Growth hormone binding sites are present in tissues of the porcupine Hystrix hodgesoni,the snakehead Channa maculata and the winter flounder Pleuronectes americanus but not in those of the lamprey Petromyzon marinus

• 1.1. Various tissues of the porcupine Hystrix hodgesoni including liver, intestine, stomach, spleen, kidney, brain and lung were examined for the presence of growth hormone binding sites.
• 2.2. Membranes were prepared from the aforementioned tissues and tested for binding to ¹²⁵I-bovine growth hormone (¹²⁵I-bGH).
• 3.3. Porcupine kidney membranes yielded 1.3 and 2.7% specific binding when tested at 1000 and 2500 μg protein, respectively. Porcupine liver membranes demonstrated approximately 1% specific binding at 3000 μg protein. The other tissues gave low specific binding. The results indicate that porcupine kidney contained binding sites for growth hormone.
• 4.4. Various tissues of two teleosts, the snakehead Channa maculata and the winter founder Pleuronectes americanus, were similarly processed and tested for binding to ¹²⁵I-bGH. It was found that among the different tissues studied, the liver membranes of Channa maculata and the gonad membranes of Pleuronectes americanus gave the highest specific binding of ¹²⁵I-bGH.
• 5.5. Liver and intestine membranes of the lamprey Petromyzon marinus did not bind ¹²⁵I-bGH.

     

Effect of bovine milk antigens and egg lysozyme on the binding of 59Fe-lactoferrin to platelet plasma membranes

• 1.1. Platelets bind specifically to lactoferrin. A significant similarity between human lactoferrin and some bovine milk proteins has been established.
• 2.2. Because of the structural homology of lactoferrin and cows milk proteins they are able to influence lactoferrins regulatory function on the level of its binding to membrane receptors on platelets.
• 3.3. An inhibitory effect of bovine α-lactalbumin and of β-lactoglobulin on lactoferrin-receptor interaction was shown.
• 4.4. Bovine α-lactalbumin competes with lactoferrin for the binding sites.
• 5.5. Scatchard plot analysis of data shows one binding site for lactoferrin in the presence of α-lactalbumin with an affinity constant, Ka = 0.46 × 10⁹ mol/1 and 335 receptors/cell.
• 6.6. The inhibitory effect of β-lactoglobulin reaches 62% and is different for the common fraction ⨿-lactoglobulin and the genetic variants β-lactoglobulin A and B.
• 7.7. β-lactoglobulin does not compete with lactoferrin for the membrane receptors.
• 8.8. Bovine casein and egg lysozyme stimulate ⁵⁹Fe-lactoferrin binding to the receptors. The mechanism of these effects is still unknown.
• 9.9. Tested alimentary antigens are able to interact with lactoferrin and also with some platelet membrane structures.
• 10.10. Established changes in lactoferrin binding to the platelet membrane might be in relation to lactoferrins regulatory function and (or) eliminating mechanisms of these alimentary antigens.

     

Some physical and chemical properties of purified nematocysts of Hydra attenuata pall. (Hydrozoa,cnidaria)

• 1.1. A method for purifying undischarged nematocysts from Hydra and other cnidarians is described.
• 2.2. Isolated cysts (relative densities 1.22–1.24) evaginate their tubular content even after previous dehydration.
• 3.3. The cyst wall is permeable to dyes of mol. wts up to 600,000.
• 4.4. Approximately two-thirds of the cyst's dry wt are soluble proteins. Eighty per cent of them are of low mol. wt and highly anionic, presumably serving as binding sites for Ca²⁺ and Mg²⁺.
• 5.5. The other 20% includes 30 different proteins amongst them toxins and enzymes (phospholipase and little proteases but no collagenase, chitinase or hyaluronidase).

     

Stearoyl-CoA desaturase activity in primary culture of chicken hepatocytes. influence of insulin,glucocorticoid, fatty acids and cordycepin

• 1.1. Stearoyl-CoA desaturase (Δ9-desaturase) activity was measured in chicken primary hepatocytes, as a function of time in culture.
• 2.2. When using fasted donor animals, the desaturase activity was low at the beginning of culture and then increased steadily to a maximum value between 30 and 70 hr of culture. When hepatocyte cultures were prepared from fed animals, enzyme activity was high at the beginning of culture and maintained thereafter at similar values to those obtained in cultured hepatocytes from fasted animals after 30 hr of culture.
• 3.3. Insulin significantly enhanced enzyme activity when added to the culture medium at a 10⁻⁹M concentration, and a small stimulating effect was also observed with 10⁻⁶M dexamethasone.
• 4.4. Linoleic acid (0.5 mM) added to the culture medium as albuminic complex partly inhibited Δ9-desaturase activity.
• 5.5. Cordycepin (3' deoxyadenosine) decreased enzyme activity when present at a 3 μg/ml concentration in the culture medium.
• 6.6. Taken together, the induction of enzyme activity in culture, its impairment by cordycepin and response to insulin and linoleic acid strongly suggest that synthesis and translation of the Δ9-desaturase mRNA occur in chicken hepatocytes in primary culture, and that this cellular model may be a useful tool for further studies on Δ9-desaturase regulatory mechanisms.

     

Lactoferrin binding to human platelets

• 1.1. Platelets bind specifically lactoferrin.
• 2.2. The lactoferrin binding to the platelets depends on the concentration of labelled lactoferrin, the number of platelets, the time of incubation and pH.
• 3.3. The binding was characterized by two types of binding site: one with high affinity and low capacity, and another with low affinity and high capacity (respectively k_{aff 1} = 13.6 × 10⁹1/mol and about 40 binding sites, and K_{aff 2} = 1.23 × 10⁹1/mol and about 135 binding sites per platelet).
• 4.4. Both human transferrin and bovine lactoferrin compete with human lactoferrin for the receptors.
• 5.5. The presence of lactoferrin receptors on the platelet membrane surface is connected most probably with the effect(s) on the cell function(s) of these cells.

     

Hormonal regulation of human apolipoprotein E gene expression in HepG2 cells

• 1.1. Hormonal regulation of apolipoprotein E (apoE) gene expression by insulin and thyroid hormone was studied in a human hepatoma cell line, HepG2.
• 2.2. Changes at the mRNA level, mRNA translation, in vivo synthesis and secretion were monitored.
• 3.3. Both insulin and triiodothyronine were found to have no significant effect on apoE mRNA levels.
• 4.4. Insulin treatment caused an inhibition of: (a) the in vitro translation of endogenous apoE mRNA in a HepG2 cell-free system (25%), and (b) the incorporation of radioactivity into newly-synthesized apoE in an in vivo pulse-chase labeling experiment (32%).
• 5.5. Interestingly, apoE secretion rate was found to be significantly reduced with insulin (84%) suggesting that a major portion of newly-synthesized apoE may be shunted into a degradative pathway.
• 6.6. Using a similar experimental approach, triiodothyronine showed no significant effect on the rate of apoE synthesis or translation (6–15% decrease), however a slight reduction (20%) in secretion rate was shown.
• 7.7. Overall, apoE gene expression does not appear to be influenced by triiodothyronine significantly but is modulated by insulin at the translational and post-translational level.

     

Insulin,glucagon and catecholamine interactions in avian liver explants

• 1.1. A mechanical tissue chopper was used to obtain liver explants (35–75 mg) from 2- to 3-week-old chickens to determine both tissue sensitivity and metabolic effects of isoproterenol, avian insulin and glucagon.
• 2.2. Avian insulin had no effect on lipogenesis; however, lipogenesis was decreased by dibutyryl cyclic AMP. Insulin did not overcome a decrease in lipogenesis caused by catecholamines. Therefore, this control mechanisms is not modulated by insulin.
• 3.3. Preincubation in the presence of glucagon decreased in vitro lipogenesis. Preincubation in the presence of a 19–29 amino acid construct that approximated the radioimmune site for glucagon did not result in a similar effect. Therefore, this site does not relate to the biopotency of the hormone.
• 4.4. A previously noted catecholamine induced decrease in in vitro lipogenesis was verified, showing that points of in vitro regulation are under phosphorylation-dephosphorylation control.
• 5.5. Preincubation of slices (1 hr) with propranolol blocked the inhibition of lipogenesis caused by α and β adrenergic agonists (arterenol or isoproterenol) during a subsequent 2-hr incubation.
• 6.6. Preincubation of slices with either of these agonists decreased lipogenesis even following an extensive washout.
• 7.7. Inhibition could be overcome with propranolol, a β adrenergic antagonist.

     

Combined effect of adenosine,alpha adrenergic and adenosine antagonists on serum insulin and insulin secretion from rat pancreatic islets

• 1.1. The effect of adenosine separately or in combination with alpha-1 adrenergic antagonist prazosin and alpha-2 adrenergic antagonist yohimbine as well as adenosine antagonists 8-phenyltheophylline and xanthine amine conjugate on glucose-induced insulin secretion from isolated rat pancreatic islets was studied.
• 2.2. Their in vivo effects on serum glucose and insulin levels were also investigated. Adenosine at 10 and 100 μM inhibited significantly, insulin secretion from the isolated islets whereas at 10 mM slightly increased the secretion of insulin.
• 3.3. Prazosin used at 100 μM inhibited insulin secretion. When it combined with adenosine (10 μM) it augmented the inhibitory effect of adenosine.
• 4.4. In vivo prazosin (21 mg/kg bodywt) caused a hyperglycaemia which was accompanied by hypoinsulinaemia.
• 5.5. Concurrent administration of this drug with adenosine neither affect the hyperglycaemic nor the hypoinsulinaemic effects of adenosine.
• 6.6. On the other hand, yohimbine (100 μM) has no effect neither separately nor in combination with adenosine (10 μM) in modulating the inhibitory effect of adenosine on insulin secretion.
• 7.7. When Yohimbine administered at 19.5 mg/kg body wt it did not alter serum glucose but it markedly increased the serum insulin level. Its combined administration with adenosine reduced the hyperglycaemic effect of adenosine with a remarkable increase in serum insulin.
• 8.8. Both adenosine-antagonists were ineffective in alteration of insulin secretion.
• 9.9. However, combination of 8-phenyltheophylline with adenosine (10 μM) totally blocked the inhibitory effect of adenosine on insulin secretion while xanthine amine conjugate failed to prevent this effect of adenosine.
• 10.10. These results indicate that the inhibitory effect of adenosine on insulin secretion is neither mediated via alpha-1 nor alpha-2 adrenoceptors. It might be via activation of specific adenosine receptors on rat islets which are sensitive to blockade by 8-phenyltheophylline.

     

Characterization of lactoferrin binding to the MAC-T bovine mammary epithelial cell line using a biotin-avidin technique

• 1.1. A non-radioisotopic method utilizing a biotin-avidin approach was used to characterize lactoferrin binding to the clonal MAC-T bovine mammary epithelial cell line.
• 2.2. Binding of lactoferrin to MAC-T cells and isolated membranes was specific and saturable.
• 3.3. Unlabeled lactoferrin competed for and displaced biotin-labeled lactoferrin from binding sites on mammary epithelial cells. In contrast, unlabeled transferrin did not compete.
• 4.4. Scatchard analysis of lactoferrin binding to MAC-T cell crude membranes was nonlinear, revealing two classes of binding sites with association constants (K_a) of 2.36 × 10⁷ and 3.36 × 10⁶M⁻¹.
• 5.5. Binding of lactoferrin to MAC-T cells may be associated with the initial events which result in decreased MAC-T cell proliferation.

     

Characterization of sheep hepatocytes in primary culture

• 1.1. Primary cultures of isolated sheep hepatocytes were used to characterize metabolic functions of liver: gluconeogenesis, ureagenesis and protein synthesis. The rates of all three metabolic activities were linear over a 20 hr culture period.
• 2.2. Hepatocytes in the presence of glucagon increased the synthesis of urea by approx 30% (P < 0.05) and increased release of glucose into the medium by 60% (P < 0.05).
• 3.3. In the absence of insulin, significantly more (35%; P < 0.05) glucose was released in the medium than in the presence of insulin.
• 4.4. Results help evaluate the primary culture of sheep hepatocytes as an appropriate experimental model to study nutritional and hormonal regulation of liver in the ruminant species.

     

Effects of transforming growth factor-β on collagen gene expression and collagen synthesis level in mineralizing cultures of osteoblast-like cell line,MC3T3-E1

• 1.1. High levels of type I collagen mRNA and [³H]proline incorporation into collagenase digestable protein by MC3T3-E1 cells were detected during the first 7 days of culture, after which they declined.
• 2.2. Type I collagen gene expression was stimulated by TGF-β in the early culture stage when the collagen gene expression was fully functioning.
• 3.3. However, these stimulatory effects disappeared at the differentiation stages. Although collagen gene expression was stimulated by TGF-β (2.0 ng/ml) in early culture, collagen synthesis in medium was not.
• 4.4. This study shows that collagen synthesis and collagen gene expression were affected by the state of differentiation in MC3T3-E1 cells and that the rate of stimulation by TGF-β in collagen gene expression decreased over time in culture.

     

Small membrane-associated gtp-binding proteins of catecholamine-secreting cells

• 1.1. Four GTP-binding proteins (23–27 kDa) were identified in membranes from PC12 cells by [α³²P]GTP binding to nitrocellulose blots of SDS-polyacrylamide gels.
• 2.2. The GTP-binding proteins remained associated with membranes during stimulation of intact cells by K⁺-depolarization or even after addition of C²⁺to digitonin-permeabilized cells.
• 3.3. By two-dimensional gel electrophoresis, six GTP-binding proteins were resolved and based on their mobility, their phosphorylation state appeared independent of Ca²⁺.
• 4.4. Fractionation of PC12 membranes showed that these GTP-binding proteins were broadly distributed in post-nuclear membranes with the plasma membranes containing the highest specific GTP-binding activity.
• 5.5. Membrane fractions from bovine adrenal medulla contain similar GTP-binding proteins with GTP-binding intensity also being highest in the plasma membrane.
• 6.6. The GTP-binding proteins could be concentrated in the detergent-rich fraction upon Triton X-114 phase separation.

     

Activation and inhibition of insulin receptor autophosphorylation by trypsin treatment of intact H35 cells

• 1.1. Treatment of intact cultured H35 cells with trypsin (1 mg/ml) for 15 min at low temperature (4°C) or for 30 sec at 37°C causes activation of the insulin receptor subsequently isolated from the cells.
• 2.2. Receptor activation was assessed by increased phosphotyrosine content of the β-subunit of the receptor, and increased autophosphorylation using [³²P]-ATP.
• 3.3. Treatment of the cells for 15 min at 37°C however completely abolished insulin binding and all insulin receptor kinase activity.
• 4.4. These data demonstrate that proteolytic damage of the extracellular domain of the insulin receptor can render the receptor kinase inactive and lead to a cell which is unresponsive to insulin.

     

Oral administration of insulin in winter-acclimatized carp (Cyprinus carpio) induces hepatic ultrastructural changes

• 1.1. The intestinal absorption of insulin in carps was assessed examining the transepithelial passage of ingested gold-labeled hormone by electron microscopy. Insulin transfer occurred mainly through the intercellular spaces between the enterocytes.
• 2.2. When reaching the lamina propria, the gold-labeled hormone gathered predominantly around the granules of the granular cells, and therefore can enter the circulatory system via the blood capillaries which are found in close contact with these cells.
• 3.3. Winter-acclimatized carp were also capable of internalizing the hormone when fed with insulin.
• 4.4. Furthermore, the absorbed hormone revealed full activity in regard to the observed changes in the ultrastructure of the liver cells of the treated cold-adapted fish.
• 5.5. The fish ingesting the hormone underwent the same type of hepatic ultrastructure reprogramming observed when winter-acclimatized carps are injected intraperitoneally with insulin, i.e. conversion to a phenotype corresponding to hepatocytes from summer-adapted carp.
• 6.6. The oral absorption of insulin by winter-acclimatized fish and its effect in reversing the cold-adaptive state might be useful for the fish culturing industry.

     

Selective modification of tryptophan-150 in ovine placental lactogen

• 1.1. Ovine placental lactogen was modified by reaction with o-nitrophenylsulfenyl chloride. Fluorescence measurements indicated that one of the two tryptophan residues of the molecule had reacted. Besides, there was some reagent not covalently bound.
• 2.2. The reagent was covalently bound to Trp-150. No evidence of modification of Trp-90 was found.
• 3.3. Binding capacity to lactogenic as well as somatogenic receptors was diminished but not abolished upon modification, indicating that absolute molecular integrity of Trp-150 is not required for binding.
• 4.4. This behavior is similar to that of the tryptophan residues of ovine prolactin.

     

Increased muscle glucose uptake in response to chronic glyburide treatment is not related to changes in glucose transporter (GLUT4) protein

• 1.1. The mechanism of action of glyburide (a sulfonylurea) on muscle has been investigated by measuring glucose uptake and glucose transporter (GLUT4) protein levels after chronic glyburide treatment.
• 2.2. A dietary induced insulin resistant rat model (4 wk of high-fat, high-sucrose feeding) was given glyburide (2mg/kg/day) for 10 days and glucose uptake was measured in a perfused hindquarter preparation.
• 3.3. Protein levels of the GLUT4 glucose transporter were determined by Western analysis.
• 4.4. After 7 days of treatment, rats fed glyburide had lower blood glucose concentrations 2 hr (72 ± 5 vs 103 ± 12 mg/dl) and 24 hr (97 ± 7 vs 123 ± 7 mg/dl) after glyburide administration with no difference in serum insulin levels compared to vehicle treated animals.
• 5.5. Glucose uptake was approx doubled in basal state (0 insulin) in response to glyburide (2.8 + 0.4 vs 1.7 ± 0.2μ mol/g per hr).
• 6.6. Maximal insulin (100 nM) stimulated glucose uptake tended to be higher in the glyburide treated group, but did not reach statistical significance (8.0 ± 0.7 vs 7.0 ± 0.6 μmol/g per hr).
• 7.7. Western analysis revealed no significant effect of glyburide on the GLUT4 protein level in skeletal muscle.
• 8.8. These results suggest that glyburide alters glucose uptake through some mechanism other than alterations in the level of the GLUT4 glucose transporter protein.

     

Insulin Induces Microtubule Stabilization and Regulates the Microtubule Plus-end Tracking Protein Network in Adipocytes

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Highlights

• •Insulin Affects the Phosphorylation of G2L1, MARK2, CLIP2, EB1, AGAP3, and CKAP5.
• •Insulin Increases CLASP2 +TIP Density and Decreases CLASP2 +TIP Velocity.
• •Insulin Stimulates CLASP2 and G2L1 Trailing Along Microtubules.
• •Insulin Stimulates α-Tubulin Acetylation at Lysine 40 and Microtubule Stabilization.

     

Biomines-stimulated phosphorylation and (Na+, K+-ATPase in the gills of the chinese crab,Eriocheir sinensis

• 1.1. Subcellular distribution of (NA⁺, K⁺-ATPase and ouabain-insensitive ATPase (Mg²⁺-ATPase) are compared in branchial tissues of the euryhaline crab, Eriocheir sinensis, acclimated to fresh water.
• 2.2. Both the anterior and posterior gills contain cAMP-dependent protein kinase and endogenous protein substrate for phosphorylation.
• 3.3. Phosphorylation occurs in both “particulate” and “soluble” subcellular fractions but its stimulation by cAMP is restricted to the “soluble” fraction.
• 4.4. serotonin (5-HT) and dopamine receptors are present only in the “light particulate” fraction isolated from the posterior gills.

• 1.(a) Serotonin and dopamine have no effect on the phosphorylation observed in a subcellular fraction alone.
• 2.(b) Activation of the phosphorylation by serotonin and dopamine is found when the soluble fraction (source of cAMP-dependent protein kinase) is added to the fraction P₃ from the posterior gills.
• 3.(c) No activation occurs with the fractions P₃ as well as P₁ or P₂ (not shown) from anterior gills of fresh water crab.
• 4.(d) Cyproheptadine, a serotonin receptor antagonist, inhibits the 5-HT dependent increase in phosphorylation.
• 5.(e) The dopamine receptor antagonist, chlorpromazine, inhibits dopamine-stimulated phosphorylation.
• 6.5. Ouabain mimics the effect of cyproheptadine on the serotonin-stimulated phosphorylation found in the posterior gills.

     

Oxidation of methionine residues in equine growth hormone by chloramine-T

• 1.1. Reactivity of methionine residues towards Chloramine-T was studied in the equine growth hormone.
• 2.2. With a 20.0-fold molar excess of reagent over methionine, full oxidation of the four residues of the protein is achieved.
• 3.3. Methionine 4 is the most reactive group, followed by methionines 72 and 178—methionine 123 being the less reactive residue.
• 4.4. As judged by circular dichroism spectra and binding assays, protein conformation and binding capacity to specific receptors remains unchanged even after full oxidation of all four methionine residues.
• 5.5. Results agree with data previously obtained with bovine growth hormone.

     

Localization of the glucocorticoid receptor in rat liver cells: Evidence for plasma membrane bound receptor

• 1.1. The cytoplasmic glucocorticoid receptor of rat liver cells is in part recovered in the plasma membrane fraction.
• 2.2. After in vivo administration of [³H]dexamethasone, 0.35% of the radioactivity recovered is bound on plasma membranes.
• 3.3. Dexamethasone also binds in vitro specifically to plasma membranes. Expressed as fmol/mg protein, binding of dexamethasone to plasma membranes is comparable to binding to the soluble cytoplasmic fraction (cytosol).
• 4.4. Using polyclonal antibody to the glucocorticoid receptor and the indirect immunofluorescence technic, an intense decoration of the plasma membranes is observed, denoting a high concentration of glucocorticoid receptor on plasma membranes.
• 5.5. The localization of the receptor on plasma membranes could be of potential importance for its interaction with agents (mitogens, growth factors) initially acting on the cell membrane, regulating subsequent cell proliferation and growth at the level of the cell nucleus.