Effects of indole-3-acetic acid on <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> survival and on symbiotic nitrogen fixation and stem dry weight production

We evaluated the effects of the main auxin phytohormone, indole-3-acetic acid (IAA), on the central metabolism of Sinorhizobium meliloti 1021. We either treated S. meliloti 1021 wild-type cells with 0.5 mM IAA, 1021+, or use a derivative, RD64, of the same strain harboring an additional pathway for IAA biosynthesis (converting tryptophan into IAA via indoleacetamide). We assayed the activity of tricarboxylic acid cycle (TCA) key enzymes and found that activity of citrate synthase and α-ketoglutarate dehydrogenase were increased in both 1021+ and RD64 as compared to the wild-type strain. We also showed that the intracellular acetyl-CoA content was enhanced in both RD64 and 1021+ strains when compared to the control strain. The activity of key enzymes, utilizing acetyl-CoA for poly-β-hydroxybutyrate (PHB) biosynthesis, was also induced. The PHB level measured in these cells were significantly higher than that found in control cells. Moreover, 4-week-long survival experiments showed that 80% of 1021 cells died, whereas 50% of RD64 cells were viable. Medicago truncatula plants nodulated by RD64 (Mt-RD64) showed an induction of both acetylene reduction activity and stem dry weight production.     

RAD 001 (everolimus) prevents mTOR and Akt late re‐activation in response to imatinib in chronic myeloid leukemia

The mammalian target of rapamycin (mTOR) is one target of BCR‐ABL fusion gene of chronic myeloid leukemia (CML). Moreover, it drives a compensatory route to Imatinib mesylate (IM) possibly involved in the progression of leukemic progenitors towards a drug‐resistant phenotype. Accordingly, mTOR inhibitors are proposed for combined therapeutic strategies in CML. The major caveat in the use of mTOR inhibitors for cancer therapy comes from the induction of an mTOR‐phosphatidylinositol 3 kinase (PI3k) feedback loop driving the retrograde activation of Akt. Here we show that the rapamycin derivative RAD 001 (everolimus, Novartis Institutes for Biomedical Research) inhibits mTOR and, more importantly, revokes mTOR late re‐activation in response to IM. RAD 001 interferes with the assembly of both mTOR complexes: mTORC1 and mTORC2. The inhibition of mTORC2 results in the de‐phosphorylation of Akt at Ser⁴⁷³ in the hydrophobic motif of C‐terminal tail required for Akt full activation and precludes Akt re‐phosphorylation in response to IM. Moreover, RAD 001‐induced inhibition of Akt causes the de‐phosphorylation of tuberous sclerosis tumor suppressor protein TSC2 at 14‐3‐3 binding sites, TSC2 release from 14‐3‐3 sigma (restoring its inhibitory function on mTORC1) and nuclear import (promoting the nuclear translocation of cyclin‐dependent kinase [CDK] inhibitor p27^Kip1, the stabilization of p27^Kip1 ligand with CDK2, and the G₀/G₁ arrest). RAD 001 cytotoxicity on cells not expressing the BCR‐ABL fusion gene or its p210 protein tyrosine kinase (TK) activity suggests that the inhibition of normal hematopoiesis may represent a drug side effect. J. Cell. Biochem. 109: 320–328, 2010. © 2009 Wiley‐Liss, Inc.     

Nonsynonymous Mutations within APOB in Human Familial Hypobetalipoproteinemia: EVIDENCE FOR FEEDBACK INHIBITION OF LIPOGENESIS AND POSTENDOPLASMIC RETICULUM DEGRADATION OF APOLIPOPROTEIN B*

Five nontruncating missense APOB mutations, namely A31P, G275S, L324M, G912D, and G945S, were identified in heterozygous carriers of familial hypobetalipoproteinemia (FHBL) in the Italian population. To test that the FHBL phenotype was a result of impaired hepatic secretion of mutant apoB proteins, we performed transfection studies using McA-RH7777 cells stably expressing wild type or mutant forms of human apolipoprotein B-48 (apoB-48). All mutant proteins displayed varied impairment in secretion, with G912D the least affected and A31P barely secreted. Although some A31P was degraded by proteasomes, a significant proportion of it (although inappropriately glycosylated) escaped endoplasmic reticulum (ER) quality control and presented in the Golgi compartment. Degradation of the post-ER A31P was achieved by autophagy. Expression of A31P also decreased secretion of endogenous apoB and triglycerides, yet the impaired lipoprotein secretion did not lead to lipid accumulation in the cells or ER stress. Rather, expression of genes involved in lipogenesis was down-regulated, including liver X receptor α, sterol regulator element-binding protein 1c, fatty acid synthase, acetyl-CoA carboxylase 1, stearoyl-CoA desaturase 1, and lipin-1. These results suggest that feedback inhibition of hepatic lipogenesis in conjunction with post-ER degradation of misfolded apoB proteins can contribute to reduce fat accumulation in the FHBL liver.     

Dexamethasone induces apoptosis in human T cell clones expressing low levels of Bcl-2

Previous results of ours have demonstrated that the same clonotype can express both a sensitive and a resistant phenotype to Dex-mediated PCD induction depending on its cell cycle phase. In particular, we demonstrated that human T lymphocytes, arrested in the G0/G1 phase of the cell cycle, are susceptible, while proliferating T cells are resistant to Dex-mediated apoptosis. In this paper, we have further characterized the sensitive and resistant phenotypes and investigated whether a different expression of the apoptotic genes Fas, FasL, Bcl-2, Bcl-x and Bax is involved in the regulation of Dex-mediated apoptosis. The results show that the amount of Bcl-2 expression, that changes during cell cycle phases, determines susceptibility or resistance to apoptosis induced by Dex. In fact, undetectable expression of Bcl-2 in sensitive cells favors Dex-mediated apoptosis while high expression of Bcl-2 in proliferating cells counterbalances apoptosis induction. Moreover, the addition of exogenous IL-2, in the presence of Dex, fails to up-regulate Bcl-2 expression and to revert Dex-mediated apoptotic phenomena.     

Vav-1 and the IKK alpha subunit of I kappa B kinase functionally associate to induce NF-kappa B activation in response to CD28 engagement

We have recently observed that CD28 engagement initiates a signaling pathway leading to the activation of I kappa B kinase (IKK) complex and, consequently, to NF-kappa B activation, and we identified Vav-1 as an important mediator of this function. Here we report for the first time that Vav-1 constitutively associates with IKK alpha in both Jurkat and primary CD4(+) T cells. Vav-1/IKK alpha association is mediated by their helix-loop-helix domains, does not involve IKK beta, and is functionally relevant in that Vav-1-associated IKK alpha kinase activity is increased following CD28 engagement by B7. Moreover, we demonstrate that CD28-induced NF-kappa B activation is augmented by both IKK alpha and Vav-1, but not IKK beta. Confocal microscopy showed that endogenous Vav-1 and IKK alpha, but not IKK beta, were recruited to the membrane and colocalized in response to CD28 stimulation. Taken together, these data evidence that Vav-1 plays a key role in the control of NF-kappa B pathway by targeting IKK alpha in the T cell membrane and favoring its activation in response to CD28 stimulation.     

Reorganization of actin cytoskeleton by the phosphoinositide metabolite glycerophosphoinositol 4-phosphate

Glycerophosphoinositol 4-phosphate (GroPIns-4P) is a biologically active, water-soluble phospholipase A metabolite derived from phosphatidylinositol 4-phosphate, whose cellular concentrations have been reported to increase in Ras-transformed cells. It is therefore important to understand its biological activities. Herein, we have examined whether GroPIns-4P can regulate the organization of the actin cytoskeleton, because this could be a Ras-related function involved in cell motility and metastatic invasion. We find that in serum-starved Swiss 3T3 cells, exogenously added GroPIns-4P rapidly and potently induces the formation of membrane ruffles, and, later, the formation of stress fibers. These actin structures can be regulated by the small GTPases Cdc42, Rac, and Rho. To analyze the mechanism of action of GroPIns-4P, we selectively inactivated each of these GTPases. GroPIns-4P requires active Rac and Rho, but not Cdc42, for ruffle and stress fiber formation, respectively. Moreover, GroPIns-4P induces a rapid translocation of the green fluorescent protein-tagged Rac into ruffles, and increases the fraction of GTP-bound Rac, in intact cells. The activation of Rac by GroPIns-4P was near maximal and long-lasting. Interestingly, this feature seems to be critical in the induction of actin ruffles by GroPIns-4P.     

Effect of caffeic acid on tert-butyl hydroperoxide-induced oxidative stress in U937

Nonvitamin phenolic compounds are ubiquitous in food plants and therefore potentially present in human plasma in a diet-dependent concentration. The aim of this study was to evaluate the ability of caffeic acid, a phenolic acid with antioxidant activity, to affect cellular response in U937 human monocytic cells to t-butyl hydroperoxide-induced oxidative stress. In our experimental conditions caffeic acid was incorporated into cells without any cytotoxic effect. Caffeic acid-treated cells showed an increased resistance to oxidative challenge, as revealed by an higher percent of survival and the maintenance of an higher proliferative capacity in respect to control cells. This effect seems to be due to the ability of caffeic acid to reduce glutathione depletion and to inhibit lipid peroxidation during tBOOH treatment. It can be concluded that caffeic acid exerts an antioxidant action inside the cell, responsible for the observed modulation of the cellular response to oxidative challenge. Due to its presence in the diet, therefore, caffeic acid may play a role in the modulation of oxidative processes in vivo.