The effects of pregnancy on myelin basic protein-induced experimental autoimmune encephalomyelitis in Lewis rats: Suppression of clinical disease, modulation of cytokine expression in the spinal cord inflammatory infiltrate and suppression of lymphocyte p

Problem: The present study was performed to explore the effects of pregnancy on experimental autoimmune encephalomyelitis (EAE) induced in Lewis rats by inoculation with myelin basic protein (MBP) (MBP-EAE). Method of study: MBP-EAE was induced in pregnant and non-pregnant rats and severity of disease evaluated. Serum from pregnant and non-pregnant rats was used in standard lymphocyte proliferation assays. Real-time polymerase chain reaction (PCR) was used to investigate the expression of cytokine mRNA in the inflammatory cells obtained from the spinal cord of rats on day 15 after inoculation. Results: Pregnant rats developed less severe disease than non-pregnant rats. Serum from pregnant rats suppressed the proliferation of T lymphocytes in response to MBP. There was significantly increased expression of IL-4. IL-10 and TNF-alpha mRNA in the spinal cord infiltrate of pregnant rats. Conclusion: Circulating humoral factors and alteration in cytokine production by inflammatory cells may contribute to the suppression of EAE in pregnant rats.

Functional annotation of a full-length mouse cDNA collection

The RIKEN Mouse Gene Encyclopaedia Project, a systematic approach to determining the full coding potential of the mouse genome, involves collection and sequencing of full-length complementary DNAs and physical mapping of the corresponding genes to the mouse genome. We organized an international functional annotation meeting (FANTOM) to annotate the first 21,076 cDNAs to be analysed in this project. Here we describe the first RIKEN clone collection, which is one of the largest described for any organism. Analysis of these cDNAs extends known gene families and identifies new ones.

Sodium iodide symporter (NIS) gene expression in human placenta

The placenta must allow the passage of iodide from the maternal to the fetal circulation for synthesis of thyroxine by the fetal thyroid. The thyroid sodium iodide symporter (NIS) was cloned in 1996 and, although widely distributed among epithelial tissues, early studies failed to detect it in placenta. We demonstrated NIS mRNA in human placenta and in the human choriocarcinoma cell line, JAr. NIS protein was localized to trophoblasts, with a tendency to apical distribution, in sections of human placenta immunostained with a monoclonal antibody against hNIS. We conclude that NIS is expressed in placenta and may mediate placental iodide transport. (C) 2001 Harcourt Publishers Ltd.

Syntaxin 7 complexes with mouse Vps10p tail interactor 1b, Syntaxin 6, vesicle-associated membrane protein (VAMP)8, and VAMP7 in B16 melanoma cells

Syntaxin 7 is a mammalian target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane transport between late endosomes and lysosomes. The aim of the present study was to use immunoaffinity techniques to identify proteins that interact with Syntaxin 7. We reasoned that this would be facilitated by the use of cells producing high levels of Syntaxin 7, Screening of a large number of tissues and cell lines revealed that Syntaxin 7 is expressed at very high levels in B16 melanoma cells. Moreover, the expression of Syntaxin 7 increased in these cells as they underwent melanogenesis. From a large scale Syntaxin 7 immunoprecipitation, we have identified six polypeptides using a combination of electrospray mass spectrometry and immunoblotting. These polypeptides corresponded to Syntaxin 7, Syntaxin 6, mouse Vps10p tail interactor 1b (mVti1b), alpha -synaptosome-associated protein (SNAP), vesicle-associated membrane protein (VAMP)8, VAMP7, and the protein phosphatase 1M regulatory subunit. We also observed partial colocalization between Syntaxin 6 and Syntaxin 7, between Syntaxin 6 and mVti1b, but not between Syntaxin 6 and the early endosomal t-SNARE Syntaxin 13. Based on these and data reported previously, we propose that Syntaxin 7/mVti1b/Syntaxin 6 may form discrete SNARE complexes with either VAMP7 or VAMPS to regulate fusion events within the late endosomal pathway and that these events may play a critical role in melanogenesis.

SOX18 and the transcriptional regulation of blood vessel development

SOX18 is a transcription factor that is transiently expressed in nascent endothelial cells during embryonic development and adult neovascularization. This protein belongs to the SOX family of transcription factors, ih,which are proving to be some of the key regulators of cell-type specification in the vertebrate embryo. Natural mutations in the Sox18 gene have been shown to result to cardiovascular dysfunction, in some cases leading to death. Available evidence thus implicates Sox18 as an important regulator of vascular development, most likely playing a key role in endothelial cell specification. However; the genetic knockout of Sox18 in mice has produced a confounding result that complicates our understanding of the molecular mode of action of the SOX18 protein. We speculate that Sox18 inky act in a redundant fashion with closely related genes such as Sox7 and/or Sox17. (C) 2001, Elsevier Science Inc.

Sox18 expression in blood vessels and feather buds during chicken embryogenesis

Sox18 encodes a transcription factor known to be important for the development of blood vessels and hair follicles in mice. In order to study the functional conservation of this gene through evolution, we have isolated and characterized Sox18 in chickens. cSox18 shows a high degree of sequence homology to both the mouse and human orthologues, particularly in the high mobility group DNA-binding domain and to a lesser extent in the transcriptional activation domain. A region of unusually high sequence conservation at the C-terminus may represent a further, previously unrecognized functional domain. Both the chicken and human proteins appear to be truncated at the N-terminus relative to mouse SOX18. In situ hybridization analyses showed expression in the developing vasculature and feather follicles, consistent with reported expression in the mouse embryo. In addition, cSox18 mRNA was observed in the retina and claw beds. (C) 2001 Elsevier Science B.V. All rights reserved.

Transcriptional modulation of mouse mu-opioid receptor distal promoter activity by Sox18

Previously, we reported the presence of dual promoters, referred to as distal (DP) and proximal, with a negative regulatory element between them in the mouse mu -opioid receptor (mor) gene. Here we have identified a positive regulatory element influencing mor DP transcription, which contains multiple consensus binding motifs for Sox factors (sex-determining Sry-like high mobility group box-containing genes). In gel supershift assays, the Sox family member Sox18 bound directly to the multiple Sox consensus binding motifs of the mor DP enhancer. Overexpression of Sox18 cDNA increased luciferase activity regulated by the mor DP, and did so in a Sox18 concentration-dependent manner. In contrast, overexpression of another Sox member, Sox5, triggered no such trans-activation of mor DP-driven luciferase activity or DNA-protein binding activity. These results suggest that Sox18 directly and specifically stimulates mor gene expression, by trans-activating the mor DP enhancer.

Cytokine regulation of syndecan-1 and-2 gene expression in human periodontal fibroblasts and osteoblasts

Cell-surface proteoglycans participate in several biological functions including interactions with a variety of growth factors and cytokines. Regulation of syndecan-1 and -2 gene expression was investigated in human periodontal ligament fibroblasts (PDLF), osteoblasts (OB) and gingival fibroblasts (GF), in response to platelet-derived growth factor (PDGF-BB), transforming growth factor (TGF-beta(1)), and interleukin (IL-1beta) by Northern blot analyses. We also compared the effect of PDGF-BB and TGF-beta(1), separately and in combination, in the prolonged presence of IL-1beta on the expression of both syndecan genes. The results demonstrated that the three cell lines regulated the expression of syndecan-1 and -2 in response to growth factors and cytokines in different manners. These cell lines increased syndecan-1 mRNA levels in response to either PDGF-BB or TGF-beta(1) and decreased levels in response to IL-1beta. The effect of IL-1beta on syndecan-1 mRNA synthesis was partially reversed after adding PDGF-BB and TGF-beta(1), separately or in combination, in the presence of IL-1beta. In contrast, syndecan-2 mRNA level was markedly upregulated in response to either TGF-beta(1) or IL-1beta in OB when compared with the other two cell lines. However, the stimulatory effect of TGF-beta(1) on syndecan-2 mRNA production in OB was abolished in the prolonged presence of IL-1beta. These findings lend support to the notion that syndecan-1 and syndecan-2 have distinct functions which correlate with their source and functions within the periodontium.

Ecological regulation of development: Induction of marine invertebrate metamorphosis

In the marine environment a wide range of invertebrates have a pelagobenthic lifecycle that includes planktonic larval and benthic adult phases. Transition between these morphologically and ecologically distinct phases typically occurs when the developmentally competent larva comes into contact with a species-specific environmental cue. This cue acts as a morphogenetic signal that induces the completion of the postlarval/juvenile/adult developmental program at metamorphosis. The development of competence often occurs hours to days after the larva is morphologically mature. In the non-feeding - lecithotrophic - larvae of the ascidian Herdmania curvata and the gastropod mollusc Haliotis asinina, gene expression patterns in pre-competent and competent stages are markedly different, reflecting the different developmental states of these larval stages. For example, the expression of Hemps, an EGF-like signalling peptide required for the induction of Herdmania metamorphosis, increases in competent larvae. Induction of settlement and metamorphosis results in further changes in developmental gene expression, which apparently is necessary for the complete transformation of the larval body plan into the adult form.

Embryogenesis and metamorphosis in a haplosclerid demosponge: gastrulation and transdifferentiation of larval ciliated cells to choanocytes

Early development and metamorphosis of Reniera sp., a haplosclerid demosponge, have been examined to determine how gastrulation occurs in this species, and whether there is an inversion of the primary germ layers at metamorphosis. Embryogenesis occurs by unequal cleavage of blastomeres to form a solid blastula consisting micro- and macromeres; multipolar migration of the micromeres to the surface of the embryo results in a bi-layered embryo and is interpreted as gastrulation. Polarity of the embryo is determined by the movement of pigment-containing micromeres to one pole of the embryo; this pole later becomes the posterior pole of the swimming larva. The bi-layered larva has a fully differentiated monociliated outer cell layer, and a solid interior of various cell types surrounded by dense collagen. The pigmented cells at the posterior pole give rise to long cilia that are capable of responding to environmental stimuli. Larvae settle on their anterior pole. Fluorescent labeling of the monociliated outer cell layer with a cell-lineage marker (CMFDA) demonstrates that the monociliated cells resorb their cilia, migrate inwards, and transdifferentiate into the choanocytes of the juvenile sponge, and into other amoeboid cells. The development of the flagellated choanocytes and other cells in the juvenile from the monociliated outer layer of this sponge's larva is interpreted as the dedifferentiation of fully differentiated larval cells-a process seen during the metamorphosis of other ciliated invertebrate larvae-not as inversion of the primary germ layers. These results suggest that the sequences of development in this haplosclerid demosponge are not very different than those observed in many cnidarians.

Evolution and developmental expression of nuclear receptor genes in the ascidian Herdmania

Nuclear receptors are a superfamily of metazoan transcription factors that have been shown to be involved in a wide range of developmental and physiological processes. A PCR-based survey of genomic DNA and developmental cDNAs from the ascidian Herdmania identifies eight members of this multigene family. Sequence comparisons and phylogenetic analyses reveal that these ascidian nuclear receptors are representative of five of the six previously defined nuclear receptor subfamilies and are apparent homologues of retinoic acid [NR1B], retinoid X [NR2B], peroxisome proliferator-activated [NR1C], estrogen related [NR3B], neuron-derived orphan (NOR) [NR4A3], nuclear orphan [NR4A], TR2 orphan [NR2C1] and COUP orphan [NR2F3] receptors. Phylogenetic analyses that include the ascidian genes produce topologically distinct trees that suggest a redefinition of some nuclear receptor subfamilies. These trees also suggest that extensive gene duplication occurred after the vertebrates split from invertebrate chordates. These ascidian nuclear receptor genes are expressed differentially during embryogenesis and metamorphosis.

Selective loss of expression of glutamate GluR2/R3 receptor subunits in cerebellar tissue from a patient with olivopontocerebellar atrophy

Expression of the mRNAs encoding the astrocytic (EAAT1, EAAT2) and neuronal (EAAT3, EAAT4) excitatory amino acid transporters and the AMPA-type glutamate receptor subunits GluR2 and GluR3 was investigated in postmortem cerebellar extracts from a patient with olivopontocerebellar atrophy (OPCA) and in material from three age-matched controls. Decreased expression in the steady state level of EAAT4 mRNA in the OPCA sample was correlated with the selective loss of Purkinje cells. Neuropathological evaluation revealed reactive gliosis and concomitantly increased expression of the mRNA encoding astrocytic glial fibrillary acidic protein (GFAP). Expression of the mRNAs encoding the AMPA receptor subunits GluR2 and GluR3 subunits was found to be decreased in OPCA suggesting that excitotoxic mechanism could play a role in the pathogenesis of the selective neuronal cell death in this disorder.

The identification of the transcriptional regulator CRP in Aeromonas hydrophila JMP636 and its involvement in amylase production and the 'acidic toxicity' effect

Aims: The physiological examination of amylase production by Aeromonas hydrophila JMP636 and identification of the mechanism of regulation. Methods and Results: Aeromonas hydrophila JMP636 was grown with single, then dual carbon sources; the growth cycle was followed and amylase activity throughout was monitored. The levels of cAMP, a known secondary messenger for the regulatory gene crp, were also examined. Amylase activity was regulated by catabolite repression. Physiological studies revealed that JMP636 exhibited both diauxic growth, with two carbon sources, and the 'acid toxicity' effect on glucose. The crp gene was cloned, expressed and inactivated from the JMP636 chromosome. Catabolite repression of amylase production and the 'acid toxicity' effect both require crp and were linked to cAMP levels. Conclusions: Regulation of amylase production was predicted to follow the model CRP-mediated cAMP-dependent Escherichia coli catabolite regulation system. Significance and Impact of the Study: This work provides an understanding of the physiology of the opportunistic pathogen Aer. hydrophila through identification of the mechanism of catabolite repression of amylase production and the existence of crp within this cell. It also provides a broader knowledge of global gene regulation and suggests regulatory mechanisms of other Aer. hydrophila gene/s.

Characterization of a chemsensory protein (ASP3c) from honeybee (Apis mellifera L.) as a brood pheromone carrier

Chemosensory proteins (CSPs) are ubiquitous soluble small proteins isolated from sensory organs of a wide range of insect species, which are believed to be involved in chemical communication. We report the cloning of a honeybee CSP gene called ASP3c, as well as the structural and functional characterization of the encoded protein. The protein was heterologously secreted by the yeast Pichia pastoris using the native signal peptide. ASP3c disulfide bonds were assigned after trypsinolysis followed by chromatography and mass spectrometry combined with microsequencing. The pairing (Cys(I)-Cys(II), Cys(III)-Cys(IV)) was found to be identical to that of Schistocerca gregaria CSPs, suggesting that this pattern occurs commonly throughout the insect CSPs. CD measurements revealed that ASP3c mainly consists of alpha-helices, like other insect CSPs. Gel filtration analysis showed that ASP3c is monomeric at neutral pH. Using ASA, a fluorescent fatty acid anthroyloxy analogue as a probe, ASP3c was shown to bind specifically to large fatty acids and ester derivatives, which are brood pheromone components, in the micromolar range. It was unable to bind tested general odorants and other tested pheromones (sexual and nonsexual). This is the first report on a natural pheromonal ligand bound by a recombinant CSP with a measured affinity constant.

Cytochrome P450(cin) (CYP176A), isolation, expression, and characterization

Cytochromes P450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiologic and xenobiotic compounds in eukaryotes and prokaryotes. Studies on bacterial P450s, particularly those involved in monoterpene oxidation, have provided an integral contribution to our understanding of these proteins, away from the problems encountered with eukaryotic forms. We report here a novel cytochrome P450 (P450(cin), CYP176A1) purified from a strain of Citrobacter braakii that is capable of using cineole 1 as its sole source of carbon and energy. This enzyme has been purified to homogeneity and the amino acid sequences of three tryptic peptides determined. By using this information, a PCR-based cloning strategy was developed that allowed the isolation of a 4-kb DNA fragment containing the cytochrome P450(cin) gene (cinA). Sequencing revealed three open reading frames that were identified on the basis of sequence homology as a cytochrome P450, an NADPH-dependent flavodoxin/ferrodoxin reductase, and a flavodoxin. This arrangement suggests that P450(cin) may be the first isolated P450 to use a flavodoxin as its natural redox partner. Sequencing also identified the unprecedented substitution of a highly conserved, catalytically, important active site threonine with an asparagine residue. The P450 gene was subcloned and heterologously expressed in Escherichia coli at similar to2000 nmol/liter of original culture, and purification was achieved by standard protocols. Postulating the native E. coli flavodoxin/flavodoxin reductase system might mimic the natural redox partners of P450,in, it was expressed in E. coli in the presence of cineole 1. A product was formed in vivo that was tentatively identified by gas chromatography-mass spectrometry as 2-hydroxycineole 2. Examination of P450(cin) by UV-visible spectroscopy revealed typical spectra characteristic of P450s, a high affinity for cineole 1 (K-D = 0.7 mum), and a large spin state change of the heme iron associated with binding of cineole 1. These facts support the hypothesis that cineole 1 is the natural substrate for this enzyme and that P450(cin) catalyzes the initial monooxygenation of cineole 1 biodegradation. This constitutes the first characterization of an enzyme involved in this pathway.