Homodimerization as a molecular switch between low and high efficiency PrPC cell surface delivery and neuroprotective activity

PrP^C is associated with a variety of functions, and its ability to interact with a multitude of partners, including itself, may largely explain PrP multifunctionality and the lack of consensus on the genuine physiological function of the protein in vivo. In contrast, there is a consensus in the literature that alterations in PrP^C trafficking and intracellular retention result in neuronal degeneration. In addition, a proteolytic modification in the late secretory pathway termed the α-cleavage induces the secretion of PrPN1, a PrP^C-derived metabolite with fascinating neuroprotective activity against toxic oligomeric Aβ molecules implicated in Alzheimer disease. Thus, studies focusing on understanding the regulation of PrP^C trafficking to the cell surface and the modulation of α-cleavage are essential. The objective of this commentary is to highlight recent evidences that PrP^C homodimerization stimulates trafficking of the protein to the cell surface and results in high levels of PrPN1 secretion. We also discuss a hypothetical model for these results and comment on future challenges and opportunities.     

Reduced tumour necrosis factor-induced cytotoxicity by inhibitors of the arachidonic acid metabolism

The mechanism of tumour necrosis factor-mediated cytotoxicity was investigated by using various inhibitors of arachidonic acid metabolism. Phospholipase A2 inhibitors with different modes of action interfered with the cytotoxic action of TNF, whereas phospholipase C inhibitors did not. Neither cyclooxygenase nor lipoxygenase-blockers had a significant effect on TNF action. Experiments with scavengers of toxic oxygen radicals gave ambiguous results. The data obtained suggest the involvement of phospholipase A2 and arachidonic acid in the cytotoxic mechanism of TNF, but the exact role of these molecules is, however, still to be determined.     

Effects of direct transfer from freshwater to seawater on respiratory and circulatory variables and acid-base status in rainbow trout

Summary The effects of increased ambient salinity (35 mg · ml^-1) were studied at 1, 6, and 24 h after direct transfer of rainbow trout from freshwater to seawater. Two series of experiments were carried out successively. The first series was designed to simultaneously study all the respiratory (except Hb affinity for O₂), circulatory, and acid-base variables in each fish. In this series, fish were fitted with catheters chronically inserted into the cardiac bulbus, the dorsal aorta, and the opercular and buccal cavities. In the second series, designed to study haemoglobin O₂ affinity, fish were fitted with only a dorsal aorta catheter. The ventilatory flow ( ) was markedly increased just after transfer (by 55% at 1 h), then more moderately (by 20% at 6 h and 32% at 24 h). The initial hyperventilation peak was associated with frequent couphing motions. These ventilatory changes resulted essentially from increase in ventilatory amplitude. Initially, standard oxygen consumption (MM}O₂) decreased slightly, the moderately increased (by 12% at 24 h), so that the oxygen convection requirement ( ) increased substantially. In spite of an increased ventilation, the partial pressure of oxygen in arterial blood (P _aO₂) decreased slightly at 1 h, prior to returning to control levels, while partial pressure of carbon dioxide in arterial blood (P _aCO₂) was not significantly decreased. Gill oxygen transfer factor decreased substantially at 1 h (by 35%) then more moderately (by 7% at 1 h and 12% at 24 h). These results suggest a decrease in gas diffusing capacity of the gills. As P _aCO₂ remained approximatively unchanged, the gradual decrease in arterial pH (pH_a) from 7.94 to 7.67 at 24 h must therefore be regarded as a metabolic acidosis. The strong ion difference decreased markedly because the concentration of plasma chloride increased more than that of sodium. Arterial O₂ content (C _aO₂) gradually decreased (by 38% at 24 h) simultaneously with the decrease in pH_a, while the ratio P _aO₂/C _aO₂ increased. In parallel, seawater exposure induced a marked decrease in affinity of haemoglobin for O₂, so that at 24 h, P₅₀ was increased by 26% above the value obtained in freshwater-adapted trout. The increase in could be ascribed initially (at 1 h) to the decrease of P _aO₂ and later to a stimulation of respiratory neurons resulting from the lowered medullary interstitial pH. The decrease in C _aO₂ could be interpreted mainly as a consequence of a decreased affinity of haemoglobin for O₂, likely to be due to the blood acidosis and a predictable increase in chloride concentration within erythrocytes. Cardiac output ( ) slightly decreased at 1 h, then progressively increased by 30% at 24 h. Branchial vascular resistance increased at 1 h by 28%, then decreased by 18% of the control value at 24 h. Systemic vascular resistance decreased markedly by 40% at 24 h. As heart rate (HR) remained significantly unchanged, the cardiac stroke volume initially decreased then increased in relation to the changes in . The increase of , allowing compensation for the effect of decreased C _aO₂ in tissue O₂ supply, was interpreted as a passive consequence of the decrease in total vascular resistance occurring during seawater exposure.Abbreviations a.u. arbitrary units - C _aO₂ arterial oxygen content - pH₅₀ arterial pH at P₅₀ - C _vO₂ venous oxygen content - Hb haemoglobin - HR heart rate - Hct hematocrit - n_Hill Hill coefficient - O₂ standard oxygen consumption - P _aCO₂ arterial partial pressure of carbon dioxide - P _aO₂ arterial partial pressure of oxygen - P _vO₂ oxygen partial pressure in mixed venous blood - P₅₀ oxygen tension at half saturation of haemoglobin - P _VA, P _DA blood pressure in ventral and dorsal aorta - pH_a arterial pH - PIO₂, PEO₂ oxygen partial pressure of inspired and expired water - PO₂ oxygen partial pressure - cardiac output - SEM standard error of mean - S.I.D. strong ion difference - SV cardiac stroke volume - TO₂ gill oxygen transfer factor - U oxygen extraction coefficient - VA ventilatory amplitude - VF ventilatory frequency - VR_G, VR_S branchial and systemic vascular resistances - ventilatory flow - ventilatory oxygen convection requirement     

Involvement of a serine protease in tumour-necrosis-factor-mediated cytotoxicity

We investigated the effect of various protease inhibitors on the anti-proliferative and cytotoxic action of tumour necrosis factor (TNF) on mouse L929 fibrosarcoma cells. 1. The following serine-type protease inhibitors led to inhibition of TNF action: phenylmethylsulfonyl fluoride, N alpha-p-tosyl-L-lysine chloromethane, N alpha-p-tosyl-L-phenylalanyl chloromethane, N alpha-p-tosyl-L-arginine methyl ester, L-leucine methyl ester, DL-phenylalanine methyl ester, N-acetyl-DL-phenylalanine-beta-naphthyl ester, p-nitrophenyl p'-guanidino-benzoate and antipain. We could not detect an effect of inhibitors specific for thiol protease on TNF. 2. Inhibition of TNF-mediated cytotoxicity was evident in both the presence and absence of actinomycin D or cycloheximide. 3. TNF itself was not found to be a protease, as it had no proteolytic activity in a sensitive colorimetric assay. [1,3-3H]Diisopropyl fluorophosphate, an effective irreversible inhibitor of serine proteases, did not bind to TNF. Pretreatment of TNF with N alpha-p-tosyl-L-lysine chloromethane did not influence its biological activity. 4. The addition of protease inhibitor to the cells at various times after TNF administration led to a gradual loss of protection, suggesting that the protease acts at a rather late stage. 5. Protease inhibitors did not influence TNF binding, internalization or metabolization. 6. No increase in supernatant protease activity or in cell-associated protease activity could be detected after treatment of L929 cells with TNF. Our results document the involvement of protease activity, acting quite late during the cytolytic and growth inhibiting processes induced by TNF.     

Widespread formation of intracellular calcium carbonates by the bloom-forming cyanobacterium Microcystis

The formation of intracellular amorphous calcium carbonates (iACC) has been recently observed in a few cultured strains of Microcystis, a potentially toxic bloom-forming cyanobacterium found worldwide in freshwater ecosystems. If iACC-forming Microcystis are abundant within blooms, they may represent a significant amount of particulate Ca. Here, we investigate the significance of iACC biomineralization by Microcystis. First, the presence of iACC-forming Microcystis cells has been detected in several eutrophic lakes, indicating that this phenomenon occurs under environmental conditions. Second, some genotypic (presence/absence of ccyA, a marker gene of iACC biomineralization) and phenotypic (presence/absence of iACC) diversity have been detected within a collection of strains isolated from one single lake. This illustrates that this trait is frequent but also variable within Microcystis even at a single locality. Finally, one-third of publicly available genomes of Microcystis were shown to contain the ccyA gene, revealing a wide geographic and phylogenetic distribution within the genus. Overall, the present work shows that the formation of iACC by Microcystis is common under environmental conditions. While its biological function remains undetermined, this process should be further considered regarding the biology of Microcystis and implications on the Ca geochemical cycle in freshwater environments.     

Bioguided Fractionation and Isolation of an Antiplasmodial Saponin from the Roots of Nauclea xanthoxylon (A.Chev.) Aubrév. (Rubiaceae)

The root extract of Nauclea xanthoxylon (A.Chev.) Aubrév. displayed significant 50 % inhibition concentration (IC₅₀s) of 0.57 and 1.26 μg/mL against chloroquine resistant and sensitive Plasmodium falciparum (Pf) Dd2 and 3D7 strains, respectively. Bio-guided fractionation led to an ethyl acetate fraction with IC₅₀s of 2.68 and 1.85 μg/mL and subsequently, to the new quinovic acid saponin named xanthoxyloside ( 1 ) with IC₅₀s of 0.33 and 1.30 μM, respectively against the tested strains. Further compounds obtained from ethyl acetate and hexane fractions were the known clethric acid ( 2 ), ursolic acid ( 3 ), quafrinoic acid ( 4 ), quinovic acid ( 5 ), quinovic acid 3-O-β-D-fucopyranoside ( 6 ), oleanolic acid ( 7 ), oleanolic acid 3-acetate ( 8 ), friedelin ( 9 ), β-sitosterol ( 10a ), stigmasterol ( 10b ) and stigmasterol 3-O-β-D-glucopyranoside ( 11 ). Their structures were characterised with the aid of comprehensive spectroscopic methods (1 and 2D NMR, Mass). Bio-assays were performed using nucleic acid gel stain (SYBR green I)-based fluorescence assay with chloroquine as reference. Extracts and compounds exhibited good selectivity indices (SIs) of >10. Significant antiplasmodial activities measured for the crude extract, the ethyl acetate fraction and xanthoxyloside ( 1 ) from that fraction can justify the use of the root of N. xanthoxylon in ethnomedicine to treat malaria.     

Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation.

Structural mitochondrial damage accompanies the cytotoxic effects of several drugs including tumor necrosis factor (TNF). Using various inhibitors of mitochondrial electron transport we have investigated the mechanism of TNF-mediated cytotoxicity in L929 and WEHI 164 clone 13 mouse fibrosarcoma cells. Inhibitors with different sites of action modulated TNF cytotoxicity, however, with contrasting effects on final cell viability. Inhibition of mitochondrial electron transport at complex III (cytochrome c reductase) by antimycin A resulted in a marked potentiation of TNF-mediated injury. In contrast, when the electron flow to ubiquinone was blocked, either at complex I (NADH-ubiquinone oxidoreductase) with amytal or at complex II (succinate-ubiquinone reductase) with thenoyltrifluoroacetone, cells were markedly protected against TNF cytotoxicity. Neither uncouplers nor inhibitors of oxidative phosphorylation nor complex IV (cytochrome c oxidase) inhibitors significantly interfered with TNF-mediated effects, ruling out the involvement of energy-coupled phenomena. In addition, the toxic effects of TNF were counteracted by the addition of antioxidants and iron chelators. Furthermore, we analyzed the direct effect of TNF on mitochondrial morphology and functions. Treatment of L929 cells with TNF led to an early degeneration of the mitochondrial ultrastructure without any pronounced damage of other cellular organelles. Analysis of the mitochondrial electron flow revealed that TNF treatment led to a rapid inhibition of the mitochondria to oxidize succinate and NADH-linked substrates. The inhibition of electron transport was dose-dependent and became readily detectable 60 min after the start of TNF treatment, thus preceding the onset of cell death by at least 3-6 h. In contrast, only minor effects were observed on complex IV activity. The different effects observed with the mitochondrial respiratory chain inhibitors provide suggestive evidence that mitochondrial production of oxygen radicals mainly generated at the ubisemiquinone site is a causal mechanism of TNF cytotoxicity. This conclusion is further supported by the protective effect of antioxidants as well as the selective pattern of damage of mitochondrial chain components and characteristic alterations of the mitochondrial ultrastructure.     

Use of freeze-cracking in ontogenetic research in <Emphasis Type="Italic">Macrostomum lignano</Emphasis> (Macrostomida,Rhabditophora)

A method for studying whole mount flatworm embryos based on freeze-cracking of the eggs is described. This method allows successful immunohistological and immunocytological studies of whole mount embryos. It does not require the use of sharpened needles or a microinjection system to puncture the eggshell. Moreover, this method is more practical and less time-consuming than classical puncturing and much cheaper than the use of a microinjection system. The advantages of this method are illustrated by results of several immunolocalisation experiments in the macrostomid flatworm Macrostomum lignano. The optimal procedure and crucial steps for this method are discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Dependence of pathogen molecule-induced Toll-like receptor activation and cell function on Neu1 sialidase

The signaling pathways of mammalian Toll-like receptors (TLR) are well characterized, but the initial molecular mechanisms activated following ligand interactions with the receptors remain poorly defined. Here, we show a membrane controlling mechanism that is initiated by ligand binding to TLR-2, -3 and-4 to induce Neu1 sialidase activity within minutes in live primary bone marrow (BM) macrophage cells and macrophage and dendritic cell lines. Central to this process is that Neu1 and not Neu2,-3 and-4 forms a complex with TLR-2,-3 and-4 on the cell surface of naïve macrophage cells. Neuraminidase inhibitors BCX1827, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA), zanamivir and oseltamivir carboxylate have a limited significant inhibition of the LPS-induced sialidase activity in live BMC-2 macrophage cells but Tamiflu (oseltamivir phosphate) completely blocks this activity. Tamiflu inhibits LPS-induced sialidase activity in live BMC-2 cells with an IC₅₀ of 1.2?μM compared to an IC₅₀ of 1015?μM for its hydrolytic metabolite oseltamivir carboxylate. Tamiflu blockage of LPS-induced Neu1 sialidase activity is not affected in BMC-2 cells pretreated with anticarboxylesterase agent clopidogrel. Endotoxin LPS binding to TLR4 induces Neu1 with subsequent activation of NFκB and the production of nitric oxide and pro-inflammatory IL-6 and TNFα cytokines in primary and macrophage cell lines. Hypomorphic cathepsin A mice with a secondary Neu1 deficiency respond poorly to LPS-induced pro-inflammatory cytokines compared to the wild-type or hypomorphic cathepsin A with normal Neu1 mice. Our findings establish an unprecedented mechanism for pathogen molecule-induced TLR activation and cell function, which is critically dependent on Neu1 sialidase activity associated with TLR ligand treated live primary macrophage cells and macrophage and dendritic cell lines.