, the same agents were now examined for their effects on human neocortical brain slices under repeated hypoxic conditions. DMSO (0.4%), with and without addition of nimodipine (40 micromol/l), did not increase the latency of anoxic depolarization (AD). This finding is not in line with our previous observations of DMSO effects, with and without nimodipine, on brain slices of guinea pigs. AD latency was significantly longer in human neocortical brain slices compared with hippocampal slices of rodents even without any pharmacological influence. A possible acute effect of DMSO-nimodipine may therefore be masked by an interspecies difference of hypoxia resistance.     

Effects of valproic acid,carbamazepine, and phenobarbitone on the fatty acid composition of erythrocyte membranes in children

PURPOSE: To investigate a possible relation between antiepileptic drugs (AEDs) and the fatty acid composition of membranes. METHODS: Fatty acid (FA) composition of erythrocytes was studied in children with epilepsy receiving AED monotherapy. Children taking valproate (VPA, n = 28), carbamazepine (CBZ, n = 17), or phenobarbitone (PB, n = 14) were compared with healthy controls (n = 25). FAs were measured by capillary-gas chromatography (GC-FID). RESULTS: Significant changes (p < 0.05) in the FA composition of membranes were found. In children treated with VPA, C13:0 was decreased (8.2 +/- 2% vs. 10.7 +/- 4% in controls) and C14:0 increased (1.4 +/- 0.5% vs.1 +/- 0.5% in controls). C17:0 again was reduced (9.9 +/- 4% vs. 13.2 +/- 6% in controls), whereas the long-chained acids were enhanced: C18:2n-6 (6 +/- 2.4% vs. 3.9 +/- 2.5% in controls), and C20:4n-6 (1.9 +/- 1.7% vs. 1.4 +/- 0.5% in controls). The nonidentified FAs also increased with VPA therapy: 2.5 +/- 0.8% versus 1.7 +/- 0.9% in controls. Children treated with CBZ showed only minimal changes of FA composition: C13:0 was decreased compared with controls (8 +/- 2% vs. 10.7 +/- 4%). No changes were seen in patients taking PB. The mean corpuscular volume (MCV) showed important differences between the study groups: MCV was 84.7 +/- 6.0 fl during VPA therapy (p < 0.001) and 85.7 +/- 4.1 fl with CBZ (p < 0.001). During PB, the MCV increased to 82.87 +/- 3.29 fl compared with controls (78.73 +/- 4.92 fl; p < 0.01). CONCLUSIONS: VPA therapy is associated with changes of the FA composition of membranes, which is not the case with PB therapy. The implications of this finding remain to be established.