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S Geisse

Publications and source records attributed to S Geisse.

18 recordsLinked to original sources
PubMed2000

Pancreatic beta-cell K(ATP) channel activity and membrane-binding studies with nateglinide: A comparison with sulfonylureas and repaglinide.

Nateglinide (A-4166) is an amino acid derivative with insulinotrophic action in clinical development for treatment of type 2 diabetes. The aim of this study was to determine whether nateglinide's interaction at the K(ATP) channel/sulfonylurea receptor underlies its more rapid onset and shorter duration of action in animal models. Binding studies were carried out with membranes prepared from RIN-m5F cells and HEK-293 cells expressing recombinant human sulfonylurea receptor 1 (SUR1). The relative order for displacement of [(3)H]glibenclamide in competitive binding experiments with RIN-m5F cell membranes was glibenclamide > glimepiride > repaglinide > glipizide > nateglinide > L-nateglinide > tolbutamide. The results with HEK-293/recombinant human SUR1 cells were similar with the exception that glipizide was more potent than repaglinide. Neither nateglinide nor repaglinide had any effect on the dissociation kinetics for [(3)H]glibenclamide, consistent with both compounds competitively binding to the glibenclamide-binding site on SUR1. Finally, the inability to measure [(3)H]nateglinide binding suggests that nateglinide dissociates rapidly from SUR1. Direct interaction of nateglinide with K(ATP) channels in rat pancreatic beta-cells was investigated with the patch-clamp method. The relative potency for inhibition of the K(ATP) channel was repaglinide > glibenclamide > nateglinide. Kinetics of the inhibitory effect on K(ATP) current showed that the onset of inhibition by nateglinide was comparable to glibenclamide but more rapid than that of repaglinide. The time for reversal of channel inhibition by nateglinide was also faster than with glibenclamide and repaglinide. These results suggest that the unique characteristics of nateglinide are largely the result of its interaction at the K(ATP) channel.

ATP-Binding Cassette Transporters↗
PubMed1998

The agonist activities of the putative antipsychotic agents, L-745,870 and U-101958 in HEK293 cells expressing the human dopamine D4.4 receptor.

1. Dopamine D4 receptor antagonists are being developed by several pharmaceutical companies as putative novel antipsychotics, possibly with low propensity to side-effects. Two such compounds, L-745,870 and U-101958 have been recently introduced. 2. The radioligand binding and functional activities of L-745,870 and U-101958 were investigated in human embryonic kidney (HEK)293 cells expressing the human recombinant dopamine D4.4 receptor (HEK293/D4 cells). [3H]-spiperone binding experiments were performed and inhibition of forskolin-stimulated cyclic AMP accumulation was used as the functional response. 3. [3H]-spiperone was found to label a homogeneous and saturable population of specific binding sites in HEK293/D4 cell homogenates (Bmax 505+/-90 fmol mg(-1) protein, pK(D) 9.5+/-0.1, n=3). Inhibition of specific [3H]-spiperone binding was observed with spiperone (pKi 9.6+/-0.1, n=3), clozapine (pKi 7.4+/-0.1, n=4), L-745,870 (pKi 8.5+/-0.1, n=3) and U-101958 (pKi 8.9+/-0.1, n=3). By contrast, raclopride was very weak (pKi < 5, n=3). 4. Dopamine inhibited forskolin-stimulated cyclic AMP accumulation in HEK293/D4 cells in a concentration-dependent fashion (Emax 71+/-2% inhibition of forskolin-stimulated levels, pEC50 8.7+/-0.1, n=10). This effect was mimicked by the dopamine D2-like receptor agonists, quinpirole and 7-hydroxy-2-dipropylaminotetralin (7-OH-DPAT). 5. L-745,870 and U-101958 also inhibited forskolin-stimulated cyclic AMP accumulation in HEK293/D4 cells in a concentration-dependent way. L-745,870 was less efficacious than dopamine (71% the efficacy of dopamine), whereas U-101958 behaved as a full agonist compared to dopamine. Potencies (pEC50) values of L-745,870 and U-101958 were 9.0+/-0.2 (n=4) and 8.7+/-0.3 (n=3), consistent with pKi values determined in radioligand binding studies. 6. Dopamine, L-745,870 and U-101958 (up to 1 microM) were devoid of effect on forskolin-stimulated cyclic AMP accumulation in control, non-transfected HEK293 cells. 7. The agonist effects of dopamine, L-745,870 and U-101958 in HEK293/D4 cells could be antagonized by spiperone (pK(B) 8.2-8.8) and clozapine (pK(B) 7.1), but not by raclopride (pK(B) < 5). None of these antagonists had any significant agonist activity at concentrations up to 10 microM. 8. These results show that the putative dopamine D4 receptor antagonists, L-745,870 and U-101958 are not devoid of intrinsic activity at human recombinant dopamine D4.4 receptors. Therefore, they may not represent the most appropriate drugs for testing the benefit of D4 receptor antagonism in schizophrenic patients, if agonism should translate in vivo.

Aminopyridines↗
PubMed1996

Eukaryotic expression systems: a comparison.

Eukaryotic expression systems are frequently employed for the production of recombinant proteins as therapeutics as well as research tools. Most commonly used expression systems are based on stably transfected adherent CHO cells or nonadherent lymphoid cell lines. An efficient alternative is the infection of insect cells by recombinant baculoviruses. Transient expression in mammalian cells, e.g., COS cells, is often used for the production of smaller quantities of proteins. The choice of a suitable expression system depends largely on the biochemical and biological properties of the protein of interest, as well as on the nature of the planned experiments and the amount of recombinant protein required. We summarize here the expression of the cytokine human Leukemia Inhibitory Factor (hu-LIF) in five of the most commonly used systems, namely in CHO, Sp2/0, MEL, COS, and insect cells, in conjunction with an outline of the principles and characteristics of each of these expression systems. In result, the stably transfected cell lines, CHO, Sp2/0, and MEL cells, gave rise to production of fully glycosylated hu-LIF at variable product titers; incompletely glycosylated, albeit biological action hu-LIF could be rapidly produced by transient expression in COS cells or by baculovirus-mediated infection of insect cells.

Animals↗
PubMed1995

A COS-cell-based system for rapid production and quantification of scFv::IgC kappa antibody fragments.

Single-chain Fv (scFv) antibody (Ab) fragments were transiently produced in COS-1 cells utilizing a mammalian expression vector featuring a murine immunoglobulin (Ig) light-chain leader sequence for efficient secretion and a murine Ig kappa constant domain (IgC kappa) for detection. Several hundred milliliters of supernatants from large-scale COS cell transfections were sufficient to purify the scFv::IgC kappa fusion proteins by one-step affinity chromatography utilizing an immobilized rat anti-mouse IgC kappa monoclonal Ab. Furthermore, the murine IgC kappa domain allowed for accurate quantification of the scFv::IgC kappa fusion protein secreted into the COS cell supernatant by a sandwich enzyme-linked immunosorbent assay (S-ELISA).

Animals↗
PubMed1992

High-level expression of a human immunoglobulin gamma 1 transgene depends on switch region sequences.

We describe that chimeric mouse-human immunoglobulin heavy chain (IgH) genes lacking a switch region and controlled by an IgH promoter and the intronic enhancer are only weakly expressed in transgenic mice. Insertion of part of the human C gamma 1 or murine Cmu switch region into the major intron of the chimeric IgH gene results in a 10(2)-to 10(3)-fold increase in transgene expression. Analysis of B cell hybridoma clones from transgenic mice suggests that switch sequences influence IgH transgene expression at the cellular level. However, the effect of switch region sequences on IgH gene expression observed in vivo is not apparent in transfected B cell lines. These results indicate that switch region sequences which are located proximal to the constant part of the gene and which are normally present in a rearranged IgH gene after class switching represent a novel type of regulatory element that plays a critical role in IgH gene expression in vivo.

Animals↗
PubMed1992

The hepatocyte-specific phenotype of murine liver cells correlates with high expression of connexin32 and connexin26 but very low expression of connexin43.

This investigation was initiated in order to find out whether expression of the hepatocyte-specific phenotype is accompanied by expression of certain connexin genes coding for gap junctional protein subunits. Several clones of mouse embryonic hepatocytes immortalized in serum-free MX83 medium by infection with recombinant retrovirus-expressed transcripts for connexin32, connexin26, albumin, alpha-fetoprotein, tyrosine aminotransferase, as well as aldolase A and B, at more than half of the levels found in primary mouse hepatocytes. In addition the immortalized hepatocyte clones contained low levels of connexin43 mRNA of which only trace amounts were detected in primary embryonic mouse hepatocytes and in rat liver. Two of the immortalized hepatocyte clones were shifted from serum-free MX83 medium to Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum and, after 2, 14, or 180 days, back to MX83 medium. We found that expression of connexin32 and connexin26 mRNAs as well as transcripts of other liver-specific proteins was reversibly decreased in serum-containing medium, whereas the expression level of connexin43 transcripts was increased in serum-containing DMEM compared to serum-free MX83 medium. The expression levels of connexin26, connexin32, or connexin43 mRNAs were altered by the addition of fetal calf serum or arginine or by the absence of hydrocortisone in MX83 medium, all of which contributed to the shift in phenotype. Furthermore several dedifferentiated cell lines derived from rat or mouse liver and cultivated in serum-containing medium were found to express little connexin32 or connexin26 mRNA but relatively high levels of connexin43 mRNA.

Animals↗
PubMed1991

Recombinant human [Cys281]insulin-like growth factor-binding protein 2 inhibits both basal and insulin-like growth factor I-stimulated proliferation and collagen synthesis in fetal rat calvariae.

It is recognized that insulin-like growth factors (IGFs) are bound to specific high-affinity insulin-like growth factor-binding proteins (IGFBPs). The role of IGFBPs in bone metabolism is not well established. The effect of recombinant human [Cys281]IGFBP-2 ([Cys281]rhIGFBP-2) on bone formation in 21-day-old fetal rat calvariae was investigated. [Cys281]rhIGFBP-2 was expressed in and purified from conditioned medium of a clonal Chinese hamster ovary cell line. IGF-I-stimulated cell proliferation was inhibited dose dependently by [Cys281]rhIGFBP-2, with half-maximal inhibition observed at 2 x 10(-8) M. Suppression of the IGF-I-stimulated DNA synthesis was observed at an apparent dose ratio of 1:10. [Cys281]rhIGFBP-2 (10(-6) M) also inhibited the basal incorporation of [3H]thymidine into DNA by up to 45%. Insulin-stimulated cell proliferation was not affected in the presence of the binding protein. In addition, [Cys281]rhIGFBP-2 inhibited bone collagen synthesis under basal and IGF-I-stimulated conditions. In contrast, [Cys281]rhIGFBP-2 did not alter the parathyroid hormone-stimulated bone cell proliferation rate. In conclusion, binding of hIGF-I to rhIGFBP-2 results in an inhibition of the actions of free IGF-I on bone cell replication and matrix synthesis. Parathyroid hormone-stimulated cell proliferation is not mediated by an increase in free IGFs.

Animals↗
PubMed1991

Cyclosporin A and FK-506 both affect DNA binding of regulatory nuclear proteins to the human interleukin-2 promoter.

The structurally unrelated immunosuppressive drugs cyclosporin A (Sandimmun) and FK-506 both interfere with the process of T-cell proliferation by blocking the transcription of the T-cell growth factor interleukin-2 (IL-2). Here we demonstrate that the transcriptional activation of this gene requires the binding of regulatory nuclear proteins to a promoter element with sequence similarity to the consensus binding site for NF-kappa B-related transcription factors. We present evidence that the binding by regulatory nuclear proteins to the kappa B element of the IL-2 promoter is affected negatively by cyclosporin A and FK-506 at concentrations paralleling their immunosuppressive activity in vivo. The decrease in DNA-protein complex formation induced by the immunosuppressive drugs correlates with a decrease in IL-2 production. FK-506 is 10 to 100 times more potent than cyclosporin A in its ability to inhibit sequence-specific DNA binding and IL-2 production. Our findings suggest that the actions of both drugs converge at the level of DNA-protein interaction.

Anti-Bacterial Agents↗
PubMed1991

Purification and characterization of natural human interleukin-3.

The human interleukin-3 gene was cloned in 1986 and the biochemical and biological properties of recombinant human interleukin-3 (rhuIL-3) protein were described. In this report we compare rhuIL-3 with nonrecombinant, natural huIL-3, purified from the supernatant of the human T cell leukemia line Jurkat. The main purification step, affinity chromatography, using a selected monoclonal anti-huIL-3 antibody, resulted in an approximately 40,000-fold enrichment of huIL-3. Combination of this step with ion-exchange and reverse-phase chromatography yielded natural huIL-3 of high purity (greater than 98%). A highly sensitive and specific sandwich ELISA, comprising two epitope-mapped monoclonal anti-huIL-3 antibodies, was used to quantitate huIL-3 during purification. Amino acid sequence determination revealed that the 38 N-terminal amino acids of Jurkat-derived huIL-3 are identical to the published sequence deduced from human fetal liver genomic DNA but differ in one residue from that derived from human T cell clones. The degree of glycosylation of Jurkat-derived huIL-3 was similar to Chinese hamster ovary cell-expressed rhuIL-3. Natural huIL-3 showed very similar biological activities to rhuIL-3 in proliferation and receptor binding assays utilizing huIL-3 responsive primary cells and cell lines, as well as in the human bone marrow colony assay.

Amino Acid Sequence↗
PubMed1987

Suppression of tumorigenicity in somatic cell hybrids does not involve quantitative changes in transcription of cellular Ha-ras, Ki-ras, myc, and fos oncogenes.

The transcriptional activity of ten cellular oncogenes was analyzed in somatic cell hybrids that had been obtained after fusion of tumorigenic Chinese hamster cells and normal mouse fibroblasts. The hybrids showed either the tumorigenic or the nontumorigenic phenotype (suppression of tumorigenicity). Out of ten c-onc genes analyzed, four (c-Ha-ras, c-Ki-ras, c-myc, and c-fos) were found to be transcriptionally active at similar levels in tumorigenic as well as in nontumorigenic (suppressed) hybrids. Thus we conclude that suppression of tumorigenicity in Chinese hamster X mouse somatic cell hybrids does not correlate with quantitative changes in expression of these cellular oncogenes. The remaining six cellular oncogenes (c-abl, c-erb A and B, c-fes, c-myb, and c-sis) were not transcriptionally active in these hybrids.

Animals↗
PubMed1985

Transforming activity of DNA fragments from normal human lymphocytes results from spontaneous activation of a c-Ha-ras1 gene.

An activated human Ha-ras gene was present in a secondary NIH 3T3 transformant isolated after serial transfection of originally low-molecular-weight DNA fragments from normal human cells. This gene appeared to have acquired its transforming properties by a spontaneous mutation in codon 12 by substitution of a deoxythymidine residue for a deoxyguanosine residue. DNA rearrangements in the flanking sequences of the transferred Ha-ras gene were not involved in the activation of the protooncogene.

Animals↗
PubMed1984

The nucleotide sequences recognized by the glucocorticoid receptor in the rabbit uteroglobin gene region are located far upstream from the initiation of transcription.

Although the expression of the uteroglobin gene in the lung is regulated by glucocorticoids, no binding sites for the glucocorticoid receptor are found in the promoter region nor can they be observed in the coding sequences. Instead a fragment situated 2.6 kb upstream from the start of transcription of the uteroglobin gene shows a high affinity for the receptor. Deoxyribonuclease I and methylation protection studies show three contiguous binding sites located within this fragment. All three sites show homology to the glucocorticoid receptor binding sites described for other genes. Two of them encompass the hexanucleotide 5'-TGTTCT-3', and the other binding site contains the homologue hexanucleotide, 5'-AGTCCT-3', but the contacts between the receptor and the hexanucleotides are equivalent to those found in other functional regulatory elements for glucocorticoids. These elements may therefore be responsible for the glucocorticoid regulation of uteroglobin gene expression by acting over a relatively long stretch of nucleotide sequences.

Animals↗
PubMed1982

Persistent protoporphyrinemia in hereditary porphobilinogen synthase (delta-aminolevulinic acid dehydrase) deficiency under low lead exposure. A new molecular basis for the pathogenesis of lead intoxication.

For several years, a 4-12-fold increase of the upper normal limit in erythrocyte protoporphyrin concentrations persisted in two men 34 and 39 years of age who were chronically exposed to lead. We are dealing with a zinc protoporphyrinemia in both cases, without lead intoxication or anemia. The 34-year-old had been a regular blood donor for 10 years and had already been treated for iron deficiency several times. Hemoglobin, red cell counts, hematocrit, and iron were at the lower normal limit. The activity of porphobilinogen synthase (PBG-S), uroporphyrinogen-synthase and -decarboxylase as well as urinary porphyrin precursors and porphyrin excretion were normal. Protoporphyrinemia was said to be due to a prelatent/latent iron deficiency. In the 39-year-old, the activity of PBG-S was lowered to 388 mumol/1 . h, as compared to the mean of controls (1,190 +/- 210, x +/- SD, n = 50), in connection with a slightly elevated excretion of delta-aminolevulinic acid and coproporphyrin in the urine and a high-normal blood lead level. In his family there was no history of either a protoporphyrinemia or a hematological disturbance. Six of eight family members in three generations showed a diminished activity of PBG-S: 600 +/- 160, P less than 0.001 compared to controls. These family members are heterozygous with regard to the PBG-S deficiency; they are clinically unobtrusive in comparison to homozygotes with an acute prophyria syndrome. Activation by zinc and reactivation by dithiothreitol were normal in contrast to PBG-S from patients with lead intoxication. The cause of biochemical symptoms of subclinical lead intoxication developed by the propositus is probably due to the hereditary PBG-S deficiency which sensitizes him to low-level lead exposure. The determination of red cell PBG-S activity can be recommended as a test detecting heterozygotes. The hereditary PBG-S deficiency is recognized as a new molecular basis for the pathogenesis of lead intoxication.

Adult↗
PubMed1982

Glucocorticoid receptors recognize DNA sequences in and around murine mammary tumour virus DNA.

In several rodent cell lines, glucocorticoids increase the transcription of murine mammary tumour virus (MMTV) proviral DNA in a process mediated by the glucocorticoid receptor. To investigate whether a direct interaction between the receptor and specific sequences on the induced genes can be implicated in the hormonal regulation of transcription, filter binding studies were performed with partially purified glucocorticoid receptor of rat liver and eight cloned MMTV proviral probes. Both the 40 000 and the 90 00 mol. wt. forms of the receptor do bind preferentially to restriction fragments containing the right 400-500 nucleotides of the MMTV long terminal repeat units (LTR). Using LTR deletion mutants, we confirm that the right end of the LTR contains at least one binding site for the glucocorticoid receptor. In addition, the receptor binds preferentially to the mouse genomic sequences flanking at least three endogenous proviral copies, and to sequences within the env genes in some of the endogenous and exogenous proviruses. These findings are compatible with the hypothesis that steroid hormones regulate specific gene expression through a direct interaction of the hormone-receptor complex with DNA sequences in and around the induced genes.

Adrenalectomy↗
PubMedDate not supplied

The glucocorticoid receptor binds to defined nucleotide sequences near the promoter of mouse mammary tumour virus.

Glucocorticoids are known to induce the transcription of integrated proviral mouse mammary tumour virus (MMTV) genes in a variety of cell lines derived from mouse mammary tumours. Chimaeric genes in which selectable markers are linked to the long terminal repeat (LTR) region of MMTV can be induced by the synthetic glucocorticoid dexamethasone after introduction into mouse fibroblasts. This suggests that the regulatory elements required for hormonal induction are located within the cloned LTR fragments. The idea is supported by the observation that glucocorticoid receptors bind to certain cloned fragments of MMTV DNA in vitro. Using filter binding studies and monoclonal antibodies to the glucocorticoid receptor we have now delimited the receptor binding region to a DNA segment of 152 base pairs (bp) that has been shown to be relevant for hormonal induction. In nuclease protection experiments we have identified partially homologous receptor binding sequences located in this region, all of which share the hexanucleotide 5'-TGTTCT-3'.

Animals↗