Potency of arachidonic acid in polyunsaturated fatty acid-induced death of human monocyte-macrophages: implications for atherosclerosis

Evidence suggests that oxidation of LDL is involved in the progression of atherosclerosis by inducing apoptosis in macrophages. Polyunsaturated fatty acids (PUFAs) are prominent components of LDL and are highly peroxidisable. We therefore tested PUFAs for induction of apoptosis in human monocyte-macrophages in vitro. Arachidonic acid (AA) induced the highest levels of apoptosis followed by docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), despite DHA and EPA being more peroxidisable than AA. alpha-Linolenic acid induced lower levels of apoptosis. Linoleic and oleic acids were innocuous. Results of experiments with AA products and enzyme inhibitors suggest roles for peroxidation, cyclooxygenase and lipoxygenase in AA-induced apoptosis. Our results further suggest activation of PPARgamma by AA and DHA associated with apoptosis induction. These findings may be relevant to potential mechanisms of fatty acid influences on plaques and may suggest strategies for combating atherosclerosis progression.     

Independent combinatorial effect of antisense oligonucleotides and RNAi-mediated specific inhibition of the recombinant rat P2X3 receptor

Synthetic 21-bp-long short interfering RNAs (siRNA) can stimulate sequence-specific mRNA degradation in mammalian cell cultures, a process referred to as RNA interference (RNAi). In the present study, the potential of RNAi was compared to the traditional antisense approach, acting mainly via RnaseH, for targeting the recombinant rat pain-related cation-channel P2X₃ expressed in CHO-K1 and a rat brain tumour-derived cell line, 33B. Downregulation of the P2X₃ receptor was evaluated at the mRNA, protein, and functional levels. In this study, four siRNA duplexes induced up to 95% sequence-specific inhibition of the P2X₃ mRNA, independent of the type of 2 nt 3′-overhang modification and the location of the targeted sequences. Furthermore, we detected and characterised an independent combinatorial effect of antisense oligonucleotides (ASOs) and RNAi-mediated specific inhibition of the P2X₃ receptor. Enhanced downregulation was observed only when siRNA was combined with nonhomologous ASO, targeting distant regions on the common P2X₃ mRNA. The two reagents resulted in more efficient downregulation of P2X₃ mRNA when administered in combination rather than separately. To our knowledge, this is the first investigation at the molecular level of the potential benefits of mixed antisense and RNAi-mediated treatment for inhibiting expression of a medically relevant pain-related gene.     

Preimaginal learning determines adult response to chemical stimuli in a parasitic wasp

The behavioural responses of parasitic wasps to chemical cues from their hosts and host plants are known to be affected by genetic and environmental components. In a previous study of the codling moth ectoparasitoid Hyssopus pallidus, we found that the response of adult parasitoids to the frass of their host caterpillars depended on a learning process involving plant cues. In the present study, we investigated how and when learning takes place. A series of experiments was conducted involving exposure of parasitoids to fruit cues at different developmental stages. While parasitoids were not able to learn the fruit cues in the adult stage, exposure to fruit odour at early preimaginal stages significantly increased the adult response to frass from fruit-fed caterpillars. The olfactory memory persisted through metamorphosis, with a retention time of 14 days. Preimaginal learning was not confined to fruit cues but was also demonstrated for a host- and fruit-independent cue, menthol. Parasitoids exposed to menthol odour at the egg and larval stages no longer showed negative responses as adults. Sensitization to fruit cues and habituation to menthol are considered to be the mechanisms involved. This study provides evidence of true preimaginal learning of olfactory cues in a parasitic wasp.     

Central role of Fas-associated death domain protein in apoptosis induction by the mitogen-activated protein kinase kinase inhibitor CI-1040 (PD184352) in acute lymphocytic leukemia cells in vitro

Because the MAPK pathway plays important roles in cell proliferation and inhibition of apoptosis, this pathway has emerged as a potential therapeutic target for solid tumors and leukemia. At the present time there is little information about activation of this pathway and the consequences of its inhibition in acute lymphocytic leukemia cells (ALL). In the present study, constitutive MAPK pathway activation, as evidenced by phosphorylation of ERK1 and ERK2, was observed in 8 of 8 human lymphoid cell lines and 33% (8:24) of pretreatment ALL bone marrows. Inhibition of this pathway by the MEK inhibitors CI-1040 and PD098059 induced apoptosis through a unique pathway involving dephosphorylation and aggregation of Fas-associated death domain protein followed by death receptor-independent caspase-8 activation. Jurkat cell variants lacking Fas-associated death domain protein or procaspase-8 were resistant to CI-1040-induced apoptosis, as were Jurkat or Molt3 cells treated with the O-methyl ester of the caspase-8 inhibitor N-(Nalpha-benzyloxycarbonylisoleucylglutamyl) aspartate fluoromethyl ketone. In contrast, CI-1040-induced apoptosis was unaffected by blocking anti-Fas antibody, soluble tumor necrosis factor-alpha-related apoptosis-inducing ligand decoy receptor, or transfection with cDNA encoding the anti-apoptotic Bcl-2 family member Mcl-1 or dominant negative caspase-9. Collectively, these results identify the MAPK pathway as a potential therapeutic target in ALL and delineate a mechanism by which MEK inhibition triggers apoptosis in ALL cells.     

Rab11b resides in a vesicular compartment distinct from Rab11a in parietal cells and other epithelial cells

The Rab11 family of small GTPases is composed of three members, Rab11a, Rab11b, and Rab25. While recent work on Rab11a and Rab25 has yielded some insights into their function, Rab11b has received little attention. Therefore, we sought to examine the distribution of endogenous Rab11b in epithelial cells. In rabbit gastric parietal cells, unlike Rab11a, Rab11b did not colocalize or coisolate with H(+)/K(+)-ATPase. In MDCK cells, endogenous Rab11b localized to an apical pericentrisomal region distinct from Rab11a. The microtubule agents nocodazole and taxol dramatically alter Rab11a's localization in the cell, while effects on Rab11b's distribution were less apparent. These results indicate that in contrast to Rab11a, the Rab11b compartment in the apical region is not as dependent upon microtubules. While Rab11a is known to regulate transferrin trafficking in nonpolarized cells and IgA trafficking in polarized cells, Rab11b exhibited little colocalization with either of these cargoes. Thus, while Rab11a and Rab11b share high sequence homology, they appear to reside within distinct vesicle compartments.     

Heterogeneous selection at specific loci in natural environments in Arabidopsis thaliana

Genetic variation for quantitative traits is often greater than that expected to be maintained by mutation in the face of purifying natural selection. One possible explanation for this observed variation is the action of heterogeneous natural selection in the wild. Here we report that selection on quantitative trait loci (QTL) for fitness traits in the model plant species Arabidopsis thaliana differs among natural ecological settings and genetic backgrounds. At one QTL, the allele that enhanced the viability of fall-germinating seedlings in North Carolina reduced the fecundity of spring-germinating seedlings in Rhode Island. Several other QTL experienced strong directional selection, but only in one site and seasonal cohort. Thus, different loci were exposed to selection in different natural environments. Selection on allelic variation also depended upon the genetic background. The allelic fitness effects of two QTL reversed direction depending on the genotype at the other locus. Moreover, alternative alleles at each of these loci caused reversals in the allelic fitness effects of a QTL closely linked to TFL1, a candidate developmental gene displaying nucleotide sequence polymorphism consistent with balancing selection. Thus, both environmental heterogeneity and epistatic selection may maintain genetic variation for fitness in wild plant species.     

Juvenile hormone catabolism and oviposition in the codling moth, Cydia pomonella, as functions of age, mating status, and hormone treatment.

In vitro catabolism of juvenile hormone (JH) in haemolymph of adult female Cydia pomonella was ascribed mainly to juvenile hormone esterase (JHE) activity. No significant differences were noted between virgin and mated females 0-96 h post-emergence. Changes in JHE activity did not appear dependent upon fluctuations in JH titre; conversely, changes in JHE activity could not explain the changes in JH titres. Maximal JHE activity was recorded at 24 h (331.47 +/- 47.25 pmol/h/microl; 355.93 +/- 36.68 pmol/h/microl, virgin; mated insects, respectively) and preceded the peak in JH titres at 48 h. Topical application of JH II (10 ng-10 microg) or fenoxycarb (50 ng) enhanced JHE activity up to 640 and 56%, respectively. Treatment upon emergence with 10 microg JH II induced enzymic activity for less than 24 h, and when 10 microg JH II or 50 ng fenoxycarb were applied, circulating JH titres returned to control levels within 24 h. Oviposition was highly sensitive to exogenous JH and declined significantly with dosages >100 pg. To allow a degree of oocyte maturation before JH treatment, the hormone was administered at 6, 12, 24, or 48 h post-emergence and/or females were mated. Neither measure "protected" the system; oviposition declined immediately after JH application.     

Reducing environmental bias when measuring natural selection

Abstract.— Crucial to understanding the process of natural selection is characterizing phenotypic selection. Measures of phenotypic selection can be biased by environmental variation among individuals that causes a spurious correlation between a trait and fitness. One solution is analyzing genotypic data, rather than phenotypic data. Genotypic data, however, are difficult to gather, can be gathered from few species, and typically have low statistical power. Environmental correlations may act through traits other than through fitness itself. A path analytic framework, which includes measures of such traits, may reduce environmental bias in estimates of selection coefficients. We tested the efficacy of path analysis to reduce bias by re-analyzing three experiments where both phenotypic and genotypic data were available. All three consisted of plant species (Impatiens capensis, Arabidopsis thaliana , and Raphanus sativus) grown in experimental plots or the greenhouse. We found that selection coefficients estimated by path analysis using phenotypic data were highly correlated with those based on genotypic data with little systematic bias in estimating the strength of selection. Although not a panacea, using path analysis can substantially reduce environmental biases in estimates of selection coefficients. Such confidence in phenotypic selection estimates is critical for progress in the study of natural selection.     

Comparison of parasitism by Cotesia glomerata with bacterial infection and wounding in Pieris brassicae: induction of new haemolymph polypeptides and changes in humoral immune response

In Pieris brassicae, parasitism by Cotesia glomerata and bacterial infection are differentiated with respect to haemolymph protein arrays, and production or suppression of antibacterial agents. Bacteriolytic activity in haemolymph from parasitized larvae was slightly, but significantly, higher 24h post-treatment than that of untreated and wounded controls. Micrococcus lysodeikticus- or lipopolysaccharide-(LPS) injected insects exhibited an 11-fold greater response than those parasitized. At 24h post-treatment, antibacterial activity against Escherichia coli was observed in haemolymph from all but untreated larvae. Injection of Grace's medium, M. lysodeikticus or LPS, caused a greater than threefold response than parasitization or wounding. The protein banding patterns of parasitized hosts did not correspond to those of the other treatments. Two parasitoid-induced proteins (38 and 128 kDa) were examined. Both were found in parasitized insects, not in those wounded, injected with Grace's medium, M. lysodeikticus or LPS. Neither protein was bacteriolytic or bacteriostatic in inhibition zone assays.     

Mitochondrial death protein Nix is induced in cardiac hypertrophy and triggers apoptotic cardiomyopathy

Loss of cardiomyocytes through programmed cell death is a key event in the development of heart failure, but the inciting molecular mechanisms are largely unknown. We used microarray analysis to identify a genetic program for myocardial apoptosis in Gq-mediated and pressure-overload cardiac hypertrophy. A critical component of this apoptotic program was Nix/Bnip3L. Nix localized to mitochondria and caused release of cytochrome c, activation of caspase-3 and apoptotic cell death, when expressed in HEK293 fibroblasts. A previously undescribed truncated Nix isoform, termed sNix, was not targeted to mitochondria but heterodimerized with Nix and protected against Nix-mediated apoptosis. Forced in vivo myocardial expression of Nix resulted in apoptotic cardiomyopathy and rapid death. Conversely, sNix protected against apoptotic peripartum cardiomyopathy in G(alpha)q-overexpressors. Thus, Nix/Bnip3L is upregulated in myocardial hypertrophy, and is both necessary and sufficient for Gq-mediated apoptosis of cardiomyocytes and resulting hypertrophy decompensation.