Synaptojanin-1 plays a key role in astrogliogenesis: possible relevance for Down's syndrome.

There is increasing interest in gliogenesis as the relevance of glia to both brain development and pathology becomes better understood. However, little is known about this process. The use of multidimensional protein identification technology (MudPIT) to identify changes in phosphoprotein levels in rat neural precursor cells treated with cytokines or retinoic acid showed that phosphorylation of the catalytic subunit of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K p110alpha) and dephosphorylation of the inositol phosphatase synaptojanin-1 were common to the gliogenic stimuli. Although PI3K was found to be involved in both neuro- and astrogliogenesis, synaptojanin-1 was specifically involved in astrogliogenesis of neural precursor cells. The role of synaptojanin-1 in astrogliogenesis was further confirmed by analysis of neuron- and glia-specific markers in synaptojanin-1 knockout mouse brain. Additional experiments showed that the Sac1-like phosphatase domain of synaptojanin-1 is responsible for the observed astrogliogenic effect. Our results strongly indicate that phosphatidylinositol metabolism plays a key role in astrogliogenesis. The relevance of our findings for Down's syndrome pathology is discussed.

Residues in the C-terminal region of activin A determine specificity for follistatin and type II receptor binding.

Activin is a secreted growth factor that signals by binding two related classes of single transmembrane receptors at the cell surface. The interaction of activin with its receptors is highly regulated by other cell surface receptors, antagonistic ligands, and high affinity extracellular binding proteins such as follistatin. Two activin A mutants, the deletion mutant des[85-109]-activin A and the point mutant K102E-activin A (K102E), were investigated with respect to their ability to bind cell surface receptors and the binding protein follistatin. The deletion mutant exhibits low affinity for both receptors and follistatin whereas the point mutant fails to bind cell surface receptors but binds follistatin-288 with high affinity. K102E is able to compete with wild type activin to bind to follistatin and can thus increase the concentration of activin available for receptor binding and signaling. These findings underline the importance of the C-terminal region of activin for binding interactions and show that different residues in this region are involved in cell surface receptor and follistatin interactions.

Directional effect of inactivation of the nucleus of the optic tract on optokinetic nystagmus in the cat.

The goal of the present investigation was to elucidate the role of the nucleus of the optic tract and the dorsal terminal nucleus of the accessory optic system (NOT-DTN) for slow eye movements other than horizontal. Retinal slip neurons in the NOT-DTN in the awake behaving cat respond direction selectively to the ipsiversive component of horizontal and oblique image motion. They are, however, influenced neither by pure vertical stimulus movement nor by eye movements in the dark. Electrical stimulation of the NOT-DTN leads to pure horizontal optokinetic nystagmus with ipsiversive slow phases and does not influence vertical eye position. In addition, unilateral reversible inactivation of the NOT-DTN with muscimol elicits spontaneous contraversive horizontal nystagmus without vertical component. During oblique optokinetic stimulation, the ipsiversive OKN component is significantly decreased in all directions. After bilateral NOT-DTN inactivation, OKN can only be elicited in a narrow range of upward directions. These data indicate that the NOT-DTN is the only source to drive the horizontal component of OKN.

Novel vertebrate nucleoporins Nup133 and Nup160 play a role in mRNA export.

RNA undergoing nuclear export first encounters the basket of the nuclear pore. Two basket proteins, Nup98 and Nup153, are essential for mRNA export, but their molecular partners within the pore are largely unknown. Because the mechanism of RNA export will be in question as long as significant vertebrate pore proteins remain undiscovered, we set out to find their partners. Fragments of Nup98 and Nup153 were used for pulldown experiments from Xenopus egg extracts, which contain abundant disassembled nuclear pores. Strikingly, Nup98 and Nup153 each bound the same four large proteins. Purification and sequence analysis revealed that two are the known vertebrate nucleoporins, Nup96 and Nup107, whereas two mapped to ORFs of unknown function. The genes encoding the novel proteins were cloned, and antibodies were produced. Immunofluorescence reveals them to be new nucleoporins, designated Nup160 and Nup133, which are accessible on the basket side of the pore. Nucleoporins Nup160, Nup133, Nup107, and Nup96 exist as a complex in Xenopus egg extracts and in assembled pores, now termed the Nup160 complex. Sec13 is prominent in Nup98 and Nup153 pulldowns, and we find it to be a member of the Nup160 complex. We have mapped the sites that are required for binding the Nup160 subcomplex, and have found that in Nup98, the binding site is used to tether Nup98 to the nucleus; in Nup153, the binding site targets Nup153 to the nuclear pore. With transfection and in vivo transport assays, we find that specific Nup160 and Nup133 fragments block poly[A]+ RNA export, but not protein import or export. These results demonstrate that two novel vertebrate nucleoporins, Nup160 and Nup133, not only interact with Nup98 and Nup153, but themselves play a role in mRNA export.

Structure and biological activity of gonadotropin-releasing hormone isoforms isolated from rat and hamster brains.

Rat and hamster brain tissues were used to investigate the possible existence of a follicle stimulating hormone (FSH)-releasing factor with similar characteristics to the lamprey gonadotropin-releasing hormone III (lGnRH-III) form proposed in previous reports. The present studies involved isolation and purification of the molecule by high-performance liquid chromatography (HPLC), identification by radioimmunoassay, sequence analysis by automated Edman degradation, mass spectrometry and examination of biological activity. Hypothalamic extracts from both species contained an HPLC fraction that was immunoreactive to GnRH and coeluted with lGnRH-III and 9-hydroxyproline mGnRH ([Hyp(9)]GnRH). Determination of primary structure from purified total brain material demonstrated that the isolated molecule was [Hyp(9)]GnRH. This is the first report showing the presence of the posttranslationally modified form already known as [Hyp(9)]GnRH by primary sequence analysis. The biological activity of distinct GnRH peptides was also tested in vitro for gonadotropin release using rat pituitary primary cell cultures. The results showed that [Hyp(9)]GnRH stimulated both luteinizing hormone and FSH release, as already reported, whereas lGnRH-III had no action on the secretion of either gonadotropin.

Chemoimmunotherapy for melanoma with dacarbazine and 2,4-dinitrochlorobenzene: results from a murine tumour model.

An empirically established chemoimmunotherapy that combines the epifocal application of the contact sensitizer dinitrochlorobenzene (DNCB) to cutaneous metastases with the systemic administration of dacarbazine (DTIC) yields high response rates and results in prolonged survival. However, despite the fact that this therapy has been in clinical use for several years, the mode of action still remains elusive. In order to overcome this limitation we established a murine model system. B16 melanoma cells were implanted subcutaneously in syngeneic C57BL/6 mice and treatment was started 7 days after. In a first set of experiments mice received intraperitoneal injections of DTIC followed by epifocal applications of DNCB 24 h later. Treatment significantly decreased tumour growth. In contrast, no significant effect was induced by DTIC or DNCB alone. Using this regimen, with varying doses of either DTIC or DNCB, we demonstrated that the therapeutic effect is dose dependent. Furthermore, the treatment of subcutaneous tumours with DTIC and DNCB influenced the course of visceral metastases: the growth of pulmonary metastases was significantly inhibited if subcutaneous tumours were treated as described. In conclusion, we have established a model system that seems to be appropriate for both the optimization of this therapeutic regimen and the characterization of effector mechanisms.

Expression, purification, and characterization of a soluble form of the first extracellular domain of the human type 1 corticotropin releasing factor receptor.

The first extracellular domain (ECD-1) of the corticotropin releasing factor (CRF) type 1 receptor, (CRFR1), is important for binding of CRF ligands. A soluble protein, mNT-CRFR1, produced by COS M6 cells transfected with a cDNA encoding amino acids 1--119 of human CRFR1 and modified to include epitope tags, binds a CRF antagonist, astressin, in a radioreceptor assay using [(125)I-d-Tyr(0)]astressin. N-terminal sequencing of mNT-CRFR1 showed the absence of the first 23 amino acids of human CRFR1. This result suggests that the CRFR1 protein is processed to cleave a putative signal peptide corresponding to amino acids 1--23. A cDNA encoding amino acids 24--119 followed by a FLAG tag, was expressed as a thioredoxin fusion protein in Escherichia coli. Following thrombin cleavage, the purified protein (bNT-CRFR1) binds astressin and the agonist urocortin with high affinity. Reduced, alkylated bNT-CRFR1 does not bind [(125)I-D-Tyr(0)]astressin. Mass spectrometric analysis of photoaffinity labeled bNT-CRFR1 yielded a 1:1 complex with ligand. Analysis of the disulfide arrangement of bNT-CRFR1 revealed bonds between Cys(30) and Cys(54), Cys(44) and Cys(87), and Cys(68) and Cys(102). This arrangement is similar to that of the ECD-1 of the parathyroid hormone receptor (PTHR), suggesting a conserved structural motif in the N-terminal domain of this family of receptors.

Increased formation of micronuclei after hormonal stimulation of cell proliferation in human breast cancer cells.

The carcinogenicity of sex hormones is considered to be the result of a combination of genotoxic and epigenetic modes of action. For estrogens, genotoxic activities include DNA damage by reactive metabolites and indirect genotoxicity by redox cycling and production of reactive oxygen species. Here, we present data on the induction of micronuclei in estrogen receptor-positive (MCF-7) and -negative (MDA) human breast cancer cell lines treated with estradiol to support an additional mechanism of chromosomal damage. MCF-7 cells, but not MDA cells, treated with estradiol in the picomolar concentration range showed an increase in micronucleus formation which correlated with the estradiol-induced cell proliferation. Addition of the specific estradiol-receptor antagonist hydroxytamoxifen suppressed the estradiol-induced formation of micronuclei in MCF-7 cells. Increased frequencies were also seen after normalization of the data to the number of cell divisions by additional treatment of the cells with cytochalasin B. Thus, formation of micronuclei was not due to the chromosomal damaging activity of estradiol. The induced genomic damage may be explained by a hormone-specific forcing of responsive cells through the cell cycle, thereby overriding checkpoints operating under homeostatic control of the cell cycle.

Primary structure of a novel gonadotropin-releasing hormone in the brain of a teleost, Pejerrey.

The neuropeptide GnRH is the major regulator of reproduction in vertebrates acting as a first signal from the hypothalamus to pituitary gonadotropes. Three GnRH molecular variants were detected in the brain of a fish, pejerrey (Odontesthes bonariensis), using chromatographic and immunological methods. The present study shows that one form is identical to chicken GnRH-II (sequence analysis and mass spectrometry) and the second one is immunologically and chromatographically similar to salmon GnRH. The third form was proven to be a novel form of GnRH by isolating the peptide from the brain and determining its primary structure by chemical sequencing and mass spectrometry. The sequence of the novel pejerrey GnRH is pGlu-His-Trp-Ser-Phe-Gly-Leu-Ser-Pro-Gly-NH(2), which is different from the known forms of the vertebrate and protochordate GnRH family. The new form of GnRH is biologically active in releasing gonadotropin and GH from pituitary cells in an in vitro assay.

Targeting of lymphotoxin-alpha to the tumor elicits an efficient immune response associated with induction of peripheral lymphoid-like tissue.

A recombinant antibody-lymphotoxin-alpha fusion protein induced an adaptive immune response protecting mice from melanoma. Importantly, this fusion protein elicited the formation of a lymphoid-like tissue in the tumor microenvironment containing L-selectin+ T cells and MHC class II+ antigen-presenting cells, as well as B and T cell aggregates. Furthermore, PNAd+/TCA4+ high endothelial venules were observed within the tumor, suggesting entry channels for naive T cell infiltrates. Over the course of therapy, a marked clonal expansion of certain TCR specificities occurred among tumor-infiltrating lymphocytes that displayed reactivity against melanoma cells and the TRP-2(180-188) peptide. Consequently, naive T cells may have been recruited to as well as primed and expanded in the lymphoid-like tissue induced by the lymphotoxin-alpha fusion protein at the tumor site.

Enzymatic glycosylation of contulakin-G, a glycopeptide isolated from Conus venom, with a mammalian ppGalNAc-transferase.

We have determined that the mammalian uridine diphospho-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase T1 (EC 2.4.1.41) has the appropriate acceptor substrate specificity to recognize the non-glycosylated form of contulakin-G (ZSEEGGSNATKKPYIL-OH where Z=pyroglutamic acid) and to transfer GalNAc to the peptide. Both [Thr(10)] contulakin-G and a pre-contulakin-G(30-66) (RGLVPDDITPQLILGSLISRRQSEEGGSNATKKPYIL-OH) were shown to be acceptors for the mammalian enzyme. The site of attachment of the GalNAc residue was determined using chemical and radioactive sequencing techniques. The mammalian enzyme was highly specific for Thr(10) residue, in which the native peptide was found to be glycosylated, compared with either Ser(2) or Ser(7). In the case of pre-contulakin-G, the enzyme was also highly specific for the equivalent threonine residue. These results suggest that the Cone snail uses an enzyme with similar acceptor specificity to that of the mammalian polypeptide N-acetylgalactosaminyltransferase for glycosylating contulakin-G.

Styelin D, an extensively modified antimicrobial peptide from ascidian hemocytes.

We isolated styelin D, a 32-residue, C-terminally amidated antimicrobial peptide, from the blood cells (hemocytes) of the solitary ascidian, Styela clava. Styelin D had remarkably extensive post-translational modifications, containing two novel amino acids, dihydroxyarginine and dihydroxylysine, and two distinctly unusual ones, 6-bromotryptophan and 3,4-dihydroxyphenylalanine. In addition, the peptide exhibited microheterogeneity because of differential mono- and dihydroxylation of several lysine residues. The primary sequence of one variant was: GW(*)LR(**)K(**)AAK(**)SVGK(**)FY(*)Y(*)K(**)HK(*)Y(*) Y(*)IK(*)AAWQIG KHAL-NH(2), where W(*) is 6-bromotryptophan, R(**) is dihydroxyarginine, Y(*) is 3,4-dihydroxyphenylalanine, K(*) is 5-hydroxylysine, and K(**) is dihydroxylysine. Styelin D exhibited activity against Gram-negative and Gram-positive bacteria, and this activity was retained in 200 mm NaCl. The role of the extensive modifications may be to preserve activity at low pH and/or high salinity because, under these conditions, the native peptide was considerably more active against the Gram-positive bacterial strains than its unmodified synthetic analogue. The peptide was also hemolytic and quite cytotoxic to eukaryotic cells. These broad ranging activities, combined with its relative abundance in ascidian hemocytes, suggest that styelin D plays a significant role in the innate immune mechanisms of S. clava.

Primary structure and function of three gonadotropin-releasing hormones, including a novel form, from an ancient teleost, herring.

The evolution of GnRH and the role of multiple forms within the brain are examined. Three forms of GnRH were purified from the brain of Pacific herring (Clupea harengus pallasi) and characterized using Edman degradation and mass spectrometry. Two forms correspond with the known structures of chicken GnRH-II and salmon GnRH that are found in many vertebrate species. The third form, designated herring GnRH (hrGnRH), has a primary structure of pGlu-His-Trp-Ser-His-Gly-Leu-Ser-Pro-Gly-NH2. This novel peptide is a potent stimulator of gonadotropin II and GH release from dispersed fish pituitary cells. The content of hrGnRH in the pituitary was 8-fold that of salmon GnRH and 43-fold that of chicken GnRH-II, which provides supporting evidence that hrGnRH is involved in the release of gonadotropin. Herring is the most phylogenetically ancient animal in which three forms of GnRH have been isolated and sequenced. Our evidence suggests that the existence of three GnRHs in the brain of one species 1) is an ancestral condition for teleosts, 2) has the potential for separate regulation of the distinct GnRHs, and 3) may be an evolutionary advantage for refined control of reproduction in different environments.

FGF-2-responsive neural stem cell proliferation requires CCg, a novel autocrine/paracrine cofactor.

We have purified and characterized a factor, from the conditioned medium of neural stem cell cultures, which is required for fibroblast growth factor 2's (FGF-2) mitogenic activity on neural stem cells. This autocrine/paracrine cofactor is a glycosylated form of cystatin C (CCg), whose N-glycosylation is required for its activity. We further demonstrated that, both in vitro and in vivo, neural stem cells undergoing cell division are immunopositive for cystatin C. Finally, we showed in vivo functional activity of CCg by demonstrating that the combined delivery of FGF-2 and CCg to the adult dentate gyrus stimulated neurogenesis. We propose that the process of neurogenesis is controlled by the cooperation between trophic factors and autocrine/paracrine cofactors, of which CCg is a prototype.

DNA contacts by protein domains of the molluscum contagiosum virus type-1B topoisomerase.

All poxviruses studied encode a type 1B topoisomerase that introduces transient nicks into DNA and thereby relaxes DNA supercoils. Here we present a study of the protein domains of the topoisomerase of the poxvirus molluscum contagiosum (MCV), which allows us to specify DNA contacts made by different domains. Partial proteolysis of the enzyme revealed two stable domains separated by a protease-sensitive linker. A fragment encoding the linker and carboxyl-terminal domain (residues 82-323) was overexpressed in Escherichia coli and purified. MCV topoisomerase (MCV-TOP)(82-323) could relax supercoiled plasmids in vitro, albeit with a slower rate than the wild-type enzyme. MCV-TOP(82-323) was sensitive to sequences in the favored 5'-(T/C)CCTT-3' recognition site and also flanking DNA, indicating that some of the sequence-specific contacts are made by residues 82-323. Assays of initial binding and covalent catalysis by MCV-TOP(82-323) identified the contacts flanking the 5'-CCCTT-3' sequence at +10, +9, -2, and -3 to be important. Tests with substrates containing a 5-bridging phosphorothiolate that trap the cleaved complex revealed that correct contacts to the flanking sequences were important in the initial cleavage step. MCV-TOP(82-323) differed from the full-length protein in showing reduced sensitivity to mutations at a position within the 5'-(T/C)CCTT-3' recognition site, consistent with a model in which the amino-terminal domain contacts this region. These findings provide insight into the division of labor within the MCV-TOP enzyme.

Function and dysfunction of CD4(+) T cells in the immune response to melanoma.

A major difficulty for tumor immunotherapy derives from the phenomenon that the encounter of the immune system with an antigen does not necessarily result in activation, but may also be followed by the induction of tolerance either by anergy or physical deletion. It is well established that the immune system becomes alerted only in the face of danger, i.e. upon ligand recognition in the context of increased expression of costimulatory molecules, adhesion molecules, and MHC molecules on antigen-presenting cells (APC). The pivotal role of CD4(+) T lymphocytes in this process has been established. However, encounter of CD4(+) T cells with either MHC class II-expressing melanoma cells or certain tumor antigen-presenting APC has been reported to induce antigen-specific tolerance. Thus, as more is learned about the molecular regulation of immune responses and the role of CD4(+) T cells in particular, additional strategies to block inhibitory pathways of T-cell activation will be developed. Such strategies are likely to be based on a modulation of the context in which antigen is encountered by the immune system, e.g. in situ cytokine therapy, induction of costimulatory molecules or the simulation of 'danger' signals.

The disulfide bond arrangement in the extracellular domain of the activin type II receptor.

The initial step in the signaling cascade of the growth factor activin involves its binding to the extracellular domain of the activin type II receptor. This receptor domain contains 10 cysteine residues which are engaged in intramolecular disulfide bonds. To elucidate the structural framework of this domain we have characterized its disulfide-bonding pattern using an extracellular fragment of the receptor which binds activin A with high affinity. By combining proteolysis with mass spectroscopy and chemical sequence analysis, the disulfide connectivity was determined to be as follows: C1-C3, C2-C4, C5-C8, C6-C7, and C9-C10. A similar disulfide arrangement occurs in a family of snake toxins for which the three-dimensional structure is known.

Activin and inhibin binding to the soluble extracellular domain of activin receptor II.

Activins and inhibins belong to the transforming growth factor-beta-like superfamily of growth and differentiation factors that exert pleiotropic effects in many target tissues. Heteromeric association of activin with two structurally related receptor serine/threonine kinases, activin receptor types I and II, initiates downstream signaling events. The extracellular domain of type II mouse activin receptor (ActRII ECD) was expressed in the baculovirus system, purified in three steps by lectin affinity, anion exchange, and reverse phase chromatography, and further characterized by mass spectrometry. The reduction in the apparent size of the purified ActRII ECD on SDS-PAGE after treatment with glycosidases provided evidence for N- and O-linked oligosaccharides. Specific receptor/ligand complexes of [125I] activin A to ActRII ECD or [125I]ActRII ECD to activin A were analyzed by cross-linking and immunoprecipitation. Two major radiolabeled bands were observed on SDS-PAGE with mobilities consistent with the expected size of ActRII ECD/betaA or ActRII ECD/betaAbetaA. When inhibin A was cross-linked to [125I]ActRII ECD, a slower migrating complex corresponding to ActRII ECD/betaAalpha was also observed. The apparent dissociation constant (Kd) for activin A binding to ActRII ECD was 2-7 nM. This Kd value is approximately an order of magnitude greater than that of the full-length membrane-associated type II receptor. Treatment of cultured rat anterior pituitary cells with ActRII ECD attenuated FSH secretion in response to exogenous activin A or endogenous activin B. These data indicate that the soluble ActRII ECD has structural determinants that are sufficient for high affinity ligand binding.

Three-finger toxin fold for the extracellular ligand-binding domain of the type II activin receptor serine kinase.

The transforming growth factor beta (TGFbeta) superfamily of cytokines elicit diverse biological responses by interacting with two distinct, but structurally related transmembrane receptor serine kinases (type I and type II). The binding of these dimeric ligands to the type II receptor is the first event in transmembrane signaling for this family. Here we report the 1.5 A resolution crystal structure of the extracellular ligand-binding domain of the type II activin receptor (ActRII-ECD), which reveals a fold similar to that of a class of toxins known as three-finger toxins. This fold is primarily dictated by disulfide bonds formed by eight conserved cysteines, with a characteristic spacing, and thus is likely to be shared by most of the type I and II receptors for the TGFbeta family. Sequence comparison with an evolutionarily distant activin binding-protein identifies several conserved residues, including two hydrophobic clusters that may form binding surfaces for activin and the type I receptor.

Regulation of IL-6 synthesis in human peripheral blood mononuclear cells by C3a and C3a(desArg).

The anaphylatoxin C3a has been reported to have immunomodulatory effects on a number of different cell types. In this study we investigated the effects of C3a and C3a(desArg) on gene expression and protein secretion of IL-6 in human PBMCs, either alone or in combination with LPS or IL-1beta. C3a or C3a(desArg) alone exhibited no effect on the expression or secretion of IL-6. However, when PBMC were stimulated with LPS or IL-1beta, both C3a and C3a(desArg) were found to enhance IL-6 release by PBMC in a dose-dependent manner. Since C3a has been shown to induce PGE2 production by monocytes, and PGE2 has been shown to influence cytokine production, we investigated the potential role of PGE2 in C3a-mediated enhancement of LPS- and IL-1beta-induced IL-6 production. Indomethacin blocked PGE2 release, but had no influence on the observed effects of C3a, suggesting that the effects of C3a on IL-6 production are independent of PGE2 formation by monocytes. Northern blot analysis showed that C3a as well as C3a(desArg) enhanced LPS-induced mRNA levels for IL-6. Pretreatment of PBMCs with pertussis toxin blocked the functions of C3a and C3a(desArg), indicating that the actions of these two molecules are mediated by a G protein-coupled pathway. Furthermore, we investigated the effects of C3a and C3a(desArg) on induction of NF-kappaB and activating protein-1 binding. Both molecules enhanced LPS-induced NF-kappaB and activating protein-1 binding activity. These results demonstrate the capacity of intact C3a and its circulating des-Arg form to exert immunmodulatory effects in vitro.