Learning chronobiology by improving Wikipedia.

Although chronobiology is of growing interest to scientists, physicians, and the general public, access to recent discoveries and historical perspectives is limited. Wikipedia is an online, user-written encyclopedia that could enhance public access to current understanding in chronobiology. However, Wikipedia is lacking important information and is not universally trusted. Here, 46 students in a university course edited Wikipedia to enhance public access to important discoveries in chronobiology. Students worked for an average of 9 h each to evaluate the primary literature and available Wikipedia information, nominated sites for editing, and, after voting, edited the 15 Wikipedia pages they determined to be highest priorities. This assignment (http://www.nslc.wustl.edu/courses/Bio4030/wikipedia_project.html) was easy to implement, required relatively short time commitments from the professor and students, and had measurable impacts on Wikipedia and the students. Students created 3 new Wikipedia sites, edited 12 additional sites, and cited 347 peer-reviewed articles. The targeted sites all became top hits in online search engines. Because their writing was and will be read by a worldwide audience, students found the experience rewarding. Students reported significantly increased comfort with reading, critiquing, and summarizing primary literature and benefited from seeing their work edited by other scientists and editors of Wikipedia. We conclude that, in a short project, students can assist in making chronobiology widely accessible and learn from the editorial process.

Serotonin 1A receptors, serotonin transporter binding and serotonin transporter mRNA expression in the brainstem of depressed suicide victims.

Suicide and depression are associated with reduced serotonergic neurotransmission. In suicides, there is a reduction in serotonin transporter (SERT) sites and an increase in postsynaptic 5-HT(1A) receptors in localized regions of the prefrontal cortex. In depression, there is a diffuse decrease in SERT binding throughout the dorsoventral extent of the prefrontal cortex. Serotonergic innervation of the prefrontal cortex arises predominantly from neurons in the brainstem dorsal raphe nucleus (DRN). We, therefore, examined postmortem SERT binding and mRNA expression, as well as 5-HT(1A) autoreceptor binding in the DRN of 10 matched pairs of controls and depressed suicide victims. The concentration of SERT sites, SERT mRNA, and 5-HT(1A) binding was not different between controls and suicides (p >.05). In the DRN of suicides, the volume of tissue defined by 5-HT(1A) binding was 40% smaller than controls. An index of the total number of 5-HT(1A) receptors (receptor binding x volume of receptor distribution) was 43.3% lower in the DRN of suicides, compared with controls. The suicide group had 54% fewer DRN neurons expressing SERT mRNA compared with controls. In the serotonin neurons that expressed the SERT gene, expression per neuron was greater in suicides. Less total 5-HT(1A) and SERT binding is consistent with results of in vivo studies in depression. Less feedback inhibition of serotonin DRN firing via 5-HT(1A) autoreceptors and enhancement of serotonin action due to less uptake of serotonin, is consistent with compensatory changes in response to hypofunction in depressed suicides.

Characteristics of silicon nitride after O2 plasma surface treatment for pH-ISFET applications.

Silicon nitride (Si3N4) sensing gate pH-ion-selective field effect transistors (ISFETs) were treated by 2.54-GHz microwave O2 plasma, the results show the ISFET sensitivity has an advantage up to 24% increment after the plasma treatment. Electron spectroscopy for chemical analysis (ESCA) is used to make sure that the plasma treatment is not just a native oxide cleaning procedure. The samples, which were immobilized with glutaraldehyde used as a bifunctional reagent and 3-aminopropyItriethoxysilane used as an adhesion promoter were studied. The binding force between the glucose oxidase and glutaraldehyde immobilized samples, and the element concentrations of nitrogen in 3-aminopropyltriethoxysilane immobilized samples are higher which were treated by plasma.

In vitro autoradiography of serotonin 5-HT(2A/2C) receptor-activated G protein: guanosine-5'-(gamma-[(35)S]thio)triphosphate binding in rat brain.

Agonist activation of G protein-coupled receptors induces an increase in the binding of guanosine 5'-(gamma-[(35)S]thio)triphosphate ([(35)S]GTPgammaS); this increase in binding has been used as a tool to investigate receptor interaction with the heterotrimer guanine nucleotide-binding regulatory protein (G protein). The present study uses agonist-stimulated [(35)S]GTPgammaS binding to characterize serotonin 5-HT(2A/2C) receptors in rat brain membrane fractions and demonstrate the anatomical localization of the receptors by in vitro autoradiography on slide-mounted sections. The stimulatory effect of the agonist [1-(2,5-dimethoxy-4-iodophenyl)]-2 aminopropane (DOI) is compared to that of serotonin (5-HT). Autoradiography revealed a similar localization of DOI- and 5-HT-stimulated binding of [(35)S]GTPgammaS in distinct areas of prefrontal and parietal cortex, consistent with previously reported 5-HT(2A) receptor distribution. Specific binding was demonstrated in the frontal and parietal cortex, medial prefrontal, and cingular and orbital-insular areas as well as in the hippocampal formation, septal areas, the nucleus accumbens, and the choroid plexus. MDL 100105, a specific 5-HT(2A) antagonist, and ketanserin, an antagonist of 5-HT(2A/2C) receptors, blocked DOI stimulation in all labeled areas, whereas 5-HT stimulation was only partially blocked (70-80%). A small but significant inhibition was observed with the specific antagonist of 5-HT(2C/2B), SB 206553. This autoradiographic technique provides a useful tool for measuring in situ changes in specific receptor-Gq protein coupling in anatomically discrete brain regions, under physiological and pathological conditions.

Ca(2+)-evoked serotonin secretion by parafollicular cells: roles in signal transduction of phosphatidylinositol 3'-kinase, and the gamma and zeta isoforms of protein kinase C.

Parafollicular (PF) cells secrete 5-HT in response to stimulation of a G-protein-coupled Ca(2+) receptor (CaR) by increased extracellular Ca(2+) (upward arrow[Ca(2+)](e)). We tested the hypothesis that protein kinase C (PKC) participates in stimulus-secretion coupling. Immunoblots from membrane and cytosolic fractions of isolated PF cells revealed conventional (alpha, betaI, and gamma), novel (delta and epsilon), and atypical (iota/lambda and zeta) PKCs. Only PKCgamma was found to have been translocated to the membrane fraction when secretion of 5-HT was evoked by upward arrow[Ca(2+)](e) or phorbol esters. Although phorbol downregulation caused PKCgamma to disappear, secretion was only partially inhibited. A similar reduction of upward arrow[Ca(2+)](e)-evoked secretion was produced by inhibitors of conventional and/or novel PKCs (Gö 6976, calphostin C, and pseudoA), and these compounds did not inhibit secretion at all when applied to phorbol-downregulated cells. In contrast, the phorbol downregulation-resistant component of secretion was abolished by pseudoZ, which inhibits the atypical PKCzeta. Stimulation of PF cells with upward arrow[Ca(2+)](e) increased the activity of immunoprecipitated PKCzeta (but not PKCiota/lambda), and the activity of this PKCzeta was inhibited by pseudoZ. PF cells were found to express regulatory (p85) and catalytic (p110alpha and p110beta) subunits of phosphatidylinositol 3'-kinase (PI3'-kinase). upward arrow[Ca(2+)](e) increased the activity of immunoprecipitated PI3'-kinase; moreover, PI3'-kinase inhibitors (wortmannin and LY294002) antagonized secretion. We suggest that PKC isoforms mediate secretion of 5-HT by PF cells in response to stimulation of the CaR. PKC involvement can be accounted for by PKCgamma and an isoform sensitive to inhibition by pseudoZ, probably PKCzeta, which is activated via PI3'-kinase.

Selective, tight-binding inhibitors of integrin alpha4beta1 that inhibit allergic airway responses.

Integrin alpha4beta1 mediates leukocyte recruitment, activation, mediator release, and apoptosis inhibition, and it plays a central role in inflammatory pathophysiology. High-affinity, selective inhibitors of alpha4beta1, based on the Leu-Asp-Val (LDV) sequence from the alternatively spliced connecting segment-1 (CS-1) peptide of cellular fibronectin, are described that employ a novel N-terminal peptide "cap" strategy. One inhibitor, BIO-1211, was approximately 10(6)-fold more potent than the starting peptide and exhibited tight-binding properties (koff = 1.4 x 10(-4) s-1, KD = 70 pM), a remarkable finding for a noncovalent, small-molecule inhibitor of a protein receptor. BIO-1211 was also 200-fold selective for the activated form of alpha4beta1, and it stimulated expression of ligand-induced epitopes on the integrin beta1 subunit, a property consistent with occupancy of the receptor's ligand-binding site. Pretreatment of allergic sheep with a 3-mg nebulized dose of BIO-1211 inhibited early and late airway responses following antigen challenge and prevented development of nonspecific airway hyperresponsiveness to carbachol. These results show that highly selective and potent small-molecule antagonists can be identified to integrins with primary specificity for peptide domains other than Arg-Gly-Asp (RGD); they confirm the generality of integrins as small molecule targets; and they validate alpha4beta1 as a therapeutic target for asthma.

Expression and development of a functional plasmalemmal 5-hydroxytryptamine transporter by thyroid follicular cells.

5-Hydroxytryptamine (5-HT) is synthesized and secreted by thyroid parafollicular (PF) cells. As all PF granules contain 5-HT, it is released whenever PF cells secrete. Because 5-HT stimulates follicular (F) cells and can modulate their response to TSH, 5-HT has been proposed to be a paracrine PF to F cell transmitter. This role would require a thyroid mechanism to rapidly inactivate 5-HT. A 5-HT transporter (SERT) in the plasma membrane of serotonergic neurons inactivates neuronal 5-HT. We thus tested the hypothesis that this molecule is expressed in the thyroid. Messenger RNA encoding SERT was demonstrated in both the human thyroid and a rat F cell line (FRTL-5). SERT immunoreactivity was detected in rat F, but not PF, cells. Transporter-mediated uptake of [3H]5-HT by F cells arose early in development (E13 in mice) and was maintained in adult life in mice, guinea pigs, bats, and rats (FRTL-5 cells). These observations indicate that a functional SERT is expressed in the thyroid, not by the 5-HT-secreting PF cells, but by their putative F cell targets.

Acidification of serotonin-containing secretory vesicles induced by a plasma membrane calcium receptor.

Parafollicular (PF) cells secrete 5-hydroxytryptamine in response to increased extracellular Ca2+ ([Ca2+]e). This stimulus causes Cl- channels in PF secretory vesicles to open, leading to vesicle acidification. PF cells express a plasmalemmal heptahelical receptor (CaR) that binds Ca2+, Gd3+, and Ba2+. We now report that the CaR mediates vesicle acidification. Ca2+, Gd3+, and Ba2+ induced vesicle acidification, which was independent of channel-mediated Ca2+ entry. Agonist-induced vesicle acidification was blocked by pertussis toxin, inhibitors of phosphatidylinositol-phospholipase C, calmodulin, NO synthase, guanylyl cyclase, or protein kinase G. PF cells contained NO synthase immunoreactivity, and vesicles were acidified by NO donors and dibutyryl cGMP. [Ca2+]e, and Gd3+ mobilized thapsigargin-sensitive internal Ca2+ stores. [35S]G alpha i and [35S]G alpha q were immunoprecipitated from PF membranes incubated with agonists in the presence of [35S]adenosine 5'-O-(thiotriphosphate). Labeling of G alpha i but not G alpha q was antagonized by pertussis toxin. Vesicles acidified in response to activation of protein kinase C; however, protein kinase C inhibition blocked calcium channel- but not CaR-dependent acidification. We propose the following signal transduction pathway: CaR -> Gi -> phosphatidylinositol-phospholipase C -> inositol 1,4,5-trisphosphate -> [Ca2+]i -> Ca2+/calmodulin -> NO synthase -> NO -> guanylyl cyclase -> cGMP -> protein kinase G -> opens vesicular Cl- channel.

Secretogogue-induced gating of chloride channels in the secretory vesicles of parafollicular cells.

Thyroid parafollicular (PF) cells are neural crest-derived endocrine cells that secrete serotonin and calcitonin. The secretory vesicles of PF cells acidify when secretion is induced by increased extracellular Ca2+ or TSH. We tested the hypothesis that acidification is regulated by secretogogue-gated Cl- channels in vesicular membranes. Cl- channel (p64) immunoreactivity was enriched in purified PF vesicles. X-Ray microanalysis showed a change in chlorine level in PF vesicles in response to secretogogue-stimulation of isolated cells. Secretogogue stimulation also altered the degree of p64 channel phosphorylation. Protein kinase and phosphatase inhibitors antagonized secretogogue-induced vesicle acidification and secretion; however, secretion could occur even when acidification was blocked. We conclude that acidification of PF vesicles is regulated by a gatable Cl- channel in vesicle membranes and that protein phosphorylation and dephosphorylation are involved in channel activation. Acidification of vesicles is not required for exocytosis.

Serotonin binding protein: synthesis, secretion, and recycling.

Serotonin binding protein (SBP) is present in all neurectodermally derived cells that store serotonin (5-HT). Three forms of SBP have been detected (68, 56, and 45 kDa), and antibodies to SBP that interfere with the binding of 5-HT react with each of these proteins. The current experiments test two hypotheses: (a) that the 56- and 45-kDa forms of SBP are produced by posttranslational cleavage of a 68-kDa precursor molecule; and (b) that 45-kDa SBP is a constituent of serotonergic secretory vesicles. Pulse-chase experiments were carried out using medullary thyroid carcinoma cells as a model. These neurectodermally derived cells produce 5-HT and all three forms of SBP. Following pulse labeling for 20 min with L-[35S]methionine, the cells were incubated in the presence of an excess of unlabeled L-methionine for 0, 30, 60, or 90 min at 37 degrees C. Alternatively, the chase was performed under conditions (20 degrees C, inhibition of ATP generation) that delay or stop transport of newly synthesized proteins from the rough endoplasmic reticulum through the Golgi apparatus. Following incubation, the cells were washed and solubilized, and SBP was immunoprecipitated. Radioactive proteins in the immunoprecipitate were electrophoretically resolved and quantified. Immediately after the pulse, each of the three forms of SBP was found to be labeled with 35S. The relative proportions of 35S-labeled 68-, 56-, and 45-kDa SBP remained the same at each interval of chase. These proportions were not changed when the chase was carried out at 20 degrees C or under conditions that blocked the biosynthesis of ATP. These observations suggest that each form of SBP is a primary product of translation, that the smaller forms of SBP are not produced by cleavage from a larger molecule, and that the size of the primary products of translation is not altered by passage to the Golgi apparatus or a post-Golgi compartment. When secretion was induced, 45-kDa SBP, but not 56- or 68-kDa SBP, was released to the medium. When antibodies to 45-kDa SBP were added to the medium at the time secretion was induced, antibody binding sites appeared as patches on the cell surfaces. Because of these sites, cells were lysed when they were stimulated to secrete in the presence of antibodies to 45-kDa SBP and guinea pig complement.(ABSTRACT TRUNCATED AT 400 WORDS)

Serotonergic signalling between thyroid cells: protein kinase C and 5-HT2 receptors in the secretion and action of serotonin.

Parafollicular (PF) cells of the thyroid gland are neural crest derivatives, which costore the neurotransmitter, 5-hydroxytryptamine (5-HT) with calcitonin. PF cells are located adjacent to follicular (F) cells within the basement membrane of thyroid follicles. It has been proposed that 5-HT serves an intercellular signalling function in the thyroid and that F cells are its target. This proposal was tested by using cell lines derived from PF (medullary thyroid carcinoma [MTC]) and F (FRTL-5) cells to study the mechanisms that mediate the secretion and action of 5-HT. Secretion of 5-HT by MTC cells was evoked by thyroid stimulating hormone, thyrotropin (TSH), elevated extracellular calcium (increases [Ca2+]e), or by agents that increase intracellular cAMP (increases [cAMP]i). When protein kinase C (PKC) was down-regulated by prolonged treatment of MTC cells with phorbol 12-myristate 13-acetate (PMA), or PKC was inhibited by staurosporin, the TSH- or PMA-evoked secretion of 5-HT was blocked; however, interference with PKC function did not affect 5-HT secretion evoked by increases [Ca2+]e or increases [cAMP]i. In the putative targets, FRTL-5 cells, 5-HT increased the turnover of phosphoinositides (PI), cytosolic calcium (increases [Ca2+]i), increases [cAMP]i, and biphasically modified the effect of TSH on cAMP. All of these 5-HT effects were inhibited by 5-HT2 receptor antagonists (spiperone and ketanserin) and by pertussis toxin (PTx), suggesting that the actions of 5-HT are mediated by 5-HT2 receptors, which are coupled to a G protein. This suggestion was supported by the following additional observations: FRTL-5 membranes bound the 5-HT2 agonist, [125I]2,5-dimethoxy-4-iodophenylisopropylamine ([125I]-DOI), and anti-idiotypic antibodies, which recognize 5-HT2 receptors. [125I]-DOI binding was inhibited by guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S) and the antibodies were displaced by spiperone. Data are consistent with the hypothesis that 5-HT serves as a PF to F cell messenger.

Identification of serotonin receptors recognized by anti-idiotypic antibodies.

Anti-idiotypic antibodies were generated by immunizing rabbits with affinity-purified antibodies to serotonin (5-hydroxytryptamine; 5-HT). Anti-5-HT activity was removed from the resulting antisera by chromatography through a 5-HT affinity column. The anti-idiotypic antibodies were demonstrated by enzyme-linked immunosorbent assay to bind to affinity-purified whole anti-5-HT antibodies and their Fab fragments. Anti-idiotypic antibodies, purified by affinity chromatography on columns to which antibodies to 5-HT were coupled, competed with 5-HT (covalently bound to protein) for the binding sites on anti-5-HT antibodies and serotonin binding protein. The anti-idiotypic antibodies antagonized the binding of [3H]5-HT to membranes isolated from the cerebral cortex, striatum, and raphe area more than to membranes from hippocampus or cerebellum. The anti-idiotypic antibodies also blocked the binding of the 5-HT1B-selective ligand (-)-[125I]iodocyanopindolol (in the presence of 30 microM isoproterenol) to cortical membranes. In contrast, anti-idiotypic antibodies failed to inhibit binding of the 5-HT1A-selective ligand 8-hydroxy-2-(di-n-[3H]propylamino)-tetralin [( 3H]8-OH-DPAT) to raphe area membranes or hippocampal membranes. These observations suggested that the anti-idiotypic antibodies may recognize some 5-HT receptor subtypes but not others. This hypothesis was tested by ascertaining the ability of anti-idiotypic antibodies to immunostain cells transfected in vitro with cDNA encoding the 5-HT1C or 5-HT2 receptor or with a genomic clone encoding the 5-HT1A receptor. Punctate sites of immunofluorescence were found on the surfaces of fibroblasts that expressed 5-HT1C and 5-HT2 receptors, but not on the surfaces of HeLa cells that expressed 5-HT1A receptors. Immunostaining of cells by the anti-idiotypic antibodies was inhibited by appropriate pharmacological agents: immunostaining of cells expressing 5-HT1C receptors was blocked by mesulergine (but not ketanserin, 8-OH-DPAT, or spiperone), whereas that of cells expressing 5-HT2 receptors was blocked by ketanserin or spiperone (but not mesulergine or 8-OH-DPAT). The anti-idiotypic antibodies failed to inhibit the uptake of [3H]5-HT by serotonergic neurons. It is concluded that the anti-idiotypic antibodies generated with anti-5-HT serum recognize the 5-HT1B, 5-HT1C, and 5-HT2 receptor subtypes; however, neither 5-HT1A receptors nor 5-HT uptake sites appear to react with these antibodies.

Multiple signal transduction mechanisms leading to the secretion of 5-hydroxytryptamine by MTC cells, a neurectodermally derived cell line.

Parafollicular (PF) cells of the thyroid gland are neural crest derivatives. These cells remain plastic even in adult animals and can be induced to exhibit neural properties when exposed to NGF in vitro. A human cell line derived from PF cells, medullary thyroid carcinoma (MTC), has previously been shown to synthesize and store 5-HT, a serotonin-binding protein (SBP), and several neuropeptides; moreover, when grown in impoverished media, MTC cells display neural properties. The purpose of the current study was to utilize MTC cells as a neurally relevant model system to investigate factors involved in mediating 5-HT secretion. Electron microscopic immunocytochemistry revealed that secretory vesicles of MTC cells costore immunoreactive 5-HT with SBP and calcitonin. The cAMP derivative, N6-2'-O-dibutyryl-adenosine 3',5'-cyclic monophosphate (dibutyryl-cAMP; 1.0 mM) increased the concentration of 5-HT in MTC cells and almost doubled the rate of synthesis of 5-HT from L-tryptophan. Dibutyryl-cAMP also significantly increased the secretion of 5-HT. Cycloheximide (20 micrograms/ml) and anisomycin (20 microM) inhibited the dibutyryl-cAMP-induced increase of 5-HT release, suggesting that this action of dibutyryl-cAMP requires protein synthesis. Cholera toxin (1.0 microgram/ml) and forskolin (0.05 mM) in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (1.0 mM) both increased 5-HT biosynthesis and secretion. Attempts were made to identify a ligand that stimulates cAMP-mediated secretion of 5-HT. Both thyroid-stimulating hormone (TSH: 50 mU/ml) and elevated [Ca2+]e (7.0 mM), each of which acts as a secretogogue for PF cells, stimulated the secretion of 5-HT. The effect of TSH was Ca2(+)-dependent. Immunocytochemistry with monoclonal antibodies to the TSH receptor confirmed that these receptors are present on MTC cells. Neither TSH nor elevated [Ca2+]e elevated cAMP levels. Measurements of Fura-2 fluorescence, however, indicated that both TSH and elevated [Ca2+]e increased cytosolic calcium ([Ca2+]i), as did elevation of [K+]e. It is concluded that exocytosis can be triggered in MTC cells by multiple signal transduction mechanisms. Either cAMP or elevated [Ca2+]i can stimulate secretion; however, a secretogogue that increases cAMP has yet to be identified.

Preparation and characterization of monoclonal antibodies to serotonin binding protein.

Serotonin binding protein (SBP) is a constituent of the synaptic vesicles of serotonergic neurons. Two types of SBP, with molecular masses of 45 kDa and 56 kDa, have been purified. To determine whether there are shared epitopes between the two forms of SBP, we raised and tested for cross-reactivity monoclonal antibodies (MAbs) against each form of SBP. We obtained 12 MAbs, all of which recognize both forms of SBP. Hybridoma clones were produced by fusing P3 X 63Ag8.653 mouse myeloma cells with spleen cells from a mouse that had been immunized with 45-kDa or 56-kDa SBP. Culture supernatants were screened for the presence of anti-SBP antibodies. MAb isotypes were determined by immunodiffusion, using immunoglobulin type-specific antisera. Each antibody to SBP consisted of only a single subclass of immunoglobulin (IgM). We obtained 12 MAbs, each of which interacted with both forms of SBP, as judged by enzyme-linked immunosorbent assay and immunoblot analysis. Ascites fluid to one clone (44-10) was obtained and affinity-purified. In the presence of goat anti-mouse IgM, the partially purified 44-10 antibodies quantitatively immunoprecipitated SBP from crude brain extracts. Immunoblotting revealed two major bands corresponding to 45 kDa and 56 kDa and a minor band corresponding to 68 kDa. MAb 44-10 blocked the binding of [3H]serotonin ([3H]5-HT) to 45-kDa and 56-kDa SBP in a concentration-dependent manner. The 68-kDa protein was found to bind [3H]5-HT. Sites reacting with MAB 44-10 were located immunocytochemically in sections of rat brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Human medullary thyroid carcinoma: characterization of the serotonergic and neuronal properties of a neurectodermally derived cell line.

Parafollicular (PF) cells have been found to be a good model system for the study of serotonergic cellular mechanisms relevant to neurons. PF cells are derived from the same region of the neural crest that gives rise to the neurons of the gut and are capable of extending neurofilament-bearing neuritic processes. PF cells also synthesize 5-hydroxytryptamine (5-HT) and costore 5-HT in the same vesicles as the specific 5-HT-binding protein, 45 kDa SBP. A hypothesis has been advanced that PF cells and enteric neurons share a common developmental precursor. The present investigation was undertaken in order to determine whether a human medullary thyroid carcinoma (MTC) cell line, which is derived from PF cells, sufficiently mimics PF cells that it can be substituted for them in investigations of serotonergic cellular biology. In contrast to PF cells, MTC cells can be propagated in vitro to provide adequate amounts of material for biochemical studies. MTC cells were found to contain neuropeptides, including calcitonin, calcitonin gene-related peptide, and somatostatin, which have also been reported to be present in PF cells and enteric neurons. MTC cells also were observed to store endogenous 5-HT, to be able to synthesize 3H-5-HT from 3H-L-tryptophan, and to take up 3H-5-HT from the ambient medium by a carrier-mediated mechanism very similar to that of serotonergic neurons. In addition, the longterm accumulation of 3H-5-HT in MTC cells was antagonized by reserpine, suggesting that the cells contain 5-HT storage vesicles that, like the synaptic vesicles of serotonergic neurons, are characterized by a reserpine-sensitive transporter of biogenic amines. MTC cells also contain type A, but not type B, monoamine oxidase. Finally, MTC cells were found to contain both 45 and 56 kDa SBP. MTC cells thus retain a great many of the properties of PF cells, and, like PF cells, they are serotonergic cells with characteristics similar to serotonergic neurons. Substantial differences were found in the content of immunoreactive 5-HT and neuropeptides in individual MTC cells. Moreover, the release of newly synthesized 5-HT to the medium exceeded the ability of the cells to store the amine. Studies of the ultrastructure of the MTC cells revealed a limited and highly variable number of secretory granules, probably accounting for their limited 5-HT storage capacity and for the heterogeneity of immunostaining with antisera to 5-HT or neuropeptides.(ABSTRACT TRUNCATED AT 400 WORDS)

A Ca2+-dependent protein kinase activity associated with serotonin binding protein.

The endogenous phosphorylation of serotonin binding protein (SBP), a soluble protein found in central and peripheral serotonergic neurons, inhibits the binding of 5-hydroxytryptamine (5-HT, serotonin). A protein kinase activity that copurifies with SBP (SBP-kinase) was partially characterized and compared with calcium/calmodulin-dependent protein kinase II (CAM-PK II). SBP itself is not the enzyme since heating destroyed the protein kinase activity without affecting the capacity of the protein to bind [3H]5-HT. SBP-kinase and CAM-PK II kinase shared the following characteristics: (1) size of the subunits; (2) autophosphorylation in a Ca2+-dependent manner; and (3) affinity for Ca2+. In addition, both forms of protein kinase phosphorylated microtubule-associated proteins well and did not phosphorylate myosin, phosphorylase b, and casein. Phorbol esters or diacylglycerol had no effect on either of the protein kinases. However, substantial differences between SBP-kinase and CAM-PK II were observed: (1) CAM enhanced CAM-PK II activity, but had no effect on SBP-kinase; (2) synapsin I was an excellent substrate for CAM-PK II, but not for SBP-kinase; (3) 5-HT inhibited both the autophosphorylation of SBP-kinase and the phosphorylation of SBP, but had no effect on CAM-PK II. These data indicate that SBP-kinase is different from CAM-PK II. Phosphopeptide maps of SBP and SBP-kinase generated by digestion with S. aureus V8 protease are consistent with the conclusion that these proteins are distinct molecular entities. It is suggested that phosphorylation of SBP may regulate the transport of 5-HT within neurons.

Prenatal development of serotonin binding protein in relation to other transmitter-related characteristics of central serotonergic neurons.

Serotonin binding protein (SBP) is a neuron-specific protein that binds serotonin (5-HT) with high affinity and is concentrated in synaptic vesicles. 5-HT has been shown to be stored in situ in a macromolecular complex with SBP. We have now investigated the ontogeny of SBP in the rat CNS. The time course of the appearance of SBP was related to the appearance of tryptophan hydroxylase, endogenous 5-HT and monoamine oxidase (MAO; types A and B). Binding of [3H]5-HT by SBP was assayed using molecular sieve chromatography. SBP had appeared by day E16; its activity then rose rapidly and reached adult levels (150 nmol [3H]5-HT/g protein) at days E18-E19. Tryptophan hydroxylase activity was measured by following the accumulation in vivo (30 min) of 5-hydroxytryptophan (5-HTP) in the brains of rat embryos whose mothers were treated with the aromatic L-amino acid decarboxylase inhibitor, NSD-1015, (100 mg/kg; i.p.). Tryptophan hydroxylase activity was first detectable at E15, remained present but at a low level through day E20 and then rapidly increased to reach 75% of the adult level at birth (747 pmol/g brain wet wt.). The development of stores of endogenous 5-HT paralleled the time course of development of tryptophan hydroxylase rather than that of SBP. 5-HT was first detected at E15, remained low until the end of intrauterine life and at birth was 50% of the adult level (2640 pmol/g brain wet wt.). MAO activity was determined in crude mitochondrial fractions by measuring 5-hydroxyindoleacetic acid produced from 5-HT as substrate. This activity was already present prior to day E15 (the activity of type B MAO was higher than that of type A) and reached adult levels at day E20 (55 pmol/mg protein/min; A, B). It is concluded that the potential of neurons to store 5-HT, as measured by the activity of SBP, develops more rapidly than their ability to produce 5-HT. Moreover, although the ratio of its two forms changes, MAO activity appears very early in development.

Purification and properties of the biotin repressor. A bifunctional protein.

Definitive evidence is presented for the bifunctional nature of the biotin repressor protein which possesses both regulatory and enzymatic activities. The repressor protein can activate biotin in the presence of ATP to form biotinyl-5'-adenylate, the co-repressor which remains tightly bound to the repressor protein. This complex can either bind to the operator site and inhibit transcription or transfer the biotinyl moiety to a lysine residue of the apoenzyme of acetyl-CoA carboxylase. The two activities were coincident throughout a purification procedure which resulted in a 3500-fold increase in activity. Gel electrophoresis of the purified preparation, under native or denaturing conditions, showed three proteins with the activity corresponding to the major protein band of apparent Mr = 34,000. On gel exclusion chromatography, the activity was also associated with a protein of Mr varying fro 37,000-44,000, indicating the protein is monomeric. The occasional appearance of multiple bands with biological activity in the native gels suggests that the repressor protein can also exist in multimeric forms. On chromatofocusing, the repressor activity and the holoenzyme synthetase activity were coincidental, with the peak of activity at pH 7.2, the isoelectric point. Only a single protein band with Mr = 34,000 was observed on SDS gel electrophoresis of all fractions showing activity.

Mode of action of the biotin antimetabolites actithiazic acid and alpha-methyldethiobiotin.

Actithiazic acid and alpha-methyldethiobiotin inhibited the conversion of dethiobiotin to biotin resting-cell suspensions of Escherichia coli. The concentrations which effected 50% inhibition were 0.45 and 1.1 microM for actithiazic acid and alpha-methyldethiobiotin, respectively. Cells grown in low concentrations of the two biotin antimetabolites showed derepression of the biotin A operon, as evidenced by the enhanced levels of the enzymes 7,8-diaminopelargonic acid aminotransferase and dethiobiotin synthetase. Derepression was not due to any direct regulatory effect of the antibiotics but was the consequence of the inhibition of the biotin synthetase enzyme; this inhibition prevented the intracellular concentration of biotin from reaching the levels required for normal regulation of the biotin A operon.