The activity of the plant mitochondrial inner membrane anion channel (PIMAC) of maize populations divergently selected for cold tolerance level is differentially dependent on the growth temperature of seedlings

The activity of the plant inner membrane mitochondrial anion channel (PIMAC) is involved in metabolite shuttles and mitochondrial volume changes and could have a role in plant temperature tolerance. Our objectives were to investigate (i) the occurrence and (ii) the temperature dependence of anion fluxes through PIMAC in mitochondria isolated from seedlings of three maize populations differing in terms of cold tolerance; and (iii) the relationships between the PIMAC activity kinetics and the level of cold tolerance. Populations were the source population (C0) and two populations divergently selected for high (C4H) and low (C4L) cold tolerance. Such divergently selected populations are expected to share most of their genes, with the main exception of those genes controlling cold tolerance. Arrhenius plots of PIMAC chloride fluxes showed a linear temperature dependence when seedlings were grown at 25 or 14°C, whereas a non-linear temperature dependence was found when seedlings were grown at 5°C, with or without acclimation at 14°C. The activation energy and other thermodynamic parameters of PIMAC activity varied depending on temperature treatments during seedling growth. When seedlings were grown at 14 and 5°C with acclimation, PIMAC activity of the C4H population increased, while that of C4L declined, as compared with the activities of seedlings grown at 25°C. These symmetric responses indicate that PIMAC activity changes are associated with genetically determined differences in the cold tolerance level of the investigated populations. We conclude that anion fluxes by PIMAC depend upon changes on growth temperature and are differentially related to the tolerance level of the tested populations.     

Docosahexaenoic acid reverts resistance to UV-induced apoptosis in human keratinocytes: involvement of COX-2 and HuR

The dramatic increase in the incidence of nonmelanoma skin cancer over the last decades has been related to the augmented exposure to ultraviolet (UV) radiation (UVR). It is known that apoptosis is induced as a protective mechanism after the acute irradiation of keratinocytes, whereas apoptotic resistance and carcinogenesis may follow the chronic exposure to UVR. We found that not all the human keratinocytes lines studied underwent apoptosis following acute exposure to UVR (10-60 mJ/cm²). Whereas UVR induced apoptosis in the HaCaT cells, NCTC 2544 and nr-HaCaT cells showed apoptosis resistance. The cytokeratin pattern of the apoptosis-resistant cells indicated that they possessed a degree of differentiation lower than that of HaCaT cells. They also showed an enhanced expression of cyclooxygenase-2 (COX-2), an early marker of carcinogenesis in various tissues, including skin. n-3 polyunsaturated fatty acids have drawn increasing interest as nutritional factors with the potential to reduce UVR carcinogenesis, and since they are apoptosis inducers and COX-2 inhibitors in cancer cells, we investigated the ability of n-3 polyunsaturated fatty acids to influence the resistance to UVR-induced apoptosis in keratinocytes. We observed that docosahexaenoic acid (DHA) reverted the resistance of nr-HaCaT cells to UVR-induced apoptosis, increasing the Bax/Bcl-2 ratio and caspase-3 activity, and reduced COX-2 levels by inhibiting the expression of the human antigen R (HuR), a known COX-2 mRNA stabilizer in keratinocytes. The transfection of nr-HaCaT cells with HuR siRNA mimicked the proapoptotic effect of DHA. Overall, our findings further support the role of DHA as a suitable anticarcinogenic factor against nonmelanoma skin cancers.     

Muscular laminopathies: role of prelamin A in early steps of muscle differentiation

Lamin A is a nuclear envelope constituent involved in a group of human disorders, collectively referred to as laminopathies, which include Emery-Dreifuss muscular dystrophy. Because increasing evidence suggests a role of lamin A precursor in nuclear functions, we investigated the processing of prelamin A along muscle differentiation. Both protein levels and cellular localization of prelamin A appears to be modulated during C2C12 mouse myoblasts activation. Similar changes also occur in the expression of two lamin A-binding proteins: emerin and LAP2α. Furthermore prelamin A forms a complex with LAP2α in differentiating myoblasts. Prelamin A accumulation in cycling myoblasts by expressing unprocessable mutants affects LAP2α and PCNA amount and increases caveolin 3 mRNA and protein levels, whilst accumulation of prelamin A in differentiated muscle cells following treatment with a farnesyl transferase inhibitor inhibits caveolin 3 expression. These data provide evidence for a critical role of lamin A precursor in the early steps of muscle cell differentiation. In fact the post-translational processing of prelamin A affects caveolin 3 expression and influences the myoblast differentiation process. Thus, altered lamin A processing could affect myoblast differentiation and/or muscle regeneration and might contribute to the myopathic phenotype.     

Proteomic Characterization of Caenorhabditis elegans Larval Development

The nematode Caenorhabditis elegans is widely used as a model organism to study cell and developmental biology. Quantitative proteomics of C. elegans is still in its infancy and, so far, most studies have been performed on adult worm samples. Here, we used quantitative mass spectrometry to characterize protein level changes across the four larval developmental stages (L1–L4) of C. elegans. In total, we identified 4130 proteins, and quantified 1541 proteins that were present across all four stages in three biological replicates from independent experiments. Using hierarchical clustering and functional ontological analyses, we identified 21 clusters containing proteins with similar protein profiles across the four stages, and highlighted the most overrepresented biological functions in each of these protein clusters. In addition, we used the dataset to identify putative larval stage‐specific proteins in each individual developmental stage, as well as in the early and late developmental stages. In summary, this dataset provides system‐wide analysis of protein level changes across the four C. elegans larval developmental stages, which serves as a useful resource for the C. elegans research community. MS data were deposited in ProteomeXchange ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository with the primary accession identifier PXD006676.     

Cooling intact and demembranated trabeculae from rat heart releases myosin motors from their inhibited conformation

Myosin filament–based regulation supplements actin filament–based regulation to control the strength and speed of contraction in heart muscle. In diastole, myosin motors form a folded helical array that inhibits actin interaction; during contraction, they are released from that array. A similar structural transition has been observed in mammalian skeletal muscle, in which cooling below physiological temperature has been shown to reproduce some of the structural features of the activation of myosin filaments during active contraction. Here, we used small-angle x-ray diffraction to characterize the structural changes in the myosin filaments associated with cooling of resting and relaxed trabeculae from the right ventricle of rat hearts from 39°C to 7°C. In intact quiescent trabeculae, cooling disrupted the folded helical conformation of the myosin motors and induced extension of the filament backbone, as observed in the transition from diastole to peak systolic force at 27°C. Demembranation of trabeculae in relaxing conditions induced expansion of the filament lattice, but the structure of the myosin filaments was mostly preserved at 39°C. Cooling of relaxed demembranated trabeculae induced changes in motor conformation and filament structure similar to those observed in intact quiescent trabeculae. Osmotic compression of the filament lattice to restore its spacing to that of intact trabeculae at 39°C stabilized the helical folded state against disruption by cooling. The myosin filament structure and motor conformation of intact trabeculae at 39°C were largely preserved in demembranated trabeculae at 27°C or above in the presence of Dextran, allowing the physiological mechanisms of myosin filament–based regulation to be studied in those conditions.     

Investigation on the Origin of Sperm DNA Fragmentation: Role of Apoptosis,Immaturity and Oxidative Stress

Sperm DNA fragmentation (sDF) represents a threat to male fertility, human reproduction and the health of the offspring. The causes of sDF are still unclear, even if apoptosis, oxidative assault and defects in chromatin maturation are hypothesized. Using multicolor flow cytometry and sperm sorting, we challenged the three hypothesized mechanisms by simultaneously evaluating sDF and signs of oxidative damage (8-hydroxy, 2′-deoxyguanosine [8-OHdG] and malondialdehyde [MDA]), apoptosis (caspase activity and cleaved poly[ADP-ribose] polymerase [cPARP]) and sperm immaturity (creatine phosphokinase [CK] and excess of residual histones). Active caspases and c-PARP were concomitant with sDF in a high percentage of spermatozoa (82.6% ± 9.1% and 53.5% ± 16.4%, respectively). Excess of residual histones was significantly higher in DNA-fragmented sperm versus sperm without DNA fragmentation (74.8% ± 17.5% and 37.3% ± 16.6%, respectively, p < 0.005), and largely concomitant with active caspases. Conversely, oxidative damage was scarcely concomitant with sDF in the total sperm population, at variance with live sperm, where 8-OHdG and MDA were clearly associated to sDF. In addition, most live cells with active caspase also showed 8-OHdG, suggesting activation of apoptotic pathways in oxidative-injured live cells. This is the first investigation on the origin of sDF directly evaluating the simultaneous presence of the signs of the hypothesized mechanisms with DNA breaks at the single cell level. The results indicate that the main pathway leading to sperm DNA breaks is a process of apoptosis, likely triggered by an impairment of chromatin maturation in the testis and by oxidative stress during the transit in the male genital tract. These findings are highly relevant for clinical studies on the effects of drugs on sDF and oxidative stress in infertile men and for the development of new therapeutic strategies.     

Parvovirus b19 DNA CpG dinucleotide methylation and epigenetic regulation of viral expression

CpG DNA methylation is one of the main epigenetic modifications playing a role in the control of gene expression. For DNA viruses whose genome has the ability to integrate in the host genome or to maintain as a latent episome, a correlation has been found between the extent of DNA methylation and viral quiescence. No information is available for Parvovirus B19, a human pathogenic virus, which is capable of both lytic and persistent infections. Within Parvovirus B19 genome, the inverted terminal regions display all the characteristic signatures of a genomic CpG island; therefore we hypothesised a role of CpG dinucleotide methylation in the regulation of viral genome expression.The analysis of CpG dinucleotide methylation of Parvovirus B19 DNA was carried out by an aptly designed quantitative real-time PCR assay on bisulfite-modified DNA. The effects of CpG methylation on the regulation of viral genome expression were first investigated by transfection of either unmethylated or in vitro methylated viral DNA in a model cell line, showing that methylation of viral DNA was correlated to lower expression levels of the viral genome. Then, in the course of in vitro infections in different cellular environments, it was observed that absence of viral expression and genome replication were both correlated to increasing levels of CpG methylation of viral DNA. Finally, the presence of CpG methylation was documented in viral DNA present in bioptic samples, indicating the occurrence and a possible role of this epigenetic modification in the course of natural infections.The presence of an epigenetic level of regulation of viral genome expression, possibly correlated to the silencing of the viral genome and contributing to the maintenance of the virus in tissues, can be relevant to the balance and outcome of the different types of infection associated to Parvovirus B19.