pLG72 modulates intracellular D-serine levels through its interaction with D-amino acid oxidase: effect on schizophrenia susceptibility

Human genes coding for pLG72 and d-amino acid oxidase have recently been linked to the onset of schizophrenia. pLG72 was proposed as an activator of the human FAD-containing flavoprotein d-amino acid oxidase (hDAAO). In the brain this oxidizes d-serine, a potent activator of N-methyl-d-aspartate receptor. We have investigated the mechanistic regulation of hDAAO by pLG72. Immunohistochemical analyses revealed that hDAAO and pLG72 are both expressed in astrocytes of the human cortex, where they most likely interact, considering their partial overlapping subcellular distribution and their coimmunoprecipitation. We demonstrated that the specific in vitro interaction of the two proteins yields a complex composed of 2 hDAAO homodimers and 2 pLG72 molecules. Binding of pLG72 did not affect the kinetic properties and FAD binding ability of hDAAO; instead, a time-dependent loss of hDAAO activity in the presence of an excess of pLG72 was found. The binding affects the tertiary structure of hDAAO, altering the amount of the active form. We finally demonstrated that overexpression of hDAAO in glioblastoma cells decreases the levels of d-serine, an effect that is null when pLG72 is coexpressed. These data indicate that pLG72 acts as a negative effector of hDAAO. Therefore, a decrease in the synaptic concentration of d-serine as the result of an anomalous increase in hDAAO activity related to hypoexpression of pLG72 may represent a molecular mechanism by which hDAAO and pLG72 are involved in schizophrenia susceptibility.     

The peptide nucleic acid targeted to a regulatory sequence of the translocated c-myc oncogene in Burkitt's lymphoma lacks immunogenicity: follow-up characterization of PNAEmu-NLS

The present study aims to evaluate the antigenicity of a PNA complementary to the Emu sequence (PNAEmu) with cancer therapeutic potential properties in Burkitt's lymphoma (BL). In BL cells, the c-myc oncogene is repositioned next to the Emu enhancer of the immunoglobulin (Ig) locus, due to chromosomal translocation, and up-regulated. PNAEmu linked to a nuclear localization signal peptide was shown specifically to block c-myc hyperexpression by inhibiting cell growth in vitro and in vivo. Recently, we reported that the administration of PNAEmu to mice, following inoculation with BL cells, hinders tumor growth without toxic effects. To investigate the potential use of PNAEmu in clinical applications further, we tested its antigenicity. Mice were inoculated with an emulsion of free PNA or PNA crosslinked to the immunogenic carrier keyhole limpet hemocyanin (KLH) with Freund's adjuvant. Antibodies to free PNA were undetected, whereas both IgG and IgM antibodies to PNA-KLH were detected in mouse serum 28 and 38 days after inoculation.     

Glutamate modulation of human lymphocyte growth: in vitro studies

Peripheral blood mononuclear cell (PBMC) proliferation induced by phytohemagglutinin, or by anti-CD3 alone or plus anti-CD28 monoclonal antibodies (mAb) was inhibited by glutamate (Glu) in a concentration-dependent manner. This inhibition was not reproduced by selective ionotropic Glu receptor agonists, whereas it was potentiated by l-buthionine-(S,R)-sulfoximine, which depletes glutathione (GSH) stores, and counteracted by 2-mercaptoethanol, a preserver of cell thiols. The inhibitory effects of Glu were related to depletion of intracellular GSH stores, since it decreased GSH levels in a concentration-dependent manner. Furthermore, Glu modulated cytokine secretion by anti-CD3 mAb activated PBMC: it increased IFN-gamma (+44.3+/-8.2%) and IL-10 (+31.6+/-9.7%) secretion, whereas that of IL-2, IL-4, IL-5, and TNF-alpha was not affected. These data suggest that high levels of Glu, which can be reached in damaged tissues, modulate lymphocyte responses to activating stimuli by favouring polarization of the T helper effector response.     

Ectopic expression of RNF168 and 53BP1 increases mutagenic but not physiological non-homologous end joining

DNA double strand breaks (DSBs) formed during S phase are preferentially repaired by homologous recombination (HR), whereas G₁ DSBs, such as those occurring during immunoglobulin class switch recombination (CSR), are repaired by non-homologous end joining (NHEJ). The DNA damage response proteins 53BP1 and BRCA1 regulate the balance between NHEJ and HR. 53BP1 promotes CSR in part by mediating synapsis of distal DNA ends, and in addition, inhibits 5’ end resection. BRCA1 antagonizes 53BP1 dependent DNA end-blocking activity during S phase, which would otherwise promote mutagenic NHEJ and genome instability. Recently, it was shown that supra-physiological levels of the E3 ubiquitin ligase RNF168 results in the hyper-accumulation of 53BP1/BRCA1 which accelerates DSB repair. Here, we ask whether increased expression of RNF168 or 53BP1 impacts physiological versus mutagenic NHEJ. We find that the anti-resection activities of 53BP1 are rate-limiting for mutagenic NHEJ but not for physiological CSR. As heterogeneity in the expression of RNF168 and 53BP1 is found in human tumors, our results suggest that deregulation of the RNF168/53BP1 pathway could alter the chemosensitivity of BRCA1 deficient tumors.     

Long-term trends of epilimnetic and hypolimnetic bacteria and organic carbon in a deep holo-oligomictic lake

We analysed the long-term dynamics (1980–2007) of hypolimnetic and epilimnetic bacterial abundances and organic carbon concentrations, both dissolved (DOC) and particulate (POC), in the deep holo-oligomictic Lake Maggiore, included in the Southern Alpine Lakes Long-Term Ecological Research (LTER) site. During the 28 years of investigation, bacterial abundance and POC concentrations did not decrease with declining phosphorus concentrations, while DOC concentrations showed a pronounced decrease in the epi- and hypolimnion. We used the annual mean total lake heat content and total annual precipitation as climate-related variables, and in-lake total phosphorus as a proxy for trophic state. The model (forward stepwise regression, FSR) showed that reduced anthropogenic pressure was more significant than climate change in driving the trend in DOC concentrations. Bacterial dynamics in the hypolimnion mirrored the fluctuations observed in the epilimnion, but average cell abundance was three times lower. The FSR model indicates that bacterial number variability was dependent on POC in the epilimnion and DOC in the hypolimnion. In the hypolimnion, cell biovolumes for rod and coccal morphotypes were significantly larger than in the epilimnion.     

A proteomic approach to the bilirubin‐induced toxicity in neuronal cells reveals a protective function of DJ‐1 protein

Unconjugated bilirubin (UCB) is a powerful antioxidant and a modulator of cell growth through the interaction with several signal transduction pathways. Although newborns develop a physiological jaundice, in case of severe hyperbilirubinemia UCB may become neurotoxic causing severe long‐term neuronal damages, also known as bilirubin encephalopathy. To investigate the mechanisms of UCB‐induced neuronal toxicity, we used the human neuroblastoma cell line SH‐SY5Y as an in vitro model system. We verified that UCB caused cell death, in part due to oxidative stress, which leads to DNA damage and cell growth reduction. The mechanisms of cytotoxicity and cell adaptation to UCB were studied through a proteomic approach that identified differentially expressed proteins involved in cell proliferation, intracellular trafficking, protein degradation and oxidative stress response. In particular, the results indicated that cells exposed to UCB undertake an adaptive response that involves DJ‐1, a multifunctional neuroprotective protein, crucial for cellular oxidative stress homeostasis. This study sheds light on the mechanisms of bilirubin‐induced neurotoxicity and might help to design a strategy to prevent or ameliorate the neuronal damages leading to bilirubin encephalopathy.     

Mitochondrial quality control: Cell-type-dependent responses to pathological mutant mitochondrial DNA

Pathological mutations in the mitochondrial DNA (mtDNA) produce a diverse range of tissue-specific diseases and the proportion of mutant mitochondrial DNA can increase or decrease with time via segregation, dependent on the cell or tissue type. Previously we found that adenocarcinoma (A549.B2) cells favored wild-type (WT) mtDNA, whereas rhabdomyosarcoma (RD.Myo) cells favored mutant (m3243G) mtDNA. Mitochondrial quality control (mtQC) can purge the cells of dysfunctional mitochondria via mitochondrial dynamics and mitophagy and appears to offer the perfect solution to the human diseases caused by mutant mtDNA. In A549.B2 and RD.Myo cybrids, with various mutant mtDNA levels, mtQC was explored together with macroautophagy/autophagy and bioenergetic profile. The 2 types of tumor-derived cell lines differed in bioenergetic profile and mitophagy, but not in autophagy. A549.B2 cybrids displayed upregulation of mitophagy, increased mtDNA removal, mitochondrial fragmentation and mitochondrial depolarization on incubation with oligomycin, parameters that correlated with mutant load. Conversely, heteroplasmic RD.Myo lines had lower mitophagic markers that negatively correlated with mutant load, combined with a fully polarized and highly fused mitochondrial network. These findings indicate that pathological mutant mitochondrial DNA can modulate mitochondrial dynamics and mitophagy in a cell-type dependent manner and thereby offer an explanation for the persistence and accumulation of deleterious variants.