Activation of multidomain and BH3-only pro-apoptotic Bcl-2 family members in p53-defective cells

In the p53-deficient human B lymphoma Namalwa cell line that quickly undergoes apoptosis after DNA topoisomerase I inhibitor (camptothecin, CPT) treatment, we observed rapid and slight induction of the pro-apoptotic BH3-only Bik, Bim-EL, Bim-L and Bim-S proteins. In contrast, the expression levels of Bad and multidomain Bax-alpha and Bak remained mostly unchanged after CPT treatment. However, multiple pro-apoptotic proteins, including Bax-alpha, Bak, Bik, Bim-EL and Bim-L, translocated rapidly to the mitochondria after CPT treatment. Gel filtration chromatography experiments demonstrated that somes of the pro-apoptotic proteins assemble themselves into high molecular weight protein complexes. The protein composition of these oligomers was further analyzed by co-immunoprecipitation experiments performed on highly purified mitochondrial fractions, which revealed the formation of Bax/Bak, Bax/VDAC1, Bak/VDAC1, Bim/VDAC1 and Bim/Bcl-2 complexes after DNA damage induction. Thus, it appeared that induction, mitochondrial translocation and assembly in multimeric protein complexes of several pro-apoptotic members of the Bcl-2 family correlated with the rapid activation of apoptosis in a p53-independent pathway after CPT-mediated DNA strand breaks.     

Dysfunction of mitochondrial complex I and the proteasome: interactions between two biochemical deficits in a cellular model of Parkinson's disease

Two biochemical deficits have been described in the substantia nigra in Parkinson's disease, decreased activity of mitochondrial complex I and reduced proteasomal activity. We analysed interactions between these deficits in primary mesencephalic cultures. Proteasome inhibitors (epoxomicin, MG132) exacerbated the toxicity of complex I inhibitors [rotenone, 1-methyl-4-phenylpyridinium (MPP+)] and of the toxic dopamine analogue 6-hydroxydopamine, but not of inhibitors of mitochondrial complex II-V or excitotoxins [N-methyl-d-aspartate (NMDA), kainate]. Rotenone and MPP+ increased free radicals and reduced proteasomal activity via adenosine triphosphate (ATP) depletion. 6-hydroxydopamine also increased free radicals, but did not affect ATP levels and increased proteasomal activity, presumably in response to oxidative damage. Proteasome inhibition potentiated the toxicity of rotenone, MPP+ and 6-hydroxydopamine at concentrations at which they increased free radical levels >/= 40% above baseline, exceeding the cellular capacity to detoxify oxidized proteins reduced by proteasome inhibition, and also exacerbated ATP depletion caused by complex I inhibition. Consistently, both free radical scavenging and stimulation of ATP production by glucose supplementation protected against the synergistic toxicity. In summary, proteasome inhibition increases neuronal vulnerability to normally subtoxic levels of free radicals and amplifies energy depletion following complex I inhibition.     

Alpha-conotoxins PnIA and [A10L]PnIA stabilize different states of the alpha7-L247T nicotinic acetylcholine receptor

The effects of the native alpha-conotoxin PnIA, its synthetic derivative [A10L]PnIA and alanine scan derivatives of [A10L]PnIA were investigated on chick wild type alpha7 and alpha7-L247T mutant nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes. PnIA and [A10L]PnIA inhibited acetylcholine (ACh)-activated currents at wtalpha7 receptors with IC50 values of 349 and 168 nm, respectively. Rates of onset of inhibition were similar for PnIA and [A10L]PnIA; however, the rate of recovery was slower for [A10L]PnIA, indicating that the increased potency of [A10L]PnIA at alpha7 receptors is conveyed by its slower rate of dissociation from the receptors. All the alanine mutants of [A10L]PnIA inhibited ACh-activated currents at wtalpha7 receptors. Insertion of an alanine residue between position 5 and 13 and at position 15 significantly reduced the ability of [A10L]PnIA to inhibit ACh-evoked currents. PnIA inhibited the non-desensitizing ACh-activated currents at alpha7-L247T receptors with an IC50 194 nm. In contrast, [A10L]PnIA and the alanine mutants potentiated the ACh-activated current alpha7-L247T receptors and in addition [A10L]PnIA acted as an agonist. PnIA stabilized the receptor in a state that is non-conducting in both the wild type and mutant receptors, whereas [A10L]PnIA stabilized a state that is non-conducting in the wild type receptor and conducting in the alpha7-L247T mutant. These data indicate that the change of a single amino acid side-chain, at position 10, is sufficient to change the toxin specificity for receptor states in the alpha7-L247T mutant.     

Exportin-5 mediates nuclear export of minihelix-containing RNAs

The adenovirus VA1 RNA (VA1), a 160-nucleotide (nt)-long RNA transcribed by RNA polymerase III, is efficiently exported from the nucleus to the cytoplasm of infected cells, where it antagonizes the interferon-induced antiviral defense system. We recently reported that nuclear export of VA1 is mediated by a cis-acting RNA export motif, called minihelix, that comprises a double-stranded stem (>14 nt) with a base-paired 5' end and a 3-8-nt protruding 3' end. RNA export mediated by the minihelix motif is Ran-dependent, which indicates the involvement of a karyopherin-related factor (exportin) that remained to be determined. Here we show using microinjection in Xenopus laevis oocytes that VA1 is transported to the cytoplasm by exportin-5, a nuclear transport factor for double-stranded RNA binding proteins. Gel retardation assays revealed that exportin-5 directly interacts with VA1 RNA in a RanGTP-dependent manner. More generally, in vivo and in vitro competition experiments using various VA1-derived, but also artificial and cellular, RNAs lead to the conclusion that exportin-5 preferentially recognizes and transports minihelix motif-containing RNAs.     

Colony structure in a plant-ant: behavioural,chemical and genetic study of polydomy in<Emphasis Type="Italic"> Cataulacus mckeyi</Emphasis> (Myrmicinae)

Social organisation of colonies of obligate plant-ants can affect their interaction with myrmecophyte hosts and with other ants competing for the resources they offer. An important parameter of social organisation is whether nest sites of a colony include one or several host individuals. We determined colony boundaries in a plant-ant associated with the rainforest understorey tree Leonardoxa africana subsp. africana, found in coastal forests of Cameroon (Central Africa). This myrmecophyte is strictly associated with two ants, Petalomyrmex phylax and Cataulacus mckeyi. Plants provide food and nesting sites for P. phylax, which protects young leaves against insect herbivores. This mutualism is often parasitised by C. mckeyi, which uses but does not protect the host. The presence of C. mckeyi on a tree excludes the mutualistic ant. Because Petalomyrmex -occupied trees are better protected, their growth and survival are superior to those of Cataulacus -occupied trees, giving P. phylax an advantage in occupation of nest sites. C. mckeyi often colonises trees that have lost their initial associate P. phylax, as a result of injury to the tree caused by disturbance. Polydomy may allow C. mckeyi to occupy small clumps of trees, without the necessity of claustral colony foundation in each tree. Investigating both the proximate (behavioural repertoire, colony odour) and the ultimate factors (genetic structure) that may influence colony closure, we precisely defined colony boundaries. We show that colonies of C. mckeyi are monogynous and facultatively polydomous, i.e. a colony occupies one to several Leonardoxa trees. Workers do not produce males. Thus, the hypothesis that polydomy allows workers in queenless nests to evade queen control for their reproduction is not supported in this instance. This particular colony structure may confer on C. mckeyi an advantage in short-distance dispersal, and this could help explain its persistence within the dynamic Leonardoxa system.     

A continuous assay of myristoyl-CoA:protein N-myristoyltransferase for proteomic analysis

Protein N-myristoylation is an important lipid modification that affects the activity and membrane-binding properties of crucial proteins belonging to signal transduction cascades. The aim of this work was to develop a rapid and easy diagnostic method to check for (i) effective N-myristoylation of any given protein and (ii) easy proteome annotation. The N-myristoylation reaction was coupled to that of pyruvate dehydrogenase, and NADH was continuously detected spectrophotometrically. This method was optimized for and applied to full-length Saccharomyces cerevisiae and Arabidopsis thaliana N-myristoyltransferases and two A. thaliana enzyme derivatives. The data were validated by comparison with a previously described discontinuous assay, modification of the chemical nature of the substrates, and use of specific inhibitors. The kinetics of N-myristoylation were determined in vitro with various compounds including a full-length polypeptide substrate, a small G protein of the RAB family already known to be N-myristoylated in vivo. This automated assay can be used for proteomic studies to determine the N-myristoylation state of any protein.     

The iodothyronine selenodeiodinases are thioredoxin-fold family proteins containing a glycoside hydrolase clan GH-A-like structure

The three iodothyronine selenodeiodinases catalyze the initiation and termination of thyroid hormone effects in vertebrates. Structural analyses of these proteins have been hindered by their integral membrane nature and the inefficient eukaryotic-specific pathway for selenoprotein synthesis. Hydrophobic cluster analysis used in combination with Position-specific Iterated BLAST reveals that their extramembrane portion belongs to the thioredoxin-fold superfamily for which experimental structure information exists. Moreover, a large deiodinase region imbedded in the thioredoxin fold shares strong similarities with the active site of iduronidase, a member of the clan GH-A-fold of glycoside hydrolases. This model can explain a number of results from previous mutagenesis analyses and permits new verifiable insights into the structural and functional properties of these enzymes.     

Cytotoxic activities of novel hexahydroindolizino[8,7-b]indole derivatives prepared by 1,3-dipolar cycloaddition reactions of 3,4-dihydro-beta-carboline ylides

A series of 1-cyano and 2-cyanohexahydroindolizino[8,7-b]indole derivatives was prepared by 1,3-dipolar cycloaddition of acrylonitrile with ylides derived from 3,4-dihydro-beta-carboline and its 6-methoxy, 6-benzyloxy, 9-methyl and 9-benzyl analogues. The products, together with their reduced 1- or 2-aminomethyl derivatives, were evaluated for cytotoxic activity in L1210 cancer cells. Compounds derived from 6-benzyloxy or 9-benzyl-3,4-dihydro-beta-carboline were found to be the most active, with IC(50)'s in the 2-50 microM range. Of these, two compounds, the 1- and 2-cyano 8-benzyloxyindolizino[8,7-b]indole derivatives 20a and 20c, respectively, were found by cytometric flux analysis to stop cancer cell growth at the G(2)M and 8N (>G(2)M) stage of the cell cycle. These two compounds also showed no loss of cytotoxic activity in K562R cancer cells resistant to doxorubicin.     

Hepatic lipase: structure/function relationship,synthesis, and regulation

Hepatic lipase (HL) is a lipolytic enzyme, synthesized by hepatocytes and found localized at the surface of liver sinusoid capillaries. In humans, the enzyme is mostly bound onto heparan-sulfate proteoglycans at the surface of hepatocytes and also of sinusoid endothelial cells. HL shares a number of functional domains with lipoprotein lipase and with other members of the lipase gene family. It is a secreted glycoprotein, and remodelling of the N-linked oligosaccharides appears to be crucial for the secretion process, rather than for the acquisition of the catalytic activity. HL is also present in adrenals and ovaries, where it might promote delivery of lipoprotein cholesterol for steroidogenesis. However, evidence of a local synthesis is still controversial. HL activity is fairly regulated according to the cell cholesterol content and to the hormonal status. Coordinate regulations have been reported for both HL and the scavenger-receptor B-I, suggesting complementary roles in cholesterol metabolism. However, genetic variants largely contribute to HL variability and their possible impact in the development of a dyslipidemic phenotype, or in a context of insulin-resistance, is discussed.     

Comparison of tissue culture and animal models for assessment of Cryptospridium parvum infection

The current increased interest for using tissue culture as a surrogate for mouse infection to assess Cryptospridium viability suggests that a comparison of the two models is essential for data interpretation. Therefore, a need remains for a statistical comparison that can demonstrate if infection and inactivation predicted by new tissue culture models are comparable with those predicted by animal models. Data from a total of 31 dose-response trials using both tissue culture and mouse models to assess C. parvum infectivity were compared. The dose needed to infect 50% of the tissue cultures (ID(50)) was also compared to each ID(50) in mice. Average ID(50)s developed using the logit dose-response method for tissue culture and mice were 8 and 107, respectively, suggesting that tissue culture was more sensitive to infection. However, correlation (r) between tissue culture and mouse infectivity was statistically significant (0.9167 [95% CI=0.8428 to 0.9594, p<0.0001]). Comparison of oocyst disinfection by UV and chlorine dioxide showed no significant difference between inactivation predicted by tissue culture and mouse models (p=0.8893; t=0.0141; n=21). These results demonstrate that tissue culture can successfully be used to measure C. parvum infection and can be used for determining inactivation in disinfection studies.