Peroxisomal and mitochondrial status of two murine oligodendrocytic cell lines (158N, 158JP): potential models for the study of peroxisomal disorders associated with dysmyelination processes

In some neurodegenerative disorders (leukodystrophies) characterized by myelin alterations, the defect of peroxisomal functions on myelin-producing cells (oligodendrocytes) are poorly understood. The development of in vitro models is fundamental to understanding the physiopathogenesis of these diseases. We characterized two immortalized murine oligodendrocyte cell lines: a normal (158N) and a jimpy (158JP) cell line mutated for the proteolipid protein PLP/DM20. Fluorescence microscopy, flow cytometry, and western blotting analysis allow to identify major myelin proteins (PLP colocalizing with mitochondria; myelin basic protein), oligodendrocyte (CNPase and myelin oligodendrocyte glycoprotein), and peroxisomal markers [adrenoleukodystrophy protein, PMP70, acyl-CoA oxidase 1 (ACOX1), l -peroxisomal bifunctional enzyme, and catalase]. Using electron microscopy, peroxisomes were identified in the two cell lines. Gene expression (ATP-binding cassette, Abcd1 , Abcd2 , Abcd3 , and Acox1 ) involved in peroxisomal transport or β-oxidation of fatty acids was evaluated using quantitative PCR. 4-phenylbutyrate treatment increases expression of ACOX1, l -peroxisomal bifunctional enzyme, PLP, myelin oligodendrocyte glycoprotein, and CNPase, mainly in 158N cells. In both cell lines, 4-phenylbutyrate-induced ACOX1 and catalase activities while only Abcd2 gene was up-regulated in 158JP. Moreover, the higher mitochondrial activity and content observed in 158JP were associated with higher glutathione content and increased basal production of reactive oxygen species revealing different redox statuses. Altogether, 158N and 158JP cells will permit studying the relationships between peroxisomal defects, mitochondrial activity, and oligodendrocyte functions.     

Substrate specificity overlap and interaction between adrenoleukodystrophy protein (ALDP/ABCD1) and adrenoleukodystrophy-related protein (ALDRP/ABCD2)

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder caused by mutations in the ABCD1 gene, which encodes a peroxisomal member of the ATP-binding cassette (ABC) transporter subfamily D called ALDP. ALDP is supposed to function as a homodimer allowing the entry of CoA-esters of very-long chain fatty acids (VLCFA) into the peroxisome, the unique site of their β-oxidation. ALDP deficiency can be corrected by overexpression of ALDRP, its closest homolog. However, the exact nature of the substrates transported by ALDRP and its relationships with ALDP still remain unclear. To gain insight into the function of ALDRP, we used cell models allowing the induction in a dose-dependent manner of a wild type or a mutated non-functional ALDRP-EGFP fusion protein. We explored the consequences of the changes of ALDRP expression levels on the fatty acid content (saturated, monounsaturated, and polyunsaturated fatty acids) in phospholipids as well as on the levels of β-oxidation of 3 suspected substrates: C26:0, C24:0, and C22:6n-3 (DHA). We found an inverse correlation between the fatty acid content of saturated (C26:0, C24:0) and monounsaturated (C26:1, C24:1) VLCFA and the expression level of ALDRP. Interestingly, we obtained a transdominant-negative effect of the inactive ALDRP-EGFP on ALDP function. This effect is due to a physical interaction between ALDRP and ALDP that we evidenced by proximity ligation assays and coimmunoprecipitation. Finally, the β-oxidation assays demonstrate a role of ALDRP in the metabolism of saturated VLCFA (redundant with that of ALDP) but also a specific involvement of ALDRP in the metabolism of DHA.     

Differential distribution of DHA-phospholipids in rat brain after feeding: A lipidomic approach

On a per-weight basis, the brain is the organ richest in lipids, including a remarkable proportion of polyunsaturated fatty acids (PUFAs) of the omega 3 series, namely eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. The cerebral effects of exogenous DHA likely depend on its degree of incorporation into neuronal phospholipids and on its distribution among the various brain structures, after intake. Hence, because PUFAs are not evenly distributed among the brain phospholipid classes and because the existence of class-specific phospholipases that regulate their turnover, we sought to investigate the incorporation of omega 3 PUFAs in selected brain areas regions and specific phospholipid classes. Rats (n=7) were administered, by oral gavage, 100mg/kg/d of a commercially available fish oil (containing ～84% of long-chain omega 3 fatty acids, of which ～38% of DHA and ～46% of EPA). Control rats (n=7) received liquid paraffin. This treatment was continued for 30 days. Thereafter, we dissected three areas, namely the hippocampus, the striatum, and the cortex. Quantization of individual phospholipid classes and their molecular species was performed by ESI-MS/MS. Principal component analysis was used to examine the variation of the molecular lipid profiles (as percentage) induced by omega 3 supplementation. Our results show that provision of omega 3 fatty acids to rats results in their incorporation into brain phospholipids, the extent of which is lower in the striatum as compared with cortex and hippocampus. These data might in part explain the mixed therapeutic results obtained in neurological disorders, many of which are likely region-specific.     

Electron paramagnetic resonance in irradiated fingernails: variability of dose dependence and possibilities of initial dose assessment

The results of electron paramagnetic resonance (EPR) measurements in irradiated fingernails are presented. In total, 83 samples of different fingernails were studied. Five different groups of samples were selected based on the collection time of fingernail samples, their level of mechanical stress, and the number and size of clippings: (1) recently (<24 h) cut, irradiated and measured with EPR without any treatment of samples, and with rigorous control of size and number of clippings (stressed–fresh, controlled); (2) recently (<24 h) cut, irradiated and measured with EPR after application of a special treatment (10 min of water soaking, 5 min of drying time) to reduce the mechanical stress caused by cutting the samples, and with rigorous control of size and number of clippings (unstressed–fresh, controlled); (3) previously (>24 h) cut, stored at room temperature, additionally cut into small pieces immediately prior to study, irradiated and measured with EPR without any treatment of samples, and with rigorous control of size and number of clippings (stressed–old, controlled); (4) previously (>24 h) cut, stored at room temperature, additionally cut into small pieces immediately prior to the study, irradiated and measured with EPR after application of a special treatment to reduce mechanical stress caused by cut, and with rigorous control of size and number of clippings (unstressed–old, controlled); and (5) recently (<24 h) cut, irradiated and measured with EPR after application of a special treatment to reduce the mechanical stress caused by cut, and without rigorous control of size and number of clippings (unstressed–fresh, uncontrolled). Except for the fifth selected group, variability of the dose dependence inside all groups was found to be not statistically significant, although the variability among the different groups was significant. Comparison of the mean dose dependences obtained for each group allowed selection of key factors responsible for radiation sensitivity (dose response per unit of mass and dose) and the shape of dose dependence in fingernails. The major factor responsible for radiation sensitivity of fingernails was identified as their water content, which can affect radiation sensitivity up to 35%. The major factor responsible for the shape of the radiation sensitivity was identified as the mechanical stress. At a significant level of mechanical stress, the shape of the dose dependence is linear in the studied dose range (<20 Gy), and in lesser-stressed samples it is of an exponential growth including saturation, which depends on the degree of mechanical stress. In view of the findings, recommendations are discussed and presented for the appropriate protocol for EPR dose measurements in fingernails.     

Filamentous condensation of DNA induced by pegylated poly-L-lysine and transfection efficiency

In this paper we propose a detailed analysis of structural and morphological properties of two poly-L-lysine (PLL)-based transfection formulations, PLL/DNA and pegylated PLL (PLL-g-PEG)/DNA, by means of atomic force microscopy (AFM) and transmission electron microscopy (TEM). Comparing PLL-g-PEG/DNA with PLL/DNA polyplexes, we demonstrate that, due to the presence of PEG, the particles differ not only in size, shape, and crystalline structure, but also in transfection efficiency. While PLL condensates DNA in large agglomerates, PLL grafted with polyethylene glycol 2000 can condensate DNA in long filaments with diameters of some nanometers (6-20 nm). These structures are dependent on the grafting ratio and are more efficient than compacted ones, showing that DNA uptake and processing by cell is directly related to physicochemical properties of the polyplexes.     

Retrospective dosimetry after criticality accidents using low-frequency EPR: a study of whole human teeth irradiated in a mixed neutron and gamma-radiation field

In the context of accidental or intentional radiation exposures (nuclear terrorism), it is essential to separate rapidly those individuals with substantial exposures from those with exposures that do not constitute an immediate threat to health. Low-frequency electron paramagnetic resonance (EPR) spectroscopy provides the potential advantage of making accurate and sensitive measurements of absorbed radiation dose in teeth without removing the teeth from the potential victims. Up to now, most studies focused on the dose-response curves obtained for gamma radiation. In radiation accidents, however, the contribution of neutrons to the total radiation dose should not be neglected. To determine how neutrons contribute to the apparent dose estimated by EPR dosimetry, extracted whole human teeth were irradiated at the SILENE reactor in a mixed neutron and gamma-radiation field simulating criticality accidents. The teeth were irradiated in free air as well as in a paraffin head phantom. Lead screens were also used to eliminate to a large extent the contribution of the gamma radiation to the dose received by the teeth. The EPR signals, obtained with a low-frequency (1.2 GHz) spectrometer, were compared to dosimetry measurements at the same location. The contribution of neutrons to the EPR dosimetric signal was negligible in the range of 0 to 10 Gy and was rather small (neutron/gamma-ray sensitivity in the range 0-0.2) at higher doses. This indicates that the method essentially provides information on the dose received from the gamma-ray component of the radiation.     

Conservation implications of the mating system of the Pampa Hermosa landrace of peach palm analyzed with microsatellite markers

Peach palm (Bactris gasipaes) is cultivated by many indigenous and traditional communities from Amazonia to Central America for its edible fruits, and is currently important for its heart-of-palm. The objective of this study was to investigate the mating system of peach palm, as this is important for conservation and breeding. Eight microsatellite loci were used to genotype 24 open-pollinated progenies from three populations of the Pampa Hermosa landrace maintained in a progeny trial for genetic improvement. Both the multi-locus outcrossing rates (0.95 to 0.99) and the progeny level multi-locus outcrossing rates (0.9 to 1.0) were high, indicating that peach palm is predominantly allogamous. The outcrossing rates among relatives were significantly different from zero (0.101 to 0.202), providing evidence for considerable biparental inbreeding within populations, probably due to farmers planting seeds of a small number of open-pollinated progenies in the same plot. The correlations of paternity estimates were low (0.051 to 0.112), suggesting a large number of pollen sources (9 to 20) participating in pollination of individual fruit bunches. Effective population size estimates suggest that current germplasm collections are insufficient for long-term ex situ conservation. As with most underutilized crops, on farm conservation is the most important component of an integrated conservation strategy.     

Effect of kinase inhibitors on the therapeutic properties of monoclonal antibodies

Targeted therapies of malignancies currently consist of therapeutic monoclonal antibodies and small molecule kinase inhibitors. The combination of these novel agents raises the issue of potential antagonisms. We evaluated the potential effect of 4 kinase inhibitors, including the Bruton tyrosine kinase inhibitor ibrutinib, and 3 PI3K inhibitors idelalisib, NVP-BEZ235 and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, on the effects of the 3 monoclonal antibodies, rituximab and obinutuzumab (directed against CD20) and trastuzumab (directed against HER2). We found that ibrutinib potently inhibits antibody-dependent cell-mediated cytotoxicity exerted by all antibodies, with a 50% inhibitory concentration of 0.2 microM for trastuzumab, 0.5 microM for rituximab and 2 microM for obinutuzumab, suggesting a lesser effect in combination with obinutuzumab than with rituximab. The 4 kinase inhibitors were found to inhibit phagocytosis by fresh human neutrophils, as well as antibody-dependent cellular phagocytosis induced by the 3 antibodies. Conversely co-administration of ibrutinib with rituximab, obinutuzumab or trastuzumab did not demonstrate any inhibitory effect of ibrutinib in vivo in murine xenograft models. In conclusion, some kinase inhibitors, in particular, ibrutinib, are likely to exert inhibitory effects on innate immune cells. However, these effects do not compromise the antitumor activity of monoclonal antibodies in vivo in the models that were evaluated.