Hartwig Schmale

Differential Responses to Osmotic Stress of Vasopressin-Neurophysin mRNA in Hypothalamic Nuclei

mRNA encoding the vasopressin-neurophysin precursor was quantitated in individual hypothalamic nuclei of rats by a liquid hybridization assay. Drinking of 2% saline for 14 days, a treatment that increased the plasma vasopressin concentration 9-fold, resulted in a 5- and 2-fold increase in mRNA levels in the supraoptic and paraventricular nucleus, respectively. This osmotic stimulus had no effect on vasopressin-neurophysin mRNA content of the suprachiasmatic nucleus. This dissociation in regulation of vasopressin-neurophysin mRNA in hypothalamic nuclei indicates the existence of two separate vasopressin systems that are independently activated.

Molecular cloning of human von Ebner's gland protein, a member of the lipocalin superfamily highly expressed in lingual salivary glands

Von Ebners glands (VEG) are small lingual salivary glands. Their ducts open into trenches of circumvallate and foliate papillae, thus influencing the milieu where the interaction between taste receptor cells and sapid molecules takes place. The major secretions of human VEG is a protein with a molecular mass of 18 kDa. The human VEG protein crossreacts with antibodies raised against the rat VEG protein, indicating sequence similarity between the rat and human VEG proteins. This was subsequently confirmed by N-terminal protein sequencing. A cDNA clone, isolated from a human VEG library, contained an insert of 735 bp including an open reading frame that encodes the human VEG protein of 176 amino acids. Comparison of the human and rat VEG proteins revealed an overall identity of 60%. Immunocytochemistry, in situ hybridization and in vitro translation studies demonstrated the human VEG protein to be highly and exclusively expressed in VEG. The VEG proteins are members of the lipocalin protein superfamily and, together with the rat odorant binding protein II, they constitute a new subfamily. Sequence similarity to proteins such as the retinol binding protein and the odorant binding protein which are lipophilic ligand carriers, suggests a possible function for the human VEG protein in taste perception.

Differential regulation of transcription and induction of programmed cell death by human p53‐family members p63 and p73

The p53 tumor suppressor acts as a transcription factor and has a central function in controlling apoptosis. With p63 and p73 two genes coding for proteins homologous to p53 have been identified. We describe the properties of seven human p63 and p73 proteins as transcriptional activators of p21WAF1/CIP1 expression and apoptotic inducers in direct comparison to p53 in the same assay systems employing DLD‐1‐tet‐off colon cells. Programmed cell death is detected in cells expressing high levels of p53 and p73α. Cells overexpressing TAp63α, TAp63γ, TA*p63α, TA*p63γ, ΔNp63α, and ΔNp63γ display low or no detectable apoptosis.

Cloning and chromosomal mapping of the human p53-related KET gene to Chromosome 3q27 and its murine homolog Ket to mouse Chromosome 16

Abstract. KET is a member of the newly discovered family of proteins that is related to the tumor suppressor p53. Here we describe the molecular cloning of a human cDNA of 4846 bp encoding a protein of 680 amino acids. The human KET protein shares 98% identity with the previously characterized rat homolog. The remarkably high degree of conservation lends support to the notion that KET proteins have important basic functions in development and differentiation. Using the GeneBridge 4 radiation hybrid panel, we have mapped KET to human Chromosome (Chr) 3q27. KET is located between the somatostatin gene SST (proximal) and the apolipoprotein D gene APOD (distal) in a region of conserved synteny to mouse Chr 16. This chromosomal region is deleted in early stages of tumorigenesis of mouse islet cell carcinomas and contains the hitherto unidentified Loh2 gene, a putative suppressor of angiogenesis. The murine homolog Ket was mapped in an interspecific backcross panel and falls into this region of loss of heterozygosity. From our mapping data we infer that KET might act as a tumor suppressor and is considered as a candidate for Loh2.

Quantitation of vasopressin mRNA and oxytocin mRNA in hypothalamic nuclei by solution hybridization assays

Abstract: Concentrations of vasopressin (VP) precursor and oxytocin (OT) precursor mRNA were measured in magno‐cellular cell groups of the rat hypothalamus by newly developed solution hybridization assays. The assays employed single‐stranded 35S‐labeled VP‐specific and OT‐specific DNA probes that were prepared by primer extension on re‐combinant M13 DNA templates. Solution hybridization assays were standardized by known amounts of cloned DNA. The detection limit was <1 pg DNA equivalent of the respective mRNA. In total RNA preparations of microdis‐sected supraoptic nucleus (SON) mean (SEM) basal levels of 1.370.18pgVP mRNA and 1.950.I4pgOT mRNA were measured. RNA of the microdissected paraventricular nucleus (PVN) contained 0.35 ± 0.02 pg VP mRNA and 1.77 ± 0.15 pg OT mRNA. Elevation of plasma osmolality induced by drinking of 2% saline for 25 days resulted in a 1.85‐fold increase in VP mRNA levels of the SON and a 1.6‐fold increase in VP mRNA levels of the PVN. The solution hybridization assays are suitable tools to study the regulation of VP and OT mRNAs in magnocellular neurons of the brain.

Transcription of the opsin gene in degenerate eyes of cave-dwelling Astyanax fasciatus (Teleostei, Characidae) and of its conspecific epigean ancestor during early ontogeny

The early morphogenesis of the degenerate eyes of the Mexican cave fish Astyanax fasciatus and of its conspecific epigean ancestor has been studied comparatively using light- and electron-microscopical techniques; the transcription of the opsin gene has been analysed during early ontogeny in both populations by in situ hybridization. The opsin protein is an integral component of the disk membranes of the photoreceptor cells. In epigean specimens, its expression is correlated with the beginning of outer segment formation on the third day of development. Morphogenesis of the cave fish eye is similar to that of the epigean eye until end of the third day. However, eye growth and morphogenesis of the retina are delayed and sporadic cell death occurs in all retinal layers at the beginning of the second day. Retinal cytodifferentiation breaks off at the point of outer segment formation. Cave specimens are not able to develop regular outer segments at any stage, but the opsin gene is nevertheless expressed in the outer nuclear layer of the developing retina for a limited period of time. On the basis of the comparative morphology and transcriptional studies of epigean and cave specimens, it is suggested that the eye regression of cave fish is primarily the result of mutations of developmental control genes and not of structural genes.

Expression of different p63 variants in healing skin wounds suggests a role of p63 in reepithelialization and muscle repair

Healing of skin wounds in mammals involves partial reconstruction of the dermis and coverage of the injured site by keratinocytes. The latter process is achieved by extensive migration and hyperproliferation of keratinocytes at the wound rim. Because the p53 protein family member p63 is expressed in human hyperproliferative epidermis, this study determined whether enhanced keratinocyte proliferation correlates with the expression of p63. Therefore, we investigated the temporal and spatial distribution of four major variants of the p63 transcription factor—TAp63α, TAp63γ, ΔNp63α and ΔNp63γ—during normal skin wound healing in mice. Transcripts encoding amino‐terminally truncated ΔNp63 variants were found at high levels in basal and suprabasal keratinocytes of the hyperproliferative wound epithelium. Interestingly, TAp63 variants, which include the conserved transactivation domain TA at their amino‐terminus, were also expressed in wound keratinocytes as well as at the edge of the injured subcutaneous muscle panniculus carnosus. These findings suggest splice‐variant specific functions of p63 in reepithelialization and muscle repair.

Postnatal development of von Ebner's glands: accumulation of a protein of the lipocalin superfamily in taste papillae of rat tongue

SummaryAntibodies produced against rat von Ebners gland (VEG) protein, a recently characterized member of a lipophilic ligand carrier protein family, detect this protein immunocytochemically in von Ebners gland acini and show that it is present at high concentrations in the clefts of circumvallate and foliate papillae. During embryonic development, von Ebners gland anlagen are innervated (as shown immunocytochemically using neuronal specific antibodies) as early as embryonic day 20, before lateral glandular outgrowth and VEG protein can be observed. Expression of the VEG protein as determined by in sity hybridization and immunocytochemistry begins at postnatal day-2 cells in differentiating and branching off from von Ebners gland ducts, and sharply increases with further enlargement and maturation of the gland. The close temporal correlation of von Ebners gland innervation and VEG protein expression with papilla innervation and taste-bud development suggests a functional relationship of both structures. VEG protein might control access of lipophilic sapid molecules, such as bitter substances, to the gustatory receptors.

Cell-free translation of bovine hypothalamic mRNA: Synthesis and processing of the prepro-neurophysin I and II

Neurophysins are synthesized in neurosecretor~ neurons of the hypo~alarnus. They are characterized by their function which is to transport the octapeptides vasopressin and oxytocin along the axons to a storage site in the posterior pituitary [ 13. Bovine neurophysin I (Np I) and II (Np II) have been purified and sequenced [Z]; they have mol. wt -10 000. Two groups have identified the tentative precursor to Np I and Np II, respectively, from the hypothalamus of mice and rats [3,4]. The precursors have mol. wts -20 000 [3,4] and at least one of them is gly~sylated [ 4 ,.5 1. The aim of this study was to investigate the cellfree biosynthesis of the reported precursors by translation of mRNA isolated from calf hypothalamus. Also we were interested whether one or both synthesized &-precursors identified by immunoprecipitation could be cleaved and glyc~sylated by microsomal membranes as known from studies of other precursor polypeptides [ls].

Vasopressin Expression in Cultured Neurons is Stimulated by Cyclic AMP

Vasopressin and oxytocin genes are expressed in mutually exclusive sets of magnocellular neurons in the hypothalamus. Cell specificity and regulation are probably controlled by extra‐ and intracellular signals acting on one or the other gene. In order to identify factors that regulate peptide expression, we have used primary dissociated cultures derived from 14‐day old foetal rats. Vasopressin expression was monitored by combined immunocytochemistry and in situ hybridization. Treatment of cultures with forskolin and/or the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX), both of which result in elevated intracellular cyclic AMP levels, increased the numbers of vasopressin‐expressing cells up to 10‐fold. The specific Vasopressin messenger ribonucleic acid accumulation was verified quantitatively by ribonuclease protection assays. Forskolin and IBMX did not change the levels of the general neuronal markers, neuron‐specific enolase and synaptophysin, suggesting that the effect of these drugs was specific for vasopressin‐expressing cells. The drugs were not mitogenic for magnocellular neurons. Furthermore, their effect was not mediated trans‐synaptically, as the drugs were also effective in cultures grown in low Ca2+/high Mg2+ medium, as well as in cultures treated with either tetanus toxin or tetrodotoxin. The presence of putative response elements for the transcription factor AP‐2 in the 5’promoter regions of all vasopressin genes sequenced so far may provide the molecular basis of the observed cyclic AMP effect. No such elements are present in the genes for oxytocin, the messenger ribonucleic acid levels of which were not measurably affected by forskolin and IBMX in our cultures.