Synthesis of diacylglycerol de novo is responsible for permanent activation and down-regulation of protein kinase C in transformed cells

We measured the synthesis of diacylglycerol de novo in normal NIH/3T3 fibroblasts and in cells transformed by ras, src, sis and abl oncogenes. Analysis of the incorporation of glucose-derived 14C into diacylglycerol indicated that neosynthesis of diacylglycerol was constitutively active in the transformed cell lines. Elevated levels of diacylglycerol and persistent activation/down-regulation of protein kinase C reduced the binding of phorbol dibutyrate to transformed cells. This phenomenon could be reversed by blocking the glycolytic pathway, thus indicating that neosynthesized diacylglycerol was responsible for persistent activation and down-regulation of protein kinase C. In transformed cells, protein kinase C activity could not be stimulated by the addition of diolein; however, inhibition of glycolysis restored the ability of transformed cells to respond to diolein. Taken together these data indicate that constitutive synthesis of diacylglycerol de novo is responsible for activation and down-regulation of protein kinase C in transformed cells, and it may play a role in altered mitogenic signalling.     

Subtle differences in two non‐native congeneric beach grasses significantly affect their colonization,spread, and impact

Comparisons of congeneric species have provided unique insights into invasion ecology. Most often, non‐native species are compared to native ones to look for traits predicting invasion success. In this study, we examine a different facet of congeneric comparisons in which both species are non‐native. Ecological variability among non‐native congeners might 1) lead to the inhibition or facilitation of either species’ ability to colonize and spread, 2) result in larger cumulative impacts due to synergies between species, and 3) depend on the physical context of the invaded habitat. To explore these possibilities, we studied the distribution and abundance of two non‐native beach grasses: European beach grass Ammophila arenaria and American beach grass Ammophila breviligulata, their interaction with one another, and their biotic and physical impacts on dune ecosystems of the Pacific coast of North America. We found that over a two‐decade period, A. breviligulata has increased its dominance over A. arenaria on dunes where it was originally planted in 1935 and has actively spread to new sites formerly dominated by A. arenaria. Our results also show that dune plant species richness was lower at A. breviligulata sites, although there was an increase in the native beach grass Elymus mollis. More significantly, we found that the two grass species are associated with significantly different foredune shapes that are likely controlled by a combination of variability in sand supply along the coast and subtle differences in the congeners’ morphology and growth form. These differences have significant implications for the coastal protection services of dunes to humans and the conservation of native species. They provide a cautionary tale on the impacts of introducing novel species based purely on analogy with closely related species.     

Interaction of peroxidized cardiolipin with rat-heart mitochondrial membranes: induction of permeability transition and cytochrome c release

Cardiolipin peroxidation plays a critical role in mitochondrial cytochrome c release and subsequent apoptotic process. Mitochondrial pore transition (MPT) is considered as an important step in this process. In this work, the effect of peroxidized cardiolipin on MPT induction and cytochrome c release in rat heart mitochondria was investigated. Treatment of mitochondria with micromolar concentrations of cardiolipin hydroperoxide (CLOOH) resulted in a dose-dependent matrix swelling, DeltaPsi collapse, release of preaccumulated Ca2+ and release of cytochrome c. All these events were inhibited by cyclosporin A and bongkrekic acid, indicating that peroxidized cardiolipin behaves as an inducer of MPT. Ca2+ accumulation by mitochondria was required for this effect. ANT (ADP/ATP translocator) appears to be involved in the CLOOH-dependent MPT induction, as suggested by the modulation by ligands and inhibitors of adenine nucleotide translocator (ANT). Together, these results indicate that peroxidized cardiolipin lowers the threshold of Ca2+ for MPT induction and cytochrome c release. This synergistic effect of Ca2+ and peroxidized cardiolipin on MPT induction and cytochrome c release in mitochondria, might be important in regulating the initial phase of apoptosis and also may have important implications in those physiopathological situations, characterized by both Ca2+ and peroxidized cardiolipin accumulation in mitochondria, such as aging, ischemia/reperfusion and other degenerative diseases.     

Chemical denaturation of the elongation factor 1alpha isolated from the hyperthermophilic archaeon Sulfolobus solfataricus

The stability against chemical denaturants of the elongation factor EF-1alpha (SsEF-1alpha), a protein isolated from the hyperthermophilic archaeon Sulfolobus solfataricus has been characterized in detail. Indeed, the atypical shape of the protein structure and the unusual living conditions of the host organism prompted us to analyze the effect of urea and guanidine hydrochloride (GuHCl) on the GDP complex of the enzyme (SsEF-1alpha x GDP) by fluorescence and circular dichroism. These studies were also extended to the nucleotide-free form of the protein (nfSsEF-1alpha). Interestingly, the experiments show that the denaturation curves of both SsEF-1alpha forms present a single inflection point, which is indicative of a cooperative unfolding process with no intermediate species. Moreover, the chemically induced unfolding process of both SsEF-1alpha x GDP and nfSsEF-1alpha is fully reversible. Both SsEF-1alpha forms exhibit remarkable stability against urea, but they do not display a strong resistance to the denaturing action of GuHCl. These findings suggest that electrostatic interactions significantly contribute to SsEF-1alpha stability.     

Melatonin inhibits cardiolipin peroxidation in mitochondria and prevents the mitochondrial permeability transition and cytochrome c release

Cardiolipin oxidation is emerging as an important factor in mitochondrial dysfunction as well as in the initial phase of the apoptotic process. We have previously shown that exogenously added peroxidized cardiolipin sensitizes mitochondria to Ca²⁺-induced mitochondrial permeability transition (MPT) pore opening and promotes the release of cytochrome c. In this work, the effects of intramitochondrial cardiolipin peroxidation on Ca²⁺-induced MPT and on the cytochrome c release from mitochondria were studied. The effects of melatonin, a compound known to protect the mitochondria from oxidative damage, on both of these processes were also tested. tert-Butylhydroperoxide (t-BuOOH), a lipid-soluble peroxide that promotes lipid peroxidation, was used to induce intramitochondrial cardiolipin peroxidation. Exposure of heart mitochondria to t-BuOOH resulted in the oxidation of cardiolipin, associated with an increased sensitivity of mitochondria to Ca²⁺-induced MPT and with the release of cytochrome c from the mitochondria. All these processes were inhibited by micromolar concentrations of melatonin. It is proposed that melatonin inhibits cardiolipin peroxidation in mitochondria, and this effect seems to be responsible for the protection afforded by this agent against the MPT induction and cytochrome c release. Thus, manipulating the oxidation sensitivity of cardiolipin with melatonin may help to control MPT and cytochrome c release, events associated with cell death, and thus, be used for treatment of those disorders characterized by mitochondrial cardiolipin oxidation and Ca²⁺ overload.     

Identification of Critical Residues of the MyD88 Death Domain Involved in the Recruitment of Downstream Kinases

MyD88 couples the activation of the Toll-like receptors and interleukin-1 receptor superfamily with intracellular signaling pathways. Upon ligand binding, activated receptors recruit MyD88 via its Toll-interleukin-1 receptor domain. MyD88 then allows the recruitment of the interleukin-1 receptor-associated kinases (IRAKs). We performed a site-directed mutagenesis of MyD88 residues, conserved in death domains of the homologous FADD and Pelle proteins, and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of residues 52 (MyD88_E52A) and 58 (MyD88_Y58A) impaired recruitment of both IRAK1 and IRAK4, whereas mutation of residue 95 (MyD88_K95A) only affected IRAK4 recruitment. Since all MyD88 mutants were defective in signaling, recruitment of both IRAKs appeared necessary for activation of the pathway. Moreover, overexpression of a green fluorescent protein (GFP)-tagged mini-MyD88 protein (GFP-MyD88-(27–72)), comprising the Glu⁵² and Tyr⁵⁸ residues, interfered with recruitment of both IRAK1 and IRAK4 by MyD88 and suppressed NF-κB activation by the interleukin-1 receptor but not by the MyD88-independent TLR3. GFP-MyD88-(27–72) exerted its effect by titrating IRAK1 and suppressing IRAK1-dependent NF-κB activation. These experiments identify novel residues of MyD88 that are crucially involved in the recruitment of IRAK1 and IRAK4 and in downstream propagation of MyD88 signaling.MyD88 was first discovered during studies addressing the differentiation of mouse myeloid cells in response to growth-inhibitory stimuli (1). Subsequent investigations revealed that MyD88 possesses a modular organization (2), with an amino-terminal death domain (DD),³ found in proteins involved in cell death (3, 4), and a carboxyl-terminal Toll-interleukin-1 receptor (TIR) domain, present in the intracytoplasmic tail of receptors belonging to the Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) superfamily (5). MyD88 also has an intermediate domain (ID) that is crucial in TLR signaling due to its interaction with IRAK4 (6). The role of MyD88 as a signal transducer was first shown in the pathways triggered by the activation of IL-1R (7, 8) and TLR4 (9). Further studies showed that all TLRs, with the sole exception of TLR3, and the IL-1R family utilize the adaptor protein MyD88 to initiate their signaling pathway (10).By virtue of its modular organization, MyD88 critically bridges activated receptor complexes to downstream adaptors/effectors. Upon activation, MyD88 is recruited through its TIR domain by the homologous domain of the activated TLR/IL-1R (11, 12). MyD88, in turn, has been shown to interact with a family of downstream kinases, namely IRAK1 (13), IRAK2 (7), IRAK-M (15), and IRAK4 (16), through the interaction of its DD with the respective DDs present in the amino-terminal region of IRAKs (17). At this stage, this multimeric complex is competent to elicit the propagation of the signal downstream of the receptor(s). Although MyD88 recruits IRAK-1 via DD-DD interactions, its recruitment of IRAK-4 appears to be rather unusual. Burns et al. (6) first demonstrated that an alternatively spliced variant of MyD88 (MyD88s), lacking the ID domain, failed to interact with IRAK-4, suggesting that residues located in both the DD and ID of MyD88 are crucially involved in the recruitment of IRAK-4. Nevertheless, no information is available on the specific residues in the DD in MyD88 required for its interaction with either IRAK1 or IRAK4.The DD was initially defined as the region of homology between the cytoplasmic tails of the FAS/Apo1/CD95 and TNF receptors required for their induction of cytotoxic signaling (18, 19). In analogy with other DD-containing proteins, this domain in MyD88 is also involved in the formation of homomeric and heteromeric interactions. Herein, we have undertaken an alanine-scanning mutational analysis to identify amino acids that are required for downstream signaling and might participate in the homomeric and heteromeric interactions. Our studies revealed that MyD88_E52A and MyD88_Y58A mutants are strongly impaired in the recruitment of both IRAK1 and IRAK4, whereas the MyD88_K95A mutant is deficient in recruiting IRAK4. These findings identify residues within the DD of MyD88 crucially involved in the formation of higher order complexes containing IRAK1 and IRAK4 and required for the propagation of the TLR/IL1-R signaling pathways.