The European strategy on low dose risk research and the role of radiation quality according to the recommendations of the “ad hoc” High Level and Expert Group (HLEG)

Health effects of exposures at low doses and/or low dose rates are recognized as requiring intensive research activity to answer several questions. To address these issues at a strategic level in Europe, with the perspective of integrating national and EC efforts (in particular those within the Euratom research programmes), a “European High Level and Expert Group (HLEG) on low dose risk research” was formed and carried out its work during 2008. The Group produced a report published by the European Commission in 2009 and available on the website . The more important research issues identified by the HLEG were as follows: (a) the shape of dose–response for cancer; (b) the tissue sensitivities for cancer induction; (c) the individual variability in cancer risk; (d) the effects of radiation quality (type); (e) the risks from internal radiation exposure; and (f) the risks of, and dose response relationships for, non-cancer diseases. In this paper, the radiation quality issues are especially considered, since they are closely linked to health problems and related radioprotection in space and in emerging radiotherapeutic techniques (i.e., hadrontherapy). The peculiar features of low-fluence, high-LET radiation exposures can question in particular the validity of the radiation-weighting factor (w _R ) approach. Specific strategies are therefore needed to assess such risks. A multi-scale/systems biology approach, based on mechanistic studies coordinated with molecular-epidemiological studies, is considered essential to elucidate differences and similarities between specific effects of low- and high-LET radiation.     

Sinapinic acid can replace ascorbate in the biotin switch assay

Background

Protein S-nitrosation is an important post-translational modification altering protein function. Interaction of nitric oxide with thiols is an active area of research, and is one of the mechanisms by which NO exerts its biological effects. Biotin switch assay is the method, which has been developed to identify S-nitrosated proteins. The major concern with biotin switch assay includes reducing disulfide which may lead to false positives. We report a modification of the biotin switch assay where sinapinic acid is utilized instead of ascorbate to eliminate potential artifacts in the detection of S-nitrosated proteins.

Methods

The denitrosation ability of sinapinic acid was assessed by monitoring either the NO or NO₂^- released by chemiluminescent NO detection or by the griess assay, respectively. DTNB assay was used to compare disulfide reduction by ascorbate and sinapinic acid. Sinapinic acid and ascorbate were compared in the biotin switch detection of S-nitrosoproteins in RAW 264.7 cells ± S-nitrosocysteine (CysNO) exposure.

Results

We show that sinapinic acid has the ability to denitrosate S-nitrosothiols at pH 7.0 and denitrate plus denitrosate at pHs 8 and 8.5. Unlike ascorbate, sinapinic acid degrades S-nitrosothiols, but it does not reduce disulfide bridges.

Conclusions

Sinapinic acid denitrosate RSNO and does not reduce disulfides. Thus can readily replace ascorbate in detection of S-nitrosated proteins in biotin switch assay.

General significance

The work described is important in view of protein S-nitrosation. In this study we provide an important modification that eliminates artifacts in widely used technique for detecting the S-nitrosoproteome, the biotin switch assay.     

Unusual evolution of a catalytic core element in CCA-adding enzymes

CCA-adding enzymes are polymerases existing in two distinct enzyme classes that both synthesize the C-C-A triplet at tRNA 3′-ends. Class II enzymes (found in bacteria and eukaryotes) carry a flexible loop in their catalytic core required for switching the specificity of the nucleotide binding pocket from CTP- to ATP-recognition. Despite this important function, the loop sequence varies strongly between individual class II CCA-adding enzymes. To investigate whether this loop operates as a discrete functional entity or whether it depends on the sequence context of the enzyme, we introduced reciprocal loop replacements in several enzymes. Surprisingly, many of these replacements are incompatible with enzymatic activity and inhibit ATP-incorporation. A phylogenetic analysis revealed the existence of conserved loop families. Loop replacements within families did not interfere with enzymatic activity, indicating that the loop function depends on a sequence context specific for individual enzyme families. Accordingly, modeling experiments suggest specific interactions of loop positions with important elements of the protein, forming a lever-like structure. Hence, although being part of the enzyme’s catalytic core, the loop region follows an extraordinary evolutionary path, independent of other highly conserved catalytic core elements, but depending on specific sequence features in the context of the individual enzymes.     

Taurine and the renal system

Taurine participates in a number of different physiologic and biologic processes in the kidney, often reflected by urinary excretion patterns. The kidney is key to aspects of taurine body pool size and homeostasis. This review will examine the renal-taurine interactions relative to ion reabsorption; renal blood flow and renal vascular endothelial function; antioxidant properties, especially in the glomerulus; and the role of taurine in ischemia and reperfusion injury. In addition, taurine plays a role in the renal cell cycle and apoptosis, and functions as an osmolyte during the stress response. The role of the kidney in adaptation to variations in dietary taurine intake and the regulation of taurine body pool size are described. Finally, the protective function of taurine against several kidney diseases is reviewed.     

Carborane-conjugated 2-quinolinecarboxamide ligands of the translocator protein for boron neutron capture therapy

Potential boron neutron capture therapy (BNCT) agents have been designed on the basis of the evidence about translocator protein (TSPO) overexpression on the outer mitochondrial membrane of tumor cells. The structure of the first TSPO ligand bearing a carborane cage (compound 2d) has been modified in order to find a suitable candidate for in vivo studies. The designed compounds were synthesized and evaluated for their potential interaction with TSPO and tumor cells. In vitro biological evaluation showed in the case of fluoromethyl derivative 4b a nanomolar TSPO affinity very similar to that of 2d, a significantly lower cytotoxicity, and a slightly superior performance as boron carrier toward breast cancer cells. Moreover, compound 4b could be used as a 1?F magnetic resonance imaging (MRI) agent as well as labeled with 11C or 1?F to obtain positron emission tomography (PET) radiotracers in order to apply the "see and treat" strategy in BNCT.     

Effects of the new adiponectin paralogous protein CTRP-3 and of LPS on cytokine release from monocytes of patients with type 2 diabetes mellitus

Aims/Hypothesis: It was the aim to investigate the hypothesis that the new C1q/TNF-family member CTRP-3 (C1q/TNF-related protein-3) acts anti-inflammatory in human monocytes from healthy controls and patients with type 2 diabetes mellitus (T2D). Methods: Monocytes were isolated from 20 healthy controls and 30 patients with T2D. IL-6 and TNF concentrations were measured by ELISA. CTRP-3 was expressed in insect cells and used for stimulation experiments. Results: Basal IL-6 and TNF were not different in control and in T2D monocytes. LPS-stimulation (1 μg/ml) significantly (p < 0.001) increased IL-6 and TNF in the supernatants of control and in T2D monocytes to a similar extent. CTRP-3 (1 μg/ml) significantly (p = 0.03) inhibited LPS-induced IL-6 in control monocytes but not in T2D monocytes. TNF upon co-stimulation with LPS and CTRP-3 was significantly (p = 0.012) lower in control than in T2D monocytes. LPS-induced TNF concentration was significantly and positively correlated with serum total cholesterol and LDL cholesterol in T2D patients. Conclusions: CTRP-3 inhibits LPS-induced IL-6 and TNF release. This anti-inflammatory effect is lost in T2D. Serum cholesterol concentration affects the pro-inflammatory potential of LPS to induce TNF release from T2D monocytes in the presence or absence of CTRP-3. CTRP-3 might partly account for the pro-inflammatory state in T2D.     

Chromocentre integrity and epigenetic marks

The epigenetic modification of histones dictates the formation of euchromatin and heterochromatin domains. We studied the effects of a deficiency of histone methyltransferase, SUV39h, and trichostatin A-dependent hyperacetylation on the structural stability of centromeric clusters, called chromocentres. We did not observe the expected disintegration of chromocentres, but both SUV39h deficiency and hyperacetylation in SUV39h+/+ cells induced the re-positioning of chromocentres closer to the nuclear periphery. Conversely, TSA treatment of SUV39h?/? cells re-established normal nuclear radial positioning of chromocentres. This structural re-arrangement was likely caused by several epigenetic events at centromeric heterochromatin. In particular, reciprocal exchanges between H3K9me1, H3K9me2, H3K9me3, DNA methylation, and HP1 protein levels influenced chromocentre nuclear composition. For example, H3K9me1 likely substituted for the function of H3K9me3 in chromocentre nuclear arrangement and compaction. Our results illustrate the important and interchangeable roles of epigenetic marks for chromocentre integrity. Therefore, we propose a model for epigenetic regulation of nuclear stability of centromeric heterochromatin in the mouse genome.     

Toward a resolution of a taxonomic enigma: first genetic analyses of Paradoxornis webbianus and Paradoxornis alphonsianus (Aves: Paradoxornithidae) from China and Italy

Paradoxornis webbianus and Paradoxornis alphonsianus naturally occur in South-East Asia. Due to a recent introduction, a mixed population currently occurs in northern Italy. A preliminary phylogeographic analysis using samples from Italy and China found little genetic differentiation between the two taxa and revealed the existence of two molecular lineages, sympatric in some part of China, that do not correspond to the morphological classification. Possible taxonomic changes and preliminary inferences on the relationships between Chinese and the Italian populations and on the likely provenance of the founders introduced in Italy are also discussed.