Plasma hsa-mir-19b is a potential LevoDopa therapy marker

Parkinson's disease (PD) is the second most common neurodegenerative disorder among the elderly, the diagnostic and prognostic of which is based mostly on clinical signs. LevoDopa replacement is the gold standard therapy for PD, as it ameliorates the motor symptoms. However, it does not affect the progression of the disease and its long-term use triggers severe complications. There are no bona fide biomarkers for monitoring the patients’ response to LevoDopa and predicting the efficacy of levodopa treatment. Here, we have combined qPCR microRNA array screening with analysis of validated miRs in naïve versus Levodopa-treated PD patients. We have identified plasma miR-19b as a possible biomarker for LevoDopa therapy and validated this result in human differentiated dopaminergic neurons exposed to LevoDopa. In silico analysis suggests that the LevoDopa-induced miR-19b regulates ubiquitin-mediated proteolysis.     

Cave bears (Ursus spelaeus) from the Peştera cu Oase (Banat, Romania): Paleobiology and taphonomy

The 2002 discovery of the earliest European modern humans in the Peştera cu Oase (Romania) has led to systematic excavations of a bone bed (two levels), containing principally the remains of cave bears (ca. 5000 bones) and dated between 50 and 40 ka cal BP. The thanatocenosis corresponds mainly to a subadult and adult male utilization of the cavity. Levels 1 and 2 are distinguishable by different conservation processes demonstrating respectively low- and high-energy hydraulic transports. The heavy component of cannibalism may indicate bone processing for trace elements and/or a more omnivorous diet than is usually ascribed to cave bears. To cite this article: J. Quilès et al., C. R. Palevol 5 (2006).     

Development and Analysis of Patient-Based Complete Conducting Airways Models

The analysis of high-resolution computed tomography (CT) images of the lung is dependent on inter-subject differences in airway geometry. The application of computational models in understanding the significance of these differences has previously been shown to be a useful tool in biomedical research. Studies using image-based geometries alone are limited to the analysis of the central airways, down to generation 6–10, as other airways are not visible on high-resolution CT. However, airways distal to this, often termed the small airways, are known to play a crucial role in common airway diseases such as asthma and chronic obstructive pulmonary disease (COPD). Other studies have incorporated an algorithmic approach to extrapolate CT segmented airways in order to obtain a complete conducting airway tree down to the level of the acinus. These models have typically been used for mechanistic studies, but also have the potential to be used in a patient-specific setting. In the current study, an image analysis and modelling pipeline was developed and applied to a number of healthy (n = 11) and asthmatic (n = 24) CT patient scans to produce complete patient-based airway models to the acinar level (mean terminal generation 15.8 ± 0.47). The resulting models are analysed in terms of morphometric properties and seen to be consistent with previous work. A number of global clinical lung function measures are compared to resistance predictions in the models to assess their suitability for use in a patient-specific setting. We show a significant difference (p < 0.01) in airways resistance at all tested flow rates in complete airway trees built using CT data from severe asthmatics (GINA 3–5) versus healthy subjects. Further, model predictions of airways resistance at all flow rates are shown to correlate with patient forced expiratory volume in one second (FEV1) (Spearman ρ = −0.65, p < 0.001) and, at low flow rates (0.00017 L/s), FEV1 over forced vital capacity (FEV1/FVC) (ρ = −0.58, p < 0.001). We conclude that the pipeline and anatomical models can be used directly in mechanistic modelling studies and can form the basis for future patient-based modelling studies.     

Modulation of α2C adrenergic receptor temperature-sensitive trafficking by HSP90

Decreasing the temperature to 30 °C is accompanied by significant enhancement of α_2C-AR plasma membrane levels in several cell lines with fibroblast phenotype, as demonstrated by radioligand binding in intact cells. No changes were observed on the effects of low-temperature after blocking receptor internalization in α_2C-AR transfected HEK293T cells. In contrast, two pharmacological chaperones, dimethyl sulfoxide and glycerol, increased the cell surface receptor levels at 37 °C, but not at 30 °C. Further, at 37 °C α_2C-AR is co-localized with endoplasmic reticulum markers, but not with the lysosomal markers. Treatment with three distinct HSP90 inhibitors, radicicol, macbecin and 17-DMAG significantly enhanced α_2C-AR cell surface levels at 37 °C, but these inhibitors had no effect at 30 °C. Similar results were obtained after decreasing the HSP90 cellular levels using specific siRNA. Co-immunoprecipitation experiments demonstrated that α_2C-AR interacts with HSP90 and this interaction is decreased at 30 °C. The contractile response to endogenous α_2C-AR stimulation in rat tail artery was also enhanced at reduced temperature. Similar to HEK293T cells, HSP90 inhibition increased the α_2C-AR contractile effects only at 37 °C. Moreover, exposure to low-temperature of vascular smooth muscle cells from rat tail artery decreased the cellular levels of HSP90, but did not change HSP70 levels. These data demonstrate that exposure to low-temperature augments the α_2C-AR transport to the plasma membrane by releasing the inhibitory activity of HSP90 on the receptor traffic, findings which may have clinical relevance for the diagnostic and treatment of Raynaud Phenomenon.     

Cholesterol depletion of hepatoma cells impairs hepatitis B virus envelopment by altering the topology of the large envelope protein

Previous reports have shown that cholesterol depletion of the membrane envelope of the hepatitis B virus (HBV) impairs viral infection of target cells. A potential function of this lipid in later steps of the viral life cycle remained controversial, with secretion of virions and subviral particles (SVP) being either inhibited or not affected, depending on the experimental approach employed to decrease the intracellular cholesterol level. This work addressed the role of host cell cholesterol on HBV replication, assembly, and secretion, using an alternative method to inhibition of the enzymes involved in the biosynthesis pathway. Growing HBV-producing cells with lipoprotein-depleted serum (LPDS) resulted in an important reduction of the amount of cholesterol within 24 h of treatment (about 40%). Cell exposure to chlorpromazine, an inhibitor of the clathrin-mediated pathway used by the low-density lipoprotein receptor for endocytosis, also impacted the cholesterol level; however, this level of inhibition was not achievable when the synthesis inhibitor lovastatin was used. HBV secretion was significantly inhibited in cholesterol-depleted cells (by ～80%), while SVP release remained unaffected. The viral DNA genome accumulated in LPDS-treated cells in a time-dependent manner. Specific immunoprecipitation of nucleocapsids and mature virions revealed an increased amount of naked nucleocapsids, while synthesis of the envelope proteins occurred as normally. Following analysis of the large envelope protein conformation in purified microsomes, we concluded that cholesterol is important in maintaining the dual topology of this polypeptide, which is critical for viral envelopment.     

Effects of the absence of procollagen C-endopeptidase enhancer-2 on myocardial collagen accumulation in chronic pressure overload

Cardiac interstitial fibrillar collagen accumulation, such as that associated with chronic pressure overload (PO), has been shown to impair left ventricular diastolic function. Therefore, insight into cellular mechanisms that mediate excessive collagen deposition in the myocardium is pivotal to this important area of research. Collagen is secreted as a soluble procollagen molecule with NH(2)- and COOH (C)-terminal propeptides. Cleavage of these propeptides is required for collagen incorporation to insoluble collagen fibrils. The C-procollagen proteinase, bone morphogenic protein 1, cleaves the C-propeptide of procollagen. Procollagen C-endopeptidase enhancer (PCOLCE) 2, an enhancer of bone morphogenic protein-1 activity in vitro, is expressed at high levels in the myocardium. However, whether the absence of PCOLCE2 affects collagen content at baseline or after PO induced by transverse aortic constriction (TAC) has never been examined. Accordingly, in vivo procollagen processing and deposition were examined in wild-type (WT) and PCOLCE2-null mice. No significant differences in collagen content or myocardial stiffness were detected in non-TAC (control) PCOLCE2-null versus WT mice. After TAC-induced PO, PCOLCE2-null hearts demonstrated a lesser collagen content (PCOLCE2-null TAC collagen volume fraction, 0.41% ± 0.07 vs. WT TAC, 1.2% ± 0.3) and lower muscle stiffness compared with WT PO hearts [PCOLCE2-null myocardial stiffness (β), 0.041 ± 0.002 vs. WT myocardial stiffness, 0.065 ± 0.001]. In addition, in vitro, PCOLCE2-null cardiac fibroblasts exhibited reductions in efficiency of C-propeptide cleavage, as demonstrated by increases in procollagen α1(I) and decreased levels of processed collagen α1(I) versus WT cardiac fibroblasts. Hence, PCOLCE2 is required for efficient procollagen processing and deposition of fibrillar collagen in the PO myocardium. These results support a critical role for procollagen processing in the regulation of collagen deposition in the heart.     

Lactosylceramide contributes to mitochondrial dysfunction in diabetes

Sphingolipids have been implicated as key mediators of cell-stress responses and effectors of mitochondrial function. To investigate potential mechanisms underlying mitochondrial dysfunction, an important contributor to diabetic cardiomyopathy, we examined alterations of cardiac sphingolipid metabolism in a mouse with streptozotocin-induced type 1 diabetes. Diabetes increased expression of desaturase 1, (dihydro)ceramide synthase (CerS)2, serine palmitoyl transferase 1, and the rate of ceramide formation by mitochondria-resident CerSs, indicating an activation of ceramide biosynthesis. However, the lack of an increase in mitochondrial ceramide suggests concomitant upregulation of ceramide-metabolizing pathways. Elevated levels of lactosylceramide, one of the initial products in the formation of glycosphingolipids were accompanied with decreased respiration and calcium retention capacity (CRC) in mitochondria from diabetic heart tissue. In baseline mitochondria, lactosylceramide potently suppressed state 3 respiration and decreased CRC, suggesting lactosylceramide as the primary sphingolipid responsible for mitochondrial defects in diabetic hearts. Moreover, knocking down the neutral ceramidase (NCDase) resulted in an increase in lactosylceramide level, suggesting a crosstalk between glucosylceramide synthase- and NCDase-mediated ceramide utilization pathways. These data suggest the glycosphingolipid pathway of ceramide metabolism as a promising target to correct mitochondrial abnormalities associated with type 1 diabetes.