Stereospecific binding of MRI contrast agents to human serum albumin: the case of Gd-(S)-EOB-DTPA (Eovist) and its (R) isomer

The water proton relaxation rate enhancement of the hepatospecific Gd-(S)-EOB-DTPA (Eovist) and of its (R) isomer in aqueous solutions free of protein, in serum and in 4% human serum albumin solution, are compared. In the absence of proteins, both compounds exhibit, as expected, the same proton relaxivity, as measured by the nuclear magnetic relaxation dispersion (NMRD) profiles. In serum and albumin solution, non-covalent binding of the paramagnetic complexes to macromolecules is observed. Both isomers are likely to bind to the same site of human serum albumin, but the affinity of the (S) isomer is larger than for the (R) isomer.     

Genome‐wide analysis of Italian sheep diversity reveals a strong geographic pattern and cryptic relationships between breeds

Italy counts several sheep breeds, arisen over centuries as a consequence of ancient and recent genetic and demographic events. To finely reconstruct genetic structure and relationships between Italian sheep, 496 subjects from 19 breeds were typed at 50K single nucleotide polymorphism loci. A subset of foreign breeds from the Sheep HapMap dataset was also included in the analyses. Genetic distances (as visualized either in a network or in a multidimensional scaling analysis of identical by state distances) closely reflected geographic proximity between breeds, with a clear north–south gradient, likely because of high levels of past gene flow and admixture all along the peninsula. Sardinian breeds diverged more from other breeds, a probable consequence of the combined effect of ancient sporadic introgression of feral mouflon and long‐lasting genetic isolation from continental sheep populations. The study allowed the detection of previously undocumented episodes of recent introgression (Delle Langhe into the endangered Altamurana breed) as well as signatures of known, or claimed, historical introgression (Merino into Sopravissana and Gentile di Puglia; Bergamasca into Fabrianese, Appenninica and, to a lesser extent, Leccese). Arguments that would question, from a genomic point of view, the current breed classification of Bergamasca and Biellese into two separate breeds are presented. Finally, a role for traditional transhumance practices in shaping the genetic makeup of Alpine sheep breeds is proposed. The study represents the first exhaustive analysis of Italian sheep diversity in an European context, and it bridges the gap in the previous HapMap panel between Western Mediterranean and Swiss breeds.     

Pertussis toxin-sensitive G-proteins are not involved in activation of T-lymphocytes.

Multiple effects of pertussis toxin (PT) on Jurkat T-cells can be distinguished on the basis of their dose-response and their kinetics. High concentrations of PT deliver to cells an activating signal resulting in a rapid rise in [Ca2+]i followed by IL-2 synthesis. This activation is accompanied (within 2 h) by a down-regulation of the CD3/TCR complex from the cell surface. Cells then become refractory towards stimulation by CD3 mAb or PHA. All these effects, referred to as 'mitogenic effects', present the same dose-response curves with an EC50 of 0.5 micrograms/ml. Short term effects (PT-induced Ca2+ movements, down-regulation of CD3/TCR complex and inhibition of PHA and CD3-induced Ca2+ signal) are observed under conditions where no PT-induced ADP-ribosylation can be detected. In contrast, ADP-ribosylation of the 40,000 alpha-subunit of G-proteins requires a sustained (18 h) incubation of intact cells in the presence of low concentration (EC50 = 0.3 ng/ml) of PT. Dose-response curves for PT-dependent ADP-ribosylation and mitogenic effects are separated by three orders of magnitude. Covalent modification of G-protein has no effect on CD3-induced increase in [Ca2+]i and IL-2 synthesis induced by a combination of phorbol ester and either CD3 mAb, PHA or calcium ionophore. These data indicate that transduction of the mitogenic signal does not involve a PT-sensitive G-protein. Furthermore, inhibition of mitogenic signals following PT treatment results from a PT-induced activation leading to a down-regulation of the CD3/T cell receptor complex.     

A procedure for identifying homologous alternative splicing events

Background  

The study of the functional role of alternative splice isoforms of a gene is a very active area of research in biology. The difficulty of the experimental approach (in particular, in its high-throughput version) leaves ample room for the development of bioinformatics tools that can provide a useful first picture of the problem. Among the possible approaches, one of the simplest is to follow classical protein function annotation protocols and annotate target alternative splice events with the information available from conserved events in other species. However, the application of this protocol requires a procedure capable of recognising such events. Here we present a simple but accurate method developed for this purpose.     

Clinical and cytokine profile evaluation in Northeast Brazilian psoriasis plaque-type patients

Objective and Design

Psoriasis is a common, enigmatic, and recurrent disease. The precise etiology and pathogenesis of psoriasis are still unclear. Psoriasis has been treated as an inflammatory disorder related to an underlying Th1/Th17-dominated immune response. Interleukins are involved in the development of psoriasis lesions through Th-17-associated inflammation. Th1 and Th17 cytokines are found in skin lesions and in the peripheral blood of psoriasis patients.We sought to analyze serum levels of IL-1-β, IL-8, IL-9, IL-27, IL-29, IL-35, IFN-γ, TNF and TGF-β in patients with psoriasis and healthy control volunteers.

Material

Blood samples were collected from fifty-three patients with psoriasis and thirty-five healthy controls.

Methods

Serum cytokines concentrations were determined using an enzyme-linked immunosorbent assay.

Results

Serum IL-8, IL-9, IL-27, IL-29 and TNF levels were statistically significant in psoriasis patients. Detectable serum IL-9 levels were found in 47 patients of the 53 in the psoriasis group.

Conclusions

Interleukins-8, 27, 29 and TNF levels measured in the serum of psoriasis patients were slightly elevated as compared to healthy controls in a weakly significant way. On the other hand, there were highly significant differences in IL-9 levels between the two groups.

     

The Impact of the HydroxyMethylCytosine epigenetic signature on DNA structure and function

We present a comprehensive, experimental and theoretical study of the impact of 5-hydroxymethylation of DNA cytosine. Using molecular dynamics, biophysical experiments and NMR spectroscopy, we found that Ten-Eleven translocation (TET) dioxygenases generate an epigenetic variant with structural and physical properties similar to those of 5-methylcytosine. Experiments and simulations demonstrate that 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) generally lead to stiffer DNA than normal cytosine, with poorer circularization efficiencies and lower ability to form nucleosomes. In particular, we can rule out the hypothesis that hydroxymethylation reverts to unmodified cytosine physical properties, as hmC is even more rigid than mC. Thus, we do not expect dramatic changes in the chromatin structure induced by differences in physical properties between d(mCpG) and d(hmCpG). Conversely, our simulations suggest that methylated-DNA binding domains (MBDs), associated with repression activities, are sensitive to the substitution d(mCpG) ➔ d(hmCpG), while MBD3 which has a dual activation/repression activity is not sensitive to the d(mCpG) d(hmCpG) change. Overall, while gene activity changes due to cytosine methylation are the result of the combination of stiffness-related chromatin reorganization and MBD binding, those associated to 5-hydroxylation of methylcytosine could be explained by a change in the balance of repression/activation pathways related to differential MBD binding.     

Tyrosine hydroxylase activity in the endocrine pancreas: changes induced by short-term dietary manipulation

BACKGROUND: Tyrosine hydroxylase (TH) activity and its possible participation in the control of insulin secretion were studied in pancreatic islets of adult Wistar rats fed a standard commercial diet (SD) or carbohydrates alone (CHD) for one week. TH activity, norepinephrine (NE) content, and glucose-induced insulin secretion were assessed. Blood glucose and insulin levels were measured at the time of sacrifice. RESULTS: CHD rats had significantly higher blood glucose and lower insulin levels than SD rats (114.5 PlusMinus; 6.7 vs 80.7 PlusMinus; 7.25 mg/dl, p < 0.001; 20.25 PlusMinus; 2.45 vs 42.5 PlusMinus; 4.99 &mgr;U/ml, p < 0.01, respectively). Whereas TH activity was significantly higher in CHD isolated islets (600 PlusMinus; 60 vs 330 PlusMinus; 40 pmol/mg protein/h; p < 0.001), NE content was significantly lower (18 PlusMinus; 1 vs 31 PlusMinus; 5 pmol/mg protein), suggesting that TH activity would be inhibited by the end-products of catecholamines (CAs) biosynthetic pathway. A similar TH activity was found in control and solarectomized rats (330 PlusMinus; 40 vs 300 PlusMinus; 80 pmol/mg protein/h), suggesting an endogenous rather than a neural origin of TH activity. CHD islets released significantly less insulin in response to glucose than SD islets (7.4 PlusMinus; 0.9 vs 11.4 PlusMinus; 1.1 ng/islet/h; p < 0.02). CONCLUSIONS: TH activity is present in islet cells; dietary manipulation simultaneously induces an increase in this activity together with a decrease in glucose-induced insulin secretion in rat islets. TH activity - and the consequent endogenous CAs turnover - would participate in the paracrine control of insulin secretion.     

Genetic and Biochemical Characterization of Human AP Endonuclease 1 Mutants Deficient in Nucleotide Incision Repair Activity

Background

Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key DNA repair enzyme involved in both base excision repair (BER) and nucleotide incision repair (NIR) pathways. In the BER pathway, APE1 cleaves DNA at AP sites and 3′-blocking moieties generated by DNA glycosylases. In the NIR pathway, APE1 incises DNA 5′ to a number of oxidatively damaged bases. At present, physiological relevance of the NIR pathway is fairly well established in E. coli, but has yet to be elucidated in human cells.

Methodology/Principal Finding

We identified amino acid residues in the APE1 protein that affect its function in either the BER or NIR pathway. Biochemical characterization of APE1 carrying single K98A, R185A, D308A and double K98A/R185A amino acid substitutions revealed that all mutants exhibited greatly reduced NIR and 3′→5′ exonuclease activities, but were capable of performing BER functions to some extent. Expression of the APE1 mutants deficient in the NIR and exonuclease activities reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to an alkylating agent, methylmethanesulfonate, suggesting that our APE1 mutants are able to repair AP sites. Finally, the human NIR pathway was fully reconstituted in vitro using the purified APE1, human flap endonuclease 1, DNA polymerase β and DNA ligase I proteins, thus establishing the minimal set of proteins required for a functional NIR pathway in human cells.

Conclusion/Significance

Taken together, these data further substantiate the role of NIR as a distinct and separable function of APE1 that is essential for processing of potentially lethal oxidative DNA lesions.