Loss of HR6B ubiquitin-conjugating activity results in damaged synaptonemal complex structure and increased crossing-over frequency during the male meiotic prophase

The ubiquitin-conjugating enzymes HR6A and HR6B are the two mammalian homologs of Saccharomyces cerevisiae RAD6. In yeast, RAD6 plays an important role in postreplication DNA repair and in sporulation. HR6B knockout mice are viable, but spermatogenesis is markedly affected during postmeiotic steps, leading to male infertility. In the present study, increased apoptosis of HR6B knockout primary spermatocytes was detected during the first wave of spermatogenesis, indicating that HR6B performs a primary role during the meiotic prophase. Detailed analysis of HR6B knockout pachytene nuclei showed major changes in the synaptonemal complexes. These complexes were found to be longer. In addition, we often found depletion of synaptonemal complex proteins from near telomeric regions in the HR6B knockout pachytene nuclei. Finally, we detected an increased number of foci containing the mismatch DNA repair protein MLH1 in these nuclei, reflecting a remarkable and consistent increase (20 to 25%) in crossing-over frequency. The present findings reveal a specific requirement for the ubiquitin-conjugating activity of HR6B in relation to dynamic aspects of the synaptonemal complex and meiotic recombination in spermatocytes.     

beta-Adrenoceptor-mediated thermogenesis and lipolysis in patients with chronic obstructive pulmonary disease

The present study investigated whether development or maintenance of a relatively increased fat mass in normal-weight patients with chronic obstructive pulmonary disease (COPD), despite periods of weight loss, may be related to impaired beta-adrenoceptor-mediated responses in lipid utilization and thermogenesis. Nine COPD patients and nine healthy controls (body mass index: 23.0 +/- 1.3 vs. 23.8 +/- 0.6 kg/m2, not significant; fat mass: 19.0 +/- 2.1 vs. 11.9 +/- 1.5 kg, P < 0.01) received consecutive 30-min infusions of 6, 12, and 24 ng x kg fat free mass(-1) x min(-1) isoproterenol. During beta-adrenergic stimulation, nonesterified fatty acid levels increased significantly less in COPD patients (P < 0.001). Respiratory exchange ratio decreased similarly in both groups, indicating a similar change in the rate of lipid to carbohydrate oxidation. Energy expenditure increased similarly in both groups during beta-adrenergic stimulation. However, because plasma isoproterenol concentrations were significantly higher in COPD patients, thermogenesis related to isoproterenol concentration was significantly reduced in this group (P < 0.05). In conclusion, beta-adrenoceptor-mediated lipolysis and thermogenesis are impaired in COPD patients. This may play a role in the development or maintenance of their relatively increased fat mass.     

Characterization of mRAD18Sc, a mouse homolog of the yeast postreplication repair gene RAD18

The RAD18 gene of the yeast Saccharomyces cerevisiae encodes a protein with ssDNA binding activity that interacts with the ubiquitin-conjugating enzyme RAD6 and plays an important role in postreplication repair. We identified and characterized the putative mouse homolog of RAD18, designated mRAD18Sc. The mRAD18Sc open reading frame encodes a 509-amino-acid polypeptide that is strongly conserved in size and sequence between yeast and mammals, with specific conservation of the RING-zinc-finger and the classic zinc-finger domain. The degree of sequence conservation between mRAD18Sc, RAD18, and homologous sequences identified in other species (NuvA from Aspergillus nidulans and Uvs-2 from Neurospora crassa) is entirely consistent with the evolutionary relationship of these organisms, strongly arguing that these genes are one another's homologs. Consistent with the presence of a nuclear translocation signal in the amino acid sequence, we observed the nuclear localization of GFP-tagged mRAD18Sc after stable transfection to HeLa cells. mRNA expression of mRAD18Sc in the mouse was observed in thymus, spleen, brain, and ovary, but was most pronounced in testis, with the highest level of expression in pachytene-stage primary spermatocytes, suggesting that mRAD18Sc plays a role in meiosis of spermatogenesis. Finally, we mapped the mRAD18Sc gene on mouse chromosome 6F.     

Targeted Inactivation of Mouse RAD52 Reduces Homologous Recombination but Not Resistance to Ionizing Radiation

The RAD52 epistasis group is required for recombinational repair of double-strand breaks (DSBs) and shows strong evolutionary conservation. In Saccharomyces cerevisiae, RAD52 is one of the key members in this pathway. Strains with mutations in this gene show strong hypersensitivity to DNA-damaging agents and defects in recombination. Inactivation of the mouse homologue of RAD52 in embryonic stem (ES) cells resulted in a reduced frequency of homologous recombination. Unlike the yeast Scrad52 mutant, MmRAD52^−/− ES cells were not hypersensitive to agents that induce DSBs. MmRAD52 null mutant mice showed no abnormalities in viability, fertility, and the immune system. These results show that, as in S. cerevisiae, MmRAD52 is involved in recombination, although the repair of DNA damage is not affected upon inactivation, indicating that MmRAD52 may be involved in certain types of DSB repair processes and not in others. The effect of inactivating MmRAD52 suggests the presence of genes functionally related to MmRAD52, which can partly compensate for the absence of MmRad52 protein.     

<Emphasis Type="Italic">TRAF1/C5</Emphasis>polymorphism is not associated with increased mortality in rheumatoid arthritis: two large longitudinal studies

Introduction  

Recently an association between a genetic variation in TRAF1/C5 and mortality from sepsis or cancer was found in rheumatoid arthritis (RA). The most prevalent cause of death, cardiovascular disease, may have been missed in that study, since patients were enrolled at an advanced disease stage. Therefore, we used an inception cohort of RA patients to investigate the association between TRAF1/C5 and cardiovascular mortality, and replicate the findings on all-cause mortality. As TRAF1/C5 associated mortality may not be restricted to RA, we also studied a large cohort of non-RA patients.     

Mesenchymal stem cells in autoimmune diseases: hype or hope?

Intervention with mesenchymal stem cells (MSCs) represents a promising therapeutic tool in treatment-refractory autoimmune diseases. A new report by Schurgers and colleagues in a previous issue of Arthritis Research & Therapy sheds novel mechanistic insight into the pathways employed by MSCs to suppress T-cell proliferation in vitro, but, at the same time, indicates that MSCs do not influence T-cell reactivity and the disease course in an in vivo arthritis model. Such discrepancies between the in vitro and in vivo effects of potent cellular immune modulators should spark further research and should be interpreted as a sign of caution for the in vitro design of MSC-derived interventions in the setting of human autoimmune diseases.     

Pentacycloundecane lactam vs lactone norstatine type protease HIV inhibitors: binding energy calculations and DFT study

Background

Novel pentacycloundecane (PCU)-lactone-CO-EAIS peptide inhibitors were designed, synthesized, and evaluated against wild-type C-South African (C-SA) HIV-1 protease. Three compounds are reported herein, two of which displayed IC₅₀ values of less than 1.00 μM. A comparative MM-PB(GB)SA binding free energy of solvation values of PCU-lactam and lactone models and their enantiomers as well as the PCU-lactam-NH-EAIS and lactone-CO-EAIS peptide inhibitors and their corresponding diastereomers complexed with South African HIV protease (C-SA) was performed. This will enable us to rationalize the considerable difference between inhibitory concentration (IC₅₀) of PCU-lactam-NH-EAIS and PCU-lactone-CO-EAIS peptides.

Results

The PCU-lactam model exhibited more negative calculated binding free energies of solvation than the PCU-lactone model. The same trend was observed for the PCU-peptide inhibitors, which correspond to the experimental activities for the PCU-lactam-NH-EAIS peptide (IC₅₀ = 0.076 μM) and the PCU-lactone-CO-EAIS peptide inhibitors (IC₅₀ = 0.850 μM). Furthermore, a density functional theory (DFT) study on the natural atomic charges of the nitrogen and oxygen atoms of the three PCU-lactam, PCU-lactim and PCU-lactone models were performed using natural bond orbital (NBO) analysis. Electrostatic potential maps were also used to visualize the electron density around electron-rich regions. The asymmetry parameter (η) and quadrupole coupling constant (χ) values of the nitrogen and oxygen nuclei of the model compounds were calculated at the same level of theory. Electronic molecular properties including polarizability and electric dipole moments were also calculated and compared. The Gibbs theoretical free solvation energies of solvation (∆G_solv) were also considered.

Conclusions

A general trend is observed that the lactam species appears to have a larger negative charge distribution around the heteroatoms, larger quadrupole constant, dipole moment and better solvation energy, in comparison to the PCU-lactone model. It can be argued that these characteristics will ensure better eletronic interaction between the lactam and the receptor, corresponding to the observed HIV protease activities in terms of experimental IC₅₀ data.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0115-5) contains supplementary material, which is available to authorized users.