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K M Gautvik

Publications and source records attributed to K M Gautvik.

At least 19 recordsLinked to original sources
PubMed1996

Overview of the most prevalent hypothalamus-specific mRNAs, as identified by directional tag PCR subtraction.

We applied the directional tag PCR subtractive hybridization method to construct a rat hypothalamic cDNA library from which cerebellar and hippocampal sequences had been depleted, enriching 20-30-fold for sequences expressed selectively in the hypothalamus. We studied a sample of 94 clones selected for enrichment in the subtracted library. These clones corresponded to 43 distinct mRNA species, about half of which were novel. Thirty-eight of these 43 mRNAs (corresponding to 85 of the clones in the sample) exhibited enrichment in the hypothalamus; 23 were highly enriched. In situ hybridization studies revealed that one novel species was restricted to cells in a small bilaterally symmetric area of the paraventricular hypothalamus. Other novel mRNAs showed substantial enrichment in basal diencephalic structures, particularly the hypothalamus, without restriction to single hypothalamic nuclei. The data suggest that the hypothalamus utilizes at least two distinct strategies for employing its selectively expressed proteins. Secretory neuropeptides utilized for intercellular communication are produced by functionally discrete nuclei, while several other proteins are shared by structures that are unrelated in their physiological roles but may share biochemical systems.

Animals↗
PubMed1996

A cortical neuropeptide with neuronal depressant and sleep-modulating properties.

Acetylcholine (ACh) plays a key role in the transitions between the different phases of sleep: Slow-wave sleep requires low ACh concentrations in the brain, whereas rapid-eye-movement (REM) sleep is associated with high levels of ACh. Also, these phases of sleep are differentially sensitive to a number of endogenous neuropeptides and cytokines, including somatostatin, which has been shown to increase REM sleep without significantly affecting other phases. Here we report the cloning and initial characterization of cortistatin, a neuropeptide that exhibits strong structural similarity to somatostatin, although it is the product of a different gene. Administration of cortistatin depresses neuronal electrical activity but, unlike somatostatin, induces low-frequency waves in the cerebral cortex and antagonizes the effects of acetylcholine on hippocampal and cortical measures of excitability. This suggests a mechanism for cortical synchronization related to sleep.

Acetylcholine↗
PubMed1995

High-level production of human parathyroid hormone in Bombyx mori larvae and BmN cells using recombinant baculovirus.

A full-length cDNA encoding human parathyroid hormone (hPTH) containing the prepro region was cloned into Bombyx mori baculovirus under the control of the polyhedrin promoter and polyadenylation sequences. After transfection and generation of the recombinant baculovirus, hPTH production was examined in silkworm larvae and BmN cell cultures. The larvae synthesized and efficiently secreted the correctly processed and authentic hPTH (9.4 kDa) with no sign of internal degradation. In BmN cells, the major secreted form was the correctly sized protein, but small amounts of degraded hPTH could also be detected in the medium by immunoblotting. Unlike the situation in larvae, prepro-hPTH could also be demonstrated intracellularly in BmN cells. The concentration of hPTH in the larval hemolymph was about 70 mg/l, as compared to approx. 55 micrograms/l in the medium per 7.5 x 10(6) cells. Recombinant hPTH (re-hPTH) from the hemolymph was purified by reverse-phase HPLC and subjected to chemical and biological analyses. The authenticity of the purified re-hPTH was confirmed by N-terminal sequencing, amino acid composition and a mass of 9425 Da, close to the theoretical value. The hormone showed high-affinity receptor binding and full biological potency in increasing cellular cAMP.

Animals↗
PubMed1995

Human parathyroid hormone as a secretory peptide in milk of transgenic mice.

In a transgenic mouse model we have targeted the expression of recombinant human parathyroid hormone (hPTH) to the mammary gland yielding hPTH as a secretory, soluble peptide in milk. A 2.5 kb upstream regulatory sequence of the murine whey acidic protein (WAP) directed the expression of the hPTH cDNA in a fusion gene construct (WAPPTHSV2) containing the SV40 small t-antigen intron and polyadenylation site in the 3' end. Established lines of transgenic mice secreted hPTH to milk in concentrations up to 415 ng/ml. Recombinant hPTH recovered from the milk was purified by HPLC and shown to be identical to hPTH standard as analyzed by SDS-PAGE followed by immunoblotting. Expression of the WAPPTHSV2 was limited to the mammary gland as analyzed by polymerase chain reaction (PCR) and Southern blot of reversed transcribed mRNA from different tissues. hPTH is an important bone anabolic hormone and may be a potentially important pharmaceutical for treatment of demineralization disorders such as osteoporosis. We present the transgenic animal as a possible production system for hPTH.

Amino Acid Sequence↗
PubMed1995

Expression and characterization of a recombinant human parathyroid hormone partial agonist with antagonistic properties: Gly-hPTH(-1-->+84).

We have produced and characterized a hPTH analogue with an amino-terminal extension of glycine, Gly-hPTH(-1-->+84) (denoted Gly-hPTH). The hormone analogue was synthesized in E. coli strain BJ5183 transformed with the expression plasmid pKKPTH, extracted from the bacterial pellet and purified by reverse-phase high performance liquid chromatography. Its chemical nature, as determined by amino acid composition analysis, N-terminal amino acid analysis, and mass spectrometry, showed the 9480-Da Gly-hPTH as the predominant species. Because f-Met-Gly-hPTH was the expected form encoded by the plasmid construct, the results indicate that the f-Met residue was efficiently removed from the precurser form. The following functional characteristics of Gly-hPTH were demonstrated. 1) In cells transfected with the human PTH/PTHrP receptor, the receptor binding affinity was reduced threefold compared to the authentic hPTH(1-84) produced by Saccharomyces cerevisiae (apparent Kds: 8.4 and 2.7 nM, respectively). 2) Using the same cells, Gly-hPTH showed 27-fold reduced potency compared to hPTH(1-84) in stimulating intracellular cAMP production (EC50: 32 and 1.2 nM, respectively). 3) Gly-hPTH demonstrated antagonist activity by reducing hPTH-induced cAMP production by 33 +/- 5% (mean +/- SD) when tested at a 1:1 molar ratio. In these studies the recombinant authentic hPTH(1-84) was used as standard for comparisons, and it showed an equal receptor binding affinity and cAMP production as the chemically synthesized peptide [Nle8,18,Tyr34]bovinePTH(1-34)-NH2.

Amino Acid Sequence↗
PubMed1994

Dexamethasone and insulin demonstrate marked and opposite regulation of the steady-state mRNA level of the peroxisomal proliferator-activated receptor (PPAR) in hepatic cells. Hormonal modulation of fatty-acid-induced transcription.

Fatty acids and the peroxisomal proliferator, 3-tetradecylthioacetic acid (TTA) stimulate transcription of peroxisomal beta-oxidation enzymes. Recently, we have shown that their actions are markedly modulated by dexamethasone and insulin which show synergistic and inhibitory effects, respectively. In this study, we describe the regulation of the peroxisomal proliferator-activated receptor (PPAR), a member of the steroid-hormone-receptor superfamily, in a similar manner by hormones and fatty acids, supporting the hypothesis that PPAR may act as a ligand-activated transcription factor. Northern-blot analysis of steady-state mRNA levels revealed three different specific transcripts for PPAR of 10.2, 4.6 and 1.8 kb, and the former two being regulated in hepatic tissue, hepatocytes and hepatoma cells. Dexamethasone produced a pronounced overall stimulatory effect (15.3-fold) in rat hepatocytes, while insulin blocked this action completely. Minor inductions of PPAR mRNA (up to twofold induction) were observed when different fatty acids were administrated alone. However, in combination with dexamethasone, additive or synergistic actions, mounting to 24-fold stimulation, were observed, while insulin always exerted an over-riding down-regulatory effect. In non-fasting rats receiving dexamethasone, elevation of serum insulin, a slight increase in serum free fatty acids accompanied by PPAR mRNA level increases of 2.4-fold and stimulation of liver peroxisomal acyl-CoA oxidase mRNA were observed. Our results suggest that PPAR mRNA expression is under strict hormonal control and that the fatty acids and hormones affect PPAR mRNA levels in a manner analogous to the regulation of the peroxisomal beta-oxidation enzymes. The PPAR gene-regulating unit apparently contains hormone-response elements (HRE) for dexamethasone and insulin, which are thus functionally important for PPAR transcription in liver cells, making a significant enhancement or inhibition of the physiological actions of fatty acids possible.

Acyl-CoA Oxidase↗
PubMed1994

Cell-specific expression and function of adenylyl cyclases in rat pituitary tumour cell lines.

The present study demonstrates cell-specific distribution and describes distinct functional regulation of different adenylyl cyclases (AC, types I-VI) in rat pituitary cell tumor cell lines (GH12C1, GH3 and GH4C1 cells) and pituitary tissue. Northern-blot analysis revealed a distinct pattern of cell-specific expression of the different AC types; Ca2+/calmodulin (CaM)-insensitive AC type II was found in all cell lines tested except GH(1)2C1 cells. The Ca(2+)-inhibitable AC type VI was found in all cell types tested. We observed a lack of the Ca2+/CaM-sensitive AC type I in GH3 and GH4C1 cells. GH(1)2C1 cells exclusively contained both Ca2+/CaM-sensitive AC types I and III, the latter previously believed to be specific for olfactory tissue. An additional transcript of AC type III was found in rat brain and rat liver tissue. AC type IV, which is Ca2+/CaM insensitive, could be detected in the prolactin-producing GH3 and GH4C1 cells and pituitary tissue but not in growth-hormone-producing GH(1)2C1 cells. Basal and vasoactive-intestinal-peptide-(VIP)-releasing-hormone, somatostatin (SRIF) and thyrotropin-releasing-hormone (TRH)-modulation of AC activity was measured in the presence of 100 microM EGTA, anti-CaM serum (dilution 1:2000) or 10 microM trifluoroperazine. Antisera against guanine-nucleotide-binding protein (G-protein) alpha subunits (G(i)-2 alpha, Gs alpha) and beta subunits (G beta 35/36) and CaM were added for functional studies of the SRIF and VIP-modulated AC in GH(1)2C1 and GH3 cells. These experiments indicate that the VIP and the SRIF receptors are coupled to a Ca2+/CaM-sensitive AC in GH(1)2C1 cells, different from the AC involved in the regulation of cAMP levels in GH3 and GH4C1 cells. In addition, the beta gamma-complex is possibly able to modulate SRIF-inhibited AC activity by potentiating the inhibitory effect. The TRH receptor in GH3 and GH4C1 cells is coupled to a Ca2+/CaM-sensitive AC which is different from the already cloned forms of AC types I and III. We, therefore, conclude that hormone regulation of pituitary tumour cell functions differs between the GH cell lines, due to specific utilisation of AC types.

Adenylyl Cyclases↗
PubMed1994

Expression of human parathyroid hormone in mammalian cells, Escherichia coli and Saccharomyces cerevisiae.

The entire human parathyroid hormone (hPTH) cDNA gene with its natural signal and pro-region is expressed in transfected mouse mammary tumor cells (C127I cells) and Chinese hamster lung cells (DON cells) under control of the murine metallothioneine-1 promoter in a vector in which replication functions are provided by the entire genome of bovine papilloma virus type I (BPV-1). Authentic hPTH is efficiently produced by the non-endocrine cells and secreted to the growth medium without any abberant processing. Immunoblots from SDS-PAGE gels of concentrated growth medium reveal one band corresponding to intact, undegraded hPTH. Purification by reversed-phase HPLC results in a peptide with an amino acid content and N-terminal sequence identical to hPTH. For comparison, hPTH cDNA with deleted prepro-region is also expressed as secretory proteins in Escherichia coli and in Saccharomyces cerevisiae. In E. coli the vector construct is based on the staphylococcal protein A promoter employing protein A signal sequence. In S. cerevisiae a mating factor alpha expression system containing the factor alpha-signal sequence is employed. The results show that intact hPTH is secreted in addition to proteolytically cleaved fragments in both microorganisms. Thus, the signal sequences promote efficient secretion, and correct N-terminal processing of hPTH in both mammalian, bacterial and yeast cells. However, the folding characteristics of hPTH make it susceptible to internal proteolytical cleavage which appears to be species specific in yeast and E. coli.

Amino Acid Sequence↗
PubMed1994

Translocation and processing of various human parathyroid hormone peptides in Escherichia coli are differentially affected by protein-A-signal-sequence mutations.

Two staphylococcal protein-A signal sequences were constructed and tested for function in Escherichia coli, after being linked to human parathyroid hormone (hPTH) cDNAS representing the intact form (1-84 amino acids) and two N-terminal (1-37 and 1-7 amino acids) peptides. One signal sequence was identical to the wild type, and the other signal contained a deletion of 12 bp at the 3' end. The truncated hPTH cDNAs were fused at their 3' ends to IgG-binding domains (ZZ) derived from protein A in order to facilitate purification and characterization. The expression plasmid pSPTH, containing the wild-type signal sequence, secreted efficiently the intact recombinant hPTH (1-84) into the medium. Plasmids containing the truncated hPTH genes after the wild-type signal, gave rise to hPTH-ZZ hybrid proteins which were correctly processed at the N-terminal, but the major fractions appeared in the periplasmic compartment. In contrast, the plasmid pS'PTH which harboured the 4-amino-acid signal deletion did not promote a uniform secretion of intact hPTH (1-84) to the medium, but released a non-processed form both into the periplasmic compartment and to the medium. The related plasmids pS'PTH37ZZ and pS'PTH7ZZ with the mutated signal sequence gave rise to small or trace amounts of unprocessed forms of fusion proteins in the medium and periplasm, thus the secretion competence was markedly reduced. Thus, for correct N-terminal processing, we conclude that the amino acid sequence in the signal adjacent to the expressed protein, is a key determinant. However, release into the medium or periplasmic space appeared to be dependent also on protein folding, irrespective of signal-sequence cleavage. Furthermore, we observed that the peptides with the wild-type signal sequence and correct N-terminal processing, were the only forms that showed internal cleavage of hPTH. Uncleaved signals may contribute to folding characteristics of the ensuing protein and e.g., prevent internal proteolysis.

Amino Acid Sequence↗
PubMed1994

Parathyroid hormone-related protein is produced by cultured endothelial cells: a possible role in angiogenesis.

Parathyroid hormone-related protein (PTHrP) is produced by various normal and neoplastic tissues. Even if the physiological function(s) of PTHrP is unclear, evidence suggests that the protein may participate in the local regulation of smooth muscle contractility. We show here that PTHrP is produced in endothelial cells cultured from human umbilical veins as demonstrated both at the mRNA and protein level. The expression of PTHrP can be upregulated by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate, which is known to stimulate endothelial cell differentiation and angiogenesis in vitro. Unlike smooth muscle cells, the endothelial cells do not express the parathyroid hormone (PTH)/PTHrP receptor mRNA, nor could specific binding of the protein be detected. We therefore suggest that PTHrP produced by endothelial cells acts on smooth muscle cells and may be of importance for the growth and development of new vasculature.

Cells, Cultured↗
PubMed1994

Differences in binding affinities of human PTH(1-84) do not alter biological potency: a comparison between chemically synthesized hormone, natural and mutant forms.

The purpose of this study was to evaluate receptor binding affinities and biological properties in vitro and in vivo of various recombinant hPTH(1-84) forms representing the natural hormone and a mutagenized hPTH form, [Gln26]hPTH(1-84) (QPTH), after expression in E. coli and Saccharomyces cerevisiae. In LLC-PK1 cells stably transformed with the rat PTH/PTHrP receptor, chemically synthesized hPTH(1-84) and QPTH showed a reduced binding affinity (apparent Kd 18 and 23 nM, respectively) than the recombinant, hPTH(1-84) (apparent Kd 9.5 nM). All recombinant hPTH forms showed a similar potency to stimulate cellular cAMP production (EC50 1.5 nM) and significantly better than chemically synthesized hPTH (EC50 5.7 nM). All hormone forms showed an about equipotent activity in causing elevation in serum calcium, increased excretion of urine phosphate, and cAMP. Thus, the natural recombinant PTH forms showed higher binding affinities and adenylate cyclase activation potencies in LLC-PK1 cells, but the reduced receptor binding affinity exerted by QPTH did not transcend differences in cAMP generation and in vivo biological activities.

Animals↗
PubMed1994

G-proteins: implications for pathophysiology and disease.

This article focuses on the involvement of G-proteins in neuroendocrine secretion, cell growth and phenotype alterations. The current concept of hormonal activation of the GTPase cycle, as well as the molecular diversity of G-proteins families and receptor*G-protein*effector coupling, are described. Also described are certain G-proteins as possible proto-oncogenes and how point mutations and frame shift mutations alter G-protein function and determine the characteristics of various endocrine diseases. The article outlines in detail how receptors and G-proteins interact in prolactin and growth-hormone-secreting pituicytes, how G-proteins are involved in the growth and differentiation of preadipocytes and osteoblasts. All in all, it seems that hormonal activation through G-proteins is modulated through direct intra- and inter-signalling system cross-talk at the plasma membrane level (short-term) and through interactions on the level of transcription (HREs) from tyrosine kinases, steroid-like hormones and metabolic pathways. Pharmacological intervention to treat diseases where G-proteins are involved should take both long and short-term regulatory phenomena into consideration.

Animals↗
PubMed1993

Molecular cloning of a functional human thyrotropin-releasing hormone receptor.

A cDNA encoding the human thyrotropin-releasing hormone receptor (hTRH-R) was isolated from a human brain cDNA library. Screening of 1.2 million clones resulted in 2 candidates. The largest clone contained TRH-R homologous sequences starting in the third transmembrane domain and included a long 3' untranslated sequence. The smaller clone contained a potential start of the open reading frame, but was interrupted by an intron in the sixth transmembrane domain. The two clones had 497 bp of overlapping identical sequences and it was possible to assemble a complete cDNA thus restoring the assumed coding sequence. Electrophysiological studies of frog oocytes injected with in vitro transcribed mRNA showed TRH-specific inward currents, demonstrating that the reconstituted cDNA encoded a functional receptor. The predicted amino acid sequence of the hTRH-R protein showed high homology with the rat and mouse TRH-Rs with the exception of their C-terminal region. The human TRH-R gene seems to contain two introns.

Amino Acid Sequence↗
PubMed1993

Synthesis of human parathyroid-hormone-related protein(1-141) in Saccharomyces cerevisiae. A correct amino-terminal processing vital for the hormone's biological activity is obtained by an ubiquitin fusion protein approach.

Gene fusions have been widely used in heterologous expression systems as a technique to stabilize the recombinant product against proteolysis, increase the translational initiation efficiency or to serve as an affinity handle for the purification of the protein. A further advantage is the potential to generate an authentic amino terminus of the foreign protein when this is vital for its biological activity, such as for the ability of human parathyroid-hormone-related protein (hPTHrP) to mediate activation of adenylate cyclase. We report here the construction and utility of a ubiquitin fusion protein system for production of the otherwise short-lived hPTHrP(1-141) as a carboxyl extension to ubiquitin in yeast. A hybrid gene containing the hPTHrP(1-141) cDNA coding region fused in-frame to the 3' end of the yeast ubiquitin cDNA was constructed and expressed under the control of the regulatable yeast metallothionein promoter. The recombinant protein was purified to homogeneity and finally characterized by N-terminal amino acid sequencing and amino acid composition analysis, demonstrating that the fusion protein was cleaved correctly and quantitatively in vivo by an ubiquitin-specific yeast endoprotease to generate authentic hPTHrP(1-141). hPTHrP(1-141) stimulated adenylate cyclase in rat osteosarcoma cell membranes to the same extent as equimolar amounts of recombinant human parathyroid hormone(1-84) and [Tyr34]hPTHrP(1-34)amide. Thus, this expression cloning strategy permits the production of authentic, biologically active recombinant hPTHrP(1-141), and the procedure can easily be adapted to make PTHrP analogues for further studies of its domain-specific activities and biological roles.

Adenylyl Cyclases↗
PubMed1993

[Growth hormone and prolactin receptors belong to a new receptor family. Biological and medical aspects].

The molecular structures of the receptors of growth hormone and prolactin have recently been identified by molecular cloning, and have been characterized in a number of species and different tissues. The receptors consist of one polypeptide chain with a single transmembrane region. The extracellular region contains cysteines for disulphide bonding and potential sites for glycosylations. The intracellular part which mediates the biological actions displays considerable heterogeneity of size. A high degree of homology is demonstrated between the extracellular part of these receptors and the receptors of a number of cytokines, interferons and growth factors. Together they comprise a new family of receptors called the cytokine/growth hormone/prolactinreceptor family. Their structure is reviewed, along with the actions of growth hormone and prolactin in vivo. In spite of numerous biological effects the molecular mechanisms of actions for this class of receptors are unknown, even though they convey important cell regulatory functions. The molecular analysis of growth hormone receptor has provided new medical insight on the results of growth hormone replacement therapy in persons with deficient growth.

Animals↗
PubMed1993

Induction of peroxisomal acyl-CoA oxidase by 3-thia fatty acid, in hepatoma cells and hepatocytes in culture is modified by dexamethasone and insulin.

The effects of tetradecylthioacetic acid (TTA) (50 microM), dexamethasone (0.25 microM) and insulin (0.4 microM) on induction of peroxisomal acyl-CoA oxidase activity and mRNA levels were studied in short term cultures of Morris 7800C1 and MH1C1 hepatoma cells and of rat hepatocytes. Dexamethasone and TTA resulted in parallel increases in the enzyme activity and the steady state mRNA content in the hepatoma cells. Combination of dexamethasone and TTA resulted in a synergistic and parallel stimulation of both the enzyme activity and the mRNA levels up to 11-12-fold and maximal changes were observed after 14 days of treatment. Semiquantitative immunoblot analyses of acyl-CoA oxidase were in concordance with enzyme and mRNA results. Insulin counteracted the inductive effects of dexamethasone and TTA on all parameters. The half-life of the acyl-CoA oxidase mRNA increased after treatment with the 3-thia fatty acid (t1/2 = 10.0 h +/- 0.4) compared to control (t1/2 = 5.9 h +/- 0.3). However, in combination with dexamethasone there was no further increase in the mRNA stability (t1/2 = 8.0 h +/- 0.3). Southern blot analysis did not reveal any changes on the oxidase gene level in any treatment group. TTA alone or in combination with dexamethasone did not affect the expression of either the glucocorticoid receptor or the peroxisomal proliferator acting receptor (PPAR) steady state mRNA levels. In cultured hepatocytes the acyl-CoA oxidase was modified in similar manner by these treatments, but the changes were less marked. We suggest that the changes in peroxisomal acyl-CoA oxidase activity in hepatoma cells are due to a major effect on the level of mRNA, involving both transcriptional effects and message stabilization.

Acyl-CoA Oxidase↗
PubMed1993

Surgically induced uremia in rats. I: Effect on bone strength and metabolism.

During the course of chronic renal failure (CRF) in man, renal osteodystrophy (osteitis fibrosa and/or osteomalacia) gradually develops. The present study aimed to establish a similar type of CRF leading to renal osteodystrophy in rats. During progressive CRF development over 225 days after 5/6 nephrectomy, the following serum variables were measured: creatinine, immunoreactive parathyroid hormone (iPTH), 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), a25-hydroxyvitamin D3, (25(OH)D3), alkaline phosphatase, albumin, phosphate, urea nitrogen, total calcium, and other blood electrolytes. Subsequent to sacrifice, mechanical properties of the rat femur, bone histomorphometry (osteoid and eroded surfaces) and bone contents of calcium, phosphate and hydroxyproline were also examined. Serum creatinine in rats with CRF gradually escalated by some 70%, while circulating 1,25(OH)2D3 was reduced beneath detection level. Total plasma calcium and phosphate concentrations were, however, almost unchanged indicating that PTH-induced bone remodeling due to moderate hyperparathyroidism sustained calcium homeostasis. Alkaline phosphatase levels were reduced by some 50%, which reflects chronically impeded bone formation. Bone histomorphometry assessment revealed substantial elevation of resorption with moderate accompanying fibrosis in about 70% of afflicted animals. Bone calcium, phosphate and hydroxypyrroline contents remained unaltered. However, hydroxyproline/calcium ratio was marginally reduced. These results, together with altered mechanical bending stress characteristics and diminished diaphysis cross section area, confirm development of mixed bone lesions in the uremic animals. Our results are compatible with the early development of CRF in man. The established rat model is therefore useful in elucidating the precipitation and early treatment of renal osteodystrophy in humans.

Alkaline Phosphatase↗