The expression pattern of a mouse doublesex-related gene is consistent with a role in gonadal differentiation

The signal for somatic sex determination in mammals, Caenorhabditis elegans and Drosophila melanogaster is chromosomal, but the overall mechanisms do not appear to be conserved between the phyla. However it has been found quite recently that the C. elegans sex-determining gene Mab-3 contains a domain highly homologous to the Drosophila sex-determining gene doublesex (dsx) and shares a similar role. These data suggest that at least some aspects of the regulation of sex determination might be conserved. In humans, a doublesex-related gene (DMRT1) was identified at less than 30 kb from the critical region for sex reversal on chromosome 9p24 (TD9). In order to get insights into the role of DMRT1 in sex determination/differentiation, we have isolated DMRT1 mouse homologue (Dmrt1) and analysed its expression pattern. The gene is expressed in the genital ridges of both sexes during the sex-determining switch and it shows male/female dimorphism at late stages of sex differentiation.     

R- and S-isomers of nonsteroidal anti-inflammatory drugs differentially regulate cytokine production

2-arylpropionic acids, a well known class of non-steroidal anti-inflammatory drugs (NSAIDs), exist as a racemic mixture of their enantiomeric forms, with S-isomers primarily responsible for inhibition of prostaglandin (PG) production and of inflammatory events. In this study we show that S-isomers are also responsible for the paradoxical up-regulation of tumor necrosis factor (TNF) induced by ketoprofen, flurbiprofen and ibuprofen in murine peritoneal macrophages stimulated by bacterial endotoxin (LPS). This effect is in close correlation with cyclooxygenase inhibitory capacity of S-isomers and, from Northern blot analysis, seems to be mediated by the up-regulation of TNF mRNA. In addition, up-regulation of TNF production by S-isomers is associated with inhibition of interleukin-10 (IL-10) production. Conversely, we have observed that S-enantiomers reduce IL-6 production at a concentration 100 times higher than that able to inhibit cyclooxygenase activity. The unwanted pro-inflammatory effects of S-isomers through TNF and IL-10 production could therefore hinder their analgesic effect, that is, at least in part, related to IL-6 inhibition. In addition, TNF amplification by S-isomers could be correlated to the clinical evidence of their gastric toxicity. On the other hand, R-isomers did not affect TNF and IL-10 production even at cyclooxygenase-blocking concentration, while they reduced IL-6 production to the same levels as S-isomers. It is concluded that the regulation of cytokine production by S-isomers of 2-arylpropionic acids could partially mask their therapeutic effects and could be correlated to the clinical evidence of their higher gastric toxicity. On the other hand, IL-6 inhibition without the unwanted effects on TNF and IL-10 production shown by R-isomers could be correlated to the analgesic effect reported for R-2-arylpropionic acids.     

Backbone dynamics of human Cu,Zn superoxide dismutase and of its monomeric F50E/G51E/E133Q mutant: the influence of dimerization on mobility and function

The backbone assignment of reduced human dimeric Cu,Zn superoxide dismutase (SOD) was performed on a sample 100% enriched in (15)N, (13)C and 70% enriched in (2)H. (15)N T(1), T(2), and T(1)(rho) and (15)N-(1)H NOE assignment was performed at 600 MHz proton frequency on both wild-type SOD and the monomeric F50E/G51E/E133Q mutant. This allowed a comparison of the mobility in the subnanosecond and in the millisecond to microsecond time scales of the two systems. Both proteins are rather rigid, although some breathing of the beta sheets is detected in the wild type dimer. The monomer displays large mobility in the loops in the first part of the sequence, in loop IVa where point mutations have been introduced and at the C-terminus. The dimeric wild type is rigidified at loop IVa and at the C-terminus. Only loop VII shows a higher mobility in the dimer (besides some individual NH moieties). Conformational equilibria are displayed in the monomeric form around cysteines 57 and 146, thus explaining the disorder of arginine 143 which is the most important residue in guiding O(2)(-) toward the copper ion. The larger mobility in the wild type form with respect to the monomer in the picosecond to nanosecond time scale of helix alpha1 and loop VIIb, which provides the correct electrostatic driving force for O(2)(-) in the active channel, has been discussed in terms of favoring the activity of SOD.     

Solution structure of reduced Clostridium pasteurianum rubredoxin

The solution structure of reduced Clostridium pasteurianum rubredoxin (MW 6100) is reported here. The protein is highly paramagnetic, with iron(II) being in the S=2 spin state. The Hβ protons of the ligating cysteines are barely observed, and not specifically assigned. Seventy-six percent of the protons have been assigned and 1267 NOESY peaks (of which 1037 are meaningful) have been observed. Nonselective T ₁ measurements have been measured by recording four nonselective 180°-τ-NOESY at different τ values, and fitting the intensity recoveries to an exponential recovery. Thirty-six metal-proton upper and lower distance constraints have been obtained from the above measurements. The use of such constraints is assessed with respect to spin delocalization on the sulfur donor atoms. The solution structure obtained with the program DYANA has been refined through restrained energy minimization. A final family of 20 conformers is obtained with no distance violations larger than 0.24?Å, and RMSD values to the mean structure of 0.58 and 1.03?Å for backbone and all heavy atoms, respectively (measured on residues 3–53). The structure is compared to the X-ray structure of the oxidized and of the zinc substituted protein, and to the available structures of other rubredoxins. In particular, the comparison with the crystal structure and the solution structure of the Zn derivative of the highly thermostable Pyrococcus furiosus rubredoxin suggested that the relatively low thermal stability of the clostridial rubredoxin may be tentatively ascribed to the loosening of its secondary structure elements. This research is a further achievement at the frontier of solution structure determinations of paramagnetic proteins.     

The CD4+AT2R+ T cell subpopulation improves post‐infarction remodelling and restores cardiac function

Myocardial infarction (MI) is a major condition causing heart failure (HF). After MI, the renin angiotensin system (RAS) and its signalling octapeptide angiotensin II (Ang II) interferes with cardiac injury/repair via the AT1 and AT2 receptors (AT1R, AT2R). Our study aimed at deciphering the mechanisms underlying the link between RAS and cellular components of the immune response relying on a rodent model of HF as well as HF patients. Flow cytometric analyses showed an increase in the expression of CD4⁺ AT2R⁺ cells in the rat heart and spleen post‐infarction, but a reduction in the peripheral blood. The latter was also observed in HF patients. The frequency of rat CD4⁺ AT2R⁺ T cells in circulating blood, post‐infarcted heart and spleen represented 3.8 ± 0.4%, 23.2 ± 2.7% and 22.6 ± 2.6% of the CD4⁺ cells. CD4⁺ AT2R⁺ T cells within blood CD4⁺ T cells were reduced from 2.6 ± 0.2% in healthy controls to 1.7 ± 0.4% in patients. Moreover, we characterized CD4⁺ AT2R⁺ T cells which expressed regulatory FoxP3, secreted interleukin‐10 and other inflammatory‐related cytokines. Furthermore, intramyocardial injection of MI‐induced splenic CD4⁺ AT2R⁺ T cells into recipient rats with MI led to reduced infarct size and improved cardiac performance. We defined CD4⁺ AT2R⁺ cells as a T cell subset improving heart function post‐MI corresponding with reduced infarction size in a rat MI‐model. Our results indicate CD4⁺ AT2R⁺ cells as a promising population for regenerative therapy, via myocardial transplantation, pharmacological AT2R activation or a combination thereof.     

Consideration of Viral Resistance for Optimization of Direct Antiviral Therapy of Hepatitis C Virus Genotype 1-Infected Patients

Different highly effective interferon-free treatment options for chronic hepatitis C virus (HCV) infection are currently available. Pre-existence of resistance associated variants (RAVs) to direct antiviral agents (DAAs) reduces sustained virologic response (SVR) rates by 3–53% in hepatitis C virus (HCV) genotype 1 infected patients depending on different predictors and the DAA regimen used. Frequencies of single and combined resistance to NS3, NS5A and NS5B inhibitors and consequences for the applicability of different treatment regimens are unknown. Parallel population based sequencing of HCV NS3, NS5A and NS5B genes in 312 treatment-naïve Caucasian HCV genotype 1 infected patients showed the presence of major resistant variants in 20.5% (NS3), 11.9% (NS5A), and 22.1% (NS5B) with important differences for HCV subtypes. In NS3, Q80K was observed in 34.7% and 2.1% of subtype 1a and 1b patients, respectively while other RAVs to second generation protease inhibitors were detected rarely (1.4%). Within NS5A RAVs were observed in 7.1% of subtype 1a and 17.6% in subtype 1b infected patients. RAVs to non-nucleoside NS5B inhibitors were observed in 3.5% and 44.4% of subtype 1a and 1b patients, respectively. Considering all three DAA targets all subtype 1a and 98.6% of subtype 1b infected patients were wildtype for at least one interferon free DAA regimen currently available. In conclusion, baseline resistance testing allows the selection of at least one RAVs-free treatment option for nearly all patients enabling a potentially cost- and efficacy-optimized treatment of chronic hepatitis C.