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991.
Endocrine cell cultures have potential in bioprocessing, for the production of biologically active hormones, and in tissue engineering, for the development of implantable artificial tissues for long-term restoration of endocrine function. To optimize such systems, it is necessary to develop a thorough understanding of how inherently present environmental stresses, such as nutrient depletion and metabolite accumulation, affect the cells. This work focuses on the effects of the metabolite ammonium on indicators of endocrine cell metabolism and on the processing, storage and secretion of regulated secretory proteins. Experiments were conducted on recombinant insulin-producing mouse pituitary AtT-20 cells and mouse insulinoma TC3 cells. Exposure for 24–48 hours to 6 mM of exogenous ammonium resulted in higher rates of glucose consumption by both AtT-20 and TC3 cells, while the formation of additional ammonium generally decreased relative to ammonium-free controls. When TC3 cells were discharged of their intracellular insulin stores, the presence of ammonium during a subsequent recharge completely inhibited addition of new insulin-related peptides to the stores, as we had observed previously for both cell lines. There was a correlation between insulin-related peptides stored in TC3 cells during recharging and the amount that could be released upon secretagogue stimulation. Using a combination of radioimmunoassay and high performance liquid chromatography, we found that intracellular insulin and insulin-related peptides changed in the same fashion. Intracellular mechanisms that may be producing the observed results are discussed.Abbreviations IRP
insulin-related peptides
- HPLC
high performance liquid chromatography
- DAMP
3-(2,4-dinitroanilino)-3 amino-N-methyldipropylamine 相似文献
992.
We have synthesised the -subunit of the chick nicotinic acetylcholine receptor (nAChR) in stable, continuous insect (Spodoptera frugiperda) cell lines. A cDNA was integrated randomly into the insect cell genome under control of a baculovius immediate early gene promoter. Transformed cells were obtained by co-transfection of the insect cells with pIEK1.nAChR, encoding the -subunit cDNA, and pIEK1.neo, encoding the neomycin resistance gene. G-418-resistant clones were selected and expanded into continuous cell lines synthesising the chick nAChR -subunit. Using fluorescence microscopy and ligand binding studies we were able to demonstrate efficient membrane targeting of the receptor subunit in the insect cell plasma membrane. Stable insect cell lines may thus have significant advantages over transient baculovirus vectors for the synthesis and characterisation of heterologous receptor proteins.Abbreviations AcNPV
Autographa californica nuclear polyhedrosis virus
- BTX
-bungarotoxin
- BSA
bovine serum albumin
- FITC
Fluoroscein isothiocyanate
- G418
geneticin-418
- hpi
hours post-infection
-
ie-1
immediate early 1 gene
- nAChR
nicotinic acetylcholine receptor alpha subunit
- Sf
Spodoptera frugiperda
- tPA
tissue plasminogen activator 相似文献
993.
Jonathan H. Davis 《Journal of biomolecular NMR》1995,5(4):433-437
Summary 2D 15N-1H correlation spectra are ideal for measuring backbone amide populations to determine amide exchange protection factors in studies of protein folding or other structural features. Most protein NMR spectroscopists use HSQC, which has been shown to be generally superior to HMQC in both resolution and sensitivity. The refocused HSQC experiment is intrinsically less sensitive than the regular HSQC, due to T2 relaxation during the refocusing delays. However, we show here that, when high 15N resolution is needed, an optimized refocused HSQC sequence that utilizes a semi-constant time evolution period and pulsed field gradients has better signal-to-noise ratio and resolution, and integrates more accurately, than a similar HSQC. The differences are demonstrated on a 20 kDa protein. The technique can also be applied to 3D NOESY experiments to eliminate strong NH2 geminal peaks and their truncation artefacts at a modest cost in sensitivity. 相似文献
994.
Carrie Haskell-Luevano Lakmal W. Boteju Victor J. Hruby 《Letters in Peptide Science》1995,1(4):163-170
Summary Nitrogen indole protection of the -methyltryptophan side-chain residue is important for avoiding undesired side reactions during peptide synthesis. Of great importance is the choice of a side-chain protecting group for orthogonal peptide synthesis and its stability under a variety of chemical conditions required for synthesis of the four isomers of this unusual amino acid. We report here the successful use of the mesitylenesulfonyl (Mts) protecting group for -methyltryptophan in the synthesis of melanotropin and CCK peptide analogues and the ready cleavage of this protecting group under HF conditions. 相似文献
995.
Izaura Yoshico Hirata Maria Helena Sedenho Cezari Clovis Ryuichi Nakaie Paulo Boschcov Amando Siuiti Ito Maria Aparecida Juliano Luiz Juliano 《Letters in Peptide Science》1995,1(6):299-308
Summary A general procedure, using the commonly employed solid-phase peptide synthesis methodology for obtaining internally quenched fluorogenic peptides with ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs, is presented. The essential feature of this procedure is the synthesis of an N
-Boc or-Fmoc derivative of glutamic acid with the -carboxyl group bound to N-(2,4-dinitrophenyl)-ethylenediamine (EDDnp), which provides the quencher moiety attached to the C-terminus of the substrate. The fluorescent donor group, ortho-aminobenzoic acid (Abz), is incorporated into the resin-bound peptide in the last coupling cycle. Depending on the resin type used, Abz-peptidyl-Gln-EDDnp or Abz-peptidyl-Glu-EDDnp is obtained. Using the procedure described above, substrates for human renin and tissue kallikreins were synthesised. Spectrofluorimetric measurements of Abz bound to the -amino group of proline showed that strong quenching of Abz fluorescence occurs in the absence of any acceptor group. 相似文献
996.
Valerie Brecx Aleksandra Misicka Patricia Verheyden Dirk Tourwé George van Binst 《Letters in Peptide Science》1995,2(3-4):165-168
Summary A new cis-peptide bond mimetic, -benzyl-o-aminomethylphenylacetic acid, was synthesized and incorporated in a homodetic somatostatin analogue. Biological binding tests and 2D NMR conformational analysis indicate that the configuration of the bridge-unit asymmetric center and the orientation of the benzyl side chain play a key role in the biological activity of this type of somatostatin analogues. 相似文献
997.
Marianne Borloo Koen Augustyns Alexander Belyaev Ingrid de Meester Anne-Marie Lambeir Filip Goossens Willy Bollaert Padinchare Rajan Simon Scharpé Achiel Haemers 《Letters in Peptide Science》1995,2(3-4):198-202
Summary A series of azaproline dipeptides with various N-substituents were synthesized as possible active-site-directed inhibitors of two proline-specific serine proteases, dipeptidyl peptidase IV and prolyl oligopeptidase. Compounds with semicarbazide, carbazate, acylhydrazine and sulphonylhydrazine structures were tested. Some compounds show moderate activity, i.e., in the millimolar range. 相似文献
998.
Autonomous division of the endosperm was induced by in vitro culture of unpollinated ovaries or placenta-attached ovules in Helleborus niger, Lupinus luteus and Melandrium album. The induction frequencies for the three species were 50%, 10–20% and 0.1%, respectively. The endosperms contained up to 20 free nuclei; only a few ovules with 80–420 endosperm nuclei were found. Induction of autonomous division of the endosperm, which is unusual in amphimictic plants, was observed in three new species. No embryos appeared in the ovules. This suggests a developmental independence of the endosperm from the embryo in the culture of unpollinated ovaries or ovules. 相似文献
999.
Summary In the present study the binding of [3H]MK-801 to glutamatergic receptors of the NMDA type was compared in spontaneously hypertensive (SHR) and normotensive (WKY) rats in various brain structures (including nucleus tractus solitarii) by quantitative receptor autoradiography. Additionally, blood pressure changes after treatment with the NMDA antagonist MK-801 were studied in both strains. There were no differences between SHR and WKY rats either in the level of [3H]MK-801 binding or in the hypertensive reaction to MK-801. 相似文献
1000.
G. Vertuani M. Boggian A. Breveglieri G. Cavicchioni S. Spisani A. Scatturin 《Amino acids》1995,9(4):375-383
Summary In order to investigate the proper peptide backbone conformation able to elicit a biological activity, HCO-Met-Pro-Phe-OMe, HCO-Met-[COO]Leu-Phe-OMe, and HCO-Met-OLeu-Phe-OMe, analogues of the prototypical chemotactic peptide HCO-Met-Leu-Phe-OMe, were studied by CD and IR techniques. The results obtained comparing biological and conformational data evidence the critical presence of (i) the NH group at position 2, (ii) a rather flexible backbone, (iii) the chemical structure of the central residue which can affect the stability of a possible active conformer. 相似文献