Dimethylarginine Dimethylaminohydrolase1 Is an Organ-Specific Mediator of End Organ Damage in a Murine Model of Hypertension

Background

The endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) is an independent predictor of cardiovascular and overall mortality. Moreover, elevated ADMA plasma concentrations are associated with the extent of hypertension. However, data from small-sized clinical trials and experimental approaches using murine transgenic models have revealed conflicting results regarding the impact of ADMA and its metabolizing enzyme dimethylarginine dimethylaminohydrolase (DDAH) in the pathogenesis of hypertension.

Methodology/Principal Findings

Therefore, we investigated the role of ADMA and DDAH1 in hypertension-induced end organ damage using the uninephrectomized, deoxycorticosterone actetate salt, and angiotensin II-induced hypertension model in human DDAH1 (hDDAH1) overexpressing and wild-type (WT) mice. ADMA plasma concentrations differed significantly between hDDAH1 and WT mice at baseline, but did not significantly change during the induction of hypertension. hDDAH1 overexpression did not protect against hypertension-induced cardiac fibrosis and hypertrophy. In addition, the hypertension-induced impairment of the endothelium-dependent vasorelaxation of aortic segments ex vivo was not significantly attenuated by hDDAH1 overexpression. However, hDDAH1 mice displayed an attenuated hypertensive inflammatory response in renal tissue, resulting in less hypertensive renal injury.

Conclusion/Significance

Our data reveal that hDDAH1 organ-specifically modulates the inflammatory response in this murine model of hypertension. The lack of protection in cardiac and aortic tissues may be due to DDAH1 tissue selectivity and/or the extent of hypertension by the used combined model. However, our study underlines the potency of hDDAH1 overexpression in modulating inflammatory processes as a crucial step in the pathogenesis of hypertension, which needs further experimental and clinical investigation.     

Impact of S-sulfocysteine on fragments and trisulfide bond linkages in monoclonal antibodies

The quality of recombinant proteins such as monoclonal antibodies produced using Chinese hamster ovary cell-based mammalian systems is dependent on many factors, including cell line, process and cell culture media. Due to these factors, the generated product is heterogeneous and may have chemically-induced modifications or post-translational modifications that affect antibody stability, functionality and, in some cases, patient safety. This study demonstrates that S-sulfocysteine, a cysteine derivative, can increase the antibody specific productivity in different cell lines cultivated with different processes while minimizing trisulfide linkages in generated mAbs, mainly between heavy and light chain. The supplementation of a cell culture feed with S-sulfocysteine also proved to be useful to reduce the percentage of antibody fragments generated from the monoclonal antibody. Overall, this new component used in the upstream process allows a reduction of product heterogeneity.     

The P2X7 carboxyl tail is a regulatory module of P2X7 receptor channel activity

P2X(7) receptors are ATP-gated cation channels composed of three identical subunits, each having intracellular amino and carboxyl termini and two transmembrane segments connected by a large ectodomain. Within the P2X family, P2X(7) subunits are unique in possessing an extended carboxyl tail. We expressed the human P2X(7) subunit as two complementary fragments, a carboxyl tail-truncated receptor channel core (residues 1-436 or 1-505) and a tail extension (residues 434-595) in Xenopus laevis oocytes. P2X(7) channel core subunits efficiently assembled as homotrimers that appeared abundantly at the oocyte surface, yet produced only approximately 5% of the full-length P2X(7) receptor current. Co-assembly of channel core subunits with full-length P2X(7) subunits inhibited channel current, indicating that the lack of a single carboxyl tail domain is dominant-negative for P2X(7) receptor activity. Co-expression of the tail extension as a discrete protein increased ATP-gated current amplitudes of P2X(7) channel cores 10-20-fold, fully reconstituting the wild type electrophysiological phenotype of the P2X(7) receptor. Chemical cross-linking revealed that the discrete tail extension bound with unity stoichiometry to the carboxyl tail of the P2X(7) channel core. We conclude that a non-covalent association of crucial functional importance exists between the carboxyl tail of the channel core and the tail extension. Using a slightly shorter P2X(7) subunit core and subfragments of the tail extension, this association could be narrowed down to include residues 409-436 and 434-494 of the split receptor. Together, these results identify the tail extension as a regulatory gating module, potentially making P2X(7) channel gating sensitive to intracellular regulation.     

Conservation genetics of the pond bat (Myotis dasycneme) with special focus on the populations in northwestern Germany and in Jutland,Denmark

Conservation genetics is important in the management of endangered species, helping to understand their connectivity and long‐term viability, thus identifying populations of importance for conservation. The pond bat (Myotis dasycneme) is a rare species classified as “Near Threatened” with a wide but patchy Palearctic distribution. A total of 277 samples representing populations in Denmark, Germany, Latvia, Hungary, and Russia were used in the genetic analyses; 224 samples representing Denmark, Germany, and Russia were analyzed at 10 microsatellite loci; 241 samples representing all areas were analyzed using mitochondrial D‐loop and cytochrome B sequences. A Bayesian clustering approach revealed two poorly resolved clusters, one representing the Danish and German groups and the other the Russian group. However, significantly different pairwise F_ST and D_EST estimates were observed between the Danish and German groups and between the Danish and Russian groups suggesting a recent population structure. These conflicting results might be attributed to the effect of migration or low resolution due to the number of microsatellite markers used. After concatenating the two mitochondrial sequences, analysis detected significant genetic differentiation between all populations, probably due to genetic drift combined with a founder event. The phylogenetic tree suggested a closer relationship between the Russian and Northern European populations compared to the Hungarian population, implying that the latter belongs to an older ancestral population. This was supported by the observed haplotype network and higher nucleotide diversity in this population. The genetic structuring observed in the Danish/German pond bat stresses the need for a cross‐border management between the two countries. Further, the pronounced mtDNA structuring, together with the indicated migration between nearby populations suggest philopatric female behavior but male migration, emphasizes the importance of protecting suitable habitat mosaics to maintain a continuum of patches with dense pond bat populations across the species' distribution range.     

Identification of quantitative trait loci associated with resistance to net form net blotch in a collection of Nordic barley germplasm

Key message

Association mapping of resistance to Pyrenophora teres f. teres in a collection of Nordic barley germplasm at different developmental stages revealed 13 quantitative loci with mostly small effects.

Abstract

Net blotch, caused by the necrotrophic fungus Pyrenophora teres, is one of the major diseases in barley in Norway causing quantitative and qualitative yield losses. Resistance in Norwegian cultivars and germplasm is generally insufficient and resistance sources have not been extensively explored yet. In this study, we mapped quantitative trait loci (QTL) associated with resistance to net blotch in Nordic germplasm. We evaluated a collection of 209 mostly Nordic spring barley lines for reactions to net form net blotch (NFNB; Pyrenophora teres f. teres) in inoculations with three single conidia isolates at the seedling stage and in inoculated field trials at the adult stage in 4 years. Using 5669 SNP markers genotyped with the Illumina iSelect 9k Barley SNP Chip and a mixed linear model accounting for population structure and kinship, we found a total of 35 significant marker-trait associations for net blotch resistance, corresponding to 13 QTL, on all chromosomes. Out of these QTL, seven conferred resistance only in adult plants and four were only detectable in seedlings. Two QTL on chromosomes 3H and 6H were significant during both seedling inoculations and adult stage field trials. These are promising candidates for breeding programs using marker-assisted selection strategies. The results elucidate the genetic background of NFNB resistance in Nordic germplasm and suggest that NB resistance is conferred by a number of genes each with small-to-moderate effects, making it necessary to pyramid these genes to achieve sufficient levels of resistance.

     

It Takes Two to Tango: Activation of Protein Kinase D by Dimerization

The recent discovery and structure determination of a novel ubiquitin-like dimerization domain in protein kinase D (PKD) has significant implications for its activation. PKD is a serine/threonine kinase activated by the lipid second messenger diacylglycerol (DAG). It is an essential and highly conserved protein that is implicated in plasma membrane directed trafficking processes from the trans-Golgi network. However, many open questions surround its mechanism of activation, its localization, and its role in the biogenesis of cargo transport carriers. In reviewing this field, the focus is primarily on the mechanisms that control the activation of PKD at precise locations in the cell. In light of the new structural findings, the understanding of the mechanisms underlying PKD activation is critically evaluated, with particular emphasis on the role of dimerization in PKD autophosphorylation, and the provenance and recognition of the DAG that activates PKD.