Induction of cytosolic and microsomal epoxide hydrolases in mouse liver by peroxisome proliferators, with special emphasis on structural analogues of 2-ethylhexanoic acid

Using dietary administration, mice were exposed to eight substances known to cause peroxisome proliferation (i.e. clofibrate clofibric acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, nafenopin, ICI-55.897, S-8527 and Wy-14.643) or the related substance p-chlorophenoxyacetic acid (group A). Other animals received di(2-ethylhexyl)phthalate, mono(2-ethylhexyl)phthalate, 2-ethylhexanoic acid, or one of 12 other metabolically and/or structurally related compounds (group B). The effects of these treatments on liver cytosolic and microsomal epoxide hydrolases, microsomal cytochrome P-450, cytosolic glutathione transferase activity, the liver-somatic index and the protein contents of the microsomal and cytosolic fractions prepared from liver were subsequently monitored. In general, peroxisome proliferation was accompanied by increases in cytosolic epoxide hydrolase activity. Many peroxisome proliferators also caused increases in microsomal epoxide hydrolase activity, although the correlation was poorer in this case. Immunochemical quantitation by radial immunodiffusion demonstrated that the increases observed in both of these enzyme activities reflected equivalent increases in enzyme protein, i.e. that induction truly occurred. Induction of total microsomal cytochrome P-450 was obtained after dietary exposure to clofibrate, clofibric acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, nafenopin, Wy-14.643, di(2-ethylhexyl)phthalate and di(2-ethylhexyl)phosphate. The most pronounced effects on cytosolic glutathione transferase activity were the decreases obtained after treatment with clofibrate, clofibric acid and Wy-14.643. Our results, together with those reported by others, suggest that the processes of peroxisome proliferation and induction of cytosolic epoxide hydrolase are intimately related. One possible explanation for this is presented.     

CDK inhibitors R-roscovitine and S-CR8 effectively block renal and hepatic cystogenesis in an orthologous model of ADPKD

Autosomal dominant polycystic kidney disease (ADPKD) and other forms of PKD are associated with dysregulated cell cycle and proliferation. Although no effective therapy for the treatment of PKD is currently available, possible mechanism-based approaches are beginning to emerge. A therapeutic intervention targeting aberrant cilia-cell cycle connection using CDK-inhibitor R-roscovitine showed effective arrest of PKD in jck and cpk models that are not orthologous to human ADPKD. To evaluate whether CDK inhibition approach will translate into efficacy in an orthologous model of ADPKD, we tested R-roscovitine and its derivative S-CR8 in a model with a conditionally inactivated Pkd1 gene (Pkd1 cKO). Similar to ADPKD, Pkd1 cKO mice developed renal and hepatic cysts. Treatment of Pkd1 cKO mice with R-roscovitine and its more potent and selective analog S-CR8 significantly reduced renal and hepatic cystogenesis and attenuated kidney function decline. Mechanism of action studies demonstrated effective blockade of cell cycle and proliferation and reduction of apoptosis. Together, these data validate CDK inhibition as a novel and effective approach for the treatment of ADPKD.     

Translational control by cytoplasmic polyadenylation in Xenopus oocytes

Elongation of the poly(A) tails of specific mRNAs in the cytoplasm is a crucial regulatory step in oogenesis and early development of many animal species. The best studied example is the regulation of translation by cytoplasmic polyadenylation elements (CPEs) in the 3' untranslated region of mRNAs involved in Xenopus oocyte maturation. In this review we discuss the mechanism of translational control by the CPE binding protein (CPEB) in Xenopus oocytes as follows: Finally we discuss some of the remaining questions regarding the mechanisms of translational regulation by cytoplasmic polyadenylation and give our view on where our knowledge is likely to be expanded in the near future.     

Hepatic autophagy and intracellular ATP. A morphometric study

In order to estimate the sensitivity of macroautophagy in liver toward changes in ATP we have analyzed the volume density of the autophagic/lysosomal system in isolated rat hepatocytes, incubated under conditions where intracellular ATP was partially depleted. (a) It appeared that reduction of the intracellular ATP concentration by 30-50% decreased the volume density of autophagic vacuoles by 70%. (b) Partial ATP depletion did not involve significant changes in the volume density of dense bodies. Together with studies showing that the rate of overall proteolysis via macroautophagy decreases with decreasing ATP concentration (P.J.A.M. Plomp, E.J. Wolvetang, A.K. Groen, A.J. Meijer, P.B. Gordon, and P.O. Seglen (1987) Eur. J. Biochem. 164, 197-203) our data indicate that changes in intracellular ATP primarily affect early steps in the autophagic/proteolytic pathway.     

Limited Promiscuity of HLA-DRB1 Presented Peptides Derived of Blood Coagulation Factor VIII

The formation of inhibitory antibodies directed against coagulation factor VIII (FVIII) is a severe complication in the treatment of hemophilia A patients. The induction of anti-FVIII antibodies is a CD4⁺ T cell-dependent process. Activation of FVIII-specific CD4⁺ T cells is dependent on the presentation of FVIII-derived peptides on MHC class II by antigen-presenting cells. Previously, we have shown that FVIII-pulsed human monocyte-derived dendritic cells can present peptides from several FVIII domains. In this study we show that FVIII peptides are presented on immature as well as mature dendritic cells. In immature dendritic cells half of the FVIII-loaded MHC class II molecules are retained within the cell, whereas in LPS-matured dendritic cells the majority of MHC class II/peptide complexes is present on the plasma membrane. Time-course studies revealed that presentation of FVIII-derived peptides was optimal between 12 and 24 hours after maturation but persisted for at least 96 hours. We also show that macrophages are able to internalize FVIII as efficiently as dendritic cells, however FVIII was presented on MHC class II with a lower efficiency and with different epitopes compared to dendritic cells. In total, 48 FVIII core-peptides were identified using a DCs derived of 8 different donors. Five HLA-promiscuous FVIII peptide regions were found – these were presented by at least 4 out of 8 donors. The remaining 42 peptide core regions in FVIII were presented by DCs derived from a single (30 peptides) or two to three donors (12 peptides). Overall, our findings show that a broad repertoire of FVIII peptides can be presented on HLA-DR.     

Rational identification of an optimal antibody mixture for targeting the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is frequently dysregulated in human malignancies and a validated target for cancer therapy. Two monoclonal anti-EGFR antibodies (cetuximab and panitumumab) are approved for clinical use. However, the percentage of patients responding to treatment is low and many patients experiencing an initial response eventually relapse. Thus, the need for more efficacious treatments remains. Previous studies have reported that mixtures of antibodies targeting multiple distinct epitopes are more effective than single mAbs at inhibiting growth of human cancer cells in vitro and in vivo. The current work describes the rational approach that led to discovery and selection of a novel anti-EGFR antibody mixture Sym004, which is currently in Phase 2 clinical testing. Twenty-four selected anti-EGFR antibodies were systematically tested in dual and triple mixtures for their ability to inhibit cancer cells in vitro and tumor growth in vivo. The results show that targeting EGFR dependent cancer cells with mixtures of antibodies is superior at inhibiting their growth both in vitro and in vivo. In particular, antibody mixtures targeting non-overlapping epitopes on domain III are efficient and indeed Sym004 is composed of two monoclonal antibodies targeting this domain. The superior growth inhibitory activity of mixtures correlated with their ability to induce efficient EGFR degradation.Key words: EGFR, antibody synergy, functional screening, epitope binning, antibody combinations     

Effects of plant-soil feedback on tree seedling growth under arid conditions

Aims Plants are able to influence their growing environment by changing biotic and abiotic soil conditions. These soil conditions in turn can influence plant growth conditions, which is called plant–soil feedback. Plant–soil feedback is known to be operative in a wide variety of ecosystems ranging from temperate grasslands to tropical rain forests. However, little is known about how it operates in arid environments. We examined the role of plant–soil feedbacks on tree seedling growth in relation to water availability as occurring in arid ecosystems along the west coast of South America.Methods In a two-phased greenhouse experiment, we compared plant–soil feedback effects under three water levels (no water, 10% gravimetric moisture and 15% gravimetric moisture). We used sterilized soil inoculated with soil collected from northwest Peru (Prosopis pallida forests) and from two sites in north-central Chile (Prosopis chilensis forest and scrublands without P. chilensis).Important findings Plant–soil feedbacks differed between plant species and soil origins, but water availability did not influence the feedback effects. Plant–soil feedbacks differed in direction and strength in the three soil origins studied. Plant–soil feedbacks of plants grown in Peruvian forest soil were negative for leaf biomass and positive for root length. In contrast, feedbacks were neutral for plants growing in Chilean scrubland soil and positive for leaf biomass for those growing in Chilean forest soil. Our results show that under arid conditions, effects of plant–soil feedback depend upon context. Moreover, the results suggest that plant–soil feedback can influence trade-offs between root growth and leaf biomass investment and as such that feedback interactions between plants and soil biota can make plants either more tolerant or vulnerable to droughts. Based on dissecting plant–soil feedbacks into aboveground and belowground tissue responses, we conclude that plant–soil feedback can enhance plant colonization in some arid ecosystems by promoting root growth.     

Molecular mechanics calculation of geometries of NAD+ derivatives, modified in the nicotinamide group, in a ternary complex with horse liver alcohol dehydrogenase

The geometry of seven NAD+ analogues bound to horse liver alcohol dehydrogenase (LADH) modified only in their nicotinamide group, have been studied using AMBER molecular mechanics energy-minimization procedures. Starting geometries were taken from X-ray crystallographic data for NAD+/Me2SO/LADH reported by Eklund and co-workers. In this study the NAD+ analogues were encaged by the constituent amino acids of the enzyme within a range of 0.6 nm from the initial NAD+/Me2SO/Zn2+ complex. The calculational method used is able to rationalize individual substituent effects and to evaluate the essential interactions between NAD+ analogue, enzyme, Me2SO and Zn2+ without the necessity of additional X-ray data. The results presented here demonstrate that the reactivity of NAD+ derivatives as reported in literature can be qualitatively related to the position of the pyridine moiety in the active site.     

Histochemical characteristics of chemoreceptor organs (Glomera)

Summary Some important histochemical characteristics of the carotid, aortic and coronary glomera have been studied in man and the rabbit.All glomera present a similar histochemical pattern. Type I glomus cells contain acetylcholinesterase, monoamine oxidase and norepinephrine. Type II glomus cells are highly positive for cholinesterase, carbonic anhydrase and nucleoside phosphatases hut they do not contain acetylcholinesterase nor catecholamines. It is postulated that the type I glomus cell is the true chemoreceptor cell. Together with the type II glomus cell, which is considered to be a special type of glial cell, a functional metabolic unit is established. Efferent nerve fibres could be adrenergic; by way of cholinergic transmission action potentials could be initiated in the afferent nerve fibres.The following Abbreviations will be used AChE acetylcholinesterase - ChE cholinesterase - iso-OMPA tetraisopropylpyrophosphoramide - DFP di-isopropylfluorophosphate - 62C47 15-bis-(4-trimethylammonium-phenyl) pentan-3-one-diiodide - CAH carbonic anhydrase - ATP-ase adenosine triphosphatase - NP-ases nucleoside phosphatases - UDP uridine diphosphate - UTP uridine triphosphate - IDP inosine diphosphate - CTP cytidine triphosphate - CaFoMa calcium-formol-macrodex - Glut glutaraldehyde - TPP-ase thiamine pyrophosphatase - MAO monoamine oxidase - CA catecholamines - NE norepinephrine