PL10 DEAD-Box Protein is Expressed during Germ Cell Differentiation in the Reptile Podarcis sicula (Family Lacertidae)

Among genes involved in the regulation of germ cell differentiation, those of DDX4/Vasa and the Ded1/DDX3 subfamilies encode for DEAD-box ATP-dependent RNA helicases, proteins involved in many mechanisms related to RNA processing. For the first time in reptiles, using specific antibodies at confocal microscopy, we analysed the localization pattern of a Ded1/DDX3 subfamily member in testis and ovary of Podarcis sicula (Ps-PL10) during the reproductive cycle. In testis, Ps-PL10 is expressed in the cytoplasm of spermatocytes and it is not detected in spermatogonia. Differently from Ps-VASA, in round spermatids, Ps-PL10 is not segregated in the chromatoid body but it accumulates in the cytoplasm of residual bodies, and mature spermatozoa are unstained. These observations suggest that in males, Ps-PL10 (1) is involved in spermatogenesis and (2) is then eliminated with residual bodies. In the ovary, Ps-PL10 is present with granules in the cytoplasm of early meiotic cells of the germinal bed (GB), while it is not present in oogonia and somatic cells of the GB stroma. In follicular cells of ovarian follicles, Ps-PL10 expression starts after their fusion with the oocyte. Numerous Ps-PL10 spots are visible in pyriform (nurse-like) cells concomitantly with the protein accumulation in the cytoplasm of differentiating oocyte. In pyriform cells, Ps-PL10 spots are present in the cytoplasm and nuclei, as observed for Ps-VASA, and in the nucleoli, suggesting for Ps-PL10 a role in rRNA processing and in the transport of molecules from the nucleus to cytoplasm and from nurse cells to the oocyte

Level of mitochondrial heteroplasmy in a natural and stable heteroplasmic system

Mitochondrial heteroplasmy is the presence of more than one type of mitochondrial genome (mtDNA) within an individual, and in most of the reported cases it seems to be an unfavourable condition. For example, heteroplasmy in humans increases the risk of common age-related disorders as neurodegenerative diseases. The only evolutionarily stable and natural heteroplasmic system in Metazoa is the Doubly Uniparental inheritance (DUI), typical of some bivalves, in which two mitochondrial lineages are present: one transmitted through eggs (F-type) and the other through sperm (M-type). While females are homoplasmic for the F-type, males have M-type-homoplasmic gametes, but heteroplasmic soma. So far, no study has investigated mitochondrial heteroplasmy at the protein level, and no analysis has been performed to clarify if it is present at tissue, cell, or organelle level. We characterized the expression of three mitochondrially-encoded proteins (ND5, CYTB, COX3) in the DUI species Ruditapes philippinarum. Specific antibodies were produced to discriminate, with immunolocalization, between the F and M form of the same protein (variants highly divergent in DUI species) in germ line and somatic tissues of females and males in different developmental stages. Unexpectedly, M-type antibodies labelled mitochondria in female primordial stem cells (PriSCs), undifferentiated germ cells, and early oocytes, while mature eggs and female somatic cells expressed only the F-type. M- and F-type expression in male somatic tissues showed mitochondrial heteroplasmy at the cell level. Interestingly, F-labelled mitochondria were detected in male PriSCs and early germ cells. We hypothesize that PriSCs and undifferentiated germ cells carry both types of mtDNA, but during gamete maturation one of the two genomes disappears, and only the sexspecific mtDNA remains, maintaining the homoplasmy of the germ line. This would imply a selective degradation of mitochondria in adulthood

Nutlin-3a Enhances Natural Killer Cell-Mediated Killing of Neuroblastoma by Restoring p53-Dependent Expression of Ligands for NKG2D and DNAM-1 Receptors

In this study, we explored whether Nutlin-3a, a well-known, nontoxic small-molecule compound antagonizing the inhibitory interaction of MDM2 with the tumor suppressor p53, may restore ligands for natural killer (NK) cell-activating receptors (NK-AR) on neuroblastoma cells to enhance the NK cell-mediated killing. Neuroblastoma cell lines were treated with Nutlin-3a, and the expression of ligands for NKG2D and DNAM-1 NK-ARs and the neuroblastoma susceptibility to NK cells were evaluated. Adoptive transfer of human NK cells in a xenograft neuroblastoma-bearing NSG murine model was assessed. Two data sets of neuroblastoma patients were explored to correlate p53 expression with ligand expression. Luciferase assays and chromatin immunoprecipitation analysis of p53 functional binding on PVR promoter were performed. Primary neuroblastoma cells were also treated with Nutlin-3a, and neuroblastoma spheroids obtained from one high-risk patient were assayed for NK-cell cytotoxicity. We provide evidence showing that the Nutlin-3a-dependent rescue of p53 function in neuroblastoma cells resulted in (i) increased surface expression of ligands for NK-ARs, thus rendering neuroblastoma cell lines significantly more susceptible to NK cell-mediated killing; (ii) shrinkage of human neuroblastoma tumor masses that correlated with overall survival upon adoptive transfer of NK cells in neuroblastoma-bearing mice; (iii) and increased expression of ligands in primary neuroblastoma cells and boosting of NK cell-mediated disaggregation of neuroblastoma spheroids. We also found that p53 was a direct transcription factor regulating the expression of PVR ligand recognized by DNAM-1. Our findings demonstrated an immunomodulatory role of Nutlin-3a, which might be prospectively used for a novel NK cell-based immunotherapy for neuroblastoma