Immunocytochemical characterisation of endophytic bacteriaAzospirillum brasilense, Herbaspirillum seropedicae, Burkholderia ambifaria andGluconacetobacter diazotrophicus

In the present work an immunocytochemical characterisation of four endophytic bacterial species has been made by using polyclonal antiserum produced against each of the four bacterial strains previously heated at 60 °C. The aim of this researchsito identify common elements among bacteria associated with their endophytic behaviour. Analysis of extracts of each strain by immunoblotting and ELISA confirmed the presence of proteins from different bacterial strains made up of common epitopes. However, antisaproduced againstHerbaspirillum seropedicae andBurkholderia ambifaria show a high number of bands recognised on each extracts, while antisera againstAzospirillum brasilense andGluconacetobacter diazotrophicus show a low number of bands recognised on each extract. Immunogold labelling showed that epitopes are located both on the cell wall and in the cytoplasm; most likely they could be preursor cell wall proteins synthesized inside the cytoplasm and subsequently transported onto cell wall. Finally, the common bands amog bacterial strains revealed by immunoblotting could play a role as active hydrolases involved in host tissue penetration.     

Presence of a drifting algal bed in the Mar Piccolo basin,Taranto (Ionian Sea,Southern Italy)

A survey is reported of the drifting algal community in Mar Piccolo, a polluted basin subject to sewage outlets. The key role was played by a few key species, mainly floridean red algae.     

Induction of globin mRNA expression by interleukin-3 in a stem cell factor-dependent SV-40 T-antigen-immortalized multipotent hematopoietic cell line

Erythropoiesis requires the stepwise action on immature progenitors of several growth factors, including stem cell factor (SCF), interleukin 3 (IL-3), and erythropoietin (Epo). Epo is required to sustain proliferation and survival of committed progenitors and might further modulate the level of expression of several erythroid genes, including globin genes. Here we report a new SCF-dependent immortalized mouse progenitor cell line (GATA-1 ts SCF) that can also grow in either Epo or IL-3 as the sole growth factor. When grown in SCF, these cells show an "open" chromatin structure of the beta-globin LCR, but do not significantly express globin. However, Epo or IL-3 induce globin expression and are required for its maintainance. This effect of IL-3 is unexpected as IL-3 was previously reported either to be unable to induce hemoglobinization, or even to antagonize it. This suggests that GATA-1 ts SCF cells may have progressed to a stage in which globin genes are already poised for expression and only require signal(s) that can be elicited by either Epo or IL-3. Through the use of inhibitors, we suggest that p38 may be one of the molecules modulating induction and maintenance of globin expression.     

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.     

Scavenger receptor CD36 mediates uptake of high density lipoproteins in mice and by cultured cells

The mechanisms of HDL-mediated cholesterol transport from peripheral tissues to the liver are incompletely defined. Here the function of scavenger receptor cluster of differentiation 36 (CD36) for HDL uptake by the liver was investigated. CD36 knockout (KO) mice, which were the model, have a 37% increase (P = 0.008) of plasma HDL cholesterol compared with wild-type (WT) littermates. To explore the mechanism of this increase, HDL metabolism was investigated with HDL radiolabeled in the apolipoprotein (¹²⁵I) and cholesteryl ester (CE, [³H]) moiety. Liver uptake of [³H] and ¹²⁵I from HDL decreased in CD36 KO mice and the difference, i. e. hepatic selective CE uptake ([³H]¹²⁵I), declined (–33%, P = 0.0003) in CD36 KO compared with WT mice. Hepatic HDL holo-particle uptake (¹²⁵I) decreased (–29%, P = 0.0038) in CD36 KO mice. In vitro, uptake of ¹²⁵I-/[³H]HDL by primary liver cells from WT or CD36 KO mice revealed a diminished HDL uptake in CD36-deficient hepatocytes. Adenovirus-mediated expression of CD36 in cells induced an increase in selective CE uptake from HDL and a stimulation of holo-particle internalization. In conclusion, CD36 plays a role in HDL uptake in mice and by cultured cells. A physiologic function of CD36 in HDL metabolism in vivo is suggested.     

Protein oxidative damage and redox imbalance induced by ionising radiation in CHO cells

Reactive oxygen species (ROS) are important mediators of the cytotoxicity induced by the direct reaction of ionising radiation (IR) with all critical cellular components, such as proteins, lipids, and nucleic acids. The derived oxidative damage may propagate in exposed tissues in a dose- and spatiotemporal dependent manner to other cell compartments, affecting intracellular signalling, and cell fate. To understand how cell damage is induced, we studied the oxidative events occurring immediately after cell irradiation by analysing the fate of IR-derived ROS, the intracellular oxidative damage, and the modification of redox environment accumulating in Chinese hamster ovary (CHO) within 1?h after cell irradiation (dose range 0–10?Gy). By using the immuno-spin trapping technique (IST), spectrophotometric methods, and electron paramagnetic resonance (EPR) spectroscopy, we showed that IR-derived ROS (i) induced an IST-detectable, antioxidant-inhibitable one-electron oxidation of specific intracellular proteins; (ii) altered the glutathione (GSH) content (which was found to increase below 2?Gy, and decrease at higher doses, leading to a redox imbalance); (iii) decreased glutathione peroxidase and glutaredoxin activity; (iv) modified neither glutathione reductase nor thioredoxin reductase activity; (v) were detected by spin trapping technique, but adduct intensity decreased due to cell competition for ROS; and (vi) induced no EPR-detectable radicals assignable to oxidised cellular components. In conclusion, our results showed that IR generated an early high oxidising potential (protein radical intermediates, redox imbalance, modified redox enzyme activity) in irradiated cells potentially able to propagate the damage and induce oxidative modification of secondary targets.     

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.     

Cytotoxicity,apoptosis, DNA damage and methylation in mammary and kidney epithelial cell lines exposed to ochratoxin A

This study aimed to investigate the in vitro damage induced by ochratoxin A (OTA) in BME-UV1 and MDCK epithelial cells. Both cells lines were treated with OTA (0 up to 10 μg/mL), and cell viability (MTT assay), membrane stability (lactate dehydrogenase (LDH) release assay) and apoptotic cell rate (Tunel assay) were investigated. Further, the effect of the incubation with OTA has been evaluated at DNA level by the determination of DNA integrity, by the quantification of DNA adduct formation (8-hydroxy-2′-deoxyguanosine (8-OHdG)) and by the assessment of the global DNA methylation status (5-methyl-cytosine (5-mC)). The obtained results showed that after 24 h of OTA treatment, BME-UV1 cell viability was reduced in a dose-dependent way. OTA significantly (P?<?0.05) increased LDH release in BME-UV1 cells at all concentrations tested. OTA (1.25 μg/mL) induced 35 % LDH release in MDCK cells (P?<?0.05). A significant (P?<?0.05) change in percentages of apoptotic BME-UV1 (10?±?0.86) and MDCK (25?±?0.88) cells was calculated when the cells were co-incubated with OTA. The level of 8-OHdG adduct formation was significantly (P?<?0.05) increased in BME-UV1 cells treated with 1.25 μg/mL of OTA. The results of the present study suggest that a different mechanism of action may occur in these cell lines.

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Graphical abstract Study results overview