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Biomedical subjects

O Saether

Publications and source records attributed to O Saether.

12 recordsLinked to original sources
PubMed2001

Differences in neurotransmitter synthesis and intermediary metabolism between glutamatergic and GABAergic neurons during 4 hours of middle cerebral artery occlusion in the rat: the role of astrocytes in neuronal survival.

Astrocytes are intimately involved in both glutamate and gamma-aminobutyric acid (GABA) synthesis, and ischemia-induced disruption of normal neuroastrocytic interactions may have important implications for neuronal survival. The effects of middle cerebral artery occlusion (MCAO) on neuronal and astrocytic intermediary metabolism were studied in rats 30, 60, 120, and 240 minutes after MCAO using in vivo injection of [1-13C]glucose and [1,2- 13C]acetate combined with ex vivo 13C magnetic resonance spectroscopy and high-performance liquid chromatography analysis of the ischemic core (lateral caudoputamen and lower parietal cortex) and penumbra (upper frontoparietal cortex). In the ischemic core, both neuronal and astrocytic metabolism were impaired from 30 minutes MCAO. There was a continuous loss of glutamate from glutamatergic neurons that was not replaced as neuronal glucose metabolism and use of astrocytic precursors gradually declined. In GABAergic neurons astrocytic precursors were not used in GABA synthesis at any time after MCAO, and neuronal glucose metabolism and GABA-shunt activity declined with time. No flux through the tricarboxylic acid cycle was found in GABAergic neurons at 240 minutes MCAO, indicating neuronal death. In the penumbra, the neurotransmitter pool of glutamate coming from astrocytic glutamine was preserved while neuronal metabolism progressively declined, implying that glutamine contributed significantly to glutamate excitotoxicity. In GABAergic neurons, astrocytic precursors were used to a limited extent during the initial 120 minutes, and tricarboxylic acid cycle activity was continued for 240 minutes. The present study showed the paradoxical role that astrocytes play in neuronal survival in ischemia, and changes in the use of astrocytic precursors appeared to contribute significantly to neuronal death, albeit through different mechanisms in glutamatergic and GABAergic neurons.

Acetate-CoA Ligase↗
PubMed1997

Studies on recombinant Acetobacter xylinum alpha-phosphoglucomutase.

The phosphoglucomutase (PGM) from Acetobacter xylinum, which had been cloned and expressed in Escherichia coli, has been studied. After expression, the enzyme was purified from the E. coli in a three-step process consisting of (NH4)2SO4 precipitation, gel filtration and anion-exchange chromatography. The purified enzyme gave one band on gel electrophoresis and was judged essentially free of impurities, although it was unstable when diluted without the addition of 15 microM BSA. The isoelectric point for A. xylinum PGM was 4.8 and the molar absorbance was 3.9 x 10(4) M-1.cm-1. The enzyme was reasonably heat-stable below 50 degrees C and was stable throughout the pH 5.5-7.4 range, but was 70% inactivated at pH 10.0 and completely inactivated after standing for 10 min at pH 3.0 or at pH 12.4. When isolated, the recombinant enzyme was fully active without the addition of extra Mg2+. The Km for glucose 1-phosphate was much higher than that of other PGM species reported, which accords with the production of extracellular cellulose in A. xylinum. Glucose 1,6-diphosphate is not considered to be a substrate or coenzyme but an activating cofactor like Mg2+. The following kinetic constants were determined: Vmax 81.1 units/mg; kcat and the turnover rate 135 s-1; Km (glucose 1,6-diphosphate) 0.2 microM; Km (glucose 1-phosphate) 2.6 mM; kcat/Km (glucose 1-phosphate) 5.2 x 10(4) M-1.s-1. The recombinant enzyme is considered to follow a characteristic substituted enzyme or Ping Pong reaction mechanism.

Anions↗
PubMed1997

[Ground water and health. Reflections based on analysis of water specimens from Hordaland and Vestfold].

Basically, Norway has an ample supply of water. The quality of Norwegian drinking water, however, is threatened, not the least because of pollution of surface water reservoirs. Ground water is better protected against pollution, and sub-surface water sources are being exploited more than before. At present, less than 15% of the Norwegian population uses ground water for household purposes, but the percentage is increasing rapidly Ground water is (normally) clean and has a good taste. A large number of elements can be traced in ground water; some of them in concentrations of significance for human health. The present paper reports elemental analyses of 150 water samples from ground water reservoirs in rock, collected in Vestfold (Eastern Norway) and Hordaland (Western Norway). Sixty-four elements were assessed using modern equipment such as ICP-MS. In most cases the chemical composition of the water was well within the limits set for good quality drinking water. For some of the elements one or more of the results exceeded the "highest acceptable concentrations" as defined by the Norwegian health authorities. This was the case for Al, As, Ba, Ca, Cd, F, Fe, Hg, K, Mg, Mn, Na, P, Pb, Rn and Zn. No drinking water standards have been established for Be, Mo, Th and U, which are of clear significance to the health. More research is needed to assess the relationship between drinking water chemistry and human health. The authors call for a systematic analysis of all Norwegian ground water wells, and emphasise the need for regular quality control, even of small, private water supplies.

Humans↗
PubMed1995

Elucidation of the primary and three-dimensional structure of the uterotonic polypeptide kalata B1.

The amino acid sequence and structure of a uterotonic polypeptide extracted from the African plant Oldenlandia affinis DC have been determined. The peptide, kalata B1, consists of 29 amino acid residues and is rich in cysteine (6), threonine (5), and glycine (5). Enzyme cleavage studies show that the polypeptide backbone is cyclic. The three-dimensional solution structure has been determined using two-dimensional nuclear magnetic resonance (NMR) spectroscopy and distance-restrained simulated annealing. Kalata B1 is composed mainly of beta-strands connected by tight turns, forming regions of beta-sheet, except in the case of one section which forms a longer, less structured loop. The tertiary fold, together with the disulfides that form a sulfur core, produces a striking and unusual surface in which the majority of the hydrophobic residues form a solvent-exposed patch. The hydrophobic side of kalata B1 is flanked by two diametrically opposed and opposite-charged residues. The structure calculations have been used to predict the previously unknown disulfide bond connectivities using two approaches. In the first, a family of structures was calculated on the basis of NOE constraints without the assumption of a specific disulfide connectivity. The resultant structures were examined to determine whether the calculated position of the sulfur atoms suggested that one set of disulfide connectivities was more likely than the other, theoretically possible, sets. In the second approach, a separate family of structures (50 per set) was calculated for each of the 15 possible disulfide-bonded molecules. The resultant families of structures were compared to see whether one was favored over the others. Both approaches led to the same global fold, and the most likely disulfide connectivity is predicted to be 5-22, 13-27, and 17-29. In the calculated structure the cyclic peptide backbone is folded back onto itself and braced with disulfide pairs across diagonally opposed beta-strands. This structure involves one of the disulfide bonds (5-22) threading through the eight amino acid loop formed by the other two disulfide bonds and the peptide fragments connecting them.

Amino Acid Sequence↗
PubMed1991

Characterization of a K26Q site-directed mutant of human parathyroid hormone expressed in yeast.

Human parathyroid hormone (hPTH) is susceptible to proteolytical cleavage both in humans and when expressed as a secretory product in Escherichia coli (Høgseth, A., Blingsmo, O. R., Saether, O., Gautvik, V. T., Holmgren, E., Hartmanis, M., Josephson, S., Gabrielsen, O. S., Gordeladze, J. O., Alestrøm, P., and Gautvik, K. M. (1990) J. Biol. Chem. 265, 7338-7344) and Saccharomyces cerevisiae (Gabrielsen, O. S., Reppe, S., Saether, O., Blingsmo, O. R., Sletten, K., Gordeladze, J. O., Høgset, A., Gautvik, V. T., Alestrøm, P., Oyen, T. B., and Gautvik, K. M. (1990) Gene (Amst.) 90, 255-262). In the latter system, one major site of cleavage was identified (Arg25-Lys26 decreased Lys27). To produce hPTH resistant to this proteolytic processing, a point mutation changing Lys26 to Gln was introduced, and the modified gene expressed in S. cerevisiae as a fusion protein with the alpha-factor leader sequence. The resulting major form of hPTH secreted to the growth medium was of full length showing that the mutation had eliminated internal processing. Consequently, the yield of the mutant hormone was significantly higher than obtained with the natural peptide. Using improved purification procedures, a significantly higher purity was also obtained. The secreted mutant hPTH-(1-84,Q26) had the correct size, full immunological reactivity with two different hPTH antisera, correct amino acid composition and N-terminal sequence, and correct mass as determined by mass spectrometry. Furthermore, the introduced mutation did not reduce the biological activity of the hormone as judged from its action in three biological assay systems: 1) a hormone-sensitive osteoblast adenylate cyclase assay; 2) an in vivo calcium mobilizing assay in rats; and 3) an in vitro bone resorption assay.

Amino Acid Sequence↗
PubMed1990

1,25-dihydroxyvitamin D-3 and 24,25-dihydroxyvitamin D-3 affect parathormone (PTH) -sensitive adenylate cyclase activity and alkaline phosphatase secretion of osteoblastic cells through different mechanisms of action.

In UMR 106 rat osteosarcoma cells, parathormone (1-34hPTH) and calcitonin (sCT) stimulated adenylate cyclase (AC) activity 5.5-and 2.8-fold, respectively. AC in osteoblasts (OB) from collagenase-treated calvaria of 3-day-old rats responded similarly to 1-34hPTH. In contrast, fibroblasts (mouse fibroblastomas) displayed a marginal 1-34hPTH sensitive AC. Osteoclasts (OC) of collagenase-treated rat calvariae, rat monocytes and mouse macrophages did not demonstrate 1-34hPTH inducable AC activity. Physiological concentrations of 24,25-dihydroxyvitamin D-3 attenuated PTH-sensitive AC in OB and UMR 106 cells within 20 min, while 1,25-dihydroxyvitamin D-3 showed no such immediate effect. In contrast, the AC response to Gpp(NH)p was unaffected by 24,25-(OH)2D3, indicating that 24,25-(OH)2D3 interrupts the coupling of the PTH receptor to the GTP binding protein Gs. OB and UMR 106 cells were also subjected to long-term (48 h) incubation with vitamin D-3 metabolites, 1-34hPTH or 20% serum from patients with secondary hyperparathyroidism (sHBT-serum), respectively. PTH-sensitive AC was markedly attenuated by pre-exposure to both 1-34hPTH and 1,25-(OH)2D3, while minimally affected by corresponding 24,25-(OH)2D3 and 20% sHPT-serum treatment. The secretion of alkaline phosphatase (Alphos) from the two cell types was strongly increased by 1-34hPTH, the effect being abolished by the presence of 24,25-(OH)2D3. Iliac crest biopsies of normal individuals exhibited a clear negative correlation between PTH-sensitive AC and corresponding serum 24,25-(OH)2D3 levels. Basal AC activity was, however, negatively correlated to serum 1,25-(OH)2D3 concentrations. In summary, the results show that 24,25-(OH)2D3 reduces PTH-stimulated AC activity in and Alphos secretion from osteoblastic bone cells by rapidly and directly interfering with the plasma membrane. These data reinforce the probable in vivo significance of 24,25-(OH)2D3. Moreover, the negative correlation between basal AC activity and serum 1,25-(OH)2D3 levels indicates a possible role for 1,25-(OH)2D3 in regulating bone cell synthesis of AC components in vivo.

24,25-Dihydroxyvitamin D 3↗
PubMed1990

Efficient secretion of human parathyroid hormone by Saccharomyces cerevisiae.

A cDNA encoding mature human parathyroid hormone (hPTH) was expressed in Saccharomyces cerevisiae, after fusion to the prepro region of yeast mating factor alpha (MF alpha). Radioimmunoassay showed high levels of hPTH immunoreactive material in the growth medium (up to 10 micrograms/ml). More than 95% of the immunoreactive material was found extracellularly as multiple forms of hormone peptides. Three internal cleavage sites were identified in the hPTH molecule. The major cleavage site, after a pair of basic amino acids (aa) (Arg25Lys26 decreases Lys27), resembles that recognized by the KEX2 gene product on which the MF alpha expression-secretion system depends. The use of a protease-deficient yeast strain and the addition of high concentrations of aa to the growth medium, however, not only changed the peptide pattern, but also resulted in a significant increase in the yield of intact hPTH (1-84) (more than 20% of the total amount of immunoreactive material). The secreted hPTH (1-84) migrates like a hPTH standard in two different gel-electrophoretic systems, co-elutes with standard hPTH on reverse-phase high-performance liquid chromatography, reacts with two hPTH antibodies raised against different parts of the peptide, has a correct N-terminal aa sequence, and has full biological activity in a hormone-sensitive osteoblast adenylate cyclase assay.

Adenylyl Cyclases↗
PubMed1990

Expression and characterization of a recombinant human parathyroid hormone secreted by Escherichia coli employing the staphylococcal protein A promoter and signal sequence.

Human parathyroid hormone (hPTH) is a peptide hormone consisting of 84 amino acids (hPTH(1-84)). Employing the promoter and signal sequence of Staphylococcus aureus-protein A we have expressed hPTH in Escherichia coli. The expressed proteins are excreted to the growth medium, allowing for rapid and easy purification of the desired products. By amino acid sequence analysis and mass spectrometry, we have shown that the major excreted product is correctly processed human identical hPTH(1-84). The purified recombinant hPTH(1-84) stimulates adenylate cyclase activity in rat osteosarcoma cell membranes to exactly the same extent as synthetic parathyroid hormone standards, indicating that the recombinant product has full biological activity.

Adenylyl Cyclases↗
PubMed1990

Expression of human parathyroid hormone in Escherichia coli.

Human parathyroid hormone (hPTH) is a peptide hormone consisting of 84 amino acids. Using the expression plasmid pKK223-3 with the strong tacpromoter, we have produced a variant of hPTH in E. coli. From the expression plasmid construct the expected product was hPTH with an N-terminal extension of Met-Gly. The peptide was extracted from E. coli cells and purified by high performance liquid chromatography. In two different gel electrophoresis systems including identification by immunoblotting the product behaved exactly as an hPTH standard. N-terminal amino acid sequence analysis of the purified product showed traces of Gly-hPTH. At least 90% of the expressed product was N-terminally blocked, suggesting the presence of N-formyl-methionine. This variant of hPTH did not stimulate adenylate cyclase activity in rat osteosarcoma cell membranes.

Adenylyl Cyclases↗
PubMed1986

Lipids of North Atlantic krill.

The seasonal variations in the total lipid content, lipid class composition, fatty acid composition, and fatty alcohol composition of Meganyctiphanes norvegica (M. Sars), Thysanoessa inermis (Krøyer), and T. raschii (M. Sars) have been examined. The total lipid content was highest in the autumn and early winter months and lowest in the spring. In M. norvegica, triacylglycerols served as the only depot lipids, whereas in T. inermis and T. raschii triacylglycerols, wax esters, and glycerophospholipids varied in proportion to the total lipid content. This suggests that glycerophospholipids, as well as wax esters and triacylglycerols, constitute depot lipids in these species. Wax esters and glycerophospholipids were the dominating depot lipids in T. inermis, whereas triacylglycerols and glycerophospholipids were most important in T. raschii. Results suggest that non-depot glycerophospholipids may constitute 3.5-4.5% of the dry weight of the three species of krill examined. T. inermis and T. raschii, from the same catches, had very similar fatty acid compositions for each of the major lipid classes, with the exception of a few minor fatty acids. The major lipid classes in all three species showed complex seasonal variations in the content of the fatty acids that typically reflect the diet, particularly in the case of the triacylglycerols. The results suggest that all the species examined are more herbivorous during the summer than during the autumn and winter. M. norvegica seemed to be significantly more carnivorous than the two Thysanoessa species. The degree of incorporation of individual fatty acids from the diet is probably specific for each lipid class in each krill species. The proportion of polyenoic fatty acids in the glycerophospholipids and the proportion of monoenoic fatty acids in the wax esters may be of importance for the temperature adaptation of T. inermis and T. raschii.

Animals↗