Misassembly of full-length Disrupted-in-Schizophrenia 1 protein is linked to altered dopamine homeostasis and behavioral deficits.

Disrupted-in-schizophrenia 1 (DISC1) is a mental illness gene first identified in a Scottish pedigree. So far, DISC1-dependent phenotypes in animal models have been confined to expressing mutant DISC1. Here we investigated how pathology of full-length DISC1 protein could be a major mechanism in sporadic mental illness. We demonstrate that a novel transgenic rat model, modestly overexpressing the full-length DISC1 transgene, showed phenotypes consistent with a significant role of DISC1 misassembly in mental illness. The tgDISC1 rat displayed mainly perinuclear DISC1 aggregates in neurons. Furthermore, the tgDISC1 rat showed a robust signature of behavioral phenotypes that includes amphetamine supersensitivity, hyperexploratory behavior and rotarod deficits, all pointing to changes in dopamine (DA) neurotransmission. To understand the etiology of the behavioral deficits, we undertook a series of molecular studies in the dorsal striatum of tgDISC1 rats. We observed an 80% increase in high-affinity DA D2 receptors, an increased translocation of the dopamine transporter to the plasma membrane and a corresponding increase in DA inflow as observed by cyclic voltammetry. A reciprocal relationship between DISC1 protein assembly and DA homeostasis was corroborated by in vitro studies. Elevated cytosolic dopamine caused an increase in DISC1 multimerization, insolubility and complexing with the dopamine transporter, suggesting a physiological mechanism linking DISC1 assembly and dopamine homeostasis. DISC1 protein pathology and its interaction with dopamine homeostasis is a novel cellular mechanism that is relevant for behavioral control and may have a role in mental illness.

Development of a capillary isoelectric focusing immunoassay to measure DJ-1 isoforms in biological samples.

We report on the development of a novel assay protocol for the separation and detection of charge isoforms of DJ-1 in biological samples by automated capillary isoelectric focusing followed by immunological detection. DJ-1 (PARK7) is considered as a biomarker candidate for Parkinson's disease and may potentially support the differentiation of clinical subtypes of the disease. The new method allows for separation and subsequent relative quantitative comparison of different isoforms of DJ-1 in biological samples. The assay was successfully applied to the analysis of DJ-1 isoform patterns in brains from mice subjected to normal or high-fat diet and revealed statistically significant group differences. Furthermore, in a pooled and concentrated sample of human cerebrospinal fluid that was depleted of albumin and immunoglobulin G, four different charge variants of DJ-1 could be detected. Taken together, the capillary isoelectric focusing immunoassay for DJ-1 represents a promising tool that may ultimately serve in clinical biomarker studies.

The proteomic signature of insulin-resistant human skeletal muscle reveals increased glycolytic and decreased mitochondrial enzymes.

AIMS/HYPOTHESIS: The molecular mechanisms underlying insulin resistance in skeletal muscle are incompletely understood. Here, we aimed to obtain a global picture of changes in protein abundance in skeletal muscle in obesity and type 2 diabetes, and those associated with whole-body measures of insulin action. METHODS: Skeletal muscle biopsies were obtained from ten healthy lean (LE), 11 obese non-diabetic (OB), and ten obese type 2 diabetic participants before and after hyperinsulinaemic-euglycaemic clamps. Quantitative proteome analysis was performed by two-dimensional differential-gel electrophoresis and tandem-mass-spectrometry-based protein identification. RESULTS: Forty-four protein spots displayed significant (p < 0.05) changes in abundance by at least a factor of 1.5 between groups. Several proteins were identified in multiple spots, suggesting post-translational modifications. Multiple spots containing glycolytic and fast-muscle proteins showed increased abundance, whereas spots with mitochondrial and slow-muscle proteins were downregulated in the OB and obese type 2 diabetic groups compared with the LE group. No differences in basal levels of myosin heavy chains were observed. The abundance of multiple spots representing glycolytic and fast-muscle proteins correlated negatively with insulin action on glucose disposal, glucose oxidation and lipid oxidation, while several spots with proteins involved in oxidative metabolism and mitochondrial function correlated positively with these whole-body measures of insulin action. CONCLUSIONS/INTERPRETATION: Our data suggest that increased glycolytic and decreased mitochondrial protein abundance together with a shift in muscle properties towards a fast-twitch pattern in the absence of marked changes in fibre-type distribution contribute to insulin resistance in obesity with and without type 2 diabetes. The roles of several differentially expressed or post-translationally modified proteins remain to be elucidated.

Establishment of "one-piece" large-gel 2-DE for high-resolution analysis of small amounts of sample using difference gel electrophoresis saturation labelling.

Large-gel two-dimensional gel electrophoresis (2-DE) is the method of choice for high-resolution proteome analysis of complex protein mixtures. Until now, however, the advantages of large 2-DE in combination with multiplexed fluorescence dye protein labelling has been complicated by the separate handling and analysis of the second-dimension gels. Therefore, we adapted the large 2-DE procedure allowing us to run "one-piece" large 2-DE gels (40 cm x 30 cm) in the second dimension for high resolution proteome analysis. Here, we show that in combination with fluorescence dye protein saturation labelling "one-piece" large 2-DE enables analysis of small amounts of sample (3 microg protein) for high-resolution proteome analysis.

Expression of MUPP1 protein in mouse brain.

Localizing cell surface receptors to specific subcellular sites can be crucial for proper functioning. PDZ proteins apparently play central roles in such protein localizations. 5-HT(2C) receptors have previously been shown to interact with MUPP1, a multi PDZ domain protein, in heterologous systems and in rat choroid plexus. We now report the generation and characterization of two independent MUPP1 antisera, which recognise distinct areas of the mouse brain in agreement with previous in-situ hybridization studies. Our results indicate that MUPP1 immunoreactivity co-localizes with 5-HT(2A) or 5-HT(2C) receptor expression in all regions of the mouse brain, including the choroid plexus where 5-HT(2C) receptors are highly enriched.

[Atrial sensing by a new VDD pacemaker]. / Atriale Wahrnehmung bei einem neuen VDD-Schrittmacher.

VDD stimulation is an alternative to DDD pacing due to the possibility of p-wave synchronous ventricular pacing without the need of an atrial lead. Mainly, the reliability of the system depends on the atrial sensing. In 22 patients the intraoperative atrial amplitude and, postoperatively, the atrial sensing threshold were measured. Furthermore, the stability of the atrial sensing threshold during follow-up was proven. The mean atrial amplitude was intraoperative by 2.4 +/- 1.2 (1.0-6.8) mV. The measurement of the atrial sensing threshold in the first 5 postoperative days showed a mean value of 1.02 +/- 0.49 (0.3-1.6) mV. The measurements of the mean atrial sensing threshold after 30, 90, and 180 days showed no statistical differences. Intraindividual variance was shown in 17/20 patients (0.55 +/- 0,42; 0.15-1.05 mV). Seventeen of the 22 patients were programmed with an atrial sensing threshold of 0.3 mV. In five patients the atrial sensing threshold was programmed at less than 0.3 mV in order to reach a twofold atrial sensitivity. Despite a programmed atrial sensitivity of 0.1 mV and isometric conditions no atrial oversensing occurred. The postoperative atrial sensing thresholds of the VDD system investigated were significantly lower than the intraoperatively measured atrial amplitudes. The mean atrial sensing threshold did not change during the follow-up period. The variation which did occur was within individual variation at different return visits.

Cardiac output in single-lead VDD pacing versus rate-matched VVIR pacing.

The importance of atrioventricular synchronous pacing compared with single-chamber rate-responsive pacing is still under discussion, especially for low-intensity workload representing daily life activities. We evaluated hemodynamics in single-lead VDD pacing versus VVIR pacing in 11 patients (8 men and 3 women, aged 58.6 +/- 13.8 years) with normal left ventricular function and a previously implanted single-lead VDDR pacemaker. A low-intensity steady-state treadmill test at 1 to 2.5 mph with a gradient of 2% to 4% was performed. Cardiac output was determined using a standard carbon dioxide rebreathing technique. Initially, the VDD mode was programmed, and after 5 minutes of exercise, cardiac output was measured in steady-state conditions. The pacemaker was then reprogrammed to the VVI mode at a rate 5 to 10 beats above the maximal atrial tracking rate to simulate rate-matched VVIR pacing (VVIRm). After 5 additional minutes of steady-state exercise, cardiac output was measured again. The maximal atrial rate in the VDD mode was 119 +/- 19 beats/min versus a programmed rate of 129 +/- 18 beats/min in the VVIRm mode. VDD pacing resulted in a significantly higher cardiac output than VVIRm pacing (10.6 +/- 1.9 vs 9.2 +/- 1.4 L/min; p < 0.002), with a mean difference of 1.6 +/- 1.2 L/min between the 2 modes. In the VDD mode, stroke volume (90.7 +/- 20.1 vs 71.6 +/- 13.0 ml; p < 0.001) and maximal oxygen uptake (1,183 +/- 264 vs 1,076 +/- 289 ml/min, p < 0.01) were also higher than in VVIRm.(ABSTRACT TRUNCATED AT 250 WORDS)

SDZ PSC 833, a non-immunosuppressive cyclosporine: its potency in overcoming P-glycoprotein-mediated multidrug resistance of murine leukemia.

Cyclosporin A (CsA, Sandimmune) is known to reverse P-glycoprotein (P-gp170)-mediated multidrug resistance as efficiently as other prototype compounds of resistance modifiers. The immunosuppressive activity and nephrotoxicity of CsA, however, may limit its clinical use. PSC-833, a new cyclosporine, exerts a similar resistance-modifying activity but lacks toxicity or immunosuppressive activity. We have tested its potency in vitro and in vivo on the L1210 leukemia cell line transfected with a full-length cDNA copy of the human mdr I gene, which showed a stable 30-fold resistance towards adriamycin as compared to the parental cell line. In vitro growth of the transfected cell was unchanged. In vivo growth was less aggressive; the survival time of inoculated mice was prolonged. In vitro, PSC-833 was at least as potent as CsA or verapamil in reversing multidrug resistance. In vivo, the drug-resistant L1210 leukemia was completely unresponsive to i.v. monotherapy with adriamycin at its maximum tolerated dose (MTD). PSC-833 enhanced the activity and toxicity of adriamycin. The MTD of adriamycin was about 3 times lower than when given alone. On this basis, the MTD of i.v. adriamycin in combination with oral PSC-833 successfully overcame refractoriness to treatment. Survival times of the mice were considerably prolonged and even some cures of leukemic mice occurred.

Phosphorylation of myelin basic proteins: an age-dependent process affected by divalent cations and Co-Dergocrine (dihydroergotoxine).

Separation of rat cortical or hippocampal myelin preparations after in vitro phosphorylation on sodium dodecyl sulfate slab gel electrophoresis revealed several phosphorylated proteins with molecular weights between 12,000 and 25,000. The major phosphate acceptors seemed to be two basic proteins. This in vitro phosphorylation of myelin uses endogenous phosphokinase activity and was not influenced by biologically relevant concentrations of cyclic AMP or cyclic GMP. However, phosphorylation was stimulated by addition of Ca2+ and completely inhibited by omission of Mg2+ from the incubation medium. In vitro phosphorylation of CNS myelin proved to be age dependent: increased phosphate incorporation into CNS myelin basic proteins was observed up to the age of about 12 weeks. A steady decline in phosphate incorporation occurred thereafter leaving little in vitro phosphorylating capacity at 80 weeks of age or more. Old animals (80 weeks), when treated with Co-Dergocrine, showed improved in vitro phosphorylating capacity of myelin preparations approaching that found in young control animals.

Effects of the epipodophyllotoxin derivative VM 26 in mitosis and in interphase.

The epipodophyllotoxin derivative VM 26 inhibits entry of mouse mastocytoma cells into mitosis in cell cultures at drug concentrations of 0.01--1 microgram/ml, the cells being arrested in G2 phase of the cell cycle. At higher concentrations, the compound exhibits spindle poison activity which manifests itself in a shortlasting rise in the number of cells arrested in metaphase of mitosis. These cells then disintegrate after a short period of time.