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At least 1,207 records · Page 67Linked to original sources
PubMed1989

The relation between phenytoin-receptor and glucocorticoid in the induction of cleft palate with phenytoin in mice.

Both phenytoin (PHT) and glucocorticoid (GC) have been reported to be an effective teratogen for the production of cleft palate. The authors suggest the possibility that a part of GC-receptor to which PHT competitively bind (common receptor with PHT), participate in the production of cleft palate. In the present study, as one of the studies for the elucidation of the mechanism of cleft palate induction with PHT, we investigated the PHT-receptors and its relation with GC in the lung tissue of mice, which were either sensitive (CF1) or resistant (C57BL/6) to PHT or GC. Scatchard analysis of the specific binding of 14C-PHT to the cytosol protein revealed that the total capacity of PHT-receptor for CF1 (1,571 fmole/mg protein) was much greater than that for C57BL/6 (994 fmole/mg). The value of the Kd's for these strains, however, were 18.1 and 16.5 nM, respectively, indicating the absence of any great difference between both strains. The capacity of the common receptors with GC for CF1 (236 fmole/mg) was greater than that for C57BL/6 (126 fmole/mg). But, the value of the Kd's for these strains were 5.4 and 3.1 nM, respectively, indicating the absence of a great difference between the strains. The analysis indicated that the lung tissue cytosols of both strains also contain the proper receptor for PHT, which is in a much greater amount than that of the common receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormalities, Drug-Induced↗
PubMed1992

Cimaterol reduces beta-adrenergic receptor density in rat skeletal muscles.

Interactions between the beta-adrenoceptor agonist cimaterol and beta-adrenoceptors on rat skeletal muscle membranes were examined in two studies. In Exp. 1, muscle samples from eight Sprague-Dawley rats (female, approximately 200 g) were used for competition binding and autoradiographic studies using [125I]cyanopindolol (ICYP) as a radioligand. The affinities or dissociation constants for binding (KD values) for cimaterol in plantaris and soleus muscles were .68 and .92 microM, respectively. Muscle areas stained for succinic dehydrogenase had propranolol-resistant ICYP binding sites; cimaterol did not seem to compete for these sites. In Exp. 2, 60 Sprague-Dawley rats (female, approximately 218 g) were fed 0 or 10 ppm of cimaterol in rat diet that was ground. Groups were killed after 1, 3, 7, 14, or 28 d of treatment. Cimaterol increased BW gain up to 14 d after commencement of treatment, with little or no improvement thereafter. Enhanced weight gain in skeletal muscles also occurred up to 14 d of cimaterol treatment. Densities of beta-adrenoceptors in plantaris and soleus muscle membrane homogenates were estimated using a radioligand binding assay with ICYP. A significant reduction in the number of binding sites (Bmax) was observed after 3 d of cimaterol treatment in plantaris muscle without a change in the KD of ICYP binding. The percentage reductions in Bmax were 26.8, 42.2, 37.7, and 37.8% at 3, 7, 14, and 28 d after cimaterol administration, respectively. In the soleus muscle, significant reductions (44.1 and 29.8%) in Bmax were observed after 3 and 14 d of cimaterol treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Agonists↗
PubMed1994

Effects of dietary insulin-like growth factor I on growth and insulin-like growth factor receptors in neonatal calf intestine.

Colostrum is rich in IGF-I and IGF-II, and the dietary effects of recombinant human (rh)IGF-I on the newborn are of interest. The objective of this study was to examine the effects of dietary rhIGF-I on intestinal tissue growth and populations of IGF receptors. Twenty-three male diary calves were fed one of three experimental diets: 1) milk replacer plus isolated colostrum-derived globulins (MR-), 2) same as 1 plus 750 ng of rhIGF-I/mL (MR+), or 3) pooled cow colostrum (COL). After the first four feedings, all calves received milk replacer without additional globulins; calves fed the MR+ diet continued to receive the addition of 750 ng of rhIGF-I/mL until the experiment ended at 7 d after birth. Calves were killed and intestinal tissue was collected for in vitro [3H]thymidine incorporation studies. Incorporation differed among intestinal regions (duodenum, jejunum, and ileum). The MR+ calves had greater (P < .01) [3H]thymidine incorporation per unit of DNA than either the COL or MR- calves (31.8 vs 18.6 and 11.5 x 10(3) dpm/microgram of DNA, respectively). Competitive binding analysis indicated the presence of specific type 1 and type 2 intestinal IGF receptors. The IGF-I was more potent than IGF-II and insulin at inhibiting [125I]rhIGF-I binding (ED50 was 1.84, 9.17, and 1.91 ng/mL, respectively). The IGF-II was the only ligand capable of inhibiting [125I]rhIGF-II binding (ED50 was .30 nmol/mL).(ABSTRACT TRUNCATED AT 250 WORDS)

Animal Feed↗
PubMed1986

Relative affinities of estramustine phosphate metabolites to the cytosol oestrogen receptor of rat testis.

A competitive binding assay was used for analysis of the affinities of ethinyl oestradiol, diethylstilboestrol and metabolites of estramustine phosphate to the oestrogen receptor of rat testis. The affinities were ranked as follows: ethinyl oestradiol greater than oestradiol-17 beta greater than diethylstilboestrol much greater than estramustine. Estromustine did not reveal any oestrogen receptor affinity. The results support the concept that estramustine and estromustine do not mediate the oestrogenic effects of estramustine phosphate. The oestrogenicity of estramustine phosphate is suggested to be mainly due to the metabolite oestradiol-17 beta.

Animals↗
PubMedDate not supplied

Characterization of endothelin receptor subtypes on airway smooth muscle cells.

Endothelin-1 (ET-1) has constrictor and mitogenic effects on airway smooth muscle strips and cultured cells, respectively. This study addresses the type of the ET receptor subtype(s) present on ovine airway smooth muscle cells and the possibility of autocrine effects. The expression of the preproendothelin-1 gene was demonstrated by Northern analysis, and the medium obtained from these cells contained immunoreactive-ET-1. Competitive binding experiments between [125I]ET-1 and ET-1, ET-3, and two ET-receptor subtype selective-ligands, BQ-123 (ETA) and sarafotoxin S6c (ETB), yielded IC50 values of 1.1 +/- 0.1, 227 +/- 13, 12 +/- 1, and 194 +/- 21 nM, respectively. ET-3 also gave a limited number of higher affinity sites. In the presence of BQ-123 (1 microM), the binding of [125I]ET-1 was decreased by 80-85%, and the IC50 values with ET-1, ET-3, and S6c were 2.0 +/- 0.4, 3.6 +/- 0.6, and 1.1 +/- 0.9 nM, respectively. In similar experiments with 0.1 microM sarafotoxin S6c, the respective IC50 values for ET-1 and ET-3 were 2.4 +/- 0.4 and 300 +/- 20 nM. These results demonstrate that about 85 +/- 5% of ET-1 binding to airway smooth muscle cells is to ETA receptors and that these cells produce ET-1 in vitro.

Animals↗
PubMed1975

Quantitation of estradiol receptors employing a sensitive radioimmunoassay.

A method aimed at the identification and quantitation of estradiol receptors has been developed. The test is based on competitive binding reaction between solubilized cell receptors and anti-estradiol antibodies bound to acrylamide beads, for 3H-estradiol. The technique can be employed both for estradiol radioimmunoassay and quantitation of specific receptors in tissues and appears to be a sensitive, specific and reproducible method which can be applied to the evaluation of any hormone and its receptor.

Animals↗
PubMedDate not supplied

Alterations of angiotensin II Receptor levels in sutured wounds in rat skin.

Angiotensin II receptor levels have been shown to vary with postoperative time in tissue harvested from full-thickness dermal excisional wounds on adult rats. This study examined the expression of AII receptors in a sutured wound model. Two full-thickness incisional wounds were made in the dorsal skin of adult Sprague-Dawley rats and sutured immediately under general anesthesia. The wound tissues were harvested at 0, 0.5, 1, 2, 4, 24 h and on days 2, 3, 4, 5, 7, and 10 after the wounding. The levels of 125I-Sar1.Ile8-AII bound to membrane preparations of the wound tissues decreased at early time points (from 0.5 to 4 h), increased from day 1 to day 7, and returned to nonsurgical levels by day 10. Competitive binding studies showed that the receptors were predominantly of the AT1 receptor subtype. These results suggest that an immediate and transient reduction in AII receptor expression occurred after wounding, followed by an increase in the number of AII receptors that was maintained for 5 to 7 days postoperatively. Because these data are consistent with those observed after excisional wounding, temporal changes in AII receptor expression may be integral to the process of wound healing.

1-Sarcosine-8-Isoleucine Angiotensin II↗
PubMed1996

Characterization of receptors for insulin-like growth factor type I on cultured human endometrial stromal cells: downregulation by progesterone.

Because insulin-like growth factor type I (IGF-I) is reputed to be involved in the endometrial decidualization, we analyzed the expression of IGF-I receptors in an in vitro system of human endometrial stromal cells. Competitive binding studies of both intact stromal cells and membrane preparation indicated the presence of specific components with high affinity for binding IGF-I. Half-maximum displacement was obtained with 2.3 nmol/l native IGF-I, whereas insulin was unable to achieve half-maximum displacement even at higher concentrations. This IGF-I binding component was found to be a saturable protein in respect of the radioligand [125I]IGF-I, with a dissociation constant of 0.16 nmol/l. Affinity cross-linking studies revealed a labelled band of approximate relative molecular mass 135000, corresponding to the known alpha-subunit of IGF-I receptor. This band was significantly inhibited dose-dependently by the IGF-I receptor monoclonal antibody alpha-IR3 or native IGF-I, suggesting that the IGF-I binding component in the membrane of stromal cells has the identity of the alpha-subunit of IGF-I receptor. Cell proliferation in vitro was stimulated by progesterone. Furthermore, progesterone downregulated the [125I]IGF-I binding activity by downregulation of the IGF-I membrane receptor of human endometrial stromal cells. These data show that the IGF-I receptor is a functionally integral component of the stromal cell membrane structure, and its expression might be directly modulated by progesterone and, therefore, might play an important role in the preparation of the stroma for successful embryo implantation.

Autoradiography↗
PubMedDate not supplied

Stable expression of the human MSH receptor in a mouse melanoma cell line.

Stable expression of the MSH receptor in a homologous system is important for the study of the function and mechanism of signalling of this receptor. This is the first report on the stable expression of the human alpha-MSH receptor in the mouse melanoma G4F clone which lacks an endogenous MSH receptor. Several stable transfectant cell lines were obtained all of which express the human MSH receptor in high numbers. Human MSH receptor mRNA expression was detected by Northern blot analysis. Competition binding experiments showed that the MSH receptors expressed in these cells have the same affinity for [Nle4,D-Phe7]-alpha-MSH as the MSH receptors of the human HBL melanoma cell line. Several of the transfectant cell lines produced melanin constitutively, some of them secreting melanin into the medium whereas other clones did not secrete melanin. MSH and cholera toxin did not or only marginally increase melanogenesis in these clones, and forskolin had an opposite effect. These results suggest that the human MSH receptor may be constitutively active in these transfected mouse melanoma cells.

Animals↗
PubMed1983

Inhibition of programmed cell death in mouse embryonic palate in vitro by cortisol and phenytoin: receptor involvement and requirement of protein synthesis.

In an in vitro model cortisol and phenytoin inhibit the precisely timed process of palatal development, the lysosomally mediated cell death of the medial edge palatal epithelium. This inhibition of programmed cell death of the palatal midline epithelium by each drug is virtually completely blocked by the antiglucocorticoid, cortexolone, whose blocking action results from competitive binding of the glucocorticoid receptor site. The inhibition produced by each of these drugs is prevented by the protein synthesis blocker, cycloheximide. Thus, blockade of programmed cell death by each of these drugs involves the glucocorticoid receptor site and requires protein synthesis.

Animals↗
PubMed2001

Human IFN-alpha protein engineering: the amino acid residues at positions 86 and 90 are important for antiproliferative activity.

Human IFN-alpha is a family of structurally related proteins that exhibit a wide range of antiproliferative activities. To understand the structural basis for these different antiproliferative activities, eight recombinant human IFN-alpha hybrids (HY) of alpha21a/alpha2c (HY-4, HY-5) and mutants (site-directed mutagenesis (SDM)-1, 2 and cassette mutagenesis (CM)-1, 2, 3, and 4) have been expressed, purified, and characterized. The data showed that the amino acid region 81-95 is important for antiproliferative activity. Site-directed mutagenesis and cassette mutagenesis studies showed that if serine (S) 86 and asparagine (N) 90 were replaced by tyrosine (Y), the antiproliferative activity was increased. We have also observed that if Y86 was replaced by isoleucine (I), the antiproliferative activity was comparable. However, if Y86 was replaced by aspartic acid (D), lysine (K), or alanine (A), the antiproliferative activity was substantially decreased. Our results indicate that Y and/or I at position 86 and Y at position 90 are very important in antiproliferative activity of human IFN-alpha. Circular dichroism spectra showed that the amino acid replacements at position 86 did not change the secondary structure. Thus the biological activity changes among those mutants do not appear to be due to conformational changes. The results also suggest that hydrophobic residue(s) at position 86 may be important for the interaction of the molecule with its receptor. The competitive binding data correlated with the antiproliferative activity. The N-terminal region of the molecule and the hydrophobic residues (including Y and I) on the C-helix region at positions 86 and/or 90 are important for binding and antiproliferative activities of human IFN-alphas.

Amino Acid Sequence↗
PubMed1990

Seroepidemiology of human T cell lymphotropic virus in the Republic of Panama.

The human T-lymphotropic virus (HTLV) and associated diseases, adult T cell leukemia and spastic paraparesis, appear to be endemic in southwestern Japan and the Caribbean. This cross-sectional population-based study was conducted to describe the seroepidemiology of HTLV in the Republic of Panama. HTLV antibody was measured by first generation and commercial ELISA tests and confirmed by competitive binding ELISA, a radioimmunoassay for anti-p 24, and Western blot. Of 3,231 subjects greater than or equal to 15 years of age, 135 (4.2%) had antibody detected in ELISA screening tests, but because only 20% were confirmed positive, HTLV seroprevalence varied from 0.2-2% throughout the Republic. Infection with HTLV clustered in Guaymi Indians living in Bocas del Toro province (9.9% prevalence rate). With the exception of Guaymi Indians, no major geographic, urban/rural, male/female or racial differences in antibody prevalence were observed; specifically, HTLV infection rates were not elevated in black Panamanians. Clustering of infection in an isolated Amerind population must be further investigated. The small proportion of screen-positive sera which confirmed positive illustrates the importance of strict uniform criteria for seropositivity.

Binding, Competitive↗
PubMed2006

Detection of multiple antibiotic-resistant Salmonella enterica serovar Typhimurium DT104 by phage replication-competitive enzyme-linked immunosorbent assay.

A phage replication-competitive enzyme-linked immunosorbent assay (PR-cELISA) was developed for the detection of multiple antibiotic-resistant Salmonella Typhimurium DT104. In the PR-cELISA procedure, a phage, BP1, was inoculated into a log-phase bacterial culture at a ratio of 1:100. After a 3-h incubation of the mixture, BP1 replication was measured by cELISA based on the competitive binding between BP1 and biotinylated BP1 to Salmonella Typhimurium smooth lipopolysaccharide. Among the 84 Salmonella strains and 9 non-Salmonella strains that were tested by PR-cELISA, BP1 detected 39 of 40 Salmonella Typhimurium strains, 2 of 10 Salmonella non-Typhimurium somatic group B strains, and 5 of 18 Salmonella somatic group D1 strains. With the addition of chloramphenicol to the culture medium, PR-cELISA detected all 27 multiple antibiotic-resistant Salmonella Typhimurium DT104 and none of the other Salmonella strains or non-Salmonella strains tested. The results demonstrated that PR-cELISA has potential applications for the detection of multiple antibiotic-resistant Salmonella Typhimurium DT104.

Anti-Bacterial Agents↗
PubMed1999

(-)-Phenylahistin arrests cells in mitosis by inhibiting tubulin polymerization.

(-)-Phenylahistin, a fungal diketopiperazine metabolite composed of phenylalanine and isoprenylated dehydrohistidine, arrested cells in mitosis and inhibited the proliferation of A549 cells. The microtubule network in A549 cells was disrupted by (-)-phenylahistin, which also inhibited the polymerization of both microtubule protein from bovine brain and phosphocellulose-purified tubulin in vitro. Competitive binding studies indicated that (-)-phenylahistin interacted with the colchicine binding site on tubulin but not with the vinblastine binding site.

Animals↗
PubMed1999

Epitope mapping of mouse monoclonal antibodies to the ppUL83 lower matrix phosphoprotein of human cytomegalovirus.

Of nine mouse monoclonal antibodies (MAbs) directed against the lower matrix protein (pp65; ppUL83) of human cytomegalovirus (HCMV), all immunoprecipitated the 65-kDa protein. Only five were reactive by Western blotting, however, and four of these mapped to linear antigenic epitopes located between amino acids 184-195 (MAb C6), 343-357 (MAb C11), 448-462 (MAb C5), and 448-459 (MAb C13). The epitope specificity of the fifth antibody (MAb C3) and the four that recognised nonlinear sites could not be determined. Competition binding studies using HCMV antigen extracted from productively infected human embryonic lung fibroblasts (HELF), in an enzyme immunoassay (EIA), showed that three of the antibodies reactive with linear epitopes and two of those reactive with conformational epitopes (MAbs C3, C6, C11, C14, and C18), were unique in their binding specificities. MAb C4 competed with MAb C8 and MAb C5 competed with MAb C13 for binding to ppUL83. One of the linear epitopes identified, corresponding to amino acids SAFVFPTKDVAL (MAb C6), was an epitope described previously for CD8+ cytotoxic T lymphocytes.

Amino Acid Sequence↗
PubMed1999

2,2',6,6'-Tetrachlorobiphenyl is estrogenic in vitro and in vivo.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants whose effects on biological systems depend on the number of and the positions of the chlorine substitutions. In the present study we examined the estrogenicity of the fully ortho-substituted PCB, 2,2',6,6'-tetrachlorobiphenyl (2,2',6,6'-TeCB). This PCB was chosen as the prototypical ortho-substituted PCB to test the hypothesis that ortho-substitution of a PCB with no para- or meta-chlorine-substitutions results in enhanced estrogenic activity. The results indicate that 2,2',6,6'-TeCB is estrogenic both in vitro, in the MCF-7 cell focus assay, and in vivo, in the rat uterotropic assay. The estrogenic activity elicited by the addition of 5 microM 2,2',6,6'-TeCB to the medium of MCF-7 cultures was inhibited by the estrogen receptor (ER) antagonist, LY156758, suggesting that 2,2',6,6'-TeCB or a metabolite is acting through an ER-dependent mechanism. Results from competitive binding assays using recombinant human (rh) ER indicate that 2,2',6,6'-TeCB does not bind rhERalpha or rhERbeta. A metabolite of 2,2',6,6'-TeCB, 2,2',6,6'-tetrachloro-4-biphenylol (4-OH-2,6,2',6'-TCB), does bind rhERalpha and rhERbeta and is also 10-fold more estrogenic than 2,2',6,6'-TeCB in the MCF-7 focus assay; however, this metabolite is not detected in the medium of MCF-7 cultures exposed to 2,2',6,6'-TeCB. Taken together, the results suggest that the estrogenicity observed in human breast cancer cells and the rat uterus may be due to 1) an undetected metabolite of 2,2',6,6'-TeCB binding to the ER, 2) 2,2',6,6'-TeCB binding directly to a novel form of the ER, or 3) an unknown mechanism involving the ER.

Animals↗
PubMed1999

Isolation and characterization of human monoclonal antibodies to digoxin.

Fab preparations of sheep polyclonal anti-digoxin Abs have proven useful for reversal of the toxic effects of digoxin overdoses in patients. Unfortunately, the use of foreign species proteins in humans is limited because of the potential for immunological responses that include hypersensitivity reactions and acute anaphylaxis. Immunization of recently developed transgenic mice, whose endogenous micro heavy and kappa light chain Ig genes are inactivated and which carry human Ig gene segments, with a digoxin-protein conjugate has enabled us to generate and isolate eight hybridoma cell lines secreting human sequence anti-digoxin mAbs. Six of the mAbs have been partially characterized and shown to have high specificity and low nanomolar affinities for digoxin. In addition, detailed competition binding studies performed with three of these mAbs have shown them to have distinct differences in their digoxin binding, and that all three structural moieties of the drug, the primary digitoxose sugar, steroid, and five-member unsaturated lactone ring, contribute to Ab recognition.

Animals↗
PubMed1999

ATP sensitive potassium channel and myocardial preconditioning.

KATP channels play an important role in physiology and pathophysiology of many tissues. As in the pancreatic beta cells, they couple the change of blood glucose with insulin release. The data coming from Baukrowitz et al. and Shyng and Nichols gave the possible answers to the two old enigmas of KATP channels, i.e., different ATP sensitivity reported in the same tissue and how the channel opened under intracellular millimolar ATP concentration, in which they showed the lipids and lipid metabolites are essential for KATP channel regulation by altering ATP sensitivity. This new information rises several further considerations. How does PIP2 reduce the sensitivity of the channel to ATP? In order to clarify the possibility of direct competing or allosteric effect on the ATP binding site, competitive binding assay should be performed. Since the PIP2 theory seems to be the key event to determine the ATP sensitivity and thus control the channel open probability, then what is the resting concentration of PIP2 in the cell membrane? Is it sufficient to account for the difference in the ATP sensitivity of the intact cell and excised patch from different tissues? Quantitative studies either immunoblotting by PIP2 antibody or fluorescence-labeled lipid assay-may obtain some basic but useful data for further studies to answer these questions. Furthermore, the ATPi mediated restoration of activity was inhibited by antibodies against PIP2. The dualistic behavior of KATP channels to intracellular NDPs should be reexamined with respect to PIP2. The vast majority of preconditioning studies has been performed in intact animals in which myocardial infarct size was used as the end point to define the cardio-protective effect of ischemic PC. These results suggest a key role for the KATP channel as both a trigger and as an end effector of both acute and delayed ischemic PC. The persistent activation of KATP channels during the early reperfusion phase is essential for a smooth and full recovery of contractile function, as well as for maintenance of electrical stability in heart that has been exposed to ischemia. Though activate adenosine A1 receptor coupled with Gi protein can open the KATP channels, adenosine is quickly released during ischemia and exerts potent coronary vasodilatation to maintain coronary blood flow through A2 receptors. This adenosine-induced coronary vasodilatation could be coupled with KATP channels based on the evidence of the augmentation effect of KCOs. Nitric oxide may also play some role in both first and second window of myocardial protection. It is possible that rapid and reversible phosphorylation and activation of constitutive expressed myocardial NOS or by direct KATP channel phosphorylation and activation leads to the first window of myocardial protection. This hypothesis can be further investigated either by using site direct mutagenesis of iNOS or KATP channel, or by applying the dominant negative iNOS in the cell ischemic model, or by building the adenosine or iNOS knock-out mice to study the relationship of these possible mechanisms. Recently, Kontos further showed that KCOs need L-lysine or L-arginine to dilate cerebral arterioles. This suggests that there may be an amino acid binding site inside the KATP channel and nitric oxide can open the KATP channel either by direct acting on the channel protein or by modulating the affinity of the amino acid binding site for L-lysine or L-arginine. Other KATP channel openers in need of additional characterization are the Type III KCOs (nicorandiol). They open the KATP channel only in the presence of elevated intracellular NDPs, which may make them specifically target to the ischemic region, because the intracellular NDP increases mostly in ischemic region. It is possible that type III KCOs can selectively improve blood flow to ischemic areas without diverting blood away to non-ischemic region, and prevents the "steal phenomenon". (ABSTRACT TRUNCATED)

Adenosine Triphosphate↗