Xeno-cannibalism: a survival "escamotage"

Several lines of evidence have demonstrated that self-cannibalism (macroautophagy) is a well regulated process of cell repair as well as of molecule and organelle recycling that allows the cells to survive. However, autophagic activity also represents a cell death pathway characterized by specific features that differentiate autophagy from other cell death processes. We found that cells that are able to exert intense autophagic activity were also able to engulf and digest entire cell siblings. This phenomenon represents a sort of xeno-cannibalism. We wonder whether these two phenomena, self and xeno-cannibalism, could be related the latter being an exacerbation of the first and providing a further survival option to the cells.     

Overexpression of sialidase NEU3 increases the cellular radioresistance potential of U87MG glioblastoma cells

The plasma membrane-associated sialidase NEU3 is known to play important roles in different physiological and pathophysiological processes such as proliferation, cellular differentiation and tumorigenesis. Up-regulation of NEU3 has been associated to several tumors and recently it was demonstrated that its down-modulation in glioblastoma cells promotes cell invasiveness. To date, no information concerning the possible role played by NEU3 in relation to tumor radioresistance is available. Here we show that overexpression of NEU3 in glioblastoma U87MG cells activates PI3K/Akt signaling pathway resulting in an increased radioresistance capacity and in an improved efficiency of double strand DNA-repair mechanisms after irradiation. Our results demonstrate for the first time that NEU3 contributes to the radioresistance features of U87MG cells, bringing to evidence a novel rand peculiar role of the enzyme in cancer biology.     

Identification of amino acid residues critical for the B cell growth-promoting activity of HIV-1 matrix protein p17 variants

Background

HIV-1 matrix protein p17 variants (vp17s) detected in HIV-1-infected patients with non-Hodgkin's lymphoma (HIV-NHL) display, differently from the wild-type protein (refp17), B cell growth-promoting activity. Biophysical analysis revealed that vp17s are destabilized as compared to refp17, motivating us to explore structure-function relationships.

Methylglyoxal accumulation de-regulates HoxA5 expression,thereby impairing angiogenesis in glyoxalase 1 knock-down mouse aortic endothelial cells

Impaired angiogenesis leads to long-term complications and is a major contributor of the high morbidity in patients with Diabetes Mellitus (DM). Methylglyoxal (MGO) is a glycolysis byproduct that accumulates in DM and is detoxified by the Glyoxalase 1 (Glo1). Several studies suggest that MGO contributes to vascular complications through mechanisms that remain to be elucidated. In this study we have clarified for the first time the molecular mechanism involved in the impairment of angiogenesis induced by MGO accumulation.Angiogenesis was evaluated in mouse aortic endothelial cells isolated from Glo1-knockdown mice (Glo1KD MAECs) and their wild-type littermates (WT MAECs). Reduction in Glo1 expression led to an accumulation of MGO and MGO-modified proteins and impaired angiogenesis of Glo1KD MAECs. Both mRNA and protein levels of the anti-angiogenic HoxA5 gene were increased in Glo1KD MAECs and its silencing improved both their migration and invasion. Nuclear NF-?B-p65 was increased 2.5-fold in the Glo1KD as compared to WT MAECs. Interestingly, NF-?B-p65 binding to HoxA5 promoter was also 2-fold higher in Glo1KD MAECs and positively regulated HoxA5 expression in MAECs. Consistent with these data, both the exposure to a chemical inhibitor of Glo1 “SpBrBzGSHCp2” (GI) and to exogenous MGO led to the impairment of migration and the increase of HoxA5 mRNA and NF-?B-p65 protein levels in microvascular mouse coronary endothelial cells (MCECs).This study demonstrates, for the first time, that MGO accumulation increases the antiangiogenic factor HoxA5 via NF-?B-p65, thereby impairing the angiogenic ability of endothelial cells.     

Monoclonal antibodies against pertussis toxin subunits

Abstract Twenty monoclonal antibodies (mAbs) reacting with cholera toxin (CT) of Vibrio cholerae strain 569B were characterized in cross-section and G_M1 ganglioside inhibition assays. MAbs were characterized by reaction with CT and Escherichia coli heat-labile porcine strain (LT_p) and human strain (LTh) enterotoxins, and by G_M1 ganglioside inhibition of mAb binding. Eight of 10 CT-A specific and 3 of 10 CT-B-specific mAbs cross-reacted with LTh and LTp. G_M1 ganglioside inhibited reactions of the CT-B cross-reacting antibodies. Results showed that these epitodes common to the B subunit of CT and LT are located in or near the G_M1 ganglioside binding region, and that the G_M1 ganglioside-binding region of LT differs from that of CT.     

ZMAT3 hypomethylation contributes to early senescence of preadipocytes from healthy first‐degree relatives of type 2 diabetics

Senescence of adipose precursor cells (APC) impairs adipogenesis, contributes to the age‐related subcutaneous adipose tissue (SAT) dysfunction, and increases risk of type 2 diabetes (T2D). First‐degree relatives of T2D individuals (FDR) feature restricted adipogenesis, reflecting the detrimental effects of APC senescence earlier in life and rendering FDR more vulnerable to T2D. Epigenetics may contribute to these abnormalities but the underlying mechanisms remain unclear. In previous methylome comparison in APC from FDR and individuals with no diabetes familiarity (CTRL), ZMAT3 emerged as one of the top‐ranked senescence‐related genes featuring hypomethylation in FDR and associated with T2D risk. Here, we investigated whether and how DNA methylation changes at ZMAT3 promote early APC senescence. APC from FDR individuals revealed increases in multiple senescence markers compared to CTRL. Senescence in these cells was accompanied by ZMAT3 hypomethylation, which caused ZMAT3 upregulation. Demethylation at this gene in CTRL APC led to increased ZMAT3 expression and premature senescence, which were reverted by ZMAT3 siRNA. Furthermore, ZMAT3 overexpression in APC determined senescence and activation of the p53/p21 pathway, as observed in FDR APC. Adipogenesis was also inhibited in ZMAT3‐overexpressing APC. In FDR APC, rescue of ZMAT3 methylation through senolytic exposure simultaneously downregulated ZMAT3 expression and improved adipogenesis. Interestingly, in human SAT, aging and T2D were associated with significantly increased expression of both ZMAT3 and the P53 senescence marker. Thus, DNA hypomethylation causes ZMAT3 upregulation in FDR APC accompanied by acquisition of the senescence phenotype and impaired adipogenesis, which may contribute to FDR predisposition for T2D.     

A rapid mini-prep method for isolation of ectomycorrhizal DNA from Tuber species

A rapid procedure has been developed to isolate DNA from the ectomycorrhizae of Tuber spp. for use in PCR experiments. The method described is fast and sensitive and can overcome the amplification problems that can arise in the presence of inhibitors. For this reason it can be used to type ectomycorrhizae even starting from a single root tip and make mycorrhizae identification much more rapid.     

Replication-mediated instability of the GAA triplet repeat mutation in Friedreich ataxia

Friedreich ataxia is caused by the expansion of a polymorphic and unstable GAA triplet repeat in the FRDA gene, but the mechanisms for its instability are poorly understood. Replication of (GAA•TTC)_n sequences (9–105 triplets) in plasmids propagated in Escherichia coli displayed length- and orientation-dependent instability. There were small length variations upon replication in both orientations, but large contractions were frequently observed when GAA was the lagging strand template. DNA replication was also significantly slower in this orientation. To evaluate the physiological relevance of our findings, we analyzed peripheral leukocytes from human subjects carrying repeats of similar length (8–107 triplets). Analysis of 9400 somatic FRDA molecules using small-pool PCR revealed a similar mutational spectrum, including large contractions. The threshold length for the initiation of somatic instability in vivo was between 40 and 44 triplets, corresponding to the length of a eukaryotic Okazaki fragment. Consistent with the stabilization of premutation alleles during germline transmission, we also found that instability of somatic cells in vivo and repeats propagated in E.coli were abrogated by (GAGGAA)_n hexanucleotide interruptions. Our data demonstrate that the GAA triplet repeat mutation in Friedreich ataxia is destabilized, frequently undergoing large contractions, during DNA replication.     

Angiopoietin decoy secreted at tumor site impairs tumor growth and metastases by inducing local inflammation and altering neoangiogenesis

The extracellular domain of the receptor tyrosine kinase Tie2/TEK (exTEK) has been used as an angiopoietin decoy to study the role of angiopoietins in the tumor–host interactions, using a syngeneic model of experimental metastases and subcutaneous tumor. Soluble exTEK secreted by transfected tumor cells inhibited HUVECs from forming tubes in Matrigel. ExTEK-transfected C26 colon carcinoma and TS/A mammary tumor cells displayed reduced growth rate when injected subcutaneously, and reduced ability to form experimental metastases when injected intravenously. Immunohistochemical analysis of tumors and metastases showed increased leukocytes infiltration and signs of inflammation in exTEK-secreting compared to parental tumor, as well as impairment in neo-vessel growth and organization. However, while neoangiogenesis eventually rescued in the subcutis, it failed to organize in the experimental metastases of exTEK-secreting tumor, contributing to the hampering of metastatic growth and to increased mice survival. The reactive infiltrate of C26TEK contained a different percentage of leukocytes and was responsible for the tumor inhibition. In fact, leukopenia induced by -irradiation of recipient mice or injection into interferon gamma (IFN-) gene knockout (GKO) mice resulted in reduced mouse survival and an increased number of lung metastases. On the other hand, interleukin (IL)-12 treatment prolonged the survival of mice bearing subcutaneous C26TEK but not of those bearing lung metastases, suggesting that IL-12 could exert further antiangiogenic effects at the site where the tumor can restore neoangiogenesis. These results show in vivo that reduced angiopoietin availability at the tumor site induces a local inflammatory response and impairment of neoangiogenesis which act synergistically to limit tumor growth and metastasis.Abbreviations AEC amino-ethylcarbazole - ELISA enzyme-linked immunosorbent assay - HRP horseradish peroxidase - HUVEC human umbilical vascular endothelial cell - i.v. intravenous - s.c. subcutaneous - TBS Tris-HCl buffered solution