Insulin and transferrin restore important cellular functions of human fetal kidney in serum-free organ culture

1991 ◽  
Vol 69 (4) ◽  
pp. 256-262 ◽  
Author(s):  
Normand Brière ◽  
Joseph Ferrari ◽  
Pierre Chailler
Keyword(s):  
Renal Cortex ◽  
Defined Medium ◽  
Dependent Manner ◽  
Direct Influence ◽  
Fetal Kidney ◽  
Cellular Functions ◽  
Insulin Effect ◽  
Serum Free ◽  
Dose Dependent ◽  
Human Fetal Kidney

A model previously developed in our laboratory to culture human fetal kidneys in serum-free chemically defined medium was used to evaluate the direct influence of potential regulators on nephrogenesis. The aim of the present work was to verify the effects of insulin and transferrin, two hormones considered as essential in other serum-free culture systems. Explants of renal cortex from human fetuses (15–21 weeks) were cultured for 2 and 5 days in serum-free Leibovitz's L-15 medium (37 °C, 95% air – 5% CO2). The addition of transferrin (5 μg/mL) had no effect, but insulin (30, 60, and 125 mU/mL) increased DNA and protein syntheses in a dose-dependent manner. The influence of insulin (125 mU/mL) was potentiated by the addition of transferrin and the combination of the two stimulated DNA synthesis by threefold on day 2 when compared with controls and by sixfold on day 5 of culture. After 5 days, synthesis was restored to values observed at day 0. Transferrin did not modify the insulin effect on protein synthesis, since the latter was already maximally stimulated as early as day 2 of culture and at levels well above that of uncultured explants (day 0). The activities of four hydrolases considered as markers of brush border differentiation were not importantly changed by any of the hormones, supplemented alone or in combination. The results indicate that proliferation rather than differentiation is the parameter mostly influenced by these two hormones. The combination of insulin plus transferrin restores cellular functions of human fetal kidney explants cultured in serum-free medium. The potentiation of insulin influence possibly results from an increase in the number of transferrin receptors. The above additions optimize the present culture system and establish its usefulness as a valuable tool to study the direct influence of different effectors involved in human metanephrogenesis.Key words: insulin, transferrin, culture, nephrogenesis.

Effects of recombinant bovine somatotrophin, insulin-like growth factor-I and insulin on bovine granulosa cell steroidogenesis in vitro

1994 ◽  
Vol 143 (1) ◽  
pp. 157-164 ◽  
Author(s):  
J G Gong ◽  
D McBride ◽  
T A Bramley ◽  
R Webb
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Dependent Manner ◽  
Serum Free ◽  
Factor I ◽  
Igf I ◽  
Serum Free Conditions ◽  
Dose Dependent ◽  
Bovine Somatotrophin ◽  
Cells Cultured

Abstract Our previous studies have demonstrated that physiological concentrations of metabolic hormones, including recombinant bovine somatotrophin (BST), insulin-like growth factor-I (IGF-I) and insulin, can significantly stimulate the proliferation of bovine granulosa cells cultured under serum-free conditions. In this study we investigated the effects of these factors on bovine granulosa cell steroidogenesis using the same culture system. Bovine granulosa cells were obtained from antral follicles classified into three size classes: small, <5 mm; medium-sized, 5–10 mm and large, >10 mm in diameter. Whilst not affecting steroidogenesis by granulosa cells from small and medium-sized follicles, BST (10–1000 ng/ml) stimulated the secretion of both oestradiol and progesterone by granulosa cells from large follicles in a dose-dependent manner. Insulin (1–1000 ng/ml) and IGF-I (10–1000 ng/ml) stimulated the secretion of oestradiol and progesterone by granulosa cells from all three size categories of follicles in a dose-dependent manner. FSH (200 ng/ml) alone increased progesterone secretion by granulosa cells from all three size classes of follicles, but had no effect on oestradiol secretion by granulosa cells. Both IGF-I (200 ng/ml) and insulin (30 ng/ml) acted in synergy with FSH (200 ng/ml) to stimulate steroidogenesis by granulosa cells from all three size categories of follicles, but no such interaction was observed between BST (50 ng/ml) and FSH (200 ng/ml). In conclusion, BST, IGF-I and insulin significantly influence the steroidogenic activity of bovine granulosa cells cultured under serum-free conditions. However, unlike their effects on cell proliferation, the minimal effective concentrations of these factors required to stimulate granulosa cell steroidogenesis were higher than those observed in our previous studies in vivo. Journal of Endocrinology (1994) 143, 157–164

scholarly journals Ferulsinaic Acid Modulates SOD, GSH, and Antioxidant Enzymes in Diabetic Kidney

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Ahmed Amir Radwan Sayed
Keyword(s):  
Antioxidant Enzymes ◽  
Renal Cortex ◽  
Weight Ratio ◽  
Diabetic Rats ◽  
Dependent Manner ◽  
Potential Drug ◽  
Body Weight Ratio ◽  
Induce Insulin Resistance ◽  
Dose Dependent ◽  
Prevention And Therapy

The efficacy of Ferulsinaic acid (FA) to modulate the antioxidant enzymes and to reduce oxidative stress induced-diabetic nephropathy (DN) was studied. Rats were fed diets enriched with sucrose (50%, wt/wt), lard (30%, wt/wt), and cholesterol (2.5%, wt/wt) for 8 weeks to induce insulin resistance. After a DN model was induced by streptozotocin; 5, 50 and 500 mg/kg of FA were administrated by oral intragastric intubation for 12 weeks. In FA-treated diabetic rats, glucose, kidney/body weight ratio, creatinine, BUN, albuminurea, and creatinine clearance were significantly decreased compared with non treated diabetic rats. Diabetic rats showed decreased activities of SOD and GSH; increased concentrations of malondialdehyde and IL-6 in the serum and kidney, and increased levels of 8-hydroxy-2′-deoxyguanosine in urine and renal cortex. FA-treatment restored the altered parameters in a dose-dependent manner. The ultra morphologic abnormalities in the kidney of diabetic rats were markedly ameliorated by FA treatment. Furthermore, FA acid was found to attenuate chronic inflammation induced by both Carrageenan and dextran in rats. We conclude that FA confers protection against injuries in the kidneys of diabetic rats by increasing activities of antioxidant enzymes and inhibiting accumulation of oxidized DNA in the kidney, suggesting a potential drug for the prevention and therapy of DN.

scholarly journals Evidence that insulin and guanosine triphosphate regulate dephosphorylation of the β-subunit of the insulin receptor in sarcolemma membranes isolated from skeletal muscle

1986 ◽  
Vol 234 (3) ◽  
pp. 527-535 ◽  
Author(s):  
R S Horn ◽  
E Lystad ◽  
A Adler ◽  
O Walaas
Keyword(s):  
Skeletal Muscle ◽  
Membrane Protein ◽  
Insulin Receptor ◽  
Beta Subunit ◽  
Regulatory Proteins ◽  
Maximal Effect ◽  
Dependent Manner ◽  
Insulin Effect ◽  
Phosphoamino Acid Analysis ◽  
Dose Dependent

When sarcolemma membranes isolated from rat skeletal muscle were incubated with [gamma-32P]ATP, a membrane protein of apparent Mr 95,000 was rapidly phosphorylated, with the 32P content reaching a maximum within 2 s. On the basis of immunoprecipitation with anti-insulin-receptor antiserum, phosphoamino acid analysis and Mr, this protein probably represents the beta-subunit of the insulin receptor. Similarly, on incubation of the membrane with adenosine 5′-[gamma-[35S]thio] triphosphate the 95 kDa protein was thiophosphorylated, indicating thiophosphorylation of the beta-subunit of the insulin receptor on the basis of immunoprecipitation studies. The effect of insulin on the phosphorylation of this protein in the membrane was studied. Insulin induced a 20% decrease in the 32P labelling of the protein when the membranes were phosphorylated for 10 s. This insulin effect was dose-dependent, with half-maximal effect obtained at 2-3 nM-insulin. Addition of GTP, but not GDP or guanosine 5′-[beta, gamma-imido]triphosphate, enhanced the effect to 35% inhibition, with half-maximal effect of GTP obtained at 0.5 microM. GTP had no effect on the phosphorylation of the protein in the absence of insulin. Analysis of this insulin effect showed that insulin increased the rate of dephosphorylation of the 95 kDa protein in the membrane. In contrast, insulin had no effect on thiophosphorylation of the 95 kDa membrane protein after incubation with adenosine 5′-[gamma-[35S]thio]triphosphate. Since thiophosphorylated proteins are less sensitive to phosphatase action, these investigations suggest that insulin stimulated a protein phosphatase activity in a GTP-dependent manner. The possibility that GTP-regulatory proteins are involved in the action of insulin on the phosphorylation of the insulin receptor and other membrane proteins is discussed.

The mechanism for the discrepancy between serum total and free thyroxine values induced by autoantibodies: Report on two patients with Graves' disease

1990 ◽  
Vol 123 (2) ◽  
pp. 123-128 ◽  
Author(s):  
Makoto I Iitaka ◽  
Nobuhiko Fukasawa ◽  
Yoshihito Hara ◽  
Morifumi Yanagisawa ◽  
Kazumasa Hase ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Light Chain ◽  
Immunoglobulin G ◽  
Specific Radioactivity ◽  
Binding Activity ◽  
High Serum ◽  
Graves Disease ◽  
Dependent Manner ◽  
Serum Free ◽  
Dose Dependent

Abstract. The sera from two patients with Graves' disease gave abnormally high serum free T4 values as compared with the total T4 and other hormone values, suggesting the presence of autoantibodies to labelled T4 analogue used in the Amersham free T4 assay kit. The autoantibodies appeared to develop after the initiation of methimazole therapy and disappeared again after the cessation of methimazole. This binding activity to labelled T4 analogue was demonstrated to be in the immunoglobulin G with a k light chain isotype in both sera, and was displaced by unlabelled T4 in a dose-dependent manner. The binding of immunoglobulin G purified from these sera to labelled T4 or T4 analogue was found to be almost identical to that of the corresponding serum binding. Since the specific radioactivity of labelled T4 analogue used in the Amersham free T4 assay kit is about 10 times higher than that of the labelled T4 in the Amersham total T4 assay kit, serum free T4 determinations are much more vulnerable to thyroid hormone autoantibodies. Thus, in the presence of autoantibodies, a large discrepancy develops between free T4 and total T4 values.

scholarly journals Comparison of the Effects of Deferiprone versus Deferoxamine on Growth and Virulence of Yersinia enterocolitica

2002 ◽  
Vol 46 (6) ◽  
pp. 1741-1745 ◽  
Author(s):  
Biliana Lesic ◽  
Jeannine Foulon ◽  
Elisabeth Carniel
Keyword(s):  
Chelation Therapy ◽  
Lethal Dose ◽  
Iron Chelation ◽  
Defined Medium ◽  
Dependent Manner ◽  
Chemically Defined Medium ◽  
Mouse Model Of Infection ◽  
Orally Active ◽  
Dose Dependent

ABSTRACT Deferoxamine, a drug used to treat patients with iron overload, has the capacity to promote systemic Y. enterocolitica infections in humans. The aim of this study was to determine whether deferiprone, the only orally active alternative treatment, has the same potential. When Y. enterocolitica IP864 was grown in an iron-poor chemically defined medium, addition of deferoxamine promoted its growth, while various concentrations of deferiprone did not display this activity. Similarly, on iron-poor agar plates, various Y. enterocolitica strains were able to grow around paper disks impregnated with deferoxamine in a dose-dependent manner, while no growth was observed around the deferiprone disks. In a mouse experimental model of infection, the 50% lethal dose (LD50) of strain IP864 was decreased by more than 5 log units in mice pretreated with deferoxamine, while a deferiprone pretreatment did not affect it. Therefore, in contrast to deferoxamine, deferiprone does not enhance growth of pathogenic Y. enterocolitica in vitro and does not have the potential to promote Y. enterocolitica septicemia in a mouse model of infection. Deferiprone may thus represent a useful alternative iron-chelation therapy during invasive Y. enterocolitica infections.

5,5'-Diphenylhydantoin decreases the entry of 3,5,3'-triiodo-L-thyronine but not L-thyroxine in cultured GH-producing cells

1988 ◽  
Vol 117 (3) ◽  
pp. 392-398 ◽  
Author(s):  
Leonard R. Zemel ◽  
David R. Biezunski ◽  
Lawrence E. Shapiro ◽  
Martin I. Surks
Keyword(s):  
Plasma Membrane ◽  
Thyroid Hormone ◽  
Dependent Manner ◽  
Exit Rate ◽  
Entry And Exit ◽  
Serum Free ◽  
Fractional Rate ◽  
Serum Free Conditions ◽  
Kinetics Of ◽  
Dose Dependent

Abstract. We determined the effect of 5,5'-diphenylhydantoin (DPH) on the kinetics of T3 and T4 uptake in cultured GH-producing (GC) cells under serum-free conditions. GC cells accumulated [125I]T3 at a greater fractional rate than [125I]T4. The t½ of exit of [125I]T4 and [125I]T3 previously equilibrated in GC cells was 28 min for T4 and 66 min for T3. T3 and T4 entry rates were not influenced by up to a 10 000-fold molar excess of nonradioactive T3, T4, d-T4, rT3, 3,5-T2 and diiodotyrosine. Thus, entry of T3 and T4 in GC cells appeared nonsaturable and was not influenced by various thyroid hormone analogues. DPH, 25–200 μmol/l, decreased the rate of T3 entry in a dose-dependent manner and did not influence the T3 exit rate. At 200 μmol/l DPH, T3 entry decreased by 40%. Rates of entry and exit of T4 were unaffected by DPH. DPH may affect T3 and T4 entry differentially at the level of the plasma membrane.

scholarly journals Phosphophloretin sensitivity of rabbit renal NaPi-IIa and NaPi-Ia

2004 ◽  
Vol 286 (5) ◽  
pp. F955-F964 ◽  
Author(s):  
Brian E. Peerce ◽  
Brandon Peerce ◽  
Rebecca D. Clarke
Keyword(s):  
Renal Cortex ◽  
Phosphate Uptake ◽  
Ph Dependence ◽  
Membrane Vesicles ◽  
Distal Tubule ◽  
Proximal Tubules ◽  
Dependent Manner ◽  
Renal Brush Border Membrane ◽  
Distal Tubules ◽  
Dose Dependent

The effect of phosphorylated phloretins on Na+-dependent phosphate uptake into rabbit renal brush-border membrane vesicles (BBMV) was examined. Na+-dependent phosphate uptake into isolated rabbit cortex BBMV was sensitive to 2′-phosphophloretin (2′-PP) and 2′-phospho-4′,4,6′-trimethoxy phloretin (PTMP) in a dose-dependent and pH-dependent manner. PTMP inhibition of Na+-dependent phosphate uptake was maximum at alkali pH, and 2′-PP inhibition of Na+-dependent phosphate uptake was maximum at acidic pH. Increasing Na+ concentrations did not increase PTMP inhibition of renal cortex BBMV Na+-dependent phosphate uptake at pH 6. The effect of phosphophloretins on Na+-dependent phosphate uptake was examined in BBMV isolated from purified proximal tubules and distal tubules. 2′-PP and PTMP inhibition of Na+-dependent phosphate uptake into BBMV isolated from purified proximal tubules was similar to the inhibition seen with BBMV from renal cortex. 2′-PP, but not PTMP, inhibited Na+-dependent phosphate uptake into BBMV isolated from purified distal tubules. The pH dependence of inhibition, the absence of PTMP inhibition of Na+-dependent phosphate uptake into distal tubule BBMV, and the inhibition of Na+-dependent phosphate uptake into distal tubule BBMV suggest that NaPi-Ia is 2′-PP sensitive and NaPi-IIa is PTMP sensitive.

scholarly journals Leishmania donovani promastigote: Effect of Adrenergic Agonists and Antagonist on Growth and Lipophosphoglycan Synthesis

2018 ◽  
Vol 14 (2) ◽  
pp. 111-115
Author(s):  
Kalipada Kar ◽  
Sujata Kar
Keyword(s):  
Leishmania Donovani ◽  
Drug Regimen ◽  
Defined Medium ◽  
Dependent Manner ◽  
Self Healing ◽  
Sds Page ◽  
Second Messenger Systems ◽  
Cutaneous Form ◽  
Dose Dependent

ABSTRACTBackground: Leishmania is the causative agent of a spectrum of diseases in human ranging in severity from self-healing cutaneous form to disfiguring mucocutaneous lesion to deadly visceral form of leishmaniasis. The treatment is still suffering from toxicity of the present drug regimen and the emergence of drug resistant leishmaniasis. Successful immunotherapy is yet to develop. It is beneficial to venture the parasite second messenger systems for the development of successful antileishmanial agents. The role of adrenergic receptor agonists and antagonist in the growth of L. donovani promastigotes and the synthesis of phosphorylated macromolecules were addressed in this report. Materials and Methods: The present work is entirely experimental. The effect of dibutyryl cAMP (db-cAMP), dibutyryl cGMP (db-cGMP), epinephrine, isoproterenol and propranolol on the growth of L. donovani promastigotes using defined medium were studied. Biosynthetically pulse labeling of promastigotes with [32P]-orthophosphate with or without experimental agents following SDS-PAGE and autoradiography was analyzed. Results: The growth of L. donovani promastigotes at 96 h culture was inhibited by 69%, 83, 52% and 95% with db-cAMP, epinephrine, isoproterenol and propranolol at 100 μM each, respectively in compared to control. Multiplication of parasite was stimulated by db-cGMP. The expression of lipophosphoglycan of the parasite was drastically affected in the following order with propranolol>epinephrine>cAMP>isoproterenol at 100 μM of each agent for 6 h exposure to the promastigotes. Conclusion: The growth of the parasite and the synthesis of lipophosphoglycan were significantly more inhibited by epinephrine or propranolol in a dose dependent manner than cAMP or isoproterenol.Keywords: cAMP; cGMP; epinephrine; Leishmania donovani; LPG; propronolol.

scholarly journals 1,6-hexanediol rapidly immobilizes and condenses chromatin in living human cells

2021 ◽  
Vol 4 (4) ◽  
pp. e202001005 ◽  
Author(s):  
Yuji Itoh ◽  
Shiori Iida ◽  
Sachiko Tamura ◽  
Ryosuke Nagashima ◽  
Kentaro Shiraki ◽  
...  
Keyword(s):  
Liquid Droplet ◽  
Chromatin Condensation ◽  
Human Cells ◽  
Dependent Manner ◽  
Liquid Droplets ◽  
Cellular Functions ◽  
Spatiotemporal Regulation ◽  
Dose Dependent ◽  
Liquid Liquid Phase Separation

Liquid droplets formed inside the cell by liquid–liquid phase separation maintain membrane-less condensates/bodies (or compartments). These droplets are important for concentrating certain molecules and facilitating spatiotemporal regulation of cellular functions. 1,6-hexanediol (1,6-HD), an aliphatic alcohol, inhibits weak hydrophobic protein–protein/protein-RNA interactions required for the droplet formation (droplet melting activity) and is used here to elucidate the formation process of cytoplasmic/nuclear condensates/bodies. However, the effect of 1,6-HD on chromatin in living cells remains unclear. We found that 1,6-HD drastically suppresses chromatin motion and hyper-condenses chromatin in human cells by using live-cell single-nucleosome imaging, which detects changes in the state of chromatin. These effects were enhanced in a dose-dependent manner. Chromatin was “frozen” by 5%, or higher, concentrations of 1,6-HD. 1,6-HD greatly facilitated cation-dependent chromatin condensation in vitro. This 1,6-HD action is distinct from its melting activity of liquid droplets. Alcohols, such as 1,6-HD, appear to remove water molecules around chromatin and locally condense chromatin. Therefore, liquid droplet results obtained using 1,6-HD should be carefully interpreted or reconsidered when these droplets are associated with chromatin.

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