Suppression of reactive oxygen species production enhances neuronal survival in vitro and in vivo in the anoxia-tolerant turtle Trachemys scripta

Hypoxia-ischemia with reperfusion is known to cause reactive oxygen species-related damage in mammalian systems, yet, the anoxia tolerant freshwater turtle is able to survive repeated bouts of anoxia/reoxygenation without apparent damage. Although the physiology of anoxia tolerance has been much studied, the adaptations that permit survival of reoxygenation stress have been largely ignored. In this study, we examine ROS production in the turtle striatum and in primary neuronal cultures, and examine the effects of adenosine (AD) on cell survival and ROS. Hydroxyl radical formation was measured by the conversion of salicylate to 2,3-dihydroxybenzoic acid (2,3-DHBA) using microdialysis; reoxygenation after 1 or 4 h anoxia did not result in increased ROS production compared with basal normoxic levels, nor did H₂O₂ increase after anoxia/reoxygenation in neuronally enriched cell cultures. Blockade of AD receptors increased both ROS production and cell death in vitro , while AD agonists decreased cell death and ROS. As turtle neurons proved surprisingly susceptible to externally imposed ROS stress (H₂O₂), we propose that the suppression of ROS formation, coupled to high antioxidant levels, is necessary for reoxygenation survival. As an evolutionarily selected adaptation, the ability to suppress ROS formation could prove an interesting path to investigate new therapeutic targets in mammals.     

Communication between RNA folding domains revealed by folding of circularly permuted ribozymes

To study the role of sequence and topology in RNA folding, we determined the kinetic folding pathways of two circularly permuted variants of the Tetrahymena group I ribozyme, using time-resolved hydroxyl radical footprinting. Circular permutation changes the distance between interacting residues in the primary sequence, without changing the native structure of the RNA. In the natural ribozyme, tertiary interactions in the P4-P6 domain form in 1 s, while interactions in the P3-P9 form in 1-3 min at 42 degrees C. Permutation of the 5' end to G111 in the P4 helix allowed the stable P4-P6 domain to fold in 200 ms at 30 degrees C, five times faster than in the wild-type RNA, while the other domains folded five times more slowly (5-8 min). By contrast, circular permutation of the 5' end to G303 in J8/7 decreased the folding rate of the P4-P6 domain. In this permuted RNA, regions joining P2, P3 and P4 were protected in 500 ms, while the P3-P9 domain was 60-80% folded within 30 s. RNase T(1) digestion and FMN photocleavage showed that circular permutation of the RNA sequence alters the initial ensemble of secondary structures, thereby changing the tertiary folding pathways. Our results show that the natural 5'-to-3' order of the structural domains in group I ribozymes optimizes structural communication between tertiary domains and promotes self-assembly of the catalytic center.     

Large-scale generation of highly enriched neural stem-cell-derived oligodendroglial cultures: maturation-dependent differences in insulin-like growth factor-mediated signal transduction

Multipotent neural stem cells (NSCs) are competent for commitment to the oligodendrocyte (OL) lineage both in vitro and in vivo. We exploited this property to develop a rat neurospheres (NS)/oligospheres (OS)-based culture system to generate large numbers of highly enriched late OL progenitors (preOLs) and mature OLs (MatOLs). CNS neuroblastoma cell line B104-derived conditioned medium promoted the generation of nearly pure populations of preOLs from dissociated OS. The subsequent culture of preOLs with ciliary neurotrophic factor (CNTF) and 3,3',5'-triiodo-L-thyronine (T(3)) generated nearly pure populations of MatOLs. OL lineage specificity was confirmed by immunocytochemistry, quantitative RT-PCR and gene expression profiling, which demonstrated large differences between preOLs and MatOLs. The insulin-like growth factors (IGFs) are potent neuro-protective agents required for OL survival. We used this system to systematically define maturation-dependent changes in IGF signaling during the course of OL differentiation. The IGF-I and insulin receptors, insulin receptor substrate-1 (IRS-1) and IRS-2, protein kinase B (PKB)/Akt and Janus kinase (JNK) were expressed at higher levels in NS and preOLs compared with OS and MatOLs. Erk expression increased markedly from NS to OS, decreased only partially upon commitment to preOLs, and, in MatOLs, returned to a low level similar to NS. IGF activation of the generally proliferative Erk pathway was gradually acquired during NSC differentiation, whereas IGF activation of the generally pro-survival, anti-apoptotic PI3K/PKB pathway was consistently robust at each developmental stage.     

Distribution of sphingosine kinase activity and mRNA in rodent brain

Sphingosine-1-phosphate (S1P) is a lipid mediator that exerts multiple cellular functions through activation of a subfamily of G-protein-coupled receptors. Although there is evidence that S1P plays a role in the developing and adult CNS, little is known about the ability of brain parenchyma to synthesize this lipid. We have therefore analyzed the brain distribution of the enzymatic activity of the S1P synthesizing enzyme, sphingosine kinase (SPHK) [EC:2.7.1.91], as well as mRNA distribution for one of the two isoforms of this enzyme, sphingosine kinase 2. SPHK activity, measured by the conversion of [(3)H]sphingosine to [(3)H]S1P, is highest in cerebellum, followed by cortex and brainstem. Lowest activities were found in striatum and hippocampus. Sensitivity to 0.1% Triton-X suggests that this activity is accounted for by SPHK2. RT-PCR and in situ hybridization studies show that mRNA for this isoform has a distribution similar to that of SPHK activity. In vivo and in vitro ischemia increase SPHK activity and SPHK2 mRNA levels. These results indicate that SPHK2 is the predominant S1P-synthesizing isoform in normal brain parenchyma. Its heterogeneous distribution, in particular laminar distribution in cortex, suggests a neuronal localization and a possible role in cortical and cerebellar functions, in normal as well as ischemic brain.     

Cytomegalovirus infection in Gambian infants leads to profound CD8 T-cell differentiation

Cytomegalovirus (CMV) infection is endemic in Gambian infants, with 62% infected by 3 months and 85% by 12 months of age. We studied the CD8 T-cell responses of infants to CMV following primary infection. CMV-specific CD8 T cells, identified with tetramers, showed a fully differentiated phenotype (CD28(-) CD62L(-) CD95(+) perforin(+) granzyme A(+) Bcl-2(low)). Strikingly, the overall CD8 T-cell population developed a similar phenotype following CMV infection, which persisted for at least 12 months. In contrast, primary infection was accompanied by up-regulation of markers of activation (CD45R0 and HLA-D) on both CMV-specific cells and the overall CD8 T-cell population and division (Ki-67) of specific cells, but neither pattern persisted. At 12 months of age, the CD8 T-cell population of CMV-infected infants was more differentiated than that of uninfected infants. Although the subpopulation of CMV-specific cells remained constant, the CMV peptide-specific gamma interferon response was lower in younger infants and increased with age. As the CD8 T-cell phenotype induced by CMV is indicative of immune dysfunction in the elderly, the existence of a similar phenotype in large numbers of Gambian infants raises the question of whether CMV induces a similarly deleterious effect.     

CARMA1 coiled-coil domain is involved in the oligomerization and subcellular localization of CARMA1 and is required for T cell receptor-induced NF-kappaB activation

T lymphocyte (T cell) activation and proliferation is induced by the activation of multiple signal transduction pathways. Earlier studies indicate that CARMA1, a Caspase Recruitment Domain (CARD) and Membrane-associated GUanylate Kinase domain (MAGUK)-containing scaffold protein, plays an essential role in NF-kappaB activation induced by the costimulation of T cell receptor (TCR) and CD28 molecules. However, the molecular mechanism by which CARMA1 mediates TCR-CD28 costimulation-induced NF-kappaB activation is not fully understood. Here we show that CARMA1 is constitutively oligomerized. This oligomerization of CARMA1 is through its Coiled-coil domain. Disruption of the predicted structure of the Coiled-coil domain of CARMA1 impaired its oligomerization and, importantly, abrogated CARMA1-mediated NF-kappaB activation. Interestingly, disruption of the CC1 domain abrogates CARMA1 localization, whereas disruption of the CC2 domain seems to inhibit CARMA1 self-association. Together, our results demonstrate that the oligomerization of CARMA1 is required for TCR-induced NF-kappaB activation.     

Sphingosine kinase type 2 activation by ERK-mediated phosphorylation

Sphingosine 1-phosphate (S1P), a potent lipid mediator, is a ligand for a family of five G protein-coupled receptors (S1P(1-5)) that have been shown to regulate a variety of biological responses important for cancer progression. The cellular level of S1P is low and tightly regulated in a spatio-temporal manner through its synthesis catalyzed by two sphingosine kinases, denoted SphK1 and SphK2. Many stimuli activate and translocate SphK1 to the plasma membrane by mechanisms that are dependent on its phosphorylation. Much less is known about activation of SphK2. Here we demonstrate that epidermal growth factor (EGF) as well as the protein kinase C activator, phorbol ester, induce rapid phosphorylation of hSphK2 which was markedly reduced by inhibition of MEK1/ERK pathway. Down-regulation of ERK1 blocked EGF-induced phosphorylation of SphK2. Recombinant ERK1 phosphorylated hSphK2 in vitro and increased its enzymatic activity. ERK1 also was found to be in a complex with hSphK2 in vivo. Site-directed mutagenesis indicated that hSphK2 is phosphorylated on Ser-351 and Thr-578 by ERK1 and that phosphorylation of these residues is important for EGF-stimulated migration of MDA-MB-453 cells. These studies provide the first clues to the mechanism of agonist-mediated SphK2 activation and enhance understanding of the regulation of SphK2 activity by phosphorylation and its role in movement of human breast cancer cells toward EGF.     

Transducing agonist binding to channel gating involves different interactions in 5-HT3 and GABAC receptors

5-hydroxytryptamine (5-HT)3 and gamma-aminobutyric acid, type C (GABAC) receptors are members of the Cys-loop superfamily of neurotransmitter receptors, which also includes nicotinic acetylcholine, GABAA, and glycine receptors. The details of how agonist binding to these receptors results in channel opening is not fully understood but is known to involve charged residues at the extracellular/transmembrane interface. Here we have examined the roles of such residues in 5-HT3 and GABAC receptors. Charge reversal experiments combined with data from activation by the partial agonist beta-alanine show that in GABAC receptors there is a salt bridge between Glu-92 (in loop 2) and Arg-258 (in the pre-M1 region), which is involved in receptor gating. The equivalent residues in the 5-HT3 receptor are important for receptor expression, but charge reversal experiments do not restore function, indicating that there is not a salt bridge here. There is, however, an interaction between Glu-215 (loop 9) and Arg-246 (pre-M1) in the 5-HT3 receptor, although the coupling energy determined from mutant cycle analysis is lower than might be expected for a salt bridge. Overall the data show that charged residues at the extracellular/transmembrane domain interfaces in 5-HT3 and GABAC receptors are important and that specific, but not equivalent, molecular interactions between them are involved in the gating process. Thus, we propose that the molecular details of interactions in the transduction pathway between the binding site and the pore can differ between different Cys-loop receptors.     

Commensal bacteria exacerbate intestinal inflammation but are not essential for the development of murine ileitis

The pathogenesis of Crohn's disease has been associated with a dysregulated response of the mucosal immune system against intraluminal Ags of bacterial origin. In this study, we have investigated the effects of germfree (GF) conditions in the SAMP1/YitFc murine model of Crohn's disease-like ileitis. We show that the bacterial flora is not essential for ileitis induction, because GF SAMP1/YitFc mice develop chronic ileitis. However, compared with disease in specific pathogen-free (SPF) mice, ileitis in GF mice is significantly attenuated, and is associated with delayed lymphocytic infiltration and defective mucosal expression of Th2 cytokines. In addition, we demonstrate that stimulation with purified fecal Ags from SPF, but not GF mice leads to the generation of IL-4-secreting effector lymphocytes. This result suggests that commensal bacteria drive Th2 responses characteristic of the chronic phase of SAMP1/YitFc ileitis. Finally, adoptive transfer of CD4-positive cells from GF, but not SPF mice induces severe colitis in SCID recipients. These effects were associated with a decreased frequency of CD4(+)CD25(+)Foxp3(+) T cells in the mesenteric lymph nodes of GF mice compared with SPF mice, as well as lower relative gene expression of Foxp3 in CD4(+)CD25(+) T cells in GF mice. It is therefore apparent that, in the absence of live intraluminal bacteria, the regulatory component of the mucosal immune system is compromised. All together, our results indicate that in SAMP1/YitFc mice, bacterial flora exacerbates intestinal inflammation, but is not essential for the generation of the chronic ileitis that is characteristic of these mice.     

Glycoprotein 120 binding to CXCR4 causes p38-dependent primary T cell death that is facilitated by, but does not require cell-associated CD4

HIV-1 infection causes the depletion of host CD4 T cells through direct and indirect (bystander) mechanisms. Although HIV Env has been implicated in apoptosis of uninfected CD4 T cells via gp120 binding to either CD4 and/or the chemokine receptor 4 (CXCR4), conflicting data exist concerning the molecular mechanisms involved. Using primary human CD4 T cells, we demonstrate that gp120 binding to CD4 T cells activates proapoptotic p38, but does not activate antiapoptotic Akt. Because ligation of the CD4 receptor alone or the CXCR4 receptor alone causes p38 activation and apoptosis, we used the soluble inhibitors, soluble CD4 (sCD4) or AMD3100, to delineate the role of CD4 and CXCR4 receptors, respectively, in gp120-induced p38 activation and death. sCD4 alone augments gp120-induced death, suggesting that CXCR4 signaling is principally responsible. Supporting that model, AMD3100 reduces death caused by gp120 or by gp120/sCD4. Finally, prevention of gp120-CXCR4 interaction with 12G5 Abs blocks p38 activation and apoptosis, whereas inhibition of CD4-gp120 interaction with Leu-3a has no effect. Consequently, we conclude that gp120 interaction with CXCR4 is required for gp120 apoptotic effects in primary human T cells.