Phosphorylation-dependent interaction between the splicing factors SAP155 and NIPP1

NIPP1 is a ubiquitously expressed nuclear protein that functions both as a regulator of protein Ser/Thr phosphatase-1 and as a splicing factor. The N-terminal part of NIPP1 consists of a phosphothreonine-interacting Forkhead-associated (FHA) domain. We show here that the FHA domain of NIPP1 interacts in vitro and in vivo with a TP dipeptide-rich fragment of the splicing factor SAP155/SF3b(155), a component of the U2 small nuclear ribonucleoprotein particle. The NIPP1-SAP155 interaction was entirely dependent on the phosphorylation of specific TP motifs in SAP155. Mutagenesis and competition studies revealed that various phosphorylated TP motifs competed for binding to the same site in the FHA domain. The SAP155 kinases in cell lysates were blocked by the Ca(2+) chelator EGTA and by the cyclin-dependent protein kinase inhibitor roscovitine. The phosphorylation level of SAP155 was dramatically increased during mitosis, and accordingly the activity of SAP155 kinases was augmented in mitotic lysates. We discuss how the interaction between NIPP1 and SAP155 could contribute to spliceosome (dis)assembly and the catalytic steps of splicing.     

Use of the Verrucomicrobia-specific probe EUB338-III and fluorescent in situ hybridization for detection of "Candidatus Xiphinematobacter" cells in nematode hosts

Fluorescent in situ hybridization with a 16S rRNA probe specific for Verrucomicrobia was used to (i) confirm the division-level identity of and (ii) study the behavior of the obligate intracellular verrucomicrobium "Candidatus Xiphinematobacter" in its nematode hosts. Endosymbionts in the egg move to the pole where the gut primordium arises; hence, they populate the intestinal epithelia of juvenile worms. During the host's molt to adult female, the endosymbionts concentrate around the developing ovaries to occupy the ovarian wall. Some bacteria are enclosed in the ripening oocytes for vertical transmission. Verrucomicrobia in males stay outside the testes because the tiny spermatozoids are not suitable for transmission of cytoplasmic bacteria.     

Glucose transport and utilization are altered in the brain of rats deficient in n-3 polyunsaturated fatty acids

Long-chain polyunsaturated (n-3) fatty acids have been reported to influence the efficiency of membrane receptors, transporters and enzymes. Because the brain is particularly rich in docosahexaenoic acid (DHA, 22:6 n-3), the present study addresses the question of whether the 22:6 n-3 fatty acid deficiency induces disorder in regulation of energy metabolism in the CNS. Three brain regions that share a high rate of energy metabolism were studied: fronto-parietal cortex, hippocampus and suprachiasmatic nucleus. The effect of the diet deficient in n-3 fatty acids resulted in a 30-50% decrease in DHA in membrane phospholipids. Moreover, a 30% decrease in glucose uptake and a 20-40% decrease in cytochrome oxidase activity were observed in the three brain regions. The n-3 deficient diet also altered the immunoreactivity of glucose transporters, namely GLUT1 in endothelial cells and GLUT3 in neurones. In n-3 fatty acid deficient rats, GLUT1-immunoreactivity readily detectable in microvessels became sparse, whereas the number of GLUT3 immunoreactive neurones was increased. However, western blot analysis showed no significant difference in GLUT1 and GLUT3 protein levels between rats deficient in n-3 fatty acids and control rats. The present results suggest that changes in energy metabolism induced by n-3 deficiency could result from functional alteration in glucose transporters.     

The protein phosphatase-1 regulator NIPP1 is also a splicing factor involved in a late step of spliceosome assembly

NIPP1 is a ubiquitous regulator of protein phosphatase-1 (PP1) and is targeted to the splicing factor storage sites (speckles) in the nucleus by its forkhead-associated domain. We show here that NIPP1 is also a component of the spliceosomes in HeLa cell-splicing extracts and that the interaction with the spliceosomes requires a functional forkhead-associated domain. The in vitro splicing of beta-globin pre-mRNA was not affected by exogenous wild type NIPP1 but was blocked by mutants that lacked residues 225-329. The inhibition by these dominant negative mutants resulted from a block in a late phase of spliceosome assembly, i.e. at the transition between the B-complex and the C-complex. Site-directed mutagenesis furthermore showed that this spliceosomal function of NIPP1 was unrelated to its ability to bind PP1 or RNA. Our data suggest that NIPP1 can function independently as a splicing factor and a phosphatase regulator.     

Membrane transport in Caenorhabditis elegans: an essential role for VPS34 at the nuclear membrane

Here we present a detailed genetic analysis of let-512/vps34 that encodes the Caenorhabditis elegans homologue of the yeast phosphatidylinositol 3-kinase Vps34p. LET-512/VPS34 has essential functions and is ubiquitously expressed in all tissues and developmental stages. It accumulates at a perinuclear region, and mutations in let-512/vps34 result in an expansion of the outer nuclear membrane as well as in a mislocalization and subsequent complete lack of expression of LRP-1, a C.elegans LDL receptor normally associated with the apical surface of hypodermal cells. Using a GFP::2xFYVE fusion protein we found that the phosphatidylinositol 3-phosphate (PtdIns 3-P) product of LET-512/VPS34 is associated with a multitude of intracellular membranes and vesicles located at the periphery, including endocytic vesicles. We propose that LET-512/VPS34 is required for membrane transport from the outer nuclear membrane towards the cell periphery. Thus, LET-512/VPS34 may regulate the secretory pathway in a much broader range of compartments than was previously suggested for the yeast orthologue.     

Antigen presentation by CD1d contributes to the amplification of Th2 responses to Schistosoma mansoni glycoconjugates in mice

During murine schistosomiasis, there is a gradual switch from a predominant Th1 cytokine response to a Th2-dominated response after egg laying, an event that favors the formation of granuloma around viable eggs. Egg-derived glycoconjugates, including glycolipids, may play a crucial role in this phenomenon. In this study, we used a model of dendritic cell sensitization to study the role of egg glycoconjugates in the induction of specific immune response to soluble egg Ag (SEA) and to investigate the possibility that CD1d, a molecule implicated in glycolipid presentation, may be involved in such a phenomenon. We show that, when captured, processed, and presented to naive T lymphocytes by dendritic cells, egg, but not larval, Ag skew the immune response toward a Th2 response. Periodate treatment reversed this effect, indicating that the sugar moiety of SEA is important in this phenomenon. Using DC treated ex vivo with a neutralizing anti-CD1d Ab or isolated from CD1d knockout mice, we show that CD1d is crucial in the priming of SEA-specific Th2 lymphocytes. We then evaluated the contribution of CD1d on the development of the SEA-specific immune response and on the formation of the egg-induced liver granuloma during murine schistosomiasis. We find that CD1d knockout mice have a reduced Th2 response after egg laying and develop a less marked fibrotic pathology compared with wild-type mice. Altogether, our results suggest that Ag presentation of parasite glycoconjugates to CD1d-restricted T cells may be important in the early events leading to the induction of Th2 responses and to egg-induced pathology during murine schistosomiasis.     

Decreased expression of glutamate transporters in genetic absence epilepsy rats before seizure occurrence

In absence epilepsy, epileptogenic processes are suspected of involving an imbalance between GABAergic inhibition and glutamatergic excitation. Here, we describe alteration of the expression of glutamate transporters in rats with genetic absence (the Genetic Absence Epilepsy Rats from Strasbourg: GAERS). In these rats, epileptic discharges, recorded in the thalamo-cortical network, appear around 40 days after birth. In adult rats no alteration of the protein expression of the glutamate transporters was observed. In 30-day-old GAERS protein levels (quantified by western blot) were lower in the cortex by 21% and 35% for the glial transporters GLT1 and GLAST, respectively, and by 32% for the neuronal transporter EAAC1 in the thalamus compared to control rats. In addition, the expression and activity of GLAST were decreased by 50% in newborn GAERS cortical astrocytes grown in primary culture. The lack of modification of the protein levels of glutamatergic transporters in adult epileptic GAERS, in spite of mRNA variations (quantified by RT-PCR), suggests that they are not involved in the pathogeny of spike-and-wave discharges. In contrast, the alteration of glutamate transporter expression, observed before the establishment of epileptic discharges, could reflect an abnormal maturation of the glutamatergic neurone-glia circuitry.     

The CXCR3 binding chemokine IP-10/CXCL10: structure and receptor interactions

The structure of IP-10 was solved by NMR spectroscopy and represents the first structure from the class of agonists toward the receptor CXCR3. CXCR3 binding chemokines are unique in their ability to bind receptors from both the CC and CXC classes of chemokine receptors. An unusual structural feature of IP-10 was identified that may provide the basis for the ability of IP-10 to bind both CXCR3 and CCR3. The surface of IP-10 that interacts with the N-terminus of CXCR3 was defined by monitoring changes in the NMR spectrum of IP-10 upon addition of a CXCR3 N-terminal peptide. These studies indicated that the interaction involves a hydrophobic cleft, formed by the N-loop and 40s-loop region of IP-10, similar to the interaction surface observed for other chemokines such as IL-8. An additional region of interaction was observed that consists of a hydrophobic cleft formed by the N-terminus of IP-10 and 30s-loop of IP-10.     

Flavonolignans from Hyparrhenia hirta

Leaves of Hyparrhenia hirta yielded the rare diastereoisomeric flavonolignans tricin 4'-O-(erythro-beta-guaiacylglyceryl) ether and tricin 4'-O-(threo-beta-guaiacylglyceryl) ether together with their 7-O-glucosides, which are the first flavonolignan glycosides to be isolated as natural products. A complete set of (1)H and (13)C NMR resonance assignments obtained for both flavonolignan aglycones indicates the need for revision of data published previously for these compounds and for a reassessment of their original stereochemical designation.