Cutting edge: microbial products elicit formation of dendritic cell aggresome-like induced structures in macrophages

In response to a maturation stimulus, dendritic cells undergo the formation of ubiquitinated protein aggregates known as dendritic cell aggresome-like induced structures (DALIS). DALIS are thought to act as Ag storage structures, allowing for the prioritized degradation of proteins during infection. In this study, we demonstrate that murine macrophages can also form ubiquitinated protein aggregates that are indistinguishable from DALIS. These were formed in a dose- and time-dependent manner, and in response to a variety of microbial products. Surprisingly, the proteasome did not accumulate on these ubiquitinated protein structures, further underlining the difference between DALIS and aggresomes. Our studies suggest that DALIS formation is important for the function of Ag-presenting immune cells during infection.     

The life span determinant p66Shc localizes to mitochondria where it associates with mitochondrial heat shock protein 70 and regulates trans-membrane potential

P66Shc regulates life span in mammals and is a critical component of the apoptotic response to oxidative stress. It functions as a downstream target of the tumor suppressor p53 and is indispensable for the ability of oxidative stress-activated p53 to induce apoptosis. The molecular mechanisms underlying the apoptogenic effect of p66Shc are unknown. Here we report the following three findings. (i) The apoptosome can be properly activated in vitro in the absence of p66Shc only if purified cytochrome c is supplied. (ii) Cytochrome c release after oxidative signals is impaired in the absence of p66Shc. (iii) p66Shc induces the collapse of the mitochondrial trans-membrane potential after oxidative stress. Furthermore, we showed that a fraction of cytosolic p66Shc localizes within mitochondria where it forms a complex with mitochondrial Hsp70. Treatment of cells with ultraviolet radiation induced the dissociation of this complex and the release of monomeric p66Shc. We propose that p66Shc regulates the mitochondrial pathway of apoptosis by inducing mitochondrial damage after dissociation from an inhibitory protein complex. Genetic and biochemical evidence suggests that mitochondria regulate life span through their effects on the energetic metabolism (mitochondrial theory of aging). Our data suggest that mitochondrial regulation of apoptosis might also contribute to life span determination.     

Examination of potential overlap in autism and language loci on chromosomes 2, 7, and 13 in two independent samples ascertained for specific language impairment

Specific language impairment is a neurodevelopmental disorder characterized by impairments essentially restricted to the domain of language and language learning skills. This contrasts with autism, which is a pervasive developmental disorder defined by multiple impairments in language, social reciprocity, narrow interests and/or repetitive behaviors. Genetic linkage studies and family data suggest that the two disorders may have genetic components in common. Two samples, from Canada and the US, selected for specific language impairment were genotyped at loci where such common genes are likely to reside. Significant evidence for linkage was previously observed at chromosome 13q21 in our Canadian sample (HLOD 3.56) and was confirmed in our US sample (HLOD 2.61). Using the posterior probability of linkage (PPL) to combine evidence for linkage across the two samples yielded a PPL over 92%. Two additional loci on chromosome 2 and 7 showed weak evidence for linkage. However, a marker in the cystic fibrosis transmembrane conductance regulator (7q31) showed evidence for association to SLI, confirming results from another group (O'Brien et al. 2003). Our results indicate that using samples selected for components of the autism phenotype may be a useful adjunct to autism genetics.     

Influence of compartmental localization on the function of yeast NADP+-specific isocitrate dehydrogenases

Three differentially compartmentalized isozymes of isocitrate dehydrogenase (mitochondrial IDP1, cytosolic IDP2, and peroxisomal IDP3) in the yeast Saccharomyces cerevisiae catalyze the NADP(+)-dependent oxidative decarboxylation of isocitrate to form alpha-ketoglutarate. These enzymes are highly homologous but exhibit some significant differences in physical and kinetic properties. To examine the impact of these differences on physiological function, we exchanged promoters and altered organellar targeting information to obtain expression of IDP2 and IDP3 in mitochondria and of IDP1 and IDP3 in the cytosol. Physiological function was assessed as complementation by mislocalized isozymes of defined growth defects of isocitrate dehydrogenase mutant strains. These studies revealed that the IDP isozymes are functionally interchangeable for glutamate synthesis, although mitochondrial localization has a positive impact on this function during fermentative growth. However, IDP2, whether located in mitochondria or in the cytosol, provided the highest level of defense against endogenous or exogenous oxidative stress.     

Structure Analysis of Two <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> and <Emphasis Type="BoldItalic">Neospora caninum</Emphasis> Satellite DNA Families and Evolution of Their Common Monomeric Sequence

A family of repetitive DNA elements of approximately 350 bp—Sat350—that are members of Toxoplasma gondii satellite DNA was further analyzed. Sequence analysis identified at least three distinct repeat types within this family, called types A, B, and C. B repeats were divided into the subtypes B1 and B2. A search for internal repetitions within this family permitted the identification of conserved regions and the design of PCR primers that amplify almost all these repetitive elements. These primers amplified the expected 350-bp repeats and a novel 680-bp repetitive element (Sat680) related to this family. Two additional tandemly repeated high-order structures corresponding to this satellite DNA family were found by searching the Toxoplasma genome database with these sequences. These studies were confirmed by sequence analysis and identified: (1) an arrangement of AB1CB2 350-bp repeats and (2) an arrangement of two 350-bp-like repeats, resulting in a 680-bp monomer. Sequence comparison and phylogenetic analysis indicated that both high-order structures may have originated from the same ancestral 350-bp repeat. PCR amplification, sequence analysis and Southern blot showed that similar high-order structures were also found in the Toxoplasma-sister taxon Neospora caninum. The Toxoplasma genome database ( ) permitted the assembly of a contig harboring Sat350 elements at one end and a long nonrepetitive DNA sequence flanking this satellite DNA. The region bordering the Sat350 repeats contained two differentially expressed sequence-related regions and interstitial telomeric sequences.     

ALIS are stress-induced protein storage compartments for substrates of the proteasome and autophagy

Misfolded proteins can be directed into cytoplasmic aggregates such as aggresomes and dendritic cell aggresome-like induced structures (DALIS). DALIS were originally identified in lipopolysaccharide-stimulated dendritic cells and act as storage compartments for polyubiquitinated Defective Ribosomal Products (DRiPs) prior to their clearance by the proteasome. Here we demonstrate that ubiquitinated protein aggregates that are similar to DALIS, and not related to aggresomes, can be observed in several cell types in response to stress, including oxidative stress, transfection, and starvation. Significantly, both immune and nonimmune cells could form these aggresome-like induced structures (ALIS). Protein synthesis was essential for ALIS formation in response to oxidative stress, indicating that DRiP formation was required. Furthermore, puromycin, which increases DRiP formation, was sufficient to induce ALIS formation. Inhibition of either proteasomes or of autophagy interfered with ALIS clearance in puromycin treated cells. Autophagy inhibition enhanced ALIS formation under a variety of stress conditions. During starvation, ALIS formation in autophagy-deficient cells was only partially inhibited by protein synthesis inhibitors, indicating that both long-lived proteins and DRiPs can be targeted to ALIS. Together, these findings demonstrate that ALIS act as generalized stress-induced protein storage compartments for substrates of the proteasome and autophagy.     

Follicular wave status at the beginning of the FSH treatment modifies reproductive features in superovulated sheep

In the current study we investigated whether the developmental status of the two largest follicles (LF1 and LF2) at the time of administration of the first two doses (0 and 12 h) of FSH of a superovulatory treatment influences periovulatory events and embryo yields in sheep. A larger size of LF1 was negatively correlated with embryo recovery (r=-0.608 for 0 h and r=-523 for 12 h, p<0.05), fertilization (r=-0.464 for 12 h, p<0.05) and viability (r=-0.775 for 12 h, p<0.005). Embryo viability rates were also lower when a higher difference between LF1 and LF2 (r=-0.839 for 0 h and r=-0.761 for 12 h, p<0.01) and a smaller size of LF2 (r=0.877 for 0 h and r=0.622 for 12 h, p<0.01) were observed. This indicates the existence of a limit in the follicular size that will be able to give rise a viable embryo. Conversely, a larger size of LF2 at the time of administration of the first two FSH doses was correlated with reduced recovery rates (r=-0.884 for 0 h and r=-0.706 for 12 h, p<0.01), whilst a decreasing size of LF1 and LF2 was correlated with an increased ovulation rate and recovered embryos. The dominance effect appeared to affect the timing of the preovulatory LH surge. Ewes with a higher difference between LF1 and LF2 displayed earlier LH surges (r=-0.420 for 0 h and r=-0.401 for 12 h, p<0.05) which were related to a higher number of non viable embryos (r=-0.777, p<0.05). The fact that superovulatory yields were affected by, both LF1 and LF2 supports the hypothesis of co-dominance effects in sheep.     

The Role of the Formamide/Zirconia System in the Synthesis of Nucleobases and Biogenic Carboxylic Acid Derivatives

We describe the one-pot synthesis of a large variety of nucleic acid bases and related compounds from formamide in the presence of zirconium minerals as catalysts. The major products observed are: purine, 2-hydroxy pyrimidine, 5-hydroxy pyrimidine, isocytosine, adenine, urea, and carbodiimide. The synthesis of low molecular weight amides and carboxylic acid derivatives (intermediates of extant metabolism) was also observed: glyoxylamide, glycolic-, lactic-, succinic-, oxalic-, fumaric-, and maleic acids. As the major problem in the origin of informational polymers is the instability of their precursors, we also investigated the effects of zirconia minerals on the stability of ribooligonucleotides in formamide and in water. The relevance of these findings with respect to the origin of informational polymers and primordial metabolism is discussed.     

Heme-binding Protein HRG-1 Is Induced by Insulin-like Growth Factor I and Associates with the Vacuolar H+-ATPase to Control Endosomal pH and Receptor Trafficking

Endocytosis and trafficking of receptors and nutrient transporters are dependent on an acidic intra-endosomal pH that is maintained by the vacuolar H⁺-ATPase (V-ATPase) proton pump. V-ATPase activity has also been associated with cancer invasiveness. Here, we report on a new V-ATPase-associated protein, which we identified in insulin-like growth factor I (IGF-I) receptor-transformed cells, and which was separately identified in Caenorhabditis elegans as HRG-1, a member of a family of heme-regulated genes. We found that HRG-1 is present in endosomes but not in lysosomes, and it is trafficked to the plasma membrane upon nutrient withdrawal in mammalian cells. Suppression of HRG-1 with small interfering RNA causes impaired endocytosis of transferrin receptor, decreased cell motility, and decreased viability of HeLa cells. HRG-1 interacts with the c subunit of the V-ATPase and enhances V-ATPase activity in isolated yeast vacuoles. Endosomal acidity and V-ATPase assembly are decreased in cells with suppressed HRG-1, whereas transferrin receptor endocytosis is enhanced in cells that overexpress HRG-1. Cellular uptake of a fluorescent heme analogue is enhanced by HRG-1 in a V-ATPase-dependent manner. Our findings indicate that HRG-1 regulates V-ATPase activity, which is essential for endosomal acidification, heme binding, and receptor trafficking in mammalian cells. Thus, HRG-1 may facilitate tumor growth and cancer progression.     

Leptin modulates the survival of autoreactive CD4+ T cells through the nutrient/energy-sensing mammalian target of rapamycin signaling pathway

Chronic inflammation can associate with autoreactive immune responses, including CD4(+) T cell responses to self-Ags. In this paper, we show that the adipocyte-derived proinflammatory hormone leptin can affect the survival and proliferation of autoreactive CD4(+) T cells in experimental autoimmune encephalomyelitis, an animal model of human multiple sclerosis. We found that myelin olygodendrocyte glycoprotein peptide 35-55 (MOG(35-55))-specific CD4(+) T cells from C57BL/6J wild-type mice could not transfer experimental autoimmune encephalomyelitis into leptin-deficient ob/ob mice. Such a finding was associated with a reduced proliferation of the transferred MOG(35-55)-reactive CD4(+) T cells, which had a reduced degradation of the cyclin-dependent kinase inhibitor p27(kip1) and ERK1/2 phosphorylation. The transferred cells displayed reduced Th1/Th17 responses and reduced delayed-type hypersensitivity. Moreover, MOG(35-55)-reactive CD4(+) T cells in ob/ob mice underwent apoptosis that associated with a downmodulation of Bcl-2. Similar results were observed in transgenic AND-TCR- mice carrying a TCR specific for the pigeon cytochrome c 88-104 peptide. These molecular events reveal a reduced activity of the nutrient/energy-sensing AKT/mammalian target of rapamycin pathway, which can be restored in vivo by exogenous leptin replacement. These results may help to explain a link between chronic inflammation and autoimmune T cell reactivity.