Distribution of IGF-I mRNA and IGF-I binding sites in the rat kidney

Summary In the present study we have investigated the distribution of IGF-I mRNA and IGF-I binding sites in the rat kidney. The distribution of IGF-I mRNA was investigated using a simple and sensitive non-radioactive in situ hybridisation technique based on probe labelling with digoxigenin labelled-UTP followed by detection with conventional immunocytochemical techniques. IGF-I mRNA was found predominantly in medullary collecting ducts and sparsely in cortical collecting duct cells. In addition IGF-I mRNA was expressed in scattered proximal tubular cells in the cortex and in cells confined to the glomerular tuft. IGF-I binding sites were studied using radiolabelled IGF-I and conventional autoradiographical techniques on tissue sections. It was found that IGF-I binding sites were widely distributed throughout the entire kidney and that the specific binding was highest in the inner medulla. These findings add further complexity to the understanding of IGF-I production and action on renal structures.     

A method for determination of the specific activity of receptor-bound human chorionic gonadotropin (hCG).

Precise knowledge of the specific activity (S.A., muc/mug) of the receptor bound radiolabelled hormone is required for study of the stoichiometry of peptide hormone-receptor interactios. A radioligand receptor assay using 131-I-hCG as tracer and transplantable mouse luteoma homogenates (Biol. Reprod. 8:550, 1973) as a source of receptor was used as a model to determine the specific activity of receptor bound 125-I-hCG. Progressive saturation of the gonadotropin receptor by 125-I-hCG suggests the presence of a high affinity-low capacity binding event (saturating between 14 and 37 ng/100 mg homogenate) that does not distinguish between non-radioactive hCG and 125-I-hCG, and a low affinity-high capacity binding event (saturating between 240 and 270 ng/100 mg homogenate) that shows a preference for non-radioactive hCG over 125-I-hCG. Parallelism between bound 125-I-hCG and non-radioactive hCG in terms of competition with tracer 131-I-hCG could only be demonstrated for the high affinity event.     

Distribution of IGF-I mRNA and IGF-I binding sites in the rat kidney.

In the present study we have investigated the distribution of IGF-I mRNA and IGF-I binding sites in the rat kidney. The distribution of IGF-I mRNA was investigated using a simple and sensitive non-radioactive in situ hybridisation technique based on probe labelling with digoxigenin labelled-UTP followed by detection with conventional immunocytochemical techniques. IGF-I mRNA was found predominantly in medullary collecting ducts and sparsely in cortical collecting duct cells. In addition IGF-I mRNA was expressed in scattered proximal tubular cells in the cortex and in cells confined to the glomerular tuft. IGF-I binding sites were studied using radiolabelled IGF-I and conventional autoradiographical techniques on tissue sections. It was found that IGF-I binding sites were widely distributed throughout the entire kidney and that the specific binding was highest in the inner medulla. These findings add further complexity to the understanding of IGF-I production and action on renal structures.     

The two NK-1 binding sites are distinguished by one radiolabelled substance P analogue

Two non-stoichiometric binding sites had previously been characterized for the NK-1 receptor using two different types of radiolabelled analogues of substance P. However, the question remained on their eventual conformational interconversion induced or not by the ligand. In this study, kinetic, saturation, and competition studies using [3H]propionyl[Pro(9)]SP demonstrate the existence of two independent binding components in CHO cells transfected with the human NK-1 receptor, with K(d) values of 0.040 nM ( approximately 20% of total sites) and 5.9 nM ( approximately 80% of total sites) that correspond to those of the two previously described binding sites. These two binding sites do not seem to interconvert since the minor one can be selectively extinguished in saturation studies in the presence of a SP analogue specific of this binding site.     

Non-invasive, kinetic measurements of [3H]nitrendipine binding to isolated rat myocytes by condensed phase radioluminescence

The binding of 3H-labelled drug molecules to membranes of living cells gives rise to photon emission from tryptophan residues at proteinaceous binding sites. This phenomenon, called condensed phase radioluminescence, has been used to measure non-invasively the kinetics of [3H]nitrendipine binding and dissociation on the same samples of cultured beating cardiac myocytes. Signal arose only from bound drug molecules. Binding was monoexponential (tau = 5.5 min) as was dissociation (14.3 min). Preincubating cells with non-radioactive nifedipine reduced the amplitude and rate of [3H]nitrendipine but not of [3H]dihydroalprenolol binding. The potential uses of this phenomenon are discussed.     

Photoaffinity labelling of a 33-35,000 dalton protein in cardiac, skeletal and smooth muscle membranes using a new 125I-labelled 1,4-dihydropyridine calcium channel antagonist

The binding sites for Ca2+ channel antagonists were probed using Bay P 8857 [2-iodoethyl isopropyl 1,4-dihydropyridine-2,6-dimethyl-4-(3-nitrophenyl)-pyridine-3,5-dicarbox ylate] that has been radiolabelled with 125I. This drug was shown to bind with high affinity to cardiac, smooth, and skeletal muscle membranes, with a KD approximately equal to 0.3 nM. A protein of molecular weight 33-35,000 daltons was specifically and irreversibly radiolabelled after irradiation of cardiac, skeletal and aortic smooth muscle membranes, incubated with the [125I]-Bay P 8857. The peptide labelled by 1,4-dihydropyridine binding therefore appears similar in size for cardiac, skeletal, and smooth muscle. This data suggests that of the three peptide subunits which reportedly comprise the skeletal and cardiac muscle 1,4-dihydropyridine receptor complex, the 33-35,000 dalton peptide contains the dihydropyridine binding site.     

Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry.

In the present study some experimental parameters for in situ hybridization histochemistry (ISHH) have been analysed using 35S-labelled and alkaline phosphatase-conjugated probes, in order to develop a reproducible double-labelling procedure. We have compared the total exclusion of tissue fixation with tissue sections fixed by immersion in formalin. In addition, the effect of dithiothreitol was assessed both when combining radiolabelled and non-radioactive probes on a single tissue section and when the probes were used separately. Hybridization of unfixed tissue resulted in stronger specific labelling and lower background both for radiolabelled and alkaline phosphatase-conjugated probes. No loss in tissue preservation was seen at the light microscopic level after hybridization of unfixed tissue. High concentrations (200 mM) of dithiothreitol strongly suppressed background when using 35S-labelled probes, whereas in the non-radioactive procedure, alkaline phosphatase labelling could only be achieved with very low dithiothreitol concentrations (less than 1 mM). This incompatibility led to a protocol using unfixed tissue sections and a sequential hybridization procedure, with the radiolabelled probe and high concentrations of dithiothreitol in the first step and the alkaline phosphatase-conjugated probe without dithiothreitol in the second step.     

Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry

Summary In the present study some experimental parameters for in situ hybridization histochemistry (ISHH) have been analysed using³⁵S-labelled and alkaline phosphatase-conjugated probes, in order to develop a reproducible double-labelling procedure. We have compared the total exclusion of tissue fixation with tissue sections fixed by immersion in formalin. In addition, the effect of dithiothreitol was assessed both when combining radiolabelled and non-radioactive probes on a single tissue section and when the probes were used separately. Hybridization of unfixed tissue resulted in stronger specific labelling and lower background both for radiolabelled and alkaline phosphatase-conjugated probes. No loss in tissue preservation was seen at the light microscopic level after hybridization of unfixed tissue. High concentrations (200 mM) of dithiothreitol strongly suppressed background when using³⁵S-labelled probes, whereas in the non-radioactive procedure, alkaline phosphatase labelling could only be achieved with very low dithiothreitol concentrations (<1 mM). This incompatibility led to a protocol using unfixed tissue sections and a sequential hybridization procedure, with the radiolabelled probe and high concentrations of dithiothreitol in the first step and the alkaline phosphatase-conjugated probe without dithiothreitol in the second step.     

Hepadnavirus infection requires interaction between the viral pre-S domain and a specific hepatocellular receptor.

To better define the molecules involved in the initial interaction between hepadnaviruses and hepatocytes, we performed binding and infectivity studies with the duck hepatitis B virus (DHBV) and cultured primary duck hepatocytes. In competition experiments with naturally occurring subviral particles containing DHBV surface proteins, these DNA-free particles were found to interfere with viral infectivity if used at sufficiently high concentrations. In direct binding saturation experiments with radiolabelled subviral particles, a biphasic titration curve containing a saturable component was obtained. Quantitative evaluation of both the binding and the infectivity data indicates that the duck hepatocyte presents about 10(4) high-affinity binding sites for viral and subviral particles. Binding to these productive sites may be preceded by reversible virus attachment to a large number of less specific, nonsaturable primary binding sites. To identify which of the viral envelope proteins is responsible for hepatocyte-specific attachment, subviral particles containing only one of the two DHBV surface proteins were produced in Saccharomyces cerevisiae. In infectivity competition experiments, only particles containing the large pre-S/S protein were found to markedly reduce the efficiency of DHBV infection, while particles containing the small S protein had only a minor effect. Similarly, physical binding of radiolabelled serum-derived subviral particles to primary duck hepatocytes was inhibited well only by the yeast-derived pre-S/S particles. Together, these results strongly support the notion that hepadnaviral infection is initiated by specific attachment of the pre-S domain of the large DHBV envelope protein to a limited number of hepatocellular binding sites.     

Effects of alloxan-induced diabetes on somatostatin binding to cytosolic components of rabbit gastric fundic mucosa

Diabetes was induced by administration of alloxan (150 mg/Kg) to 24 h-fasted rabbits. Somatostatinlike immunoreactivity (SLI) and cytosolic binding sites for somatostatin in gastric fundic mucosa were studied using radiolabelled Tyr¹¹-somatostatin. Three months after the onset of the disease, the specific binding of somatostatin to these sites was seen to be significantly lower, due to a reduction in the number (but not the affinity) of specific somatostatin binding sites of high-affinity and a disappearance of the specific, somatostatin binding sites of low-affinity. These changes were associated with an increase in the SLI concentration in both gastric fundic mucosa and plasma.     

Characterization of binding sites for acetylated low density lipoprotein in the rat liver in vivo and in vitro.

Acetylated low density lipoprotein (acetyl-LDL) binding to hepatic membrane proteins of rats was analysed in vitro by ligand blotting. Specific binding could be demonstrated to two hepatic proteins with an apparent mol. wt. of 250 kd and 220 kd. Polyanionic competitors and maleylated bovine serum albumin inhibited the binding of acetyl-LDL effectively. To determine the sites of the catabolism of acetyl-LDL, [131I]-acetyl-LDL was injected intravenously into control rats and rats pre-treated with the known competitors of the acetyl-LDL binding. Distribution of the radiolabelled acetyl-LDL was followed by a scintillation camera. Six minutes after injection, the radioactivity was concentrated in the liver. The competitors and unlabelled acetyl-LDL but not native LDL reduced the hepatic uptake of [131I]acetyl-LDL dramatically. Thus, the sensitivity of the 220- and 250-kd membrane binding sites to the competitors for the acetyl-LDL binding resembled that of the hepatic compartment in vivo. Finally, an application of scintigraphy with radiolabelled low density lipoproteins for diagnostic evaluation of tumor compartments is presented.     

Desipramine Elicits the Expression of Opiate Receptors and Sulfogalactosylceramide Synthesis in Rat C6 Glioma Cells

In the course of our studies on lipidoses induced by amphiphilic drugs, we have investigated the ef- of desipramine, a tricyclic antidepressant, on glial cells in culture. We noted that the addition of desipramine to the culture medium of C6 glioma cells resulted in the modification of the lipid profile of the cell membranes. Of particular interest was the presence, in the desipramine-treated cells, of an additional lipid comigrating on thin layer chromatography with sulfogalactosylceramide (S-GalCer). Addition of radiolabelled sulfuric acid in the culture medium of the desipramine-treated cells resulted in the incorporation of [35S]sulfate in the newly synthesized lipid. Furthermore, this lipid was localized selectively by indirect immunofluorescence using a specific rabbit anti-S-GalCer antibody on the cell surface of desipramine-treated, but not control, C6 cells. Desipramine also increased the activity of 3'-phosphoadenosine-5'-phosphosulfate sulfotransferase (the enzyme responsible for the synthesis of S-GalCer). Since it has been suggested that S-GalCer may be involved in opiate receptors, we looked for opiate binding sites on C6 glioma cells after exposure to desipramine. We found that dihydromorphine was able to bind to the desipramine-treated C6 cell membrane. The binding of [3H]dihydromorphine (180 fmol/mg protein) was stereospecific and had a KD of 30-60 nM. Furthermore, morphine reduced both the basal and isoproterenol-stimulated cyclic AMP levels of the desipramine-treated C6 cells. This effect was blocked by naloxone. In these respects, the opiate binding sites induced after treatment of C6 glioma cells with desipramine fulfill the requirements of a true opiate receptor.     

Use of digoxigenin-labelled probes for detection and host-range studies of tomato yellow leaf curl geminivirus

We studied the host range of tomato yellow leaf curl virus (TYLCV) in some agronomically important tomato species. Transmission tests with the natural vector Bemisia tabaci from tomato to sweet pepper, eggplant, cucumber, melon, zucchini and spinach showed that these species did not develop symptoms and did not support viral replication. These species therefore do not constitutive a reservoir of the virus and can be cultivated as alternative to tomato in the most affected areas.For host-range studies, we used a quick and sensitive dot-blot assay employing non-radioactive DNA probes. This technique, developed for detecting TYLCV in plant extracts, is easily used for diagnosis. The sensitivity of this non-radioactive test was comparable to that of radiolabelled probes.     

Potential targets for glucagon-like peptide 2 (GLP-2) in the rat: distribution and binding of i.v. injected (125)I-GLP-2

Glucagon-like peptide 2 (GLP-2) is a 33-amino acid (1-33) intestinotrophic peptide. In this study, the distribution and binding of i.v. injected radiolabeled GLP-2 (1-33) were investigated in rats using autoradiography in order to target possible binding sites. The major part of (125)I-GLP-2 (1-33) was distributed to kidneys, liver, and the gastrointestinal tract. In the small intestine, a high density of grains was localized in the epithelium with a predominance in the luminal part of the villus. The saturability of (125)I-GLP-2 (1-33) was investigated by administration of excess amounts of non-radioactive GLP-2 (1-33) or the primary metabolite of GLP-2 degradation, GLP-2 (3-33). In the small intestine, (125)I-GLP-2 was displaced both by non-radioactive GLP-2 (1-33) and (3-33), suggesting that the uptake of GLP-2 (1-33) in the small intestine is receptor-specific and that the metabolite GLP-2 (3-33) may interact with the GLP-2 receptor.     

Photoaffinity labeling of the benzodiazepine receptor in bovine cerebral cortex

Clonazepam, nitrazepam and flunitrazepam were found to engage in an irreversible interaction with benzodiazepine binding sites in bovine cerebral cortex homogenates upon irradiation with ultraviolet light. Photoaffinity labeling with [³H]flunitrazepam could be substantially (approx. 85%) inhibited by a number of different benzodiazepines, including clonazepam, lorazepam, Ro5-3027, and non-radioactive flunitrazepam. Spiroperidol, atropine, naltrexone, propranolol and GABA had no effect on irreversible [³H]flunitrazepam binding, indicating that this binding is to the benzodiazepine receptor as defined in previous studies.     

In Vitro Modulation by Avermectin B1a of the GABA/Benzodiazepine Receptor Complex of Rat Cerebellum

Avermectin B1a, a macrocyclic lactone anthelmintic agent, causes a concentration-dependent increase of [3H]flunitrazepam binding to membranes from rat cerebellum by increasing the affinity and the number of binding sites. This effect appears to be independent of the concentration of chloride ions. The effects of avermectin B1a occur with high affinity (EC50 = 70 nM), and they persist after washing of the membranes with drug-free buffer. Pretreatment of the membranes with Triton X-100 completely abolishes the action of avermectin B1a. GABA and the GABA-mimetic compounds piperidine-4-sulfonic acid and THIP diminish the effects of avermectin B1a on benzodiazepine receptor binding in a bicuculline-methiodide-sensitive mode. In addition, the stimulation of [3H]flunitrazepam binding by avermectin B1a is decreased by the pyrazolopyridines etazolate and cartazolate. These observations suggest that avermectin B1a stimulates benzodiazepine receptor binding by acting on a modulatory site which is independent of the GABA recognition site and of the drug receptor for the pyrazolopyridines, but which is in functional interaction with these sites.     

Studies on the interaction between Oxaprozin-E and bovine serum albumin by spectroscopic methods

The interaction between Oxaprozin-E and bovine serum albumin (BSA) was studied by spectroscopic methods including fluorescence and UV–vis absorption spectroscopy. The quenching mechanism of fluorescence of BSA by Oxaprozin-E was discussed to be a dynamic quenching procedure. The number of binding sites n and apparent binding constant K was measured by fluorescence quenching method. The thermodynamics parameter ΔH, ΔG, ΔS were calculated. The results indicate the binding reaction was mainly entropy-driven and hydrophobic forces played major role in the binding reaction. The distance r between donor (BSA) and acceptor (Oxaprozin-E) was obtained according to Förster theory of non-radioactive energy transfer.     

SMMC-7721肝癌细胞67kD层粘连蛋白受体的分离纯化

分离纯化肝癌细胞的 6 7kD层粘连蛋白受体 (6 7LR) ,以便进一步研究 6 7LR的结构、功能及其在肝癌浸润、转移过程中的作用 .以SMMC 772 1肝癌细胞和L 0 2正常肝细胞为材料 ,采用13 1I标记的层粘连蛋白测定其与细胞的结合能力 ;亲和层析法分离纯化层粘连蛋白受体 ,用SDS PAGE、放射自显影及体外竞争结合实验进行鉴定 .在相同条件下SMMC 772 1肝癌细胞与层粘连蛋白特异结合量为 17 5 4± 0 4 9ng 10 5细胞 ,而L 0 2正常肝细胞与层粘连蛋白的特异结合量为 8 36± 0 4 8ng 10 5细胞 .经过亲和层析 ,从SMMC 772 1肝癌细胞和L 0 2正常肝细胞均可获得纯化受体 ,SDS PAGE显示为单一条带 ,分子量为 6 7kD ,放射自显影及体外竞争结合实验表明其具有较强的与层粘连蛋白结合的活性 .体外竞争结合实验表明 ,SMMC 772 1肝癌细胞层粘连蛋白受体 (772 1LnR)的抑制率可达到 96 2 7± 2 2 9% ,而L 0 2正常肝细胞层粘连蛋白受体 (L 0 2LnR)的抑制率为 4 8 71± 3 79% ,这说明 772 1LnR与层粘连蛋白的亲和力明显高于L 0 2LnR(P <0 0 0 1) .结果表明 ,与L 0 2肝细胞比较 ,SMMC 772 1肝癌细胞具有与层粘连蛋白较强结合能力的特异受体 ,并从肝癌细胞膜上分离纯化到与层粘连蛋白有较强亲和力的 6 7LR     

Interactions between discrete neuronal membrane binding sites for the putative K+-channel ligands beta-bungarotoxin, dendrotoxin and mast-cell-degranulating peptide

1. beta-Bungarotoxin, a presynaptically active neurotoxin from the venom of Bungarus multicinctus, was radiolabelled with 125I and its binding to synaptic membranes from rat brain was analyzed. The interaction of these binding sites with those for dendrotoxin (a convulsant polypeptide from mamba venom) and mast-cell-degranulating peptide (from bee venom) was examined in the light of the known effects of all three toxins on voltage-dependent K+ currents. 2. When measured in Krebs/phosphate buffer, the binding appeared monotonic at low concentrations of radioiodinated beta-bungarotoxin (Kd 0.4 nM; Bmax 0.42 pmol/mg protein); higher concentrations of labelled toxin revealed an additional binding component of lower affinity, but computer analysis of the data failed to provide well-defined estimates of its Kd and Bmax values. 3. Equilibrium binding experiments conducted in imidazole-based buffers yielded distinctly biphasic Scatchard plots; computer analysis of the data revealed two populations of sites [Kd 0.26 (+/- 0.30) nM and 6.14 (+/- 5.68) nM; Bmax 0.16 (+/- 0.20) and 2.65 (+/- 1.21) pmol/mg protein]. 4. In Krebs medium, beta-bungarotoxin was a very weak antagonist of the binding of 125I-labelled dendrotoxin. In imidazole medium, however, the efficacy of the inhibition was markedly increased; analysis of this inhibition showed it to be non-competitive. 5. Dendrotoxin inhibited the binding of radioiodinated beta-bungarotoxin in Krebs medium with high potency, although the interaction was by a complex, non-competitive mechanism. 6. Mast-cell-degranulating peptide inhibited non-competitively the binding of both radiolabelled dendrotoxin and beta-bungarotoxin but with relatively low potency. 7. A speculative schematic model of the dendrotoxin/beta-bungarotoxin/mast-cell-degranulating peptide binding component(s) is proposed. Findings are discussed in terms of the likely involvement of these sites with voltage-dependent K+-channel proteins.     

Differential Effects of Iodide and Chloride on Allosteric Interactions of the GABAA Receptor

t-[35S]Butylbicyclophosphorothionate [( 35S]TBPS) has been shown to bind to the GABAA receptor complex. The binding is modulated allosterically by drugs that interact at components of the receptor complex. The present studies were designed to evaluate the influence of ionic environment and state of equilibrium on the allosteric modification of [35S]TBPS binding. In both I- and Cl- under nonequilibrium conditions, diazepam, gamma-aminobutyric acid (GABA), and pentobarbital (PB) stimulate and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) inhibits [35S]TBPS binding. In addition, there is an inhibitory component to the effect of GABA and PB at higher drug concentrations. These effects are blocked by the appropriate antagonists for each drug. In Cl-, the stimulation of [35S]TBPS binding by drugs disappears at equilibrium, whereas the inhibition by GABA and PB persists. The inhibitory effect of DMCM in Cl- also disappears at equilibrium. When assayed in I- at equilibrium, however, DMCM stimulates [35S]TBPS binding. In addition, bicuculline, which is without effect under nonequilibrium conditions in either Cl- or I-, stimulates [35S]TBPS binding in I- at equilibrium. The persistent effects of DMCM, bicuculline, and GABA in I- are accompanied by alterations in the affinity of [35S]TBPS for its receptor. In addition, the stimulation of [35S]TBPS binding by GABA is associated with a decreased number of [35S]TBPS binding sites. These data demonstrate that receptor complex interactions with anions influence the responsiveness to drug binding.