Frataxin and mitochondrial carrier proteins, Mrs3p and Mrs4p, cooperate in providing iron for heme synthesis

Frataxin is a conserved mitochondrial protein implicated in cellular iron metabolism. Deletion of the yeast frataxin homolog (YFH1) was combined with deletions of MRS3 and MRS4, mitochondrial carrier proteins implicated in iron homeostasis. As previously reported, the Deltayfh1 mutant accumulated iron in mitochondria, whereas the triple mutant (DeltaDeltaDelta) did not. When wild-type, Deltamrs3/4, Deltayfh1, and DeltaDeltaDelta strains were incubated anaerobically, all strains were devoid of heme and protected from iron and oxygen toxicity. The cultures were then shifted to air for a short time (4-5 h) or a longer time (15 h), and the evolving mutant phenotypes were analyzed (heme-dependent growth, total heme, cytochromes, heme proteins, and iron levels). A picture emerges from these data of defective heme formation in the mutants, with a markedly more severe defect in the DeltaDeltaDelta than in the individual Deltamrs3/4 or Deltayfh1 mutants (a "synthetic" defect in the genetic sense). The defect(s) in heme formation could be traced to lack of iron. Using a real time assay of heme biosynthesis, porphyrin precursor and iron were presented to permeabilized cells, and the appearance and disappearance of fluorescent porphyrins were followed. The Mrs3/4p carriers were required for rapid iron transport into mitochondria for heme synthesis, whereas there was also evidence for an alternative slower system. A different role for Yfh1p was observed under conditions of low mitochondrial iron and aerobic growth (revealed in the DeltaDeltaDelta), acting to protect bioavailable iron within mitochondria and to facilitate its use for heme synthesis.     

Akt-dependent expression of NAIP-1 protects neurons against amyloid-{beta} toxicity

Neurotrophins are a family of growth factors that attenuate several forms of pathological neuronal cell death and may represent a putative therapeutic approach to neurodegenerative diseases. In Alzheimer disease, amyloid-beta (Abeta) is thought to play a central role in the neuronal death occurring in brains of patients. In the present study, we evaluate the ability of neurotrophin-3 (NT-3) to protect neurons against the toxicity induced by aggregated Abeta. We showed that in primary cultures of cortical neurons, NT-3 reduces Abeta-induced apoptosis by limiting caspase-8, caspase-9, and caspase-3 cleavage. This neuroprotective effect of NT-3 was concomitant to an increased level of Akt phosphorylation and was abolished by an inhibitor of the phosphatidylinositol-3 kinase (PI-3K), LY294002. In parallel, NT-3 treatment reduced Abeta induced caspase-3 processing to control levels. In an attempt to link PI-3K/Akt to caspase inhibition, we evaluated the influence of the PI-3K/Akt axis on the expression of a member of the inhibitors of apoptosis proteins (IAPs), the neuronal apoptosis inhibitory protein-1. We demonstrated that NT-3 induces an up-regulation of neuronal apoptosis inhibitory protein-1 expression in neurons that promotes the inhibition of Abeta-induced neuronal apoptosis. Together, these findings demonstrate that NT-3 signaling counters Abeta-dependent neuronal cell death and may represent an innovative therapeutic intervention to limit neuronal death in Alzheimer disease.     

Bacillus cereus produces several nonproteinaceous insecticidal exotoxins

Bacillus cereus is mainly known as a human food-borne opportunistic pathogen. Here, we used biological assays and HPLC to investigate the ability of B. cereus to produce insecticidal exotoxins during the stationary growth phase. None of the 575 B. cereus strains screened produced detectable levels of β-exotoxin I, a small, heat-stable insecticidal nucleotide analogue. However, six out of a subset of 270 B. cereus strains produced several small, nonproteinaceous insecticidal exotoxins different from β-exotoxin I. Thus, B. cereus can secrete a large array of proteinaceous and nonproteinaceous toxins acting on insects and mammals.     

Angiotensin-converting enzyme inhibitor captopril prevents volume overload cardiomyopathy in experimental chronic aortic valve regurgitation

The efficacy of angiotensin-converting enzyme inhibitors (ACEIs) in the treatment of chronic aortic regurgitation (AR) is not well established and remains controversial. The mechanisms by which ACEIs may protect against left-ventricular (LV) volume overload are not well understood, and clinical trials performed until now have yielded conflicting results. This study was therefore performed to assess the effectiveness of two different doses of the ACEI captopril in a rat model of chronic AR. We compared the effects of a 6-month low-dose (LD) (25 mg/kg) or higher dose (HD) (75 mg/kg) treatment with captopril on LV function and hypertrophy in Wistar rats with severe AR. Untreated animals developed LV eccentric hypertrophy and systolic dysfunction. LD treatment did not prevent hypertrophy and provided modest protection against systolic dysfunction. HD treatment preserved LV systolic function and dimensions and tended to slow hypertrophy. The cardiac index remained high and similar among all AR groups, treated or not. Tissue renin-angiotensin system (RAS) analysis revealed that ACE activity was increased in the LVs of AR animals and that only HD treatment significantly decreased angiotensin II receptor mRNA levels. Fibronectin expression was increased in the LV or AR animals, but HD treatment almost completely reversed this increase. The ACE inhibitor captopril was effective at high doses in this model of severe AR. These effects might be related to the modulation of tissue RAS and the control of fibrosis.     

Targeting of the dual oxidase 2 N-terminal region to the plasma membrane

Dual oxidase 2 (Duox2) is a cell surface glycoprotein that probably provides thyroperoxidase with the H2O2 required to catalyze thyroid hormone synthesis. No functional H2O2-generating system has yet been obtained after transfecting Duox2 into non-thyroid cell lines, because it is retained in the endoplasmic reticulum (ER). We investigated the level of maturation of various Duox2 truncated proteins in an attempt to identify the region of Duox2 responsible for its remaining in the ER. Duox2-Q686X mutant, corresponding to the N-terminal ectodomain including the first putative transmembrane domain, was expressed in different cell lines. Carbohydrate content analysis revealed that complex type-specific Golgi apparatus (GA) oligosaccharides were present on pig Duox2-Q686X, whereas human truncated Duox2 carried only high mannose-type sugar chains characteristic of the ER. Further characterization using surface biotinylation and flow cytometry assays indicated that pig Duox2-Q686X was present at the plasma membrane, whereas human Duox2-Q686X remained inside the cell. The replacement of the last 90 residues of the human Duox2-Q686X with the pig equivalent region allowed the chimerical peptide to reach the Golgi apparatus. Pig mutants containing the complete first intracellular loop with or without the second transmembrane domain accumulated in the ER. These findings show that 1) the human Duox2-Q686X region encompassing residues 596-685 prevents mutant exportation from the ER and 2) there is a pig Duox2 retention domain in the first intracellular loop. In addition, missense mutations of four cysteines (Cys-351, -370, -568, or -582) completely inhibited the emergence of pig Duox2-Q686X from the ER compartment, indicating their importance in Duox2 maturation.     

Quantitative 2H NMR at natural abundance can distinguish the pathway used for glucose fermentation by lactic acid bacteria

For any given metabolic pathway, isotope redistribution coefficients (a(ij)) that characterize the specific derivation of each hydrogen atom can be defined. By using quantitative deuterium NMR, the redistribution of deuterium at natural abundance in lactic acid produced by the bacterial fermentation of glucose has been determined for each non-labile hydrogen atom of glucose or water and the hydrogen atoms of lactic acid. Distinct differences are observed in the lactic acid isolated from Lactococcus lactis and Leuconostoc mesenteroides that can be interpreted in terms of the different fermentative pathways used. Specifically, the affiliations observed between the H1, H3, and H4 positions of glucose with methyl and hydroxymethylene of lactic acid can give quantitative information on whether the glycolytic or the reductive pentose-phosphate pathway was involved in glucose catabolism.     

Organization of functional domains in the docking protein p130Cas

The docking protein p130Cas becomes phosphorylated upon cell adhesion to extracellular matrix proteins, and is thought to play an essential role in cell transformation. Cas transmits signals through interactions with the Src-homology 3 (SH3) and Src-homology 2 domains of FAK or v-Crk signaling molecules, or with 14-3-3 protein, as well as phosphatases PTP1B and PTP-PEST. The large (130kDa), multi-domain Cas molecule contains an SH3 domain, a Src-binding domain, a serine-rich protein interaction region, and a C-terminal region that participates in protein interactions implicated in antiestrogen resistance in breast cancer. In this study, as part of a long-term goal to examine the protein interactions of Cas by X-ray crystallography and nuclear magnetic resonance spectroscopy, molecular constructs were designed to express two adjacent domains, the serine-rich domain and the Src-binding domain, that each participate in intermolecular contacts dependent on protein phosphorylation. The protein products are soluble, homogeneous, monodisperse, and highly suitable for structural studies to define the role of Cas in integrin-mediated cell signaling.     

FGF6 mediated expansion of a resident subset of cells with SP phenotype in the C2C12 myogenic line

Fibroblast growth factor 6 (FGF6) is selectively expressed during muscle development and regeneration. We examined its effect on muscle precursor cells (mpc) by forcing stable FGF6 expression in C2C12 cells in vitro. FGF6 produced in genetically engineered mpc was active, inducing strong morphological changes, altering cell adhesion and compromising their ability to differentiate into myotubes. Expression of MyoD and myogenin, but not of Myf5, was abrogated in FGF6 engineered mpc. These effects were reversed by FGF inhibitors. Ectopic expression of MyoD also restored fiber formation indicating that FGF6 interferes with the myogenic differentiation pathway upstream of MyoD. We also report that in the presence of FGF6, the minor (0.5-2%) subpopulation of cells actively excluding Hoechst 33342 in a verapamil-dependent manner (SP phenotype) was increased to 15-20% and the expression of the mdr1a gene (but not mdr1b) was upregulated by 400-fold. Our data establish a previously undescribed link between FGF6--a muscle specific growth factor--and a multidrug resistance gene expressed in stem cells, and suggest a role for FGF6 in the maintenance of a reserve pool of progenitor cells in the skeletal muscle.     

Intratumoral CC chemokine ligand 5 overexpression delays tumor growth and increases tumor cell infiltration

Chemokines participate in the antitumor immune response by regulating the movement and positioning of lymphocytes as well as effector functions and may thus be candidates for use in antitumor therapy. To test whether CCL5, a chemokine involved in the recruitment of a wide spectrum of immunocompetent cells, can control tumor growth, we forced its expression at mouse tumor sites. Tumor growth was reduced in mice with s.c. syngeneic CCL5-EL-4 compared with EL-4-injected mice, whereas both reduced tumor growth and incidence were observed in mice with OVA-expressing EG-7 transfected with CCL5 compared with EG-7-injected mice. Significant antitumor effects were observed soon after intratumoral injection of DNA plasmid coding for chimeric CCL5-Ig. Importantly, quantitative RT-PCR assays showed that the amount of CCL5 expression at the tumor site determined the effectiveness of the antitumor response, which was associated with infiltration of increased numbers of NK, CD4, and CD8 cells at the tumor site. This effect was lost in mice deficient for T/B lymphocytes (RAG-2 knockout) or for CCR5 (CCR5 knockout). Together, these data demonstrate the antitumor activity of intratumoral CCL5 overexpression, due to its recruitment of immunocompetent cells, and the potential usefulness of chimeric CCL5-Ig DNA as an agent in cancer therapy.