Identification of cell-specific messenger ribonucleic acids in oxytocinergic and vasopressinergic magnocellular neurons in rat supraoptic nucleus by single-cell differential hybridization.

Magnocellular neurons (MCNs) in the hypothalamo-neurohypophysial system synthesize high levels of the peptides oxytocin (OT) and vasopressin (VP) in separate cells. We used RT-PCR amplification of the RNA from single-cells dissected from supraoptic nuclei of lactating rats to produce cDNAs from identified OT or VP MCNs, which were used to construct OT- and VP-MCN-specific cDNA libraries. These cDNA libraries were then screened using labeled probes from the OT- and VP-cells' amplified cDNAs. Differentially hybridized colonies were isolated and characterized by slot blot hybridization, Southern blot hybridization, DNA sequencing, and in situ hybridization histochemistry. Using this approach, several novel cell-specific mRNAs were identified in the MCNs. One cell-specific clone, phosphofructokinase-C, was isolated from the OT-cell library, and five cell-specific clones were isolated from the VP-cell library. These were identified as paternally expressed gene (Peg)5/neuronatin, metallothionein III, Peg3, synaptotagmin V, and a 3'-phosphoadenosine 5'-phosphosulfate synthase 2-related mRNA. None of these genes would have been predicted to be differentially expressed in OT and VP MCNs, based on our current knowledge; and hence, this single cell differential gene expression approach has begun to further define the MCN phenotypes by identifying selectively expressed molecules in them.

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title: Morphological and electrophysiological classification of hypothalamic supraoptic neurons
abstract: In mammals, the magnocellular neurons of the supraoptic nucleus (SON) have been classified into vasopressin- (VP) and oxytocin- (OT) producing subtypes. The degree to which these neurons have distinguishable characteristics is considered in the present review. Most of the cytoarchitectonic diversity observed in some Golgi studies has yet to be attributed to differences between OT and VP neurons. The predominant SON cell type is a large bipolar neuron with relatively short and simply branching dendrites. Based on intracellular filling, large multipolar neurons probably represent a small subset of neurosecretory cells. Parvicellular multipolar and bipolar neurons may represent interneurons or subsets of neurosecretory cells. Suggestive evidence that axonal origin and spine density may differ between OT and VP neurons remains to be confirmed in rat. Different fiber systems are thought to preferentially innervate VP or OT subgroups, but only rarely have inputs to OT and VP neurons been compared at the ultrastructural level. Potentially selective inputs to OT somata may derive from the raphe system and the nucleus of the solitary tract, whereas the apparent preferential innervation of VP neurons (e.g. from the A1 region of the ventrolateral medulla) is less certain because of the overlapping dendritic fields of OT and VP neurons. Electrophysiologically, OT and VP neurons are best distinguished in vivo by their reaction to gastric, cardiovascular and suckling stimuli. The firing patterns of activated OT and VP neurons often differ, but can transiently appear indistinguishable in vivo and especially in vitro. Classification in vitro without immunochemical labelling may be aided by the presence of phasic bursting (mostly in VP neurons) and by the differential response of these neurons to certain neurochemicals or to stimulation of certain inputs. The membrane properties of OT and VP neurons are generally similar in vitro, but the range of tests has not been extensive. The depolarizing afterpotential is more often exhibited by, but is not exclusive to, VP neurons.

title: Emerging concepts of structure-function dynamics in adult brain: The hypothalamo-neurohypophysial system
abstract: As the first known of the mammalian brain's neuropeptide systems, the magnocellular hypothalamo-neurohypophysial system has become a model. A great deal is known about the stimulus conditions that activate or inactivate the elements of this system, as well as about many of the actions of its peptidergic outputs upon peripheral tissues. The well-characterized actions of two of its products, oxytocin and vasopressin, on mammary, uterine, kidney and vascular tissues have facilitated the integration of newly discovered, often initially puzzling, information into the existing body of knowledge of this important regulatory system. At the same time, new conceptions of the ways in which neuropeptidergic neurons, or groups of neurons, participate in information flow have emerged from studies of the hypothalamo-neurohypophysial system. Early views of the SON and PVN nuclei, the neurons of which make up approximately one-half of this system, did not even associate these interesting, darkly staining anterior hypothalamic cells with hormone secretion from the posterior pituitary. Secretion from this part of the pituitary, it was thought, was neurally evoked from the pituicytes that made the oxytocic and antidiuretic "principles" and then released them upon command. When these views were dispelled by the demonstration that the hormones released from the posterior pituitary were synthesized in the interesting cells of the hypothalamus, the era of mammalian central neural peptidergic systems was born. Progress in developing an ever more complete structural and functional picture of this system has been closely tied to advancements in technology, specifically in the areas of radioimmunoassay, immunocytochemistry, anatomical tracing methods at the light and electron microscopic levels, and sophisticated preparations for electrophysiological investigation. Through the judicious use of these techniques, much has been learned that has led to revision of the earlier held views of this system. In a larger context, much has been learned that is likely to be of general application in understanding the fundamental processes and principles by which the mammalian nervous system works.(ABSTRACT TRUNCATED AT 400

title: Function-related plasticity in hypothalamus.
abstract: Physiological activation of the magnocellular hypothalamo-neurohypophysial system induces a coordinated astrocytic withdrawal from between the magnocellular somata and the parallel-projecting dendrites of the supraoptic nucleus. Neural lobe astrocytes release engulfed axons and retract from their usual positions along the basal lamina. Occurring on a minutes-to-hours time scale, these changes are accompanied by increased direct apposition of both somatic and dendritic membrane, the formation of dendritic bundles, the appearance of novel multiple synapses in both the somatic and dendritic zones, and increased neural occupation of the perivascular basal lamina. Reversal, albeit with varying time courses, is achieved by removing the activating stimuli. Additionally, activation results in interneuronal coupling increases that are capable of being modulated synaptically via second messenger-dependent mechanisms. These changes appear to play important roles in control and coordination of oxytocin and vasopressin release during such conditions as lactation and dehydration.

title: Central oxytocin and reproductive behaviours.
abstract: Oxytocin is a neurohypophyseal hormone that has long been associated with uterine contraction during parturition and milk ejection during nursing. Recent studies have suggested that oxytocin is also a neurotransmitter that has central effects important for reproduction, including the initiation of parental and sexual behaviours. This review describes oxytocin pathways in the brain and examines their regulation by gonadal steroids. Brain oxytocin receptors are remarkable for their plasticity and for striking species differences in their distribution. The molecular characterization of this receptor has provided several clues to the regulation of its expression. Comparative and transgenic studies suggest that central oxytocin release may influence reproductive behaviours, but the importance of these central effects depends on the pattern of expression of the receptor--a pattern that is species-specific.

title: Oxytocin and Vasopressin Receptors and Species-Typical Social Behaviors
abstract: Over the past 50 years, a great deal of behavioral endocrinological research has established a central role for gonadal steroid hormones in the control of sexual behaviors in a wide range of vertebrate taxa. The underlying mechanisms controlling these behaviors appear to be well conserved throughout evolution. Both the neural structures regulating male and female sexual behaviors and the distribution of sex steroid receptors within the brain are similar from reptiles to mammals (Meisel and Sachs, 1994; Pfaff and Schwartz-Giblin, 1994). More recently, the roles of the neuropeptides oxytocin (OT) and vasopressin (AVP) in modulating reproductive and social behaviors have begun to be elucidated. Comparative studies focusing on these hormones suggest that unlike gonadal steroids, the specific behavioral roles of these neuropeptides may be quite species specific. Here I review some of the behaviors associated with OT and AVP systems and discuss the idea that OT and AVP receptor systems are phylogenetically plastic, perhaps facilitating the evolution of species-typical social behavior patterns. Oxytocin and vasopressin are related nonapeptide hormones which differ in structure at only two amino acid positions (Gainer and Wray, 1994). Both peptides are synthesized in magnocellular neurons of the paraventricular (PVN) and supraoptic nuclei of the hypothalamus, which project to the neurohypophysis and are released into circulation where they modulate peripheral physiology (Brownstein, Russell, and Gainer, 1980). The peptides are also synthesized in parvocelluar neurons of the PVN and other limbic nuclei which project throughout the brain where they influence behavior. Vasopressin synthesis by neurons in the amygdala and bed nucleus of the stria terminalis is sexually dimorphic, with males producing more than females (DeVries and Al-Shamma, 1990).

title: Functional domains in the oxytocin gene for regulation of expression and biosynthesis of gene products.
abstract: In the oxytocin (OT) gene several regions can be discerned that have a function in regulating its expression. Firstly, in the proximal 5' flanking region regulatory elements have been discovered that are targets for transcription factors of the nuclear hormone receptor family. Through these elements the OT gene of rat and man is responsive to estrogens, thyroid hormones and retinoids. Furthermore, these elements can be employed by the nuclear hormone orphan receptor family for repressive or inductive actions. In the distal 5' flanking region the POU class III proteins Brn-1, Brn-2, Brn-4, that are expressed in magnocellular neurons, and Oct-6 are able to bind, but do not display a significant regulatory activity on the OT gene in heterologous expression systems. Secondly, the OT precursor harbours both the biologically active hormone and the protein neurophysin that is able to associate with the hormone. Heterologous expression of wild-type and mutant vasopressin cDNAs in peptidergic cell lines shows that the highly homologous vasopressin-associated neurophysin domain associates with the hormone domain within the prohormone. This intramolecular interaction between two prohormone domains serves an essential intracellular function, i.e. the proper sorting of the prohormone into the regulated secretory pathway.

title: Gene regulation in the magnocellular hypothalamo-neurohypophysial system.
abstract: The hypothalamo-neurohypophysial system (HNS) is the major peptidergic neurosecretory system through which the brain controls peripheral physiology. The hormones vasopressin and oxytocin released from the HNS at the neurohypophysis serve homeostatic functions of water balance and reproduction. From a physiological viewpoint, the core question on the HNS has always been, "How is the rate of hormone production controlled?" Despite a clear description of the physiology, anatomy, cell biology, and biochemistry of the HNS gained over the last 100 years, this question has remained largely unanswered. However, recently, significant progress has been made through studies of gene identity and gene expression in the magnocellular neurons (MCNs) that constitute the HNS. These are keys to mechanisms and events that exist in the HNS. This review is an inventory of what we know about genes expressed in the HNS, about the regulation of their expression in response to physiological stimuli, and about their function. Genes relevant to the central question include receptors and signal transduction components that receive and process the message that the organism is in demand of a neurohypophysial hormone. The key players in gene regulatory events, the transcription factors, deserve special attention. They do not only control rates of hormone production at the level of the gene, but also determine the molecular make-up of the cell essential for appropriate development and physiological functioning. Finally, the HNS neurons are equipped with a machinery to produce and secrete hormones in a regulated manner. With the availability of several gene transfer approaches applicable to the HNS, it is anticipated that new insights will be obtained on how the HNS is able to respond to the physiological demands for its hormones.

title: Transgenic and Transcriptional Studies on Neurosecretory Cell Gene Expression
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title: Quantitative analysis of oxytocin and vasopressin messenger ribonucleic acids in single magnocellular neurons isolated from supraoptic nucleus of rat hypothalamus.
abstract: Oxytocin (OT) and vasopressin (VP) are peptide hormones that are derived from genes predominantly expressed in distinct magnocellular neurons in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. Recent evidence suggests that some magnocellular neurons coexpress both peptides. Our qualitative RT-PCR experiments on single cells show that the majority of magnocellular neurons coexpress both peptide messenger RNAs (mRNAs) in varying amounts. Using a competitive RT-PCR method combined with a standard calibration curve, we quantitatively determined OT and VP mRNA in single magnocellular neurons from the normal female rat SON, with a detection sensitivity of less than 30 mRNA molecules/cell. We defined the phenotypes of the single magnocellular neurons according to their ratios of these two peptide mRNAs. Using this approach, we identified three major phenotypes: oxytocin neurons, where the average OT to VP mRNA ratio is about 256; vasopressin neurons, where the average VP to OT mRNA ratio is about 182; and one oxytocin/vasopressin coexisting neuron, where the OT/VP mRNA ratio is 2. Thus, there is some OT and VP mRNA coexpression in virtually all of the magnocellular neurons in supraoptic nuclei of hypothalamus. However, clear phenotypes are identifiable by considering quantitative as opposed to qualitative differences.

title: Single cell reverse transcription-polymerase chain reaction analysis of rat supraoptic magnocellular neurons: neuropeptide phenotypes and high voltage-gated calcium channel subtypes.
abstract: Magnocellular neurosecretory cells (MNCs) in the hypothalamo-neurohypophysial system that express and secrete the nonapeptides oxytocin (OT) and vasopressin (VP) were evaluated for the expression of multiple genes in single magnocellular neurons from the rat supraoptic nucleus using a single cell RT-PCR protocol. We found that all cells representing the two major phenotypes, the OT and VP MNCs, express a small, but significant, amount of the other nonapeptide's messenger RNA (mRNA). In situ hybridization histochemical analyses confirmed this observation. A third phenotype, containing equivalent amounts of OT and VP mRNA, was detected in about 19% of the MNCs from lactating female supraoptic nuclei. Analyses of these phenotypes for other coexisting peptide mRNAs (e.g. CRH, cholecystokinin, galanin, dynorphin, and the calcium-binding protein, calbindin) generally confirmed expectations from the literature, but revealed cell to cell variation in their coexpression. Our results also show that the high voltage-activated calcium channel subunit genes, alpha1A-D, alpha2, and beta1-4 are expressed in virtually all MNCs. However, the alpha1E subunit gene is not expressed at detectable levels in these cells. The expression of all of the beta-subunit genes in each MNC may account for the variations in physiological and pharmacological properties of the high voltage-activated channels found in these neurons.

title: A novel family of genes encoding putative pheromone receptors in mammals
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title: Electrophysiological distinctions between oxytocin and vasopressin neurons in the supraoptic nucleus.
abstract: Oxytocin and vasopressin neurons can be differentiated from one another, and from neurons in the immediately adjacent perinuclear zone, by their electrophysiological properties. In both sexes, oxytocin and vasopressin neurons are characterized by a prominent transient outward rectification which is conspicuously lacking in most perinuclear neurons. In addition, perinuclear neurons, some of which project to the supraoptic nucleus, exhibit a transient depolarization which underlies short bursts of spikes. Oxytocin neurons are characterized by: 1) the presence of a sustained outward rectifier above -50 mV, active below spike threshold; 2) a rebound depolarization following deactivation of the sustained rectification which can sustain short spike trains; and 3) a smaller transient outward rectification, probably associated with the potassium current, Ia. Vasopressin neurons show little of the sustained outward rectification and rebound depolarization, but have a stronger transient outward rectification. Although both cell types exhibit depolarizing afterpotentials, in vasopressin neurons these lead to plateau potentials underlying prolonged discharges. In oxytocin neurons, the depolarizing potential usually sustains a short spike discharge, but less often leads to prolonged bursts. These data suggest that the intrinsic properties of oxytocin and vasopressin neurons lead to quantitatively different forms of burst discharges, both of which may facilitate hormone release.

title: Molecular Cloning: A Laboratory Manual
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title: Sulfate-activating enzymes in normal and brachymorphic mice: evidence for a channeling defect.
abstract: The severe reduction in the amount of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) in cartilage from homozygous brachymorphic mice results from a decrease in the activities of both ATP sulfurylase (50%) and adenosine 5'-phosphosulfate (APS) kinase (14% of normal). In order to better understand the etiology of this double enzyme defect, a dual approach to elucidating the nature of the enzyme complex as well as its mechanistic properties was undertaken. Antibody reagents that react with both activities provide evidence for a single, bifunctional protein in both normal and mutant cartilage. Quantitative Western blot analysis indicates that a normal amount of a dysfunctional protein is produced in mutant cartilage. Kinetic studies show that the Vmax for mutant kinase is significantly reduced and that mutant sulfurylase and kinase appear to have lower KmAPS values than normal. Interestingly, the mutation appears to disrupt the channeling mechanism that has recently been demonstrated for this pathway [Lyle et al. (1994) Biochemistry 33, 6822-6827]. APS kinase from normal mouse cartilage utilizes APS supplied by ATP sulfurylase much more efficiently than APS which is added exogenously; i.e., channeling efficiency is > 90%. In contrast, the mutant enzymes exhibit only 54% channeling efficiency. Lastly, isotope dilution and enrichment experiments show directly that the APS binding sites of the mutant enzymes are more accessible to free APS than are those of the normal enzymes. These data suggest that the mutation primarily affects the catalytic properties of the PAPS activation system by altering the function of the novel coupling mechanism between the two activities, causing a decrease in the ability to channel APS and produce PAPS efficiently.

title: Coexpression of vasopressin and oxytocin in hypothalamic supraoptic neurons of lactating rats.
abstract: Magnocellular hypothalamic neurons in the rat supraoptic nucleus (SON) normally produce either vasopressin (VP) or oxytocin (OT). Here we demonstrate that many magnocellular neurons in the SON of lactating rats synthesize both hormones at the same time. We show the colocalization of the messenger (m) RNA that encodes the VP precursor with OT-neurophysin; OT mRNA with VP-neurophysin, the C-terminal glycopeptide of the VP precursor, and VP itself, and the presence of both mRNAs in the same cell. At the light microscopic level quantitative studies show that on the second day of lactation, 17% of the SON neurons produce both hormones, on the fifth day 13%, and on the ninth day 9%. Two days after lactation the number of cells that are positive for both hormones returns to the control level (2-3%). We also show by means of electron microscopic immunohistochemistry that both peptides (or their precursors) are present in the same neurosecretory vesicles in nerve endings in the posterior lobe of lactating rats. At the electron microscopic level quantitative studies show that on the second day of lactation 21% of the terminals contain mixed vesicles; this number increases to 24% by the fourth day and is down to 5% by the 15th day, a level similar to that found in control rats. Since the double-labeled cells seemed to be producing additional VP as opposed to OT, we hypothesized that the former should affect urinary osmolality. Urine samples of lactating rats show a significant (5-fold) increase in urine osmolality during lactation (highest on the second day). The increase in osmolality correlated with the increase in the number of VP positive cells during lactation. We suggest that magnocellular neurons that ordinarily synthesize little or no VP can produce this antidiuretic hormone to help the animal compensate for the loss of water associated with lactation.

title: Peg5/Neuronatin is an imprinted gene located on sub-distal chromosome 2 in the mouse.
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title: Expression of the vasopressin and oxytocin genes in rats occurs in mutually exclusive sets of hypothalamic neurons
abstract: The genes for the hypothalamic hormones vasopressin and oxytocin are located in close proximity to each other within the rat genome. They are separated by only approx. 11 kbp of DNA sequence and oriented in such a way that their transcription occurs on opposite DNA strands. Although the two genes are structurally very similar including common potential regulatory elements in their putative promotor regions, they are expressed in discrete populations of magnocellular neurons of the hypothalamus. In rats placed under osmotic stress, the vasopressin gene is unregulated; concomitantly transcription of the oxytocin gene is also stimulated. To address the question of whether this coordinated rise in oxytocin‐encoding mRNA is the result of switching on oxytocin gene transcription in vasopressinergic neurons, in situ hybridization with double labelled cRNA probes was carried out. Biotinylated and [α‐35S]CTP labelled antisense cRNA probes specific for either vasopressin or oxytocin mRNA were constructed and hybridized to hypothalamic sections from salt‐loaded rats. The results demonstrate that upregulation of oxytocin gene transcription is restricted solely to oxytocinergic cells; no oxytocin gene transcripts can be detected in vasopressinergic neurons.

title: Identification of the vasopressin producing and of the oxytocin producing neurons in the hypothalamic magnocellular neurosecretory system of the rat
abstract: SummaryImmuno-enzyme histochemical investigations showed that, in the magnocellular hypothalamo-hypophysial neurosecretory system of the rat, vasopressin and oxytocin are synthetized in separate neurons. Both the vasopressin neurons and the oxytocin neurons are present in both the supraoptic and the paraventricular nuclei in about the same number. Preferential location of the two kinds of rat neurosecretory neurons is not as obvious as in the bovine hypothalamus. Their perikarya do not show distinct morphological differences. The two kinds of neurosecretory perikarya are the origin of separate vasopressin-containing and oxytocin-containing axons respectively. In the neural lobe, the distribution of the two different types of axons is described.

title: Coexisting peptides in hypothalamic neuroendocrine systems: Some functional implications
abstract: Summary1.Coexisting with oxytocin or vasopressin in the cell bodies and nerve terminals of the hypothalamic-neurohypophysial system are smaller amounts of other peptides. For a number of these “copeptides” there is strong evidence of corelease with the major magnocellular hormones. Guided by the location of their specific receptors we have studied the effects of three copeptides, dynorphin, cholecystokinin (CCK), and corticotropin releasing hormone (CRH), on the secretion of oxytocin and vasopressin from isolated rat neural lobe or neurointermediate lobe preparationsin vitro.2.Dynorphin is coreleased with vasopression from neural lobe nerve terminals and acts on neural lobe kappa-opiate receptors to inhibit the electrically stimulated secretion of oxytocin. Naloxone augments oxytocin release from the neural lobe in a manner directly proportional to the amount of vasopressin (and presumably dynorphin) released.3.Cholecystokinin, coreleased with oxytocin by neural lobe terminals, has been shown to have high-affinity receptors located in the NL and to stimulate secretion of both oxytocin and vasopressin. CCK's secretagogue effect was independent of electrical stimulation and extracellular Ca2+ and was blocked by an inhibitor of protein kinase C.4.CRH, coreleased with OT from the neural lobe, has receptors in the intermediate lobe of the pituitary, but not in the neural lobe itself. CRH stimulates the secretion of oxytocin and vasopressin from combined neurointermediate lobes but not from isolated neural lobes. Intermediate lobe peptides, alpha and gamma melanocyte stimulating hormone, induced secretion of oxytocin and vasopressin from isolated neural lobes. Their effect was, like that of CCK, independent of electrical stimulation and extracellular Ca2+ and blocked by an inhibitor of protein kinase C.5.Among the CRH-producing parvocellular neurons of the paraventricular nucleus, in the normal rat, approximately half also produce and store vasopressin. After removal of glucocorticoid influence by adrenalectomy, virtually all of the CRH neurons contain vasopressin.6.The two subtypes of CRH neurosecretory cells found in the normal rat possess different topographical distributions in the paraventricular nucleus, suggesting the possibility of differential innervation. Stress selectively activates the vasopression containing subpopulation of CRH neurons, indicating that there are separate channels of regulatory input controlling the two components of the parvocellular CRH neurosecretory system.

title: Evidence for the Co‐Expression of Oxytocin and Vasopressin Messenger Ribonucleic Acids in Magnocellular Neurosecretory Cells: Simultaneous Demonstration of Two Neurohypophysin Messenger Ribonucleic Acids by Hybridization Histochemistry
abstract: A novel enzyme‐labelled oligonucleotide probe specific for oxytocin messenger ribonucleic acid (mRNA) and a radiolabelled oligonucleotide probe specific for vasopressin mRNA were used together to visualize both oxytocin and vasopressin mRNA's in hypothalamus sections from salt loaded (2% saline) rats. The results demonstrate that the majority of magnocellular neurons contain only oxytocin or vasopressin mRNA, however a small number of neurons, 1% to 2%, contained both oxytocin and vasopressin transcripts.

title: A review of animal phosphofructokinase isozymes with an emphasis on their physiological role
abstract: SummaryPhosphofructokinase (PFK) isozymes and their physiological significance have been the focus of extensive research. The majority of this work has been centered around the PFK isozymes of rat, human and rabbit tissues. Consequently, this review emphasizes these studies. Additionally, a review of PFK isozymes in chickens, mice, guinea pig, and pig is presented. The relationship of the properties of each PFK isozyme in different tissues to the rates of glycolysis and/ or gluconeogenesis in those tissues is discussed where possible. Moreover, the contribution of the different PFK isozymes to alterations of the glycolytic rate in various tissues is discussed in relationship to variations in nutritional, hormonal, developmental or pathological status of the animal.

title: Isolation and characterization of phosphofructokinase C from rabbit brain.
abstract: Phosphofructokinase from rabbit brain consists of hybrids of the A, B, and C isozymes. Phosphofructokinase C was isolated from a purified mixture of such hybrids in a 2-step procedure. In the first step, phosphofructokinase B was removed by chromatography on DEAE-Sephadex. In the second step, subunits of phosphofructokinases A and C were separated by dissociation at pH 5.0 followed by chromatography on carboxymethylcellulose. The separated isozymes were then reassociated by neutralization. Phosphofructokinase C was structurally distinct from phosphofructokinases A (obtained from muscle or brain) and B (obtained from liver) as shown by one-dimensional chymotryptic and staphylococcal V8 protease fingerprints of all three isozymes. In addition, phosphofructokinase C cross-reacted weakly or not at all with antisera raised against phosphofructokinase B or phosphofructokinase A. Phosphofructokinase C was also kinetically distinct from the A and B isozymes. The C isozyme was more sensitive than the A isozyme but less sensitive than the B isozyme to inhibition by ATP, was less sensitive than the A isozyme but more sensitive than the B isozyme to inhibition by citrate, and was less sensitive than either of the other two isozymes to activation by inorganic phosphate, AMP, and fructose 2,6-bisphosphate. The self-association properties of phosphofructokinase C differed from those of the A and B isozymes in that at pH 8.0, the C isozyme did not form oligomers larger than a tetramer under conditions where the other two isozymes did. Thus the properties of phosphofructokinase C are in general quite distinct from those of the other two phosphofructokinase isozymes.

title: Genomic organization, 5'flanking region and tissue-specific expression of mouse phosphofructokinase C gene.
abstract: Using a combination of mouse bacterial artificial chromosome (BAC) genomic library screening, long-range polymerase chain reaction (PCR) amplification, genomic walking and DNA sequencing, we have characterized the intron/exon boundaries, the sizes of each intron and 5' flanking region of the mouse PFK-C gene. The gene spans approximately 55 kb and comprises 22 exons separated by 21 introns. All intron/exon splice junctions conform to the GT/AG rule. The mouse PFK-C gene organization is similar to that of the human and rabbit PFK-A and human and mouse PFK-B genes. However, PFK-C has much larger intronic sequences throughout the gene. Anchored PCR was performed to amplify about 1.0 kb of genomic DNA upstream of the translational start site. Sequence analysis of the PFK-C 5' flanking region revealed that it is devoid of TATA and CAAT boxes at the usual positions, but it contained several putative binding sites for transcription factors AP1, GATA1, NKX2.5 and STAT. The 5' flanking region was not enriched in GC dinucleotides and lacked CpG islands and putative binding sites for SP1. Four transcription initiation sites have been identified by full-length RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) between -61 and -32 bp from the translation initiation codon. Reverse transcription-PCR analysis revealed that PFK-A, PFK-B and PFK-C genes were expressed, in all mouse tissues tested, at varying levels. PFK-A mRNA was more abundantly expressed in all tissues than were the PFK-B and PFK-C genes. Based on the mouse PFK-C signal normalized to 18S rRNA, the PFK-C mRNA was expressed at the highest levels in the brain, heart, thymus and testicles, whereas low levels were observed in the kidney, liver, muscle, and lung.

title: Characterization of the rat heart 6-phosphofructo-1-kinase isozymes.
abstract: By elution with a discontinuous gradient from QAE-Sephadex, the rat muscle 6-phosphofructo-1-kinase (PFK) isozyme (M4) and the major rat liver PFK isozyme (L4) could be completely separated. Subjecting heart supernatant fluid to this treatment indicated that all of the heart PFK activity was found in the first wash, where M4 eluted, indicating little, if any, L4 was present. However, about 50% of the heart PFK activity was immunoprecipitated by L4 anti-IgG demonstrating the presence of the L-type subunit. A purification procedure was developed which yielded an enzyme preparation of high specific activity and resulted in a recovery of 50% to 60% of the original PFK activity. Three proteins were detected in this PFK preparation that exhibited apparent mol wt of 87 500, 85 000 and 80 000. The 85 000 and 80 000 Dalton components corresponded to the subunits of M4 and L4, respectively. The third protein was thought to be a distinct subunit (C-type) since it exhibited the highest molecular weight and was present in an exhaustively washed immunoprecipitate of purified heart PFK. From seven different heart PFK preparations, the relative distributions of the L-, M-, and C-type subunits were 0.80 +/- 0.1, 4.5 +/- 0.7, and 0.7 +/- 0.1, respectively. A comparison of the kinetic properties of L4, M4, and purified heart PFK isozymes clearly demonstrated that all three preparations exhibited different regulatory properties. In ventricular and atrial preparations the total PFK activity and the relative amounts of each subunit were drastically different suggesting regional differences between the distributions of PFK isozymes and consequently in regulation of PFK activities and glycolysis.

title: Allosteric regulatory properties of muscle phosphofructokinase
abstract: SummaryWe have reviewed the allosteric regulatory properties of skeletal muscle phosphofructokinase and recent results on the phosphorylation of this enzyme. The number and affinities of various ligand binding sites are described, and a simple three state model is presented to explain the kinetic and ligand-binding properties of the enzyme. Data describing a lack of fit to a concerted transition model are presented. The widespread occurrence of partial phosphorylation of phosphofructokinase at a specific site near the carboxyl terminus is documented, as well as the lack of significant kinetic consequences of such phosphorylation.

title: Alteration of PFK subunit protein, synthesis, and mRNA during neonatal brain development.
abstract: During neonatal maturation of rat brain, a similar biphasic relationship exists between the previously reported pattern of glucose utilization and levels of each type of 6-phosphofructo-1-kinase (PFK) subunit protein, relative synthesis, and mRNA. The increasing amounts of each subunit isoform generally correlated with elevated protein synthesis which was promoted by greater amounts of each type of subunit mRNA. For each parameter, the early phase, 1 to 10 days after birth, was characterized by small increases, and the subsequent period from ten to thirty days postpartum was characterized by a much greater rate of increase. By 30 days after birth, adult values were observed. The apparent efficiency of translation of each type of PFK subunit mRNA in brain suggests that the M-type subunit mRNA is the most efficient and that the L-type subunit mRNA is the least. The greatest relative increases in subunit protein, mRNA, and synthesis were observed for the C-type subunit. Since enhanced translation apparently makes little, if any, contribution, a possible explanation of these phenomena could be increased transcription of the PFK genes. These neonatal changes could involve age-dependent alteration of methylation of the PFK gene promotor(s) and/or activity of effectors of the transcription of the PFK genes.

title: Enzymology of human cytosolic sulfotransferases
abstract: Conjugation of many xenobiotics, drugs, and endogenous compounds with a sulfonate moiety is an important reaction in their biotransformation. Sulfation of these compounds generally results in a decrease in biological activity and an increase in their urinary excretion. However, in certain instances, sulfation results in bioactivation to reactive electrophilic or therapeutically active forms. At least four cytosolic sulfotransferases (STs) have been identified and characterized from human tissues. These enzymes are two forms of phenol ST (PST), the phenol‐sulfating and the monoamine‐sulfating forms of PST (P‐PST and M‐PST, respectively), an estrogen sulfotransferase (EST), and a hydroxysteroid ST, dehydroepiandrosterone ST (DHEA‐ST). Although four cytosolic STs have been well characterized in human tissues, evidence is accumulating for the presence of allelic forms or additional distinct forms of the STs in human tissues. The STs possess distinct but overlapping substrate specificities, and all of the STs are capable of conjugating both xenobiotic and endogenous compounds. The individual forms of ST may display distinct patterns of tissue specific expression and different mechanisms of regulation. Although the role of sulfation in drug metabolism is well recognized, an increased understanding of the biochemistry and molecular biology of the STs should also provide additional information as to their functions in many normal physiologic processes.—Falany, C. N. Enzymology of human cytosolic sulfotransferases. FASEB J. 11, 206‐216 (1997)

title: Undersulfated chondroitin sulfate in the cartilage matrix of brachymorphic mice.
abstract: Homozygous brachymorphic (bmbm) mice have a disproportionately short stature, similar to human achondroplasia. We previously showed that each zone of growth in young bmbm epiphyseal cartilages is smaller than normal and that the extracellular matrix appears to contain normal collagen fibrils, but smaller and reduced numbers of proteoglycan matrix granules. Our studies reported here indicate that mutant, like normal cartilage, synthesizes type II collagen and contains normal quantities of glycosaminoglycans as judged by uronic acid content. However, the glycosaminoglycans from the mutant differ from the normal in their chromatographic and electrophoretic properties. Further studies established that glycosaminoglycans from cartilages of brachymorphic animals were undersulfated. Whereas chondroitinase digests of glycosaminoglycans from cartilage of normal C57Bl6J 5-day-old mice contained predominantly disaccharides sulfated in the 4-position, that of the mutant contained appreciable unsulfated disaccharides as well.

title: Defective PAPS-synthesis in epiphyseal cartilage from brachymorphic mice.
abstract: Activity levels of sulfotransferases, requisite for the sulfation of chondroitin sulfate proteoglycan, were measured in cell-free homogenates prepared from neonatal epiphyseal cartilage of normal C57B1/6J or homozygous brachymorphic mice. In the presence of [35S]-PAPS only or [35S]-PAPS plus an exogenous sulfate acceptor, comparable amounts of 35 SO 4 2− were incorporated into chondroitin sulfate by the normal and mutant types of cartilage. In contrast, the mutant cartilage catalyzed the conversion of only 30% of the 35 SO 4 2− into chondroitin sulfate as compared to normal mouse cartilage when synthesis was initiated from ATP and H235SO4. These results suggest that the production of an undersulfated proteoglycan which has previously been reported in brachymorphic mice (Orkin, R.W. et al . (1976) ) may result from a defect in the synthesis of the sulfate donor PAPS.

title: A member of a family of sulfate-activating enzymes causes murine brachymorphism.
abstract: Sulfation is critical to the function of a wide variety of biomolecules. This common modification requires the enzymatic synthesis of an activated sulfate donor, phosphoadenosine-phosphosulfate (PAPS). In higher organisms PAPS synthesis is catalyzed by a bifunctional sulfurylase kinase (SK) polypeptide having both ATP-sulfurylase and adenosine-phosphosulfate kinase activities. We report the identification of a gene family encoding murine SK proteins with these two activities. A family member, SK2, colocalizes with the locus for the autosomal recessive murine phenotype brachymorphism. Brachymorphic mice have normal lifespans, but abnormal hepatic detoxification, bleeding times, and postnatal growth, the latter being attributed to undersulfation of cartilage proteoglycan. A missense mutation in the SK2 coding sequence of bm mice that alters a highly conserved amino acid residue destroys adenosine-phosphosulfate kinase activity and therefore the ability of SK2 to synthesize PAPS. We conclude that a family of SK genes are responsible for sulfate activation in mammals, that a mutation in SK2 causes murine brachymorphism, and that members of this gene family have nonredundant, tissue-specific roles.

title: Mutations in orthologous genes in human spondyloepimetaphyseal dysplasia and the brachymorphic mouse
abstract: The osteochondrodysplasias are a genetically heterogeneous group of disorders affecting skeletal development, linear growth and the maintenance of cartilage and bone. We have studied a large inbred Pakistani family with a distinct form of recessively inherited spondyloepimetaphyseal dysplasia (SEMD) and mapped a gene associated with this dwarfing condition to chromosome 10q23–24, a region syntenic with the locus for the brachymorphic mutation on mouse chromosome 19. We identified two orthologous genes, ATPSK2 and Atpsk2, encoding novel ATP sulfurylase/APS kinase orthologues in the respective regions of the human and mouse genomes. We characterized a nonsense mutation in ATPSK2 in the SEMD family and a missense mutation in the region of Atpsk2 encoding the APS kinase activity in the brachymorphic mouse. ATP sulfurylase/APS kinase catalyses the metabolic activation of inorganic sulfate to PAPS, the universal donor for post-translational protein sulfation in all cell types. The cartilage-specificity of the human and mouse phenotypes provides further evidence of the critical role of sulfate activation in the maturation of cartilage extracellular matrix molecules and the effect of defects in this process on the architecture of cartilage and skeletogenesis.

title: Human 3'-phosphoadenosine 5'-phosphosulfate synthetase 1 (PAPSS1) and PAPSS2: gene cloning, characterization and chromosomal localization.
abstract: Sulfae conjugation is an important pathway in the metabolism of a large number of exogenous and endogenous compounds. These reactions are catalyzed by sulfotransferase (SULT) enzymes that utilize 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as a sulfate donor. PAPS is synthesized from ATP and inorganic sulfate by PAPS synthetase (PAPSS). Two separate PAPSS cDNAs, PAPSS1 and PAPSS2, have been identified in human tissues. We have cloned and characterized the genes for human PAPSS1 and PAPSS2 to make it possible to study the pharmacogenomics of these enzymes. Both genes consisted of 12 exons with virtually identical exon-intron splice junction locations. All splice junctions conformed to the "GT-AG" rule. The total length of PAPSS1 was approximately 108 kb, while that of PAPSS2 was greater than 37 kb. The 5'-flanking region of PAPSS1 did not include a TATA box sequence near the site of transcription initiation, but PAPSS2 had a TATA motif located 21 bp upstream from the site of transcription initiation. Northern blot analysis showed that the major PAPSS1 and PAPSS2 transcripts were approximately 2.7 and 4.2 kb in length, respectively. PAPSS1 mapped to human chromosome band 4q24 while PAPSS2 mapped to 10q22-23 by fluorescence in situ hybridization analysis. Cloning and structural characterization of PAPSS1 and PAPSS2 will make it possible to perform molecular genetic and pharmacogenomic studies of these important enzymes in humans.

title: Synaptotagmins: C2-Domain Proteins That Regulate Membrane Traffic
abstract: We wish to thank Drs. M. S. Brown, J. L. Goldstein, Y. Goda, R. Jahn, E. Kandel, E. Liebe, and C. F. Stevens for invaluable advice and discussions. The work described in this review was partially supported by the National Institutes of Health, Human Frontier Science Project, and W. M. Keck Foundation.

title: Synaptotagmin V: a novel synaptotagmin isoform expressed in rat brain
abstract: Regulated Ca2+‐dependent release of transmitters from synaptic vesicles is an important characteristic of chemical neurotransmission. Synaptotagmins are abundant synaptic vesicle transmembrane proteins that probably function as Ca2+ sensors. Molecular cloning has identified four different synaptotagmin isoforms in mammals. We report here the cloning and sequencing of a novel isoform of 386 amino acids. Synaptotagmin V is 54% identical in sequence to synaptotagmin I and possesses all the domains that characterise this multigene family. It is expressed at high levels in rat brain, but not in spinal cord or a number of peripheral non‐neuronal tissues.

title: Human synaptotagmin V (SYT5): sequence, genomic structure, and chromosomal location.
abstract: We have determined the sequence, genomic structure, and chromosomal location of the human synaptotagmin V (SYTV) gene. The human SYTV gene encodes a 386-amino-acid product which is 91% identical to rat Syt V. The human SYTV open reading frame is interrupted by seven introns which can be alternatively spliced. Human SYTV was found to lie very close to SYTIII on chromosome 19q13.4 by PCR analysis of somatic cell hybrid DNA and by DNA hybridization to arrayed cosmids of the chromosome 19 metric physical map. This provides the first report of linked synaptotagmin genes.

title: Conserved N-terminal Cysteine Motif Is Essential for Homo- and Heterodimer Formation of Synaptotagmins III, V, VI, and X*
abstract: The synaptotagmins now constitute a large family of membrane proteins characterized by one transmembrane region and two C2 domains. Dimerization of synaptotagmin (Syt) I, a putative low affinity Ca2+ sensor for neurotransmitter release, is thought to be important for expression of function during exocytosis of synaptic vesicles. However, little is known about the self-dimerization properties of other isoforms. In this study, we demonstrate that a subclass of synaptotagmins (III, V, VI, and X) (Ibata, K., Fukuda, M., and Mikoshiba, K. (1998)  forms β-mercaptoethanol-sensitive homodimers and identify three evolutionarily conserved cysteine residues at the N terminus (N-terminal cysteine motif, at amino acids 10, 21, and 33 of mouse Syt III) that are not conserved in other isoforms. Site-directed mutagenesis of these cysteine residues and co-immunoprecipitation experiments clearly indicate that the first cysteine residue is essential for the stable homodimer formation of Syt III, V, or VI, and heterodimer formation between Syts III, V, VI, and X. We also show that native Syt III from mouse brain forms a β-mercaptoethanol-sensitive homodimer. Our results suggest that the cysteine-based heterodimerization between Syt III and Syt V, VI, or X, which have different biochemical properties, may modulate the proposed function of Syt III as a putative high affinity Ca2+ sensor for neurotransmitter release.

title: Identification of a nonneuronal isoform of synaptotagmin.
abstract: Synaptotagmins, which have been found exclusively in neuroendocrine or exocrine tissues, have been implicated in the regulation of secretory vesicle fusion with the plasma membrane. The present paper describes a synaptotagmin isoform (synaptotagmin-5) which exhibits 49% amino acid identity to synaptotagmin-1 and -2. Synaptotagmin-5 mRNA is expressed in rat kidney, adipose tissue, lung, and heart, as well as at higher levels in brain and PC12 cells. Antiserum specific for the synaptotagmin-5 isoform recognizes a protein of about 50 kDa which is about 6-fold more abundant in brain synaptic vesicles than in whole brain membranes.

title: Expression of growth inhibitory factor (GIF) in normal and injured rat brains
abstract: Immunohistochemical study on growth inhibitory factor (GIF) in rat brain has revealed that a glial cell layer on the surface of cerebral cortex and the cells surrounding Purkinje cells has been reported. In addition, neurons in gray matter were weakly immunostained for GIF. In situ hybridization using digoxigenin-labeled single-strand RNA probes also demonstrated that most of the neurons and small round cells, which were presumably astrocytes, expressed GIF mRNA in the cerebral cortex of rat brain. These findings indicate that GIF is produced in neurons as well as in astrocytes. The most prominent findings in this study are, a very strong reaction of GIF and GIF mRNA in the reactive astrocytes around the site of injury induced by stab wound or kainic acid injection. These results raised the possibility that GIF may act as an acute-phase protein in reactive astrocytes and have a role in tissue repair.

title: Brain Injury and Growth Inhibitory Factor (GIF)—a Minireview
abstract: Growth inhibitory factor (GIF) is a small (7 kDa), heat-stable, acidic, hydrophilic metallothionein (MT)-like protein. GIF inhibits the neurotrophic activity in Alzheimer's disease (AD) brain extracts on neonatal rat cortical neurons in culture. GIF has been shown to be drastically reduced and down-regulated in AD brains. In neurodegenerative diseases in humans, GIF expression levels are reduced whereas GFAP expression levels are markedly induced in reactive astrocytes. Both GIF and GIF mRNA are present at high levels in reactive astrocytes following acute experimental brain injury. In chronological observations the level of GIF was found to increase more slowly and remain elevated for longer periods than that of glial fibrillary acidic protein (GFAP). These differential patterns and distribution of GIF and GFAP seem to be important in understanding the mechanism of brain tissue repair. The most important point concerning GIF in AD is not simply the decrease in the level of expression throughout the brain, but the drastic decrease in the level of expression in reactive astrocytes around senile plaques in AD. Although what makes the level of GIF decrease drastically in reactive astrocytes in AD is still unknown, supplements of GIF may be effective for AD, based on a review of current evidence. The processes of tissue repair following acute brain injury are considered to be different from those in AD from the viewpoint of reactive astrocytes.

title: Different protective roles in vitro of α- and β-domains of growth inhibitory factor (GIF) on neuron injuries caused by oxygen free radicals
abstract: It was well known that beta-amyloid (Abeta) and tau protein play an important role in pathological procedure of Alzheimer's disease (AD), a senile dementia. The growth inhibitory factor (GIF, also named metallothionein-3, MT-3) had been demonstrated to inhibit the outgrowth of cortex neurons in the medium with extract of the AD patient brain. In our experiments, it was found that the neurons of cortex and the PC12 (pheochromocytoma) cells could be protected from the cytotoxicity of beta-amyloid 25-35 in presence of GIF and its domains. Additionally, GIF can scavenge the hydroxyl radical efficiently in CytC-VitC radical producing system and its alpha-domain shown more effective potentials than its beta-domain. The electron paramagnetic resonance spectra also show that the alpha-domain has more potential ability for eliminating reactive oxygen free radicals than its beta-domain. The results suggest that GIF could act as an efficient scavenger against free radicals in vitro and the alpha-domain in GIF molecule shows more potential in protecting against reactive oxygen species injury than the beta-domain.

title: Free zinc increases at the site of injury after cortical stab wounds in mature but not immature rat brain
abstract: The accumulation of free zinc (Zn2+) appears to play a role in the neuronal degeneration that occurs after brain injury. Given that neonates respond to brain injury with increased plasticity compared to adults, this study compared the effect of age on free Zn2+ and the Zn2+-binding protein metallothionein-3 (MT-3) after injury. Unilateral cortical stab wounds were produced in 3-day-old and adult rats. Four weeks later, brains were removed for in situ visualization of free Zn2+ and measurement of MT-3 mRNA. Free Zn2+ and MT-3 mRNA accumulated after 4 weeks at the site of injury site when injury occurred in adults. However, 4 weeks after neonatal injury there was no increase in free Zn2+ or MT-3 mRNA in or around the site of injury.

title: Effect of stress on mouse and rat brain metallothionein I and III mRNA levels.
abstract: The effect of immobilization stress on brain and liver metallothionein (MT) mRNA levels has been studied in mice and rats. Stress increased brain and liver MT-I mRNA levels in mice in a time-dependent manner, in agreement with the MT-I+II protein levels, suggesting an increased gene transcription during stress. In contrast, the brain-specific isoform, MT-III, tended to decrease during stress. In selected brain areas of rats, the overall tendency for both MT-I and MT-III mRNA levels was to be transiently decreased by stress in hippocampus, and increased in hypothalamus, cerebellum and the remaining brain tissues; adrenalectomy significantly affected MT mRNA levels either in basal conditions or during stress, with very different temporal patterns of response depending on the brain area studied. These results suggest that glucocorticoids could be involved in MT-I but also MT-III regulation. In both rats and mice, the subtle response to stress observed in the brain contrasts with the robust response in the liver, suggesting that the factors involved in MT regulation in both tissues differ substantially. In primary cultures enriched in astrocytes or neurons, MT-III mRNA was clearly detected by Northern blotting in both cases, suggesting that it is expressed in both types of cells. Dexamethasone appeared to decrease MT-III mRNA levels in cultured neurons and to increase them in astrocytes, which indicates that glucocorticoids have a different role in MT-III regulation in both cell types.

title: Ischemia induces metallothionein III expression in neurons of rat brain.
abstract: Metallothionein III (MT-III) is a brain-specific member of the metallothionein family and binds zinc in vivo. In order to confirm the precise localization of MT-III in normal rat brain and the change of MT-III expression after transient whole brain ischemia, we raised a high affinity phagemid-antibody specific for rat MT-III. Immunohistochemical analysis revealed that MT-III in normal brain is localized abundantly in neuronal cell bodies in CA1-3 regions of hippocampus, dentate gyrus, cerebral cortex, olfactory bulb and Purkinje cells in cerebellum. This expression pattern of MT-III was similar to that of MT-III mRNA observed by in situ hybridization studies. ELISA and Northern blot analysis revealed that MT-III protein as well as mRNA levels were up-regulated in cerebrum soon after ischemic stress. Immunohistochemical analysis also demonstrated intense staining in neurons in injured brain after ischemia, which distributed in the same regions as in normal brain. These results suggest that MT-III plays an important role in protecting neurons from ischemic insult by reducing neurotoxic zinc levels and inhibits uncontrolled growth of neurites after ischemia.

title: Disruption of the Metallothionein-III Gene in Mice: Analysis of Brain Zinc, Behavior, and Neuron Vulnerability to Metals, Aging, and Seizures
abstract: Metallothionein-III (MT-III), a brain-specific member of the metallothionein family of metal-binding proteins, is abundant in glutamatergic neurons that release zinc from their synaptic terminals, such as hippocampal pyramidal neurons and dentate granule cells. MT-III may be an important regulator of zinc in the nervous system, and its absence has been implicated in the development of Alzheimer’s disease. However, the roles of MT-III in brain physiology and pathophysiology have not been elucidated. Mice lacking MT-III because of targeted gene inactivation were generated to evaluate the neurobiological significance of MT-III. MT-III-deficient mice had decreased concentrations of zinc in several brain regions, including hippocampus, but the pool of histochemically reactive zinc was not disturbed. Mutant mice exhibited normal spatial learning in the Morris water maze and were not sensitive to systemic zinc or cadmium exposure. No neuropathology or behavioral deficits were detected in 2-year-old MT-III-deficient mice, but the age-related increase in glial fibrillary acidic protein expression was more pronounced in mutant brain. MT-III-deficient mice were more susceptible to seizures induced by kainic acid and subsequently exhibited greater neuron injury in the CA3 field of hippocampus. Conversely, transgenic mice containing elevated levels of MT-III were more resistant to CA3 neuron injury induced by seizures. These observations suggest a potential role for MT-III in zinc regulation during neural stimulation.

title: 
 Neuronatin mRNA in PC12 cells: downregulation by nerve growth factor
abstract: Neuronatin was recently cloned from neonatal rat brain ( (1994) 1227-1234). In subsequent studies, we noted neuronatin mRNA was brain-specific and that there were two alternatively spliced forms, alpha and beta  (1995) 92-98). Furthermore, on sequencing the human neuronatin gene, it was determined that the alpha-form was encoded by three exons, and the beta-form was encoded by the first and third exons only (Genomics, 33 (1996) 292-297). The middle exon was spliced out in the beta-form. The human neuronatin gene is located in single copy of chromosome 20q 11.2-12 (Brain Res., 723 (1996) 8-22). These studies called for an understanding of the function of this gene. Therefore, we studied the expression of neuronatin in PC12 cells, an established model of neuronal growth and differentiation. Neuronatin mRNA expression was found to be abundant in undifferentiated PC12 cells. Treatment with nerve growth factor (NGF), resulting in neuronal differentiation, was associated with a downregulation of neuronatin mRNA expression. Removal of NGF was associated with a return of neuronatin mRNA levels towards baseline. These effects appear to be specific for NGF as they were not seen with transforming growth factor, epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate or dexamethasone. Although, basic fibroblast growth factor also reduced neuronatin mRNA levels, the effect was less pronounced than with NGF. The NGF-induced decreased in neuronatin mRNA occurred even in the presence of protein and RNA syntheses inhibitors. Of the two spliced forms, only the alpha-form was expressed in PC12 cells. In conclusion, we report the presence of neuronatin mRNA in PC12 cells, and that NGF downregulates its expressions. These findings provide a basis for investigating the role of neuronatin in neuronal growth and differentiation.

title: Molecular cloning of a novel mRNA (neuronatin) that is highly expressed in neonatal mammalian brain.
abstract: As differential gene expression governs the progression of development into senescence, we attempted to define the genes that are selectively expressed during postnatal brain development. A cDNA fragment selectively expressed in neonatal rat brain was identified by differential display and used to screen a cDNA library prepared from the same mRNA sample. The full length cDNA, neuronatin, was 1195bp long and coded for a novel protein of 81 amino acids. The cDNA detected an mRNA species of similar size that was highly expressed in rat neonatal and human fetal brain. The deduced protein exhibited a hydrophobic N-terminal and hydrophilic C-terminal, suggesting that it is membrane bound and might function in signal transduction. The selective expression of this novel mRNA in late fetal and early postnatal brain development, and loss of expression in adulthood and senescence, suggests that downregulation of neuronatin may be involved in terminal brain differentiation.

title: Neuronatin mRNA: alternatively spliced forms of a novel brain-specific mammalian developmental gene
abstract: Neurogenesis begins with the closure of the neural tube around mid gestation and continues in the rat for about two weeks postnatally. Therefore, we investigated the role of neuronatin, a novel cDNA that we cloned from neonatal rat brain (Joseph et al.,  (1994) 1227-1234), in brain development. Further studies described in the present manuscript, lead to the identification of two alternatively spliced forms of neuronatin mRNA, alpha and beta, with the same open reading frame. Neuronatin-alpha encoded a novel protein of 81 aa, and the beta-form encoded 54 aa. Both forms were identical, except that the alpha-form had an additional 81 bp sequence inserted into the middle of the coding region. On Northern analyses, neuronatin mRNA was relatively selective for the brain. It first appeared at E11-14, a time when the neural tube has closed and neuroepithelial proliferation initiated, became pronounced at E16-19 with a surge in neurogenesis, and declined postnatally to adult levels with the completion of neurogenesis. In order to determine whether there were other forms of neuronatin mRNA, and to study the expression of the alpha and beta forms separately during development, reverse transcriptase-polymerase chain reaction was carried out using primers flanking the coding region of the alpha and beta forms. The RT-PCR results clearly indicated that there were only two forms of neuronatin. The beta-form first appeared at E11-14, whereas the alpha-form was present even earlier at E7-10. Together, these findings indicate that the two forms of neuronatin mRNA are regulated differently during brain development.(ABSTRACT TRUNCATED AT 250 WORDS)

title: Genetic and functional analysis of neuronatin in mice with maternal or paternal duplication of distal Chr 2.
abstract: Functional differences between parental genomes are due to differential expression of parental alleles of imprinted genes. Neuronatin (Nnat) is a recently identified paternally expressed imprinted gene that is initially expressed in the rhombomeres and pituitary gland and later more widely in the central and peripheral nervous system mainly in postmitotic and differentiating neuroepithelial cells. Nnat maps to distal chromosome (Chr) 2, which contains an imprinting region that causes morphological abnormalities and early neonatal lethality. More detailed mapping analysis of Nnat showed that it is located between the T26H and T2Wa translocation breakpoints which is, surprisingly, proximal to the reported imprinting region between the T2Wa and T28H translocation breakpoints, suggesting that there may be two distinct imprinting regions on distal chromosome 2. To investigate the potential role of Nnat, we compared normal embryos with those which were PatDp.dist2.T26H (paternal duplication/maternal deficiency of chromosome 2 distal to the translocation breakpoint T26H) and MatDp.dist2.T26H. Expression of Nnat was detected in the PatDp.dist2.T26H embryos, where both copies of Nnat are paternally inherited, and normal embryos but no expression was detected in the MatDp.dist2.T26H embryos with the two maternally inherited copies. The differential expression of Nnat was supported by DNA methylation analysis with the paternally inherited alleles being unmethylated and the maternal alleles fully methylated. Although experimental embryos appeared grossly similar phenotypically in the structures where expression of Nnat was detected, differences in folding of the cerebellum were observed in neonates, and other more subtle developmental or behavioral effects due to gain or loss of Nnat cannot be ruled out.

title: Localisation of the imprinted gene neuronatin, Nnat, confirms and refines the location of a second imprinting region on mouse chromosome 2
abstract: Nine regions on six mouse autosomes are subject to imprinting and uniparental inheritance of any one of these regions results in mice with phenotypic anomalies. So far on distal Chromosome (Chr) 2 there is a unique imprinting region between 2H3 and 2H4 associated with two behavioural disorders and neonatal lethality. A maternally imprinted gene, Nnat, has been identified which is expressed in the nervous system and maps to distal Chr 2. Nnat has been excluded as a candidate for either or both the behavioural phenotypes as it lies proximal to the 2H3–2H4 imprinting region. Here we have mapped Nnat to band 2H1 which is at least 18 Mb proximal to the previously described imprinting region. It maps close to agouti, some alleles of which show differential expression according to parental origin. The localisation of Nnat to band H1 confirms and refines the map location of a second imprinting region on mouse Chr 2.

title: Segment-specific expression of the neuronatin gene during early hindbrain development.
abstract: The developing hindbrain is segmented in a series of repetitive bulges called neuromeres or rhombomeres. In the mouse, first molecular evidence for segmentation of the hindbrain came from rhombomeres 3- and 5-specific expression of the Krox-20 gene. The hindbrain segments are linked with the expression of different Hox genes which have a role in patterning the hindbrain and branchial region of the vertebrate head. Here we identified by subtractive hybridization a gene, mouse neuronatin, that is downregulated in P19 embryo carcinoma cells that have undergone a partial differentiation process. Neuronatin encodes putative transmembrane proteins of 54, 55, and 81 amino acids that might serve as protein ligands, cofactors, or small cell adhesion molecules. The neuronatin gene is transiently expressed in rhombomeres 3 and 5 during early hindbrain development and in the floor of the foregut pocket. In addition, expression is observed in the early Rathke's pouch, in the derived adenohypophysis, and in the developing inner ear. During later embryogenesis the neuronatin gene is strongly expressed in the major part of the central and peripheral nervous system. These results suggest that neuronatin participates in the maintenance of segment identity in the hindbrain and pituitary development and maturation or maintenance of the overall structure of the nervous system.

title: The human homolog of a mouse-imprinted gene, Peg3, maps to a zinc finger gene-rich region of human chromosome 19q13.4.
abstract: Peg3 (paternally expressed gene 3) is the first imprinted gene detected in the proximal region of mouse chromosome 7. Because imprinting is a trait that is generally conserved among mammals, and imprinted domains generally encompass several adjacent genes, expression patterns and chromosomal environment of the human counterpart of Peg3 are of special interest. In this study we have localized human PEG3 approximately 2 Mb proximal of the telomere of chromosome 19q, within a region known to carry large numbers of tandemly clustered Krüppel-type zinc finger-containing (ZNF) genes. Peg3 also encodes a Krüppel-type ZNF protein but one that is distinguished from other ZNF gene products by the fact that it carries two novel proline-rich motifs. Comparison between mouse Peg3 and partial human PEG3 gene sequences revealed a high level of conservation between the two species, despite the fact that one of the two proline-rich repeats is absent from the human gene. Our data demonstrate that the human gene is expressed at highest levels in ovary and placenta; mouse Peg3, by contrast, is transcribed at highest levels in the adult brain. These comparative mapping, sequencing, and expression data provide the first clues to the potential activities of PEG3, and generate new tools to aid in the analysis of structure and function of a potentially new imprinted domain located in human chromosome 19q13.4 and mouse chromosome 7.

title: Peg3 imprinted gene on proximal chromosome 7 encodes for a zinc finger protein
abstract: Genetic and embryological studies in the mouse demonstrated functional differences between parental chromosomes during development1–3. This is due to imprinted genes whose expression is dependent on their parental origin4,5. In a recent systematic screen for imprinted genes, we detected Peg3 (paternally expressed gene 3)6. Peg3 is not expressed in parthenogenones. In interspecific hybrids, only the paternal copy of the gene is expressed in the embryos, individual tissues examined in d9.5–13.5 embryos, neonates and adults. Peg3 mRNA is a 9 kb transcript encoding an unusual zinc finger protein with eleven widely spaced C2H2 type motifs and two groups of amino acid repeats. Peg3 is expressed in early somites, branchial arches and other mesodermal tissues, as well as in the hypothalamus. Peg3 maps to the proximal region of chromosome 7. Consistent with our findings, maternal duplication of the proximal chromosome 7 causes neonatal lethality7–9. This region is syntenic with human chromosome 19q13.1–13.3 (refs 10,11), where the genes for myotonic dystrophy and a putative tumour suppressor gene are located12,13.

title: Genomic Imprinting , Maternal Care , and Brain Evolution
abstract: The majority of autosomal genes are inherited as two copies, one from each parent, and such biallelically inherited genes have identical functions. Genomic imprinting confers different functions on the two parental genomes during development by silencing one allele of each imprinted gene in a parent-oforigin-dependent manner. Hence imprinted genes usually function when inherited either from the mother or from the father, but, unlike sex-linked genes, are expressed in both sons and daughters. Imprinted genes are not expressed along the lines of classic Mendelian inheritance since the expression of an imprinted allele is dependent on whether it resided in a male or a female in the previous generation. The transcriptional expression of imprinted genes is brought about by epigenetic modifications that are erased and reestablished during germ cell development. Methylation of the dinucleotide CpG has been extensively studied as a candidate marker for imprinting because of its association with transcriptional repression (Brenton et al., 1999). The biological consequences of methylation are mediated by a family of methy-CpG binding proteins, the best characterized of which is MeCp2, a transcriptional repressor that recruits histone diacetylases (Ng et al., 1999; Hendrich et al., 1999). The molecular mechanisms of genomic imprinting, nuclear reprogramming on passage through the germline, and the resistance to genomewide demethylation that occurs during preimplantation development are currently under intensive investigation (Brenton et al., 1999; Ben-Porath and Cedar, 2000; Reik and Murrell, 2000; Saitoh et al., 1996). The realization that male and female autosomal genes do not contribute equally to mammalian development derives from experiments in mice where normal development was found to require the presence of both a maternal and paternal genome (Surani et al., 1984; McGrath and Solter, 1984). In these experiments both parthenogenetic embyros (diploid with two sets of maternal chromosomes) and androgenetic embryos failed to show normal development, and survival extended for only a few days beyond implantation. Parthenogenetic conceptuses developed embryos up to the 25-somite stage but the extraembryonic tissues which form the placenta were rudimentary. Androgenetic conceptuses possessed overdeveloped placental tissue, but the embryonic development rarely progressed beyond the 6to 8-somite stage. Hence placental development came strongly under the influence of the expression of paternally inherited alleles, while embryonic growth was influenced by alleles of maternal origin, and these differing phenotypes resulted from the disruption of parent-of-origin-imprinted genes. These early observations have been substantiated by the identification of certain imprinted genes and by producing null mutations that experimentally produce some features of the placental/embryonic phenotype (De Chiara et al., 1991).

title: Regulation of maternal behavior and offspring growth by paternally expressed Peg3.
abstract: Imprinted genes display parent-of-origin-dependent monoallelic expression that apparently regulates complex mammalian traits, including growth and behavior. The Peg3 gene is expressed in embryos and the adult brain from the paternal allele only. A mutation in the Peg3 gene resulted in growth retardation, as well as a striking impairment of maternal behavior that frequently resulted in death of the offspring. This result may be partly due to defective neuronal connectivity, as well as reduced oxytocin neurons in the hypothalamus, because mutant mothers were deficient in milk ejection. This study provides further insights on the evolution of epigenetic regulation of imprinted gene dosage in modulating mammalian growth and behavior.

title: Tumour suppressor activity of human imprinted gene PEG3 in a glioma cell line
abstract: Mouse imprinted gene Peg3 encodes a large C2H2 type zinc finger protein with unique characteristics. Peg3 knockout mice were found to show an impairment in maternal behaviour of the adult female. Mouse Peg3 is located on the proximal region of chromosome 7 which is syntenic to the long arm of human chromosome 19. It has been reported that a loss of heterozygosity (LOH) of chromosome 19q occurs frequently in several glioma types.

title: Epigenetic silencing of PEG3 gene expression in human glioma cell lines *
abstract: Genomic imprinting, the phenomenon in which alleles of genes are expressed differentially depending on their parental origins, has important consequences for mammalian development, and disturbance of normal imprinting leads to abnormal embryogenesis and some inherited diseases and is also associated with various cancers. In the context of screening for novel imprinted genes on human chromosome 19q13.4 with mouse A9 hybrids, we identified a maternal allele–specific methylated CpG island in exon 1 of paternally expressed imprinted gene 3 (PEG3), a gene that exhibits paternal allele–specific expression. Because PEG3 expression is downregulated in some gliomas and glioma cell lines, despite high‐level expression in normal brain tissues, we investigated whether the loss of PEG3 expression is related to epigenetic modifications involving DNA methylation. We found monoallelic expression of PEG3 in all normal brain tissues examined and five of nine glioma cell lines that had both unmethylated and methylated alleles; the remaining four glioma cell lines exhibited gain of imprinting with hypermethylated alleles. In addition, treatment of glioma cell lines with the DNA demethylating agent 5‐aza‐2′‐deoxycytidine reversed the silencing of PEG3 biallelically. In this article, we report that the epigenetic silencing of PEG3 expression in glioma cell lines depends on aberrant DNA methylation of an exonic CpG island, suggesting that PEG3 contributes to glioma carcinogenesis in certain cases.

title: Diversity of glutamate receptor subunit mRNA expression within live hippocampal CA1 neurons.
abstract: Glutamate-mediated neurotransmission occurs through the activation of multimeric postsynaptic receptors. One mechanism by which functional diversity of glutamate responsiveness may occur is by a single cell expressing multiple receptors containing different subunits. In a direct test of this hypothesis, we examined the glutamate receptor subunit mRNA composition of several individual CA1 neurons in hippocampal slices. Experiments used amplified antisense RNA coupled with expression profiling and polymerase chain reaction amplification to identify and determine the relative amounts of subunit mRNAs co-localized in single cells. The results demonstrate that each CA1 neuron contains varying amounts of most glutamate receptor mRNAs. In addition to relative mRNA levels, the single-cell approach also highlighted other possible sources of receptor diversity. This included the existence of novel, alternatively spliced forms of the N-methyl-D-aspartate receptor type 1 and glutamate-kainate receptor type 2 subunits. Surprisingly, levels of N-methyl-D-aspartate receptor type 1 mRNA were relatively low, compared with those of other glutamate receptor mRNAs. One postulated source of potential heterogeneity, RNA editing, was not a general cellular mechanism. There was no evidence that glutamate receptor type 5 mRNA was edited in any of the cells that were examined. These data show that individual CA1 neurons, in the intact synaptic network of hippocampal slices, generate glutamate receptor mRNA diversity in several ways, which together contribute to the diversity of functional receptors observed electrophysiologically.

title: Generating a phage display antibody library against an identified neuron
abstract: The generation of monoclonal antibodies by conventional hybridoma technology is limited by the diversity of the clones and the difficulties of screening the antibodies and of their large-scale production from isolated clones. As an alternative approach, we have investigated the suitability of phage display libraries for the production of recombinant antibodies against an identified neuron. Mice were immunized with isolated leech Retzius (R) neurons. The spleen poly A+ RNA was isolated and first-strand cDNA was prepared. The variable regions of light- and heavy-chain IgG molecules were amplified by the polymerase chain reaction (PCR) and separate libraries of each were constructed and combined in the pComb8 vector to yield a combinatorial library of approximately 10(7) transformants. Single R neurons that were plated in culture bound approximately 100 phages on a first screen and several thousand when the first batch was re-screened. Of these, 96 individual phage colonies were isolated and used for immunocytochemistry with leech CNS ganglia: 41 exhibited general staining, 20 showed no detectable staining and 30 stained selective subsets of neurons including (but not specific for) the R neuron. The phage display library approach thus simplifies the screening of large libraries with small numbers of (and even single) cells. However, the combinatorial antibodies with binding activity must still be tested individually by immunocytochemistry, which is further limited by their apparently low affinity.

title: cDNA libraries from identified neurons
abstract: One approach to studying the changes in gene expression which underlie differentiation is to construct cDNA libraries from different tissues or at different stages of development. However, generating representative cDNA libraries from heterogeneous tissues such as the nervous system is often a real problem. Here, we describe a reproducible method for the construction of large and complex cDNA libraries from a few leech Retzius or P neurons (equivalent to about 50 pg of mRNA) using polymerase chain reaction-based technology. The libraries contain about 106independant recombinants and are remarkably free from contaminating rRNA or polymerase chain reaction artefacts. Sequence analysis of randomly picked clones shows that the libraries contain a high proportion (more than 90%) of cDNAs larger than 500 b. p. As expected, many of the clones are novel, but two (α-tubulin and cyclophilin-A) have been extensively characterized in other species. To our knowledge, this is the first report of a cDNA library from identified neurons.

title: Analysis of gene expression in single live neurons.
abstract: We present here a method for broadly characterizing single cells at the molecular level beyond the more common morphological and transmitter/receptor classifications. The RNA from defined single cells is amplified by microinjecting primer, nucleotides, and enzyme into acutely dissociated cells from a defined region of rat brain. Further processing yields amplified antisense RNA. A second round of amplification results in greater than 10(6)-fold amplification of the original starting material, which is adequate for analysis--e.g., use as a probe, making of cDNA libraries, etc. We demonstrate this method by constructing expression profiles of single live cells from rat hippocampus. This profiling suggests that cells that appear to be morphologically similar may show marked differences in patterns of expression. In addition, we characterize several mRNAs from a single cell, some of which were previously undescribed, perhaps due to "rarity" when averaged over many cell types. Electrophysiological analysis coupled with molecular biology within the same cell will facilitate a better understanding of how changes at the molecular level are manifested in functional properties. This approach should be applicable to a wide variety of studies, including development, mutant models, aging, and neurodegenerative disease.

title: Preparation of cDNA from single cells and subcellular regions.
abstract: Phenotypic characterization of cells in conjunction with single-cell mRNA analysis, which yields information regarding expression of multiple genes in individual neurons, facilitates a detailed and comprehensive view of neuronal cell biology. More specifically, the aRNA amplification method has provided an approach to analyze mRNA levels in single cells that have been phenotypically characterized on the basis of electrophysiology, morphology, and/or protein expression. In this way, relative mRNA abundances can be directly assayed from a well-defined population of neurons. The concept of expression profiling led to the development of robotics methods for arraying thousands of cDNAs on microarrays. These cDNA arrays can be screened with labeled aRNA or cDNA to generate a molecular fingerprint of a specific cell type, disease state, or therapeutic efficacy. A broad view of how gene expression is altered in single neurons affected by a particular disease process may provide clues to pathogenetic disease mechanisms or avenues for therapeutic interventions. The use of mRNA profiles to produce diagnostics and therapeutics is called transcript-aided drug design (TADD). When coupled with single-cell resolution, TADD promises to be an important tool in diagnosis of disease states, as well as provide a blueprint on which to develop therapeutic strategies. For example, mRNA abundances in an individual diseased cell may increase, decrease, or remain constant, and thus it is possible that a pharmaceutical alone or in combination with other drugs may be specifically designed to restore mRNA abundances to a normal state. Alternatively, if functional protein levels parallel the mRNA level changes, then drugs targeting the function of the proteins translated from these altered mRNAs may prove to be therapeutic. One promise of such an approach is that information about mRNA abundances that are altered in a diseased cell may provide new therapeutic indications for existing drugs. For example, if the abundance of mRNA for the beta-adrenergic receptor is altered as shown by the microarrays for a particular disease, already available adrenergic receptor agonists or antagonists that had not previously been used in this particular disease paradigm may prove to be therapeutically efficacious. The expression profile of a given cell is a measure of the potential for protein expression. Proteins are generally the functional entities within cells and differences in protein function often result in disease. The ability to monitor the coordinate changes in gene expression, in single phenotypically identified cells, that correlate with disease will provide unique insight into the expressed genetic variability of cells and will likely furnish unforeseen insight into the underlying cellular mechanisms that produce disease etiology.

title: Genomic Imprinting, Mammalian Evolution, and the Mystery of Egg-Laying Mammals
abstract: The evolutionary theories concerning imprinting have mainly focused on imprinted gene functions, and on growth regulation in particular. It is important to consider in much greater detail aspects of early embryonic development. For example, the necessity for the trophectoderm lineage in marsupials and eutherians and its impact on early development, as well as brain development and function, should also be considered. There are distinct physiological requirements concerning reproduction and embryonic development in utero that may be regulated centrally by the brain.A deeper insight into the evolution of genomic imprinting and its link with mammalian evolution could also emerge from a close examination of the mechanisms of imprinting amongst the mammalian groups. In particular, a detailed analysis of the cis-control elements, their mode of action in the initiation, the molecular character of the imprints, and their inheritance could illuminate how the phenomenon may have originated and played a part in the emergence of mammals.*To whom correspondence should be addressed (e-mail: as10021@mole.bio.cam.ac.uk).

title: Genomic imprinting in mammals.
abstract: A handful of autosomal genes in the mammalian genome are inherited in a silent state from one of the two parents, and in a fully active form from the other, thereby rendering the organism functionally hemizygous for imprinted genes. To date 19 imprinted genes have been identified; 5 are expressed from the maternal chromosome while the rest are expressed from the paternal chromosome. Allele-specific methylation of CpG residues, established in one of the germlines and maintained throughout embryogenesis, has been clearly implicated in the maintenance of imprinting in somatic cells. Although the function of imprinting remains a subject of some debate, the process is thought to have an important role in regulating the rate of fetal growth.