Lars Ohman - Academia.edu (original) (raw)

Papers by Lars Ohman

Journal of the Chemical Society-Dalton Transactions, 2002

Nature, Jan 16, 1997

Oestrogens are involved in the growth, development and homeostasis of a number of tissues. The ph... more Oestrogens are involved in the growth, development and homeostasis of a number of tissues. The physiological effects of these steroids are mediated by a ligand-inducible nuclear transcription factor, the oestrogen receptor (ER). Hormone binding to the ligand-binding domain (LBD) of the ER initiates a series of molecular events culminating in the activation or repression of target genes. Transcriptional regulation arises from the direct interaction of the ER with components of the cellular transcription machinery. Here we report the crystal structures of the LBD of ER in complex with the endogenous oestrogen, 17beta-oestradiol, and the selective antagonist raloxifene, at resolutions of 3.1 and 2.6 A, respectively. The structures provide a molecular basis for the distinctive pharmacophore of the ER and its catholic binding properties. Agonist and antagonist bind at the same site within the core of the LBD but demonstrate different binding modes. In addition, each class of ligand induc...

Journal of Medicinal Chemistry, 2004

Hepatic blockade of glucocorticoid receptors (GR) suppresses glucose production and thus decrease... more Hepatic blockade of glucocorticoid receptors (GR) suppresses glucose production and thus decreases circulating glucose levels, but systemic glucocorticoid antagonism can produce adrenal insufficiency and other undesirable side effects. These hepatic and systemic responses might be dissected, leading to liver-selective pharmacology, when a GR antagonist is linked to a bile acid in an appropriate manner. Bile acid conjugation can be accomplished with a minimal loss of binding affinity for GR. The resultant conjugates remain potent in cell-based functional assays. A novel in vivo assay has been developed to simultaneously evaluate both hepatic and systemic GR blockade; this assay has been used to optimize the nature and site of the linker functionality, as well as the choice of the GR antagonist and the bile acid. This optimization led to the identification of A-348441, which reduces glucose levels and improves lipid profiles in an animal model of diabetes.

Journal of Biotechnology, 1997

The physiology of cultured animal cells, in particular hybridoma, myeloma and insect cells, with ... more The physiology of cultured animal cells, in particular hybridoma, myeloma and insect cells, with respect to growth and proliferation, amino acid metabolism, energy metabolism and cellular responses to environmental stress is discussed in this paper. The rate of proliferation of hybridoma cells in serum-containing media is limited by growth factors at a surprisingly early stage of growth. To maintain exponential growth in a batch culture, it is necessary to stimulate cell proliferation with repeated additions of serum or pure growth factor. It is further suggested that proliferation of Spodoptera frugiperda (Sf9 insect cells), a normal cell line able to grow in a serum-free medium without any added growth factors, is regulated by autocrine growth factors and possibly by other regulatory mechanisms, as Sf9 cells secrete a growth factor (IGF-I) and the medium still appears nutritionally sufficient at the time of cessation of growth. The uptake and metabolism of amino acids is one of the determinants of growth and production. Wasteful overproduction of amino acids in myeloma and hybridoma cells is a result of excess glutamine, and can be avoided by glutamine limitation. Synthesis of amino acids may be conditional, as in Sf9 cells which synthesise glutamine provided that ammonium is supplied to the medium; and cysteine (from methionine) provided that a sufficiently young inoculum is used. Uptake of amino acids in Sf9 cells appears regulated in relation to the proliferative status as there is a distinct cessation of uptake even before growth ceases. The energy metabolism in myeloma, hybridoma and insect cells is a typically substrate-concentration-dependent overflow metabolism. Substrate limitation (glucose and glutamine) decreases by-product formation and increases metabolic efficiency in all these cell lines. However, glutamine limitation, as used in fed-batch cultures (or chemostat cultures) provokes cell death (in parallel to growth) in hybridoma cells in the concentration range below 0.05 mM.

Annals of the New York Academy of Sciences, 1996

Acta Crystallographica Section A Foundations of Crystallography, 2002

Nuclear receptors bind to DNA and activate in general the transcription of sets of genes in respo... more Nuclear receptors bind to DNA and activate in general the transcription of sets of genes in response to the binding of cognate ligands, usually small lipophilic molecules such as steroids, vitamins, and fatty acid derivatives. About 50 nuclear receptor genes have been identified in the human genome, but most of them have no known ligand and are therefore called orphans; their biological functions are also poorly understood. Among the orphan nuclear receptors, some appear to be constitutively active. We have solved the structure of the ligand-binding domain (LBD) of two such receptors: RORβ and ERR3. The human RORβ LBD was found in the transcriptionally active conformation thanks to the combined action of stearate, a fortuitous ligand, and a coactivator peptide. Mutagenesis based on the structure showed that RORβ transcriptional activity is in fact ligand-dependent, and thus RORβ constitutive activity is only apparent. The human ERR3 LBD in complex with a coactivator peptide was also found in the transcriptionally active conformation, but this time in the absence of any ligand, suggesting that ERR3 is indeed constitutively active. References: 1

Journal of Pharmacology and Experimental Therapeutics, 2005

Glucocorticoids amplify endogenous glucose production in type 2 diabetes by increasing hepatic gl... more Glucocorticoids amplify endogenous glucose production in type 2 diabetes by increasing hepatic glucose output. Systemic glucocorticoid blockade lowers glucose levels in type 2 diabetes, but with several adverse consequences. It has been proposed, but never demonstrated, that a liver-selective glucocorticoid receptor antagonist (LSGRA) would be sufficient to reduce hepatic glucose output (HGO) and restore glucose control to type 2 diabetic patients with minimal systemic side effects. A-348441 [(3b,5b,7a,12a)-7,12-dihydroxy-3-{2-[{4-[(11b,17b)-17-hydroxy-3-oxo-17-prop-1-ynylestra-4,9-dien-11-yl] phenyl}(methyl)amino]ethoxy}cholan-24-oic acid] represents the first LSGRA with significant antidiabetic activity. Financial support for A-348441 research completed at Vanderbilt University was paid for by Abbott Laboratories. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

Journal of Biological Chemistry, 2003

Applied Microbiology and Biotechnology, 1995

Insect cell metabolism was studied in substrate-limited fed batch cultures of Spodoptera frugiper... more Insect cell metabolism was studied in substrate-limited fed batch cultures of Spodoptera frugiperda (Sf-9) cells. Results from a glucose-limited culture, a glutamine-limited culture, a culture limited in both glucose and glutamine and a batch culture were compared. A stringent relation between glucose excess and alanine formation was found. In contrast, glucose limitation induced ammonium formation, while, at the same time, alanine formation was completely suppressed. Simultaneous glucose and glutamine limitation suppressed both alanine and ammonium formation. Although the metabolism was influenced by substrate limitation, the specific growth rate was similar in all cultures. Alanine formation must involve incorporation of free ammonium, if ammonium formation is mediated by glutaminase and glutamate dehydrogenase, as our data suggest. On the basis of the results, two possible pathways for the formation of alanine in the intermediary metabolism in insect cells are suggested. The cellular yield on glucose was increased 6.6 times during glucose limitation, independently of the cellular yield on glutamine, which was increased 50-100 times during glutamine limitation. The results indicate that alanine overflow metabolism is energetically wasteful and that glutamine is a dispensable amino acid for cultured Sf-9 cells. Preliminary data confirm that glutamine can be synthesised by the cells themselves in amounts sufficient to support growth.

Cancer Immunology Immunotherapy Cii, Jan 2, 1999

The tumour-associated antigen (TAA) GA733-2 is expressed as a non-secreted surface molecule on th... more The tumour-associated antigen (TAA) GA733-2 is expressed as a non-secreted surface molecule on the majority of human colorectal carcinoma cells. The antigen has been used as a target for passive and active immunotherapy during the last decade. To determine the incidence of autoantibodies against this antigen, sera from 1068 patients with colorectal carcinoma were analysed for naturally occurring IgG antibodies against the baculovirus-produced GA733-2E protein. A total of 14.5% of the patients had IgG antibodies against the antigen. In 519 patients, sera were collected at the time of diagnosis and 15% of those patients had anti-GA733-2E IgG antibodies. There was a tendency to a higher frequency of patients with antibodies among those in the advanced Dukes stages: 11% in stage A and 32% in stage D respectively (P 0.06). Antibodies could be detected for up to 10 years after the diagnosis. Patients with Crohn's disease or colitis ulcerosa (n 20) did not elicit anti-GA733-2E antibodies. No healthy control donor (n 45) had detectable antibodies against the antigen. The speci®city of GA733-2E-reactive serum IgG was indicated by signi®cant inhibition of mAb17-1A (originally used to de®ne GA733-2) binding to the GA733-2E antigen. Sera of positive patients bound to the GA733-2-expressing human colorectal carcinoma cell line, SW948. No sig-ni®cant correlation was found between the presence of antibodies and survival in the present patient population. However, the high incidence of autoantibodies against this tumour antigen in colorectal carcinoma patients con®rms its antigenicity in humans and supports the use of the GA733-2 antigen as a target for immunotherapy.

Biotechnol Lett, 1996

Spodopterafrugiperda (Sf-9) insect cells are fully capable of growth and proliferation in a gluta... more Spodopterafrugiperda (Sf-9) insect cells are fully capable of growth and proliferation in a glutamine, glutamate and aspartate-free medium, provided that ammonium ions are supplied. S. frugiperda (Sf-21) and Mamestra brassicae cells (IZD-MB-0503) also grow in glutamine-free media but not Trichoplusia ni cells (BTI-TN 5B 1-4). The yield of 13-galactosidase in Sf-9 cells infected with a recombinant baculovirus under glutamine-free conditions was even higher than the yield obtained in glutamine containing cultures.

Metabolism, 2006

A liver-selective glucocorticoid (GC) receptor antagonist (A-348441) was used to determine the ef... more A liver-selective glucocorticoid (GC) receptor antagonist (A-348441) was used to determine the effect of reduced hepatic GC signaling on hepatic glucose production. Fasted conscious dogs were studied in the presence (GRA, n = 6) or absence (CON, n = 6) of the intraduodenally administered GC receptor antagonist (100 mg/kg). All dogs were maintained on a pancreatic clamp and in a euglycemic state for 7 hours to ensure that any changes in glucose metabolism were the direct result of the effects of A-348441, which was given at the start of a 5-hour experimental period. In the GRA group, the arterial plasma insulin level was 4.6 F 0.7 and 4.8 F 0.6 lU/mL during the basal and the last 30 minutes of the experimental periods, respectively. In the CON group, it was 4.0 F 0.3 and 4.5 F 0.5 lU/mL in the 2 periods, respectively. The arterial plasma glucagon level was 49 F 4 and 46 F 3 pg/mL in the 2 periods in the GRA group, and 45 F 3 and 42 F 3 pg/mL in the CON group. Net hepatic glucose balance progressively decreased in the GRA group from 1.31 F 0.18 to 0.49 F 0.30 mg/kg per minute, whereas in the CON group, net hepatic glucose balance was 1.17 F 0.09 and 1.43 F 0.18 mg/kg per minute during the basal and last 30 minutes of the experimental periods, respectively. No significant change in net renal or gut glucose balance or nonhepatic glucose uptake was observed in either group. This study demonstrates that the GC receptor plays an important role in the regulation of basal hepatic glucose production and represents a significant potential therapeutic target.

Journal of Biotechnology, 2000

1 H/ 15 N and 13 C NMR were used to investigate metabolism in Spodoptera frugiperda (Sf9) cells. ... more 1 H/ 15 N and 13 C NMR were used to investigate metabolism in Spodoptera frugiperda (Sf9) cells. Labelled substrates ([2-15 N]glutamine, [5-15 N]glutamine, [2-15 N]glutamate, 15 NH 4 Cl, [2-15 N]alanine, and [1-13 C]glucose) were added to batch cultures and the concentration of labelled excreted metabolites (alanine, NH 4 + , glutamine, glycerol, and lactate) were quantified. Cultures with excess glucose and glutamine produce alanine as the main metabolic by-product while no ammonium ions are released. 1 H/ 15 N NMR data showed that both the amide and amine-nitrogen of glutamine was incorporated into alanine in these cultures. The amide-nitrogen of glutamine was not transferred to the amine-position in glutamate (for further transamination to alanine) via free NH 4 + but directly via an azaserine inhibitable amidotransfer reaction. In glutamine-free media 15 NH 4 + was consumed and incorporated into alanine. 15 NH 4 + was also incorporated into the amide-position of glutamine synthesised by the cells. These data suggest that the nitrogen assimilation system, glutamine synthetase/glutamate synthase (NADH-GOGAT), is active in glutaminedeprived cells. In cultures devoid of glucose, ammonium is the main metabolic by-product while no alanine is formed. The ammonium ions stem both from the amide and amine-nitrogen of glutamine, most likely via glutaminase and glutamate dehydrogenase. 13 C NMR revealed that the [1-13 C] label from glucose appeared in glycerol, alanine, lactate, and in extracellular glutamine. Labelling data also showed that intermediates of the tricarboxylic acid cycle were

Cancer Immunology, Immunotherapy, 1999

Biotechnology Progress, 2000

Cell cycle progression was studied in serum-free batch cultures of Spodoptera frugiperda (Sf9) in... more Cell cycle progression was studied in serum-free batch cultures of Spodoptera frugiperda (Sf9) insect cells, and the implications for proliferation and productivity were investigated. Cell cycle dynamics in KBM10 serum-free medium was characterized by an accumulation of 50-70% of the cells in the G 2 /M phase of the cell cycle during the first 24 h after inoculation. Following the cell cycle arrest, the cell population was redistributed into G 1 and in particular into the S phase. Maximum rate of proliferation (µ N,max) was reached 24-48 h after the release from cell cycle arrest, coinciding with a minimum distribution of cells in the G 2 /M phase. The following declining µ N could be explained by a slow increase in the G 2 /M cell population. However, at approximately 100 h, an abrupt increase in the amount of G 2 /M cells occurred. This switch occurred at about the same time point and cell density, irrespective of medium composition and maximum cell density. An octaploid population evolved from G 2 /M arrested cells, showing the occurrence of endoreplication in this cell line. In addition, conditioned medium factor(s) were found to increase µ N,max , decrease the time to reach µ N,max , and decrease the synchronization of cells in G 2 /M during the lag and growth phase. A conditioned medium factor appears to be a small peptide. On basis of these results we suggest that the observed cell cycle dynamics is the result of autoregulatory events occurring at key points during the course of a culture, and that entry into mitosis is the target for regulation. Infecting the Sf9 cells with recombinant baculovirus resulted in a linear increase in volumetric productivity of-galactosidase up to 68-75 h of culture. Beyond this point almost no product was formed. Medium renewal at the time of infection could only partly restore the lost hypertrophy and product yield of cultures infected after the transition point. The critical time of infection correlated to the time when the mean population cell volume had attained a minimum, and this occurred 24 h before the switch into the G 2 /M phase. We suggest that the cell density dependent decrease in productivity ultimately depends on the autoregulatory events leading to G 2 /M cell cycle arrest.

Biotechnology Letters, 1996

Metabolism, 2007

It is unclear how hepatic glucocorticoid receptor (GR) function and hypothalamic-pituitary-adrena... more It is unclear how hepatic glucocorticoid receptor (GR) function and hypothalamic-pituitary-adrenal axis tone contribute to the diabetic state and in particular whole-body glucose fluxes. We have previously demonstrated that long-term exposure to hepatic GR inhibition ...