Detection of de- and hyperpolarization of mitochondria of cultured astrocytes and neurons by the cationic fluorescent dye rhodamine 123

The mitochondrial potential is an essential regulator in cellular physiology and detection of this parameter in living cells is still under discussion. Here we present a protocol which allows the use of rhodamine 123 as a probe for quantifying the mitochondrial potential. To avoid dequenching artefacts the detection area is limited to the area above the nucleus. In co-cultured rat hippocampal astrocytes and neurons, we analysed the mitochondrial accumulation of the cationic fluorescent dye rhodamine 123 (Rh123). Application of the uncoupler carbonyl cyanide 4-(trifluoro-methoxy)phenylhydrazone (FCCP, 4 μmol/L) together with the ATP-synthase inhibitor oligomycin (Oli, 10 μmol/L) induced an immediate fluorescence increase of Rh123-loaded mitochondria. This effect is due to the well-known fluorescence dequenching caused by the reduction in concentration of Rh123 in the mitochondria after depolarization. However, above the nucleus an increase in fluorescence was registered. Due to the absence of mitochondria in the area above the nucleus this fluorescence increase is most likely caused by the Rh123 release from mitochondria. Pre-treatment of cells with antimycin A abolished the response to FCCP/Oli. Furthermore, a 10-min exposure to 50 mmol/L K⁺, which causes a plasma membrane depolarization in neurons, did not significantly change the FCCP/Oli-mediated Rh123 release measured above the nucleus of neurons. However, application of 100 μmol/L glutamate enhanced the effect of FCCP/Oli both in astrocytes and neurons. This enhancement is interpreted as an increase in mitochondrial potential above the control potential. Thus, this Rh123 method described here allows a cell type-specific determination of changes of mitochondrial polarization.     

Multiple system atrophy: a primary oligodendrogliopathy

To this day, the cause of multiple system atrophy (MSA) remains stubbornly enigmatic. A growing body of observations regarding the clinical, morphological, and biochemical phenotypes of MSA has been published, but the interested student is still left without a clue as to its underlying cause. MSA has long been considered a rare cousin of Parkinson's disease and cerebellar degeneration; it is rich in acronyms but poor in genetic and environmental leads. Because of the worldwide research efforts conducted over the last two decades and the discovery of the alpha-synuclein-encoding SNCA gene as a cause of rare familial Parkinson's disease, the MSA field has seen advances on three fronts: the identification of its principal cellular target, that is, oligodendrocytes; the characterization of alpha-synuclein-rich glial cytoplasmic inclusions as a suitable marker at autopsy; and improved diagnostic accuracy in living patients resulting from detailed clinicopathological studies. The working model of MSA as a primary glial disorder was recently strengthened by the finding of dysregulation in the metabolism of myelin basic protein and p25alpha, a central nervous system-specific phosphoprotein (also called tubulin polymerization promoting protein, TPPP). Intriguingly, in early cases of MSA, the oligodendrocytic changes in myelin basic protein and p25alpha processing were recorded even before formation of glial cytoplasmic inclusions became detectable. Here, we review the evolving concept that MSA may not just be related to Parkinson's disease but also share traits with the family of demyelinating disorders. Although these syndromes vary in their respective cause of oligodendrogliopathy, they have in common myelin disruption that is often followed by axonal dysfunction.     

The Initial Tangent of the Aortic Pressure Increase is an Estimate of Left Ventricular Contractility in Pigs

The aim was, to identify an estimate of left ventricular contractility derived from the aortic pressure wave without load changing manoeuvres. For this purpose, left ventricular contractility was assessed with several aortic pressure wave form derived parameters and was compared to standard parameters of left ventricular contractility (conductance technique) in an experimental study. Measurements were taken during baseline, after β-stimulation and after injection of a β-antagonist. The initial and the secondary tangent, the area under the aortic pressure, and the stroke volume were correlated with the endsystolic elastance, a mainly load independent measure of left ventricular contractility: The initial tangent of the aortic pressure increase correlated significantly with the endsystolic elastance (r = 0.54, P < 0.05). The initial tangent of the aortic pressure increase was significantly increased from baseline at β-stimulation (from 20.2 ± 4.7 to 36.4 ± 6.8 mmHg  s⁻¹, P < 0.05) and decreased after injection of a β-antagonist (from 20.2 ± 4.7 to 12.3 ± 2.0, P < 0.05). Thus, we conclude that the initial tangent of the aortic pressure increase is a valid estimate of left ventricular contractility in piglets. Kisch-Wedel H, Kemming G, Meisner F, Flondor M, Bruhn S, Koehler C, Zwissler B. The initial tangent of the aortic pressure increase is an estimate of left ventricular contractility in pigs.     

Levosimendan improves renal function in patients with advanced chronic heart failure awaiting cardiac transplantation

BackgroundLong-term impact of levosimendan on renal function remains undefined. Prospectively, we evaluated effects of levosimendan on renal function in patients with advanced chronic heart failure awaiting cardiac transplantation.Methods and ResultsOf 40 patients, 20 were randomized to receive levosimendan (10-minute bolus 12 μg/kg, followed by 0.1 μg/kg/min for 24 hours; LS Group), and 20 received no levosimendan (Controls). The groups did not differ in age, heart failure etiology, left ventricular ejection fraction, and plasma brain natriuretic peptide. Patients were followed for 3 months. At baseline, the groups did not differ in serum creatinine (1.92 ± 0.13 mg/dL in LS Group versus 1.91 ± 0.12 mg/dL in Controls, P = .81) and creatinine clearance (43.7 ± 2.9 mL/min versus 43.9 ± 2.8 mL/min, P = .84). At 3 months, we found a decrease in serum creatinine and an increase in creatinine clearance in LS Group, but not in Controls, leading to a significant intergroup difference in serum creatinine (1.60 ± 0.26 mg/dL in LS Group versus 1.90 ± 0.14 mg/dL in Controls, P = .005) and creatinine clearance (53.6 ± 8.6 mL/min versus 44.0 ± 3.3 mL/min, P = .005). An improvement in creatinine ≥0.5 mg/dL occurred in 50% patients from LS Group compared with 10% of Controls (P = .005).ConclusionsLevosimendan improves long-term renal function in advanced chronic heart failure patients awaiting cardiac transplantation.     

Olfactory sensitivity through the course of psychosis: Relationships to olfactory identification, symptomatology and the schizophrenia odour

There is some evidence for an unusual body odour in schizophrenia that has been linked to a hexenoic acid derivative (trans-3-methyl-2-hexenoic acid; MHA). Poor body odour has been linked to increased negative symptoms and reduced olfactory identification ability. However, the relationship between these findings and MHA, including olfactory sensitivity for MHA, has not been examined. Olfactory sensitivity thresholds were assessed for MHA and n-butyl-alcohol (NBA), in normal controls (CTL; n=24), patients with chronic schizophrenia (CHR; n=32) and a first-episode psychosis cohort (FE; n=31). In addition, forced choice detection of the pheromonal steroids 5-alpha-androst-16-en-3-one, androsterone-sulphate and estrone-3-sulphate was performed along with a measure of olfactory identification. CHR patients had significantly reduced sensitivity to MHA, but not NBA, compared to FE and CTL subjects. While sensitivity to pheromones was not different between the groups, CHR patients who could not detect them also showed poorer sensitivity to MHA. Further, the CHR group showed a significant association between reduced MHA sensitivity and greater levels of disorganised and negative symptoms. No relationships between identification and sensitivity for any substance were found. Our findings are the first to report reduced sensitivity for MHA in chronic schizophrenia patients, in the absence of similar impairment for more traditionally used substances. This may be linked to olfactory habituation effects, abnormal chemical processing or a genetic predisposition.     

Tissue factor-positive microparticles: cellular origin and association with coagulation activation in patients with colorectal cancer

The pathogenesis of hypercoagulability in cancer is not entirely understood. We hypothesized that in cancer patients circulating tissue factor-positive microparticles (TF (+) MPs) are increased and associated with hemostatic system activation. In 20 patients with advanced colorectal cancer and in 20 age- and sex-matched controls, number and cellular origin of TF (+) MPs were determined in plasma by flow cytometry. D-dimer was determined as an indicator of hemostatic system activation. Compared to controls, the median (interquartile range) number of TF (+) MPs was two-fold higher in cancer patients: 25.9 (15.4 - 42.0) x 10 (3) /ml plasma versus 13.1 (11.9 - 19.7) x 10 (3) /ml plasma, p = 0.007. This was mainly due to a higher amount of TF (+) MPs from platelets (13.4 [5.0 - 17.4] x 10 (3) /ml plasma vs. 5.8 [4.5 - 7.5] x 10 (3) /ml plasma, p = 0.017). TF (+) MPs correlated with D-dimer ( ? = 0.48, p = 0.002). High levels of TF (+) MPs in cancer patients and their correlation with D-dimer suggest that TF (+) MPs might be involved in hemostasis activation in cancer patients.     

Circulating P-selectin and the risk of recurrent venous thromboembolism

The clinical relevance of high P-selectin levels in venous thrombosis is unknown. We prospectively followed 544 patients with first unprovoked venous thromboembolism (VTE) after cessation of anticoagulation and evaluated P-selectin as a risk factor of recurrent VTE. VTE recurred in 63 (12%) patients. Patients with recurrence had significantly higher P-selectin levels than those without (45.8 mg/dl +/- 16.4 vs. 40.1 mg/dl +/- 13.3; p = 0.006). After four years, the probability of recurrence was 20.6% (95% confidence interval [CI] 12.6-28.5) among patients with P-selectin values above the 75(th) percentile of the patient population and was 10.8% (95% CI 7.2-14.3) among patients with lower values (p = 0.046). Compared to patients with low P-selectin, adjusted risk of recurrence was 1.7-fold (95% CI 1.0-2.9, p = 0.045) increased among patients with P-selectin levels exceeding the 75(th) percentile. We conclude that high circulating P-selectin is a risk factor of recurrent VTE.     

The dietary soy flavonoid genistein abrogates tissue factor induction in endothelial cells induced by the atherogenic oxidized phospholipid oxPAPC

INTRODUCTION: Tissue factor (TF) plays a pivotal role in the generation of thrombin in atherothrombotic disease. The oxidized phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC), an active compound of minimally oxidized low-density lipoprotein (MM-LDL), induces TF in endothelial cells (EC). The dietary soybean isoflavonoid genistein has been claimed to reverse several processes leading to atherosclerosis and related cardiovascular events via binding to estrogen receptors, generating nitric oxide (NO) or inhibiting tyrosine kinase-dependent pathways. METHODS AND MATERIALS: The effects and mechanisms of genistein on activity, antigen expression and mRNA levels of oxPAPC-induced TF were studied in human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC). RESULTS AND CONCLUSIONS: Genistein abrogated oxPAPC-induced TF activity in arterial and venous human EC to basal levels, as measured by functional clotting assay, and downregulated oxPAPC-induced antigen expression measured by flow cytometry and mRNA levels quantified by real-time PCR. Western blotting and inhibitor experiments with the estrogen-receptor inhibitor ICI 182,780 and the NO-synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) showed that the effect may be mediated via inhibition of phosphorylation of ERK, but not upstream MEK1/2. The effect is not mediated by the tyrosine kinase inhibitor activity of genistein, as another tyrosine kinase inhibitor (tyrphostin 25) had no effect. Binding to the estrogen receptor or generation of NO are not involved in the action of genistein on TF. In conclusion genistein reduces oxPAPC-induced TF expression and thereby the prothrombotic phenotype of EC, further substantiating and explaining the beneficial effects of dietary genistein in preventing atherosclerosis and related cardiovascular events.