scholarly journals Increased glucose oxidation and contents of insulin and ATP in polyamine-depleted rat insulinoma cells (RINm5F)

1991 ◽  
Vol 277 (2) ◽  
pp. 533-540 ◽  
Author(s):  
A Sjöholm ◽  
N Welsh ◽  
V Hoftiezer ◽  
P W Bankston ◽  
C Hellerström
Keyword(s):  
Total Protein ◽  
Glucose Oxidation ◽  
Total Protein Content ◽  
Cell Nuclei ◽  
Rinm5f Cells ◽  
Electron Microscopic ◽  
Insulin Synthesis ◽  
Insulinoma Cells ◽  
The Impact ◽  
Rat Insulinoma

In order to elucidate the role of polyamines in the replication and insulin production of insulin-secreting cells, we have investigated the impact of partial polyamine depletion on the proliferation, metabolism, insulin synthesis and ultrastructure of clonal rat insulinoma cells (RINm5F). For this purpose RINm5F cells were exposed for 4 days to the specific ornithine decarboxylase (ODC) inhibitor difluoromethylornithine (DFMO). This resulted in a profound decrease in ODC activity and cytoplasmic polyamine contents. The polyamine content of cell nuclei was, however, not altered by DFMO. Addition of small amounts of putrescine during culture elevated the intracellular content of this diamine and suppressed ODC activity. The decrease in polyamine contents was accompanied by a pronounced inhibition of the cellular proliferative activity. The rates of glucose utilization, oxygen uptake and activity of the pentose cycle were decreased in DFMO-treated cells, whereas the glucose oxidation rate, oxidation/utilization ratio, ATP content and ATP/ADP ratio were increased. Insulin mRNA content and synthesis of proinsulin, insulin and total protein were not altered by DFMO. In contrast, there was a sizeable increase in the cellular insulin content, despite a lowered total protein content. Electron-microscopic analysis revealed an accumulation of insulin-secretion granules in the DFMO-treated cells. In addition, short-term insulin release was increased after DFMO exposure, but was not rendered glucose-sensitive. It is concluded that polyamines are necessary for the maintenance of rapid insulinoma-cell replication and that DFMO-treated RINm5F cells acquire an enhanced substrate oxidation and increased content of insulin and ATP.

scholarly journals Inhibition of adenylate cyclase activity by galanin in rat insulinoma cells is mediated by the G-protein Gi3

1994 ◽  
Vol 303 (2) ◽  
pp. 369-375 ◽  
Author(s):  
P de Mazancourt ◽  
P K Goldsmith ◽  
L S Weinstein
Keyword(s):  
Adenylate Cyclase ◽  
G Protein ◽  
Pancreatic Beta Cells ◽  
Protein S ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Rinm5f Cells ◽  
Insulinoma Cells ◽  
Galanin Receptors ◽  
Rat Insulinoma

Galanin inhibits adenylate cyclase activity and insulin secretion and modulates ion channels in pancreatic beta-cells through pertussis-toxin-sensitive G-protein(s). Antibodies directed against the C-terminal region of specific G-protein alpha-subunits were used to determine which G-protein(s) couple galanin receptors to inhibition of adenylate cyclase in the rat insulinoma cell line RINm5F. Preincubation of membranes with EC antibody (anti-alpha i3) decreased the inhibition of forskolin-stimulated adenylate cyclase activity by galanin (100 nM) by 45% compared with control IgG (P < 0.05) whereas preincubation with AS (anti-alpha i1, alpha i2) or GO (anti-alpha o) antibodies had no significant effect. To confirm these results, RINm5F cells were exposed intermittently over a 4-day period to phosphorothioate oligodeoxynucleotides that were either sense or antisense to alpha i1, alpha i2, alpha i3 or alpha o. Oligodeoxynucleotides antisense to alpha i2, alpha i3 and alpha o specifically decreased the levels of the targeted alpha-subunit in membranes. alpha i1 was undetectable in these cells. Inhibition of adenylate cyclase activity by galanin was largely abolished in membranes from cells exposed to the oligodeoxynucleotide antisense to alpha i3, whereas all other oligodeoxynucleotides had no significant effect on this pathway. Indirect immunofluorescence and immunoblotting of specific membrane fractions with EC antibody show significant localization of alpha i3 to intracellular membrane compartments. These results suggest that Gi3 is the G protein that couples galanin receptors to inhibition of adenylate cyclase activity in RINm5F cells.

Dexamethasone pretreatment of rat insulinoma cells decreases binding of glucagon-like peptide-1(7–36)amide

1990 ◽  
Vol 126 (3) ◽  
pp. 445-450 ◽  
Author(s):  
G. Richter ◽  
R. Göke ◽  
B. Göke ◽  
R. Arnold
Keyword(s):  
Biological Action ◽  
Inhibitory Effects ◽  
Rinm5f Cells ◽  
Receptor Number ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
Insulinoma Cells ◽  
Camp Formation ◽  
Rat Insulinoma ◽  
Stimulation Of

ABSTRACT The effect of dexamethasone on binding of glucagonlike peptide-1(7–36)amide (GLP-1(7–36)amide) to rat insulinoma-derived cells (RINm5F) was investigated. Preincubation of RINm5F cells with dexamethasone (100 nmol/l) for 24 h resulted in a decrease of GLP1(7-36)amide binding to 55·0±8·16% (mean ± s.e.m.), incubation for 48 h to 39·1±1·76%, and for 72 h to 15·5±4·35% of maximal binding. The GLP-1(7–36)amide-induced stimulation of cyclic AMP (cAMP) production was significantly decreased to 61·03±7·4% of maximum production in cells pretreated with dexamethasone (100 nmol/l) for 48 h. The decreased binding was due to a reduction of the receptor number while the receptor affinity remained unchanged. These inhibitory effects on binding and cAMP formation induced by dexamethasone were completely abolished when the antiglucocorticoid RU 38486 (100 nmol/l) was added during preincubation with dexamethasone. RU 38486 alone had no effects. Our data suggest that the biological action of GLP-1(7–36) amide at the B-cell may be modified by glucocorticoids. Journal of Endocrinology (1990) 126, 445–450

scholarly journals Effects of genotype and bradyrhizobium inoculation on morphological traits, grain yield and protein content of soybean varieties

Genetika ◽  
2021 ◽  
Vol 53 (2) ◽  
pp. 911-925
Author(s):  
Vladimir Miladinovic ◽  
Stefan Kolasinac ◽  
Ilinka Pecinar ◽  
Biljana Kiprovski ◽  
Dragosav Mutavdzic ◽  
...  
Keyword(s):  
Raman Spectroscopy ◽  
Grain Yield ◽  
Protein Content ◽  
Plant Height ◽  
Total Protein ◽  
Morphological Traits ◽  
Crop Production ◽  
Total Protein Content ◽  
Plant Seed ◽  
The Impact

Soybean crop production in Serbia involves seed inoculation by N-fixing bacteria just before sowing time. The main objective of the current work was to assess the impact of the genotype and inoculation on range of morphological and yield traits of soybean (Glycine max L. Merrill), as well as the total protein content. The experiment was conducted on chernozem soil, where soybean was previously grown. The six local varieties were used, where each variety was sown, in three replicates for both inoculated and non-inoculated treatment. The following morphological traits were analysed: the plant height, number of lateral branches, distance to the first pod, number of pods per plant, pods (containing seeds) weight per plant, seed weight per plant, and the total grain yield. The total protein content in seeds was determined by standard analytical method, while subtle differences in qualitative protein composition were assessed using Raman spectroscopy. The total protein content varied from 39.6 to 42.15 %. Performance of inoculation resulted in an increase of the plant height and the distance to the first pod, although not in all tested varieties. The highest and the lowest plant height values were observed for non-inoculated variety Dana (59.23cm) and Sava (80.03cm), respectively. The effect of genotype was much more expressed causing differences in almost all tested characters, except for the total protein content. However, Raman spectroscopy analyses revealed distinct discrimination among surveyed varieties, and differences between inoculated and non-inoculated plants in qualitative composition of seed proteins.

TEM comparison of osmium vs osmium with potassium ferricyanide secondary fixatives and the impact of secondary fixative temperature on tissue preservation/contrast quality

1996 ◽  
Vol 54 ◽  
pp. 910-911
Author(s):  
J. W. Horn ◽  
B. J. Dovey-Hartman ◽  
V. P. Meador
Keyword(s):  
Osmium Tetroxide ◽  
Potassium Ferricyanide ◽  
Electron Microscopic ◽  
Transmission Electron ◽  
Microscopic Evaluation ◽  
Cacodylate Buffer ◽  
Transmission Electron Microscopic ◽  
Glutaraldehyde Solution ◽  
Temperature Impact ◽  
The Impact

Osmium tetroxide (OsO4) is a universally used secondary fixative for routine transmission electron microscopic evaluation of biological specimens. Use of OsO4 results in good ultrastructural preservation and electron density but several factors, such as concentration, length of exposure, and temperature, impact overall results. Potassium ferricyanide, an additive used primarily in combination with OsO4, has mainly been used to enhance the contrast of lipids, glycogen, cell membranes, and membranous organelles. The purpose of this project was to compare the secondary fixative solutions, OsO4 vs. OsO4 with potassium ferricyanide, and secondary fixative temperature for determining which combination gives optimal ultrastructural fixation and enhanced organelle staining/contrast.Fresh rat liver samples were diced to ∼1 mm3 blocks, placed into porous processing capsules/baskets, preserved in buffered 2% formaldehyde/2.5% glutaraldehyde solution, and rinsed with 0.12 M cacodylate buffer (pH 7.2). Tissue processing capsules were separated (3 capsules/secondary fixative.solution) and secondarily fixed (table) for 90 minutes. Tissues were buffer rinsed, dehydrated with ascending concentrations of ethanol solutions, infiltrated, and embedded in epoxy resin.

scholarly journals Profiling of Cerebrospinal Fluid Lipids and Their Relationship with Plasma Lipids in Healthy Humans

Metabolites ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 268
Author(s):  
Kosuke Saito ◽  
Kotaro Hattori ◽  
Shinsuke Hidese ◽  
Daimei Sasayama ◽  
Tomoko Miyakawa ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Total Protein ◽  
Plasma Lipids ◽  
Plasma Lipid ◽  
Lipid Profiles ◽  
Brain Diseases ◽  
Lipid Homeostasis ◽  
Total Protein Content ◽  
Lipid Profiling ◽  
Healthy Humans

Lipidomics provides an overview of lipid profiles in biological systems. Although blood is commonly used for lipid profiling, cerebrospinal fluid (CSF) is more suitable for exploring lipid homeostasis in brain diseases. However, whether an individual’s background affects the CSF lipid profile remains unclear, and the association between CSF and plasma lipid profiles in heathy individuals has not yet been defined. Herein, lipidomics approaches were employed to analyze CSF and plasma samples obtained from 114 healthy Japanese subjects. Results showed that the global lipid profiles differed significantly between CSF and plasma, with only 13 of 114 lipids found to be significantly correlated between the two matrices. Additionally, the CSF total protein content was the primary factor associated with CSF lipids. In the CSF, the levels of major lipids, namely, phosphatidylcholines, sphingomyelins, and cholesterolesters, correlated with CSF total protein levels. These findings indicate that CSF lipidomics can be applied to explore changes in lipid homeostasis in patients with brain diseases.

Prediction of Human Acute Toxicity by the Hep G2/24-hour/Total Protein Assay, with Protein Measurement by the CBQCA Method

2005 ◽  
Vol 33 (3) ◽  
pp. 207-213 ◽  
Author(s):  
Paul J. Dierickx
Keyword(s):  
Protein Content ◽  
Total Protein ◽  
Total Protein Content ◽  
Human Toxicity ◽  
Hep G2 ◽  
Vitro Assay ◽  
Protein Assay ◽  
Lowry Method ◽  
Total Protein Assay

In our previously described Hep G2/24-hour/total protein assay, protein levels were measured by using the Lowry method. This assay was the best acute in vitro assay for the prediction of human toxicity within the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) study. In order to increase the MEIC data-base with a wider range of chemicals, we were interested in introducing the more practical 3-(4-carboxybenzoyl)-quinoline-2-carboxaldehyde (CBQCA) method for the quantification of the total protein content. Therefore, we investigated whether the same good results for the prediction of acute human toxicity would be obtained with the CBQCA method. The cells were treated for 24 hours, then cytotoxicity was determined by measuring the total protein content with CBQCA. The results were quantified by using the PI50c: the concentration (in mM) of test compound required to reduce the total protein content measured with the CBQCA-method by 50% as compared to the control cells. The results were compared with the PI50, the corresponding value when the Lowry method was used. A relatively low correlation was observed between PI50 and PI50c, reflecting the large and unexpected, differences when using the two protein assays. However, when comparing the log PI50c with the human toxicity, a correlation coefficient of r2 = 0.761 ( n = 44) was obtained for exactly the same series of MEIC chemicals. This value is clearly higher than that for the Lowry method ( r2 = 0.695). Compared to the Lowry method originally used, the Hep G2/24-hour/CBQCA total protein assay has the additional important advantage that it can be very easily adapted for large-scale analyses with robotic systems, including the on-line calculation of the results.

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