Identification and relevance of the CD95-binding domain in the N-terminal region of ezrin

The CD95 (Fas/APO-1) linkage to the actin cytoskeleton through ezrin is an essential requirement for susceptibility to the CD95-mediated apoptosis in CD4+ T cells. We have previously shown that moesin was not involved in the binding to CD95. Here we further support the specificity of the ezrin/CD95 binding, showing that radixin did not bind CD95. The ezrin region specifically and directly involved in the binding to CD95 was located in the middle lobe of the ezrin FERM domain, between amino acids 149 and 168. In this region, ezrin, radixin, and moesin show 60-65% identity, as compared with the 86% identity in the whole FERM domain. Transfection of two different human cell lines with a green fluorescent protein-tagged ezrin mutated in the CD95-binding epitope, induced a marked inhibition of CD95-mediated apoptosis. In these cells, the mutated ezrin did not co-localize or co-immunoprecipitate with CD95. Further analysis showed that the mutated ezrin, while unable to bind CD95, was fully able to bind actin, thus preventing the actin linkage to CD95. Altogether, our results support the specificity of ezrin in the association to CD95 and the importance of the ezrin-to-CD95 linkage in CD95-mediated apoptosis. Moreover, this study suggests that a major role of ezrin is to connect CD95 to actin, thus allowing the CD95 polarization on the cells and the occurrence of the following multiple cascades of the CD95 pathway.     

Effects of two biorational insecticides, spinosad and methoxyfenozide, on Spodoptera littoralis (Lepidoptera: Noctuidae) under laboratory conditions

The toxicity of two biorational insecticides, spinosad (Tracer) and methoxyfenozide (RH-2485), was tested against eggs, larvae, and pupae of the noctuid Spodoptera littoralis (Boisduval). In the first experiment, filter paper circles containing egg masses of two different age classes, young (<24 h old) and old (24-48 h old), were dipped in different concentrations of each insecticide diluted in either water or acetone. No ovicidal activity was recorded when insecticides were diluted in water. In contrast, when insecticides were diluted in acetone, both egg age classes generally showed a concentration-dependent response for both compounds. Mortality of larvae that hatched from both egg age classes was significantly increased, compared with control larvae, at all concentrations of both insecticides when diluted in water or acetone alike. The prevalence of mortality was similar with each insecticide. In the second experiment, third instars of S. littoralis were fed semisynthetic diet containing different concentrations of both insecticides. According to LC50 values, no significant differences were observed between spinosad (2.11 mg [AI]/kg diet) and methoxyfenozide (3.98 mg [AI]/kg diet) after 48 h of treatment, based on the overlap of 95% CL. Toxic effects on the mortality of pupae, adult emergence, and the prevalence of deformed adults after topical application on young pupae also were examined. Only methoxyfenozide caused pupal mortality and deformed adults. Our results suggest that spinosad and methoxyfenozide are potentially potent compounds for control of S. littoralis.     

Up-regulation of CD1d expression restores the immunoregulatory function of NKT cells and prevents autoimmune diabetes in nonobese diabetic mice

The immunoregulatory function of NKT cells is crucial for prevention of autoimmunity. The prototypical NKT cell Ag alpha-galactosylceramide is not present in mammalian cells, and little is known about the mechanism responsible for NKT cell recruitment and activation. Up-regulation of CD1d, the NKT cell restriction molecule, expressed on mononuclear cells infiltrating the target organ, could represent the physiological trigger for NKT cells to self-contain T cell immunity and to prevent autoimmune disease. Recognition of CD1d, either by itself or bound to self-ligands (selfCD1d), could drive NKT cells toward an immunoregulatory phenotype. Hence, ineffective NKT cell-mediated immunoregulation in autoimmune-prone individuals including nonobese diabetic (NOD) mice could be related to defective signals that regulate CD1d expression at time and site of autoimmunity. To test this hypothesis, we transgenically overexpressed CD1d molecules under the control of the insulin promoter within the pancreatic islets of NOD mice (insCD1d). Recognition of overexpressed CD1d molecules rescued NKT cell immunoregulatory function and prevented autoimmune diabetes in insCD1d transgenic NOD mice. Protection from diabetes was associated with a biased IL-4-secreting cytokine phenotype of NKT cells and alteration of the cytokine microenvironment in the pancreatic lymph nodes of transgenic mice. The net effect was a reduced development of the autoimmune T cell repertoire. Our findings suggest that up-regulation of CD1d expression during inflammation is critical to maintain T cell homeostasis and to prevent autoimmunity.     

Coenzyme site-directed mutants of photosynthetic A4-GAPDH show selectively reduced NADPH-dependent catalysis, similar to regulatory AB-GAPDH inhibited by oxidized thioredoxin

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants uses both NADP(H) and NAD(H) as coenzyme and consists of one (GapA) or two types of subunits (GapA, GapB). AB-GAPDH is regulated in vivo through the action of thioredoxin and metabolites, showing higher kinetic preference for NADPH in the light than in darkness due to a specific effect on kcat(NADPH). Previous crystallographic studies on spinach chloroplast A4-GAPDH complexed with NADP or NAD showed that residues Thr33 and Ser188 are involved in NADP over NAD selectivity by interacting with the 2'-phosphate group of NADP. This suggested a possible involvement of these residues in the regulatory mechanism. Mutants of recombinant spinach GapA (A4-GAPDH) with Thr33 or Ser188 replaced by Ala (T33A, S188A and double mutant T33A/S188A) were produced, expressed in Escherichia coli, and compared to wild-type recombinant A4-GAPDH, in terms of crystal structures and kinetic properties. Affinity for NADPH was decreased significantly in all mutants, and kcat(NADPH) was lowered in mutants carrying the substitution of Ser188. NADH-dependent activity was unaffected. The decrease of kcat/Km of the NADPH-dependent reaction in Ser188 mutants resembles the behaviour of AB-GAPDH inhibited by oxidized thioredoxin, as confirmed by steady-state kinetic analysis of native enzyme. A significant expansion of size of the A4-tetramer was observed in the S188A mutant compared to wild-type A4. We conclude that in the absence of interactions between Ser188 and the 2'-phosphate group of NADP, the enzyme structure relaxes to a less compact conformation, which negatively affects the complex catalytic cycle of GADPH. A model based on this concept might be developed to explain the in vivo light-regulation of the GAPDH.     

Direct phage to intrabody screening (DPIS): demonstration by isolation of cytosolic intrabodies against the TES1 site of Epstein Barr virus latent membrane protein 1 (LMP1) that block NF-kappaB transactivation

The expression of intracellular antibodies (intrabodies) in eukaryotic cells has provided a powerful tool to manipulate microbial and cellular signaling pathways in a highly precise manner. However, there have been several technical issues that have restricted their more widespread use. In particular, single-chain antibodies (sFv) have been reported to fold poorly in the reducing environment of the cytoplasm and as such there has been a reluctance to use sFv-phage libraries as a source of intrabodies unless a pre-selection step to identify these rare sFvs from natural libraries or libraries of engineering sFvs that could fold properly in the absence of disulfide bonds were used. Here, we investigated whether target specific sFvs that are isolated from a 15 billion member non-immune human sFv-phage display library could be directly screened in pools as intrabodies without prior knowledge of their individual identity or purity within pools of antigen-specific sFvs. As the target, we used a synthetic transformation effector site 1 (TES1) polypeptide comprising the membrane-most proximal 34 amino acid residues of the carboxy-terminal cytoplasmic tail of the oncogenic latent membrane protein 1 (LMP1) of Epstein Barr virus, which serves as a docking site for adapter proteins of the tumor necrosis factor (TNF) receptor (TNFR)-associated factor (TRAF) family. Anti-TES1 sFvs, initially identified by phage ELISA screens, were grouped into pools according to the absorbance reading of the antigen-specific phage ELISA assays and then transferred as pools into eukaryotic expression vectors and expressed as cytoplasmic intrabodies. Using the pooling strategy, there was no loss of individual anti-TES1 sFvs in the transfer from prokaryotic to eukaryotic expression vectors. In addition, the initial assignments into sFv pools based on phage ELISA readings allowed the segregation of individual anti-TES1 sFvs into discrete or minimally overlapping intrabody pools. Further assessment of the biological activity of the anti-TES1 intrabody pools demonstrated that they were all able to selectively block F-LMP1-induced NFkappaB activity that was mediated through the TES1-site and to bind LMP1 protein with high efficiency. This direct phage to intrabody screening (DPIS) strategy should allow investigators to bypass much of the in vitro sFv characterization that is often not predictive of in vivo intrabody function and provide a more efficient use of large native and synthetic sFv phage libraries already in existence to identify intrabodies that are active in vivo.     

3-Nitropropionic acid-induced neurotoxicity--assessed by ultra high resolution positron emission tomography with comparison to magnetic resonance spectroscopy

To explore acute and long-term effects of 3-nitropropionic acid (3-NP)-induced neurotoxicity, longitudinal positron emission tomography (PET) studies of energy metabolism and magnetic resonance spectroscopic (MRS) studies of neurochemicals were conducted in a rat model. The first injection of 3-NP (20 mg/kg i.p.) was followed by MRS study of neurochemicals and PET study of glucose utilization using [(18)F]2-fluorodeoxy-D-glucose ((18)F-FDG). After that, 3-NP administration was done two times a day with a dose of 10 mg/kg i.p. until animals were symptomatic or for a maximum of 5 days combined with daily PET studies. Long-term effects were investigated 4 weeks and 4 months after cessation of 3-NP. These studies showed a significant inter-animal variation in response of 3-NP toxicity. Animals that developed large striatal lesions had decreased glucose utilization in the striatum and cortex 1 day after starting 3-NP injections. Similarly succinate and lactate/macromolecule levels were enhanced; these changes being, however, reversible. Progressive degeneration was observed by decreasing striatal glucose utilization and N-acetylaspartate (NAA) and increasing choline. These observations paralleled with weight loss and deficits in behavior. Animals that did not develop lesions showed reversible enhancement in cortical glucose utilization and no change in striatal glucose utilization or neurochemicals or locomotor activity.     

Enhanced anandamide degradation is associated with neuronal apoptosis induced by the HIV-1 coat glycoprotein gp120 in the rat neocortex

Human immunodeficiency virus type-1 coat glycoprotein gp120 causes delayed apoptosis in rat brain neocortex. Here, we investigated the possible role of the endocannabinoid system in this process. It is shown that gp120 causes a time-dependent increase in the activity and immunoreactivity of the anandamide (AEA)-hydrolyzing enzyme fatty acid amide hydrolase (FAAH), paralleled by increased activity of the AEA membrane transporter and decreased endogenous levels of AEA. The AEA-synthesizing phospholipase D and the AEA-binding receptors were not affected by gp120. None of the changes induced by gp120 in the cortex were induced by bovine serum albumin, nor were they observed in the hippocampus of the same animals. Also, the activity of 5-lipoxygenase, which generates AEA derivatives able to inhibit FAAH, decreased down to approximately 25% of the control activity upon gp120 treatment, due to reduced protein level ( approximately 45%). In addition, the FAAH inhibitor methyl-arachidonoyl fluorophosphonate significantly reduced gp120-induced apoptosis in rat brain neocortex, whereas selective blockers of AEA membrane transporter or of AEA-binding receptors were ineffective. Taken together, these results suggest that gp120, by activating FAAH, decreases endogenous levels of AEA, and the latter effect seems instrumental in the execution of delayed neuronal apoptosis in the brain neocortex of rats.     

Acetylcholinesterase inhibitors increase ADAM10 activity by promoting its trafficking in neuroblastoma cell lines

Acetylcholinesterase inhibitors (AChEIs) are the only currently available drugs for treating Alzheimer's Disease (AD). Some authors have suggested a function of AChEIs not only in the induction of AChE overproduction and alternative splicing shifts but also a possible role of these drugs in amyloid metabolism beyond their well-known symptomatic effect. Here, we investigate the mechanisms of action of the AChEI donepezil on APP (amyloid precursor protein) metabolism and on the activity/trafficking of the alpha-secretase candidate ADAM 10, in differentiated human neuroblastoma cells (SH-SY5Y). In these cells, the activity of AChE is significantly decreased after 2 h of donepezil treatment. Further, SH-SY5Y cells released significantly more sAPPalpha into the medium, whereas total APP levels in cell lysates were unchanged. Interestingly, treated cells showed increased ADAM 10 levels in membrane compartments. This effect was prevented by pretreatment with tunicamycin or brefeldin, suggesting that donepezil affects trafficking and/or maturation of ADAM 10; additionally, this pretreatment significantly decreased sAPPalpha levels. Pre-incubation with atropine decreased release of sAPPalpha significantly but did not revert ADAM 10 activity to control levels further suggesting that donepezil acts not solely through a purely receptor mediated pathway. These findings indicate that donepezil exerts multiple mechanisms involving processing and trafficking of key proteins involved in AD pathogenesis.     

Rat brain arachidonic acid metabolism is increased by a 6-day intracerebral ventricular infusion of bacterial lipopolysaccharide

In a rat model of acute neuroinflammation, produced by a 6-day intracerebral ventricular infusion of bacterial lipopolysaccharide (LPS), we measured brain activities and protein levels of three phospholipases A2 (PLA2) and of cyclo-oxygenase-1 and -2, and quantified other aspects of brain phospholipid and fatty acid metabolism. The 6-day intracerebral ventricular infusion increased lectin-reactive microglia in the cerebral ventricles, pia mater, and the glial membrane of the cortex and resulted in morphological changes of glial fibrillary acidic protein (GFAP)-positive astrocytes in the cortical mantel and areas surrounding the cerebral ventricles. LPS infusion increased brain cytosolic and secretory PLA2 activities by 71% and 47%, respectively, as well as the brain concentrations of non-esterified linoleic and arachidonic acids, and of prostaglandins E2 and D2. LPS infusion also increased rates of incorporation and turnover of arachidonic acid in phosphatidylethanolamine, plasmenylethanolamine, phosphatidylcholine, and plasmenylcholine by 1.5- to 2.8-fold, without changing these rates in phosphatidylserine or phosphatidylinositol. These observations suggest that selective alterations in brain arachidonic acid metabolism involving cytosolic and secretory PLA2 contribute to early pathology in neuroinflammation.     

The herpes simplex virus JMP mutant enters receptor-negative J cells through a novel pathway independent of the known receptors nectin1, HveA, and nectin2

The herpes simplex virus type 1(JMP) [HSV-1(JMP)] mutant was selected for its ability to grow and form plaques in receptor-negative J cells. It enters J cells through a novel gD-dependent pathway, independent of all known HSV receptors, nectin1, nectin2, and HveA. Evidence that the pathway is dependent on a nectin3 binding site on HSV-1(JMP) and requires three mutations in gD rests on the following. We derived monoclonal antibodies to nectin3 and show that J cells express nectin3. HSV-1(JMP) entry and cell-to-cell spread were inhibited by soluble nectin3-Fc, demonstrating that virions carry a binding site for nectin3. The site is either directly involved in HSV-1(JMP) entry, or nectin3 binding to its site affects the gD domains involved in entry (entry site). HSV-1(JMP) entry and cell-to-cell spread in J cells were also inhibited by soluble nectin1-Fc, showing that the nectin1 binding site on gD(JMP) overlaps with the entry site or that nectin1 binding to gD affects the entry site. gD(JMP) carries three mutations, S140N, R340H, and Q344R. The latter two lie in the C tail and are present in the parental HSV-1(MP). HSV-1 strain R5000 carrying the S140N substitution was not infectious in J cells, indicating that this substitution was not sufficient. We constructed two recombinants, one carrying the three substitutions and the other carrying the two C-tail substitutions. Only the first recombinant infected J cells with an efficiency similar to that of HSV-1(JMP), indicating that the three mutations are required for the novel entry pathway. The results highlight plasticity in gD which accounts for changes in receptor usage.