Human IgA Fc receptor FcαRI (CD89) triggers different forms of neutrophil death depending on the inflammatory microenvironment

FcαRI (CD89), the human Fc receptor for IgA, is highly expressed on neutrophil granulocytes. In this study, we show that FcαRI induces different forms of neutrophil death, depending on the inflammatory microenvironment. The susceptibility of inflammatory neutrophils from sepsis or rheumatoid arthritis toward death induced by specific mAb, or soluble IgA at high concentrations, was enhanced. Although unstimulated cells experienced apoptosis following anti-FcαRI mAb stimulation, preactivation with cytokines or TLR agonists in vitro enhanced FcαRI-mediated death by additional recruitment of caspase-independent pathways, but this required PI3K class IA and MAPK signaling. Transmission electron microscopy of FcαRI-stimulated cells revealed cytoplasmic changes with vacuolization and mitochondrial swelling, nuclear condensation, and sustained plasma membrane. Coculture experiments with macrophages revealed anti-inflammatory effects of the partially caspase-independent death of primed cells following FcαRI engagement. Our data suggest that FcαRI has the ability to regulate neutrophil viability and to induce different forms of neutrophils depending on the inflammatory microenvironment and specific characteristics of the ligand-receptor interactions. Furthermore, these findings have potential implications for FcαRI-targeted strategies to treat neutrophil-associated inflammatory diseases.

Unstable argininosuccinate lyase in variant forms of the urea cycle disorder argininosuccinic aciduria.

Loss of function of the urea cycle enzyme argininosuccinate lyase (ASL) is caused by mutations in the ASL gene leading to ASL deficiency (ASLD). ASLD has a broad clinical spectrum ranging from life-threatening severe neonatal to asymptomatic forms. Different levels of residual ASL activity probably contribute to the phenotypic variability but reliable expression systems allowing clinically useful conclusions are not yet available. In order to define the molecular characteristics underlying the phenotypic variability, we investigated all ASL mutations that were hitherto identified in patients with late onset or mild clinical and biochemical courses by ASL expression in human embryonic kidney 293 T cells. We found residual activities >3 % of ASL wild type (WT) in nine of 11 ASL mutations. Six ASL mutations (p.Arg95Cys, p.Ile100Thr, p.Val178Met, p.Glu189Gly, p.Val335Leu, and p.Arg379Cys) with residual activities ≥16 % of ASL WT showed no significant or less than twofold reduced Km values, but displayed thermal instability. Computational structural analysis supported the biochemical findings by revealing multiple effects including protein instability, disruption of ionic interactions and hydrogen bonds between residues in the monomeric form of the protein, and disruption of contacts between adjacent monomeric units in the ASL tetramer. These findings suggest that the clinical and biochemical course in variant forms of ASLD is associated with relevant residual levels of ASL activity as well as instability of mutant ASL proteins. Since about 30 % of known ASLD genotypes are affected by mutations studied here, ASLD should be considered as a candidate for chaperone treatment to improve mutant protein stability.

Comparison of the in vitro effects of saline, hypertonic hydroxyethyl starch, hypertonic saline, and two forms of hydroxyethyl starch on whole blood coagulation and platelet function in dogs.

OBJECTIVE To compare the in vitro effects of hypertonic solutions and colloids to saline on coagulation in dogs. DESIGN In vitro experimental study. SETTING Veterinary teaching hospital. ANIMALS Twenty-one adult dogs. INTERVENTIONS Blood samples were diluted with saline, 7.2% hypertonic saline solution with 6% hydroxyethylstarch with an average molecular weight of 200 kDa and a molar substitution of 0.4 (HH), 7.2% hypertonic saline (HTS), hydroxyethyl starch (HES) 130/0.4 or hydroxyethyl starch 600/0.75 at ratios of 1:22 and 1:9, and with saline and HES at a ratio of 1:3. MEASUREMENTS AND MAIN RESULTS Whole blood coagulation was analyzed using rotational thromboelastometry (extrinsic thromboelastometry-cloting time (ExTEM-CT), maximal clot firmness (MCF) and clot formation time (CFT) and fibrinogen function TEM-CT (FibTEM-CT) and MCF) and platelet function was analyzed using a platelet function analyzer (closure time, CTPFA ). All parameters measured were impaired by saline dilution. The CTPFA was prolonged by 7.2% hypertonic saline solution with 6% hydroxyethylstarch with an average molecular weight of 200 kDa and a molar substitution of 0.4 (HH) and HTS but not by HES solutions. At clinical dilutions equivalent to those generally administered for shock (saline 1:3, HES 1:9, and hypertonic solutions 1:22), CTPFA was more prolonged by HH and HTS than other solutions but more by saline than HES. No difference was found between the HES solutions or the hypertonic solutions. ExTEM-CFT and MCF were impaired by HH and HTS but only mildly by HES solutions. At clinically relevant dilutions, no difference was found in ExTEM-CFT between HTS and saline or in ExTEM-MCF between HH and saline. No consistent difference was found between the 2 HES solutions but HH impaired ExTEM-CFT and MCF more than HTS. At high dilutions, FibTEM-CT and -MCF and ExTEM-CT were impaired by HES. CONCLUSIONS Hypertonic solutions affect platelet function and whole blood coagulation to a greater extent than saline and HES. At clinically relevant dilutions, only CTPFA was markedly more affected by hypertonic solutions than by saline. At high dilutions, HES significantly affects coagulation but to no greater extent than saline at clinically relevant dilutions.

Distinctive insular forms of threespine stickleback (Gasterosteus aculeatus) from western Mediterranean islands

Neutral and adaptive variation among populations within a species is a major component of biological diversity and may be pronounced among insular populations due to geographical isolation and island specific evolutionary forces at work. Detecting and preserving potential evolutionary significant units below the species rank has become a crucial task for conservation biology. Combining genetic, phenotypic and ecological data, we investigated evolutionary patterns among the enigmatic threespine stickleback populations from western Mediterranean islands, all of which are threatened by habitat deterioration and climate change. We find indications that these populations derive from different genetic lineages, being genetically highly distinct from the stickleback of mainland Europe and the northern Atlantic as well as from each other. Mediterranean island stickleback populations are also phenotypically distinct from mainland populations but interestingly stickleback from Iceland have converged on a similar phenotype. This distinctive island stickleback phenotype seems to be driven by distinct selective regimes on islands versus continents. Overall, our results reveal the status of western Mediterranean island stickleback as evolutionarily distinct units, important for conservation of biodiversity.

Anticipatory and reactive forms of blame avoidance: of foxes and lions

Blame avoidance behavior (BAB) encompasses all kinds of integrity-protecting activities by officeholders in the face of potentially blame-attracting events. Although considered essential for a realistic understanding of politics and policymaking, a general understanding of this multi-faceted behavioral phenomenon and its implications has been lacking to date. We argue that this is due to the lack of careful conceptualization of various forms of BAB. Crucially, the difference between anticipatory and reactive forms of BAB is largely neglected in the literature. This paper links anticipatory and reactive forms of BAB as two consecutive decision situations. It exposes dependence relationships between the situations that trigger BAB, the rationalities at work, the resources and strategies applied by blame-avoiding actors, and the various consequences thereof. The paper concludes that anticipatory and reactive BAB are distinct phenomena that require specific research approaches to assess their relevance for the workings of polities.

DRUG METABOLISM IN LIVER TUMORS: PURIFICATION AND CHARACTERIZATION OF NADPH-CYTOCHROME-P450 REDUCTASE AND THE RESOLUTION OF MULTIPLE FORMS OF CYTOCHROME P-450

The hydroxylation of N- and O-methyl drugs and a polycyclic hydrocarbon has been demonstrated in microsomes prepared from two transplantable Morris hepatomas (i.e., 7288C. t.c. and 5123 t.c.(H). The hydroxylation rates of the drug benzphetamine and the polycyclic hydrocarbon benzo {(alpha)} pyrene by tumor microsomes were inducible 2 to 3-fold and 2-fold, respectively by pretreatment of rats with phenobarbital/hydrocortisone. Hepatoma 5123t.c.(h) microsomal hydroxylation activities were more inducible after these pretreatments than hepatoma 7288C.t.c. Two chemotherapeutic drugs (cyclophosphamide and isophosphamide) were shown to be mutagenic after activation by the tumor hemogenate with the TA100 strain of Salmonella typhimurium bacteria. NADPH-cytochrome P-450 was purified from phenobarbital/hydrocortisone treated rat hepatoma 5123t.c.(H) microsomes 353-fold with a specific activity 63.6 nmol of cytochrome c reduced per min per mg of protein. The purified enzyme, has an apparent molecular weight of 79,500 daltons, and contained an equal molar ratio of FMN and FAD, with a total flavin content of 16.4 nmol per mg of protein. The purified enzyme also catalyzed electron transfer to artificial electron acceptors with the K(,m) values of the hepatoma reductase similar to those of purified liver reductase. The K(,m) value of the hepatoma reductase (13 uM) for NADPH was similar to that of purified liver reductase (5.0 uM). In addition the purified hepatoma reductase was immunochemically similar to the liver reductase.^ Hepatoma cytochrome P-450, the hemeprotein component of the hepatoma microsomes of rats pretreated with phenobarbital/hydrocortisone. The resolution of the six forms was achieved by the DE-53 ion-exchange chromatography, and further purified by hydroxyapatite. The six different fractions that contained P-450 activity, had specific contents from 0.47 to 1.75 nmol of cytochrome P-450 per mg of protein, and indicated a 2 to 9-fold purification as compared to the original microsomes. In addition, difference spectra, molecular weights and immunological results suggest there are at least six different forms of cytochrome P-450 in hepatoma 5123 t.c.(H). ^

Analysis of the Phosphorylated Forms of Protein Kinase R

Protein Kinase R (PKR) is induced by interferon and activated by dsRNA. Subsequent autophosphorylation and phosphorylation of eIF2alpha inhibits viral replication. In the latent state PKR exists as an unphosphorylated monomer. Work in the Cole laboratory has shown two additional states, a phosphorylated monomeric state (pPKRm) and a phosphorylated dimeric state (pPKRd). RNA serves as a scaffold bringing two PKRs together allowing dimerization and autophosphorylation to occur. The contribution of each state to the function of PKR remains unclear. Western blots were performed to examine the phosphorylation states of the essential residues, T446 and T451. Activity assays have shown activation of pPKRm at a level comparable to pPKRd in its ability to phosphorylate eIF2alpha. Phosphorylation of eIF2alpha by both pPKRm and pPKRd was shown to be RNA independent. Despite reaching similar terminal levels of eIF2alpha phosphorylation, kinetic measurements revealed a faster reaction from pPKRd. Therefore, pPKRm and pPKRd may both contribute to the activity of PKR.

Concentrations of labile dissolved forms of Cu, Zn, Pb, and Cu in waters of the Kara Sea and Ob and Yenisey estuaries

Concentrations of labile dissolved forms of Cu, Zn, Pb, and Cu in waters of the Kara Sea and Ob and Yenisey estuaries measured on board during Cruise 49 of R/V Dmitry Mendeleev.