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PubMed1990

The importance of residues 195-206 of human blood clotting factor VII in the interaction of factor VII with tissue factor.

Previous studies indicated that human and bovine factor VII exhibit 71% amino acid sequence identity. In the present study, competition binding experiments revealed that the interaction of human factor VII with cell-surface human tissue factor was not inhibited by 100-fold molar excess of bovine factor VII. This finding indicated that bovine and human factor VII are not structurally homologous in the region(s) where human factor VII interacts with human tissue factor. On this premise, we synthesized three peptides corresponding to regions of human factor VII that exhibited marked structural dissimilarity to bovine factor VII; these regions of dissimilarity included residues 195-206, 263-274, and 314-326. Peptide 195-206 inhibited the interaction of factor VII with cell-surface tissue factor and the activation of factor X by a complex of factor VIIa and tissue factor half-maximally at concentrations of 1-2 mM. A structurally rearranged form of peptide 195-206 containing an aspartimide residue inhibited these reactions half-maximally at concentrations of 250-300 microM. In contrast, neither peptide 263-274 nor peptide 314-326, at 2 mM concentration, significantly affected either factor VIIa interaction with tissue factor or factor VIIa-mediated activation of factor X. Our data provide presumptive evidence that residues 195-206 of human factor VII are involved in the interaction of human factor VII with the extracellular domain of human tissue factor apoprotein.

Amino Acid Sequence↗
PubMed1995

Cell cycle regulation of metallothionein in human colonic cancer cells.

Elevated levels of metallothionein (MT) found in rapidly growing tissues such as neonatal liver and various types of human tumors have suggested a role for MT in cell proliferation. To further explore this possibility we investigated the concentration of MT in human colonic cancer (HT-29) cells at different stages of proliferation by means of immunocytochemistry and competitive binding. MT is increased in subconfluent proliferating cells relative to growth-inhibited confluent cells, much as it is in growing tissues. Cycling cells synchronized with compactin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, revealed an oscillation of cytoplasmic MT that reached a maximum in successive late G1 phases and at the G1/S transition. Individual phase of the cell cycle were assessed by [3H]thymidine incorporation and by immunofluorescence employing an antibody that detects a nuclear antigen associated with proliferation. An enzyme-linked immunosorbent assay was used to quantify the relative amounts of MT in homogenate supernatants of HT-29 cells. A 2- to 3-fold increase in MT in actively proliferating cells and the regulation of the protein during the mitotic cell cycle point to a physiological role for MT in cellular proliferation and suggest that it may also serve as a proliferation marker.

Antibodies, Monoclonal↗
PubMed1996

Cloning and functional expression of cDNAs encoding human and rat pancreatic polypeptide receptors.

PCR was used to isolate nucleotide sequences that may encode novel members of the neuropeptide Y receptor family. By use of a PCR product as a hybridization probe, a full-length human cDNA was isolated that encodes a 375-aa protein with a predicted membrane topology identifying it as a member of the G-protein-coupled receptor superfamily. After stable transfection of the cDNA into human embryonic kidney 293 cells, the receptor exhibited high affinity (Kd = 2.8 nM) for 125I-labeled human pancreatic polypeptide (PP). Competition binding studies in whole cells indicated the following rank order of potency: human PP = bovine PP > or = human [Pro34]peptide YY > rat PP > human peptide YY = human neuropeptide Y. Northern blot analysis revealed that human PP receptor mRNA is most abundantly expressed in skeletal muscle and, to a lesser extent, in lung and brain tissue. A rat cDNA clone encoding a high-affinity PP receptor that is 74% identical to the human PP receptor at the amino acid level was also isolated. These receptor clones will be useful in elucidating the functional role of PP and designing selective PP receptor agonists and antagonists.

Amino Acid Sequence↗
PubMed1995

Structure-function analysis of the p35 subunit of mouse interleukin 12.

Mouse IL-12 acts on both mouse and human cells; human IL-12 acts only on human cells. This species specificity is determined by the p35 subunit of the IL-12 heterodimer. Since mouse and human p35 sequences are 60% identical, the determinants for the species specificity most likely residue in the nonhomologous sequences of mouse p35. To identify the regions on the p35 subunit interacting with the mouse IL-12 receptor, we constructed a series of chimeric mouse-human p35 molecules by replacing mouse sequences with the nonhomologous human counterparts. An IL-12 heterodimer containing a mouse-human p35 chimera with five residues changed in three discontinuous sites had drastically reduced (750-3000-fold) bioactivities on mouse cells. However, the competitive binding activity of the same mutant IL-12 heterodimer on mouse cells was only reduced 30-fold relative to wild-type IL-12. These findings therefore suggest that 1) the mouse p35 subunit participates in both receptor binding and signaling, 2) the mutations introduced into p35 affect signaling to a much greater extent than receptor binding, and 3) the five residues identified on p35 are required for interacting with the mouse, but not with the human IL-12 receptor and as such contribute extensively to the observed species specificity of IL-12.

Amino Acid Sequence↗
PubMed1995

Regulation of the uncoupling protein gene (Ucp) by beta 1, beta 2, and beta 3-adrenergic receptor subtypes in immortalized brown adipose cell lines.

Immortalized brown adipocyte cell lines derived from a mouse hibernoma express all three beta-adrenergic receptor subtypes, including beta 3-adrenergic receptor (AR). In response to norepinephrine, cAMP production by plasma membranes from four clonal cell lines was stimulated to levels comparable with brown adipocytes isolated from interscapular brown adipose tissue (72.8-89.6 versus 97.8 pmol cAMP/min/mg of protein, respectively). All cell lines responded to the highly selective beta 3-adrenergic receptor agonist CL316,243 by stimulating adenylyl cyclase activity (3-10-fold over basal). beta 1-, beta 2-, and beta 3-adrenergic receptor mRNA was detected by Northern blotting and/or reverse transcriptase-polymerase chain reaction. Competition binding assays with the antagonists CGP20712A and 125I-cyanopindolol showed the proportions of beta 1AR and beta 2AR in immortalized cells to be similar to brown adipocytes from tissue (cells: 35% beta 1AR, 65% beta 2AR; brown adipocytes from tissue: beta 1AR 41%, 59% beta 2AR). Expression of brown fat-specific mitochondrial uncoupling protein (Ucp) was stimulated by beta-adrenergic agonists in two of the four cell lines. The ability of individual beta AR subtypes to regulate Ucp expression was examined with combinations of selective beta-adrenergic agonists and antagonists. Expression of Ucp could be induced by any of the beta-adrenergic receptor subtypes. However, the greatest response was obtained by stimulating all three beta-adrenergic receptor subtypes simultaneously (100 microM isoproterenol). Incubation of membranes from cultured cells or brown adipocytes from tissue with CL316,243 at an optimal concentration (5 microM) did not prevent norepinephrine from further stimulating adenylyl cyclase activity, suggesting that the combined activation of beta 1AR/beta 2AR, plus beta 3AR, together produced an additive cAMP response. Multiple forms of adenylyl cyclase were identified in brown and white adipocyte cell lines and tissues. Northern blot analysis detected adenylyl cyclase types 5, 6, and 10. Screening of reverse transcriptase-PCR products by DNA sequencing confirmed the identities of these forms and lower levels of additional isoforms, raising the possibility that beta-adrenergic receptor subtypes in adipocytes couple to distinct adenylyl cyclases. Because these cell lines display functional and phenotypic similarities to interscapular brown adipocytes, they will be a useful model to study the regulation of beta-adrenergic receptor expression and function, and the control of Ucp expression and activity.

Adenylyl Cyclases↗
PubMed1995

Calcium-dependent conformation of a mouse macrophage calcium-type lectin. Carbohydrate binding activity is stabilized by an antibody specific for a calcium-dependent epitope.

We established monoclonal antibodies (mAbs) against the mouse macrophage galactose/N-acetylgalactosamine-specific lectin (MMGL) that is a 42-kDa calcium-dependent lectin, using a solid phase carbohydrate binding assay as a novel strategy for screening mAbs. The specificity of six mAbs were investigated by antibody binding to native or recombinant forms (rML) of MMGL, flow cytometry, and immunoprecipitation using a macrophage cell line RAW264.7. Four of these mAbs strongly inhibited the binding of fluorescein 5-isothiocyanate-labeled galactosylated polylysine to immobilized rML, one inhibited moderately, and one did not inhibit binding. The competitive binding study revealed that the binding sites of these four blocking mAbs were closely related to each other but were different from the rest of these mAbs. A non-blocking mAb having a unique binding specificity (LOM-11) exhibited calcium-dependent binding to rML, suggesting that calcium-dependent epitope was not situated in the vicinity of the ligand binding site. Furthermore, pretreatment of rML with the mAb LOM-11 preserved ligand binding activity, especially in a low calcium environment. The four blocking mAbs mentioned above facilitated the binding of the mAb LOM-11 to rML. These results indicate that there is a positive cooperativity between the lectin's ligand binding site and its physically distinct calcium-dependent epitope.

Animals↗
PubMed1996

CXC chemokines bind to unique sets of selectivity determinants that can function independently and are broadly distributed on multiple domains of human interleukin-8 receptor B. Determinants of high affinity binding and receptor activation are distinct.

Human interleukin-8 receptors A (IL-8RA) and B (IL-8RB) are seven-transmembrane domain (TMD) neutrophil chemokine receptors with similar sequences (77% amino acid identity) and similar G protein selectivity, but markedly different selectivity for CXC chemokines. IL-8RB is selective for IL-8, growth-related oncogene alpha (GRO alpha) and neutrophil-activating peptide-2 (NAP-2), whereas IL-8RA is selective only for IL-8. To identify selectivity determinants, we made eight chimeric receptors exchanging: 1) the three main regions of sequence divergence between IL-8RA and IL-8RB (the N-terminal segment before TMD1, the region from TMD4 to the end of the second extracellular (e2) loop, and the C-terminal tail), and 2) the N-terminal segment of CC chemokine receptor 1, which does not bind CXC chemokines. Chimeras were tested by direct 125I-IL-8, 125I-GRO alpha, and 125I-NAP-2 binding, heterologous competition binding, and calcium flux assays using human embryonic kidney 293 cells stably transfected with receptor DNAs. The following results were obtained: 1) chimeric receptors had binding sites for IL-8, GRO alpha and NAP-2 distinct from those on IL-8RA and IL-8RB; 2) IL-8, GRO alpha and NAP-2 bound to overlapping but distinct sites that mapped differentially to multiple domains on IL-8RB; 3) high affinity radioligand binding and high agonist potency were separable functions for IL-8, GRO alpha and NAP-2, suggesting that the determinants of high affinity binding may not be critical for receptor activation; and 4) determinants of GRO alpha and NAP-2 selectivity were found in both the N-terminal segment before TMD1 and the region from TMD4 to the end of the e2 loop of IL-8RB, and functioned independently of each other. Stated reciprocally, the N-terminal segment of IL-8RA was not a dominant selectivity determinant. These data suggest that both narrow and broad spectrum chemokine antagonists can be developed to block functions mediated by IL-8RB.

Amino Acid Sequence↗
PubMed1996

Mechanism of action cryptophycin. Interaction with the Vinca alkaloid domain of tubulin.

Cryptophycin is a potent antitumor agent that depletes microtubules in intact cells, including cells with the multidrug resistance phenotype. To determine the mechanism of action of cryptophycin, its effects on tubulin function in vitro were analyzed. Cryptophycin reduced the in vitro polymerization of bovine brain microtubules by 50% at a drug:tubulin ratio of 0.1. Cryptophycin did not alter the critical concentration of tubulin required for polymerization, but instead caused substoichiometric reductions in the amount of tubulin that was competent for assembly. Consistent with its persistent effects on intact cells, cryptophycin-treated microtubule protein remained polymerization-defective even after cryptophycin was reduced to sub-inhibitory concentrations. The effects of cryptophycin were not due to denaturation of tubulin and were associated with the accumulation of rings of microtubule protein. The site of cryptophycin interaction with tubulin was examined using functional and competitive binding assays. Cryptophycin blocked the formation of vinblastine-tubulin paracrystals in intact cells and suppressed vinblastine-induced tubulin aggregation in vitro. Cryptophycin inhibited the binding of [3H]vinblastine and the hydrolysis of [gamma32P]GTP by isolated tubulin, but did not block the binding of colchicine. These results indicate that cryptophycin disrupts the Vinca alkaloid site of tubulin; however, the molecular details of this interaction are distinct from those of other antimitotic drugs.

Animals↗
PubMed1998

Expression, characterization, and purification of C-terminally hexahistidine-tagged thromboxane A2 receptors.

Thromboxane A2 (TxA2) receptors belong to the class of G-protein-coupled receptors. Knowledge of the relationship of structure to function for TxA2 receptors is limited because of their low levels of expression, lengthy purification procedures and poor recoveries. A C-terminal hexahistidine-tag (C-His) was ligated to the alpha-isoform of TxA2 receptors and expressed in COS-7 and Chinese hamster ovary cells. The C-His-TxA2 receptors bound the radioligands 125I-7-[(1R,2S,3S,5R)-6, 6-dimethyl-3-(4-benzenesulfonylamino)bicyclo[3.1. 1]hept-2-yl]-5(Z)-heptenoic acid, an antagonist, and 125I-[1S-1alpha, 2beta(5Z),3alpha(1E,3S*), 4alpha]-7-[3[(3-hydroxy-4-(4'-phenoxy)-1butenyl)-7-oxabicycl o-[2.2. 1]heptan-2-yl]-5-heptanoic acid, an agonist, with affinities not significantly different from those of the wild type (wt)-TxA2 receptors. LipofectAMINE transfection of the cDNAs resulted in high levels of expression (Bmax = 95 +/- 6 pmol/mg) of the C-His-TxA2 receptors. In competition binding studies the IC50 values of five different ligands were not significantly different between C-His-TxA2 and wt-TxA2 receptors. Agonist-induced stimulation of cAMP and total inositol phosphate formation were not significantly different between the two receptors. Purification on a Ni2+-NTA column resulted in a rapid (within 4 h) purification with a 36 +/- 2% recovery and a 30 +/- 6-fold purification (n = 5). The partially purified receptors were resolved on SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, dissolved in acetone/trifluoroacetic acid/hexafluoroisopropanol/sinapinic acid, and successfully subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. The results suggest that the combination of a high level of expression of C-His-TxA2 receptors and a rapid purification procedure followed by SDS- polyacrylamide gel electrophoresis may provide a useful approach for mass-spectrometry based structure-function and other studies of TxA2 receptors.

Affinity Labels↗
PubMed1999

RNA editing of the human serotonin 5-hydroxytryptamine 2C receptor silences constitutive activity.

RNA transcripts encoding the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor (5-HT2CR) undergo adenosine-to-inosine RNA editing events at up to five specific sites. Compared with rat brain, human brain samples expressed higher levels of RNA transcripts encoding the amino acids valine-serine-valine (5-HT2C-VSV) and valine-glycine-valine (5-HT2C-VGV) at positions 156, 158, and 160, respectively. Agonist stimulation of the nonedited human receptor (5-HT2C-INI) and the edited 5-HT2C-VSV and 5-HT2C-VGV receptor variants stably expressed in NIH-3T3 fibroblasts demonstrated that serotonergic agonists were less potent at the edited receptors. Competition binding experiments revealed a guanine nucleotide-sensitive serotonin high affinity state only for the 5-HT2C-INI receptor; the loss of high affinity agonist binding to the edited receptor demonstrates that RNA editing generates unique 5-HT2CRs that couple less efficiently to G proteins. This reduced G protein coupling for the edited isoforms is primarily due to silencing of the constitutive activity of the nonedited 5-HT2CR. The distinctions in agonist potency and constitutive activity suggest that different edited 5-HT2CRs exhibit distinct responses to serotonergic ligands and further imply that RNA editing represents a novel mechanism for controlling physiological signaling at serotonergic synapses.

3T3 Cells↗
PubMed2000

Dipeptidyl peptidase IV (DPIV/CD26) degradation of glucagon. Characterization of glucagon degradation products and DPIV-resistant analogs.

Over the past decade, numerous studies have been targeted at defining structure-activity relationships of glucagon. Recently, we have found that glucagon(1-29) is hydrolyzed by dipeptidyl peptidase IV (DPIV) to produce glucagon(3-29) and glucagon(5-29); in human serum, [pyroglutamyl (pGlu)(3)]glucagon(3-29) is formed from glucagon(3-29), and this prevents further hydrolysis of glucagon by DPIV (H.-U. Demuth, K. Glund, U. Heiser, J. Pospisilik, S. Hinke, T. Hoffmann, F. Rosche, D. Schlenzig, M. Wermann, C. McIntosh, and R. Pederson, manuscript in preparation). In the current study, the biological activity of these peptides was examined in vitro. The amino-terminally truncated peptides all behaved as partial agonists in cyclic AMP stimulation assays, with Chinese hamster ovary K1 cells overexpressing the human glucagon receptor (potency: glucagon(1-29) > [pGlu(3)]glu- cagon(3-29) > glucagon(3-29) > glucagon(5-29) > [Glu(9)]glu- cagon(2-29)). In competition binding experiments, [pGlu(3)]glucagon(3-29) and glucagon(5-29) both demonstrated 5-fold lower affinity for the receptor than glucagon(1-29), whereas glucagon(3-29) exhibited 18-fold lower affinity. Of the peptides tested, only glucagon(5-29) showed antagonist activity, and this was weak compared with the classical glucagon antagonist, [Glu(9)]glucagon(2-29). Hence, DPIV hydrolysis of glucagon yields low affinity agonists of the glucagon receptor. As a corollary to evidence indicating that DPIV degrades glucagon (Demuth, et al., manuscript in preparation), DPIV-resistant analogs were synthesized. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry was used to assess DPIV resistance, and it allowed kinetic analysis of degradation. Of several analogs generated, only [D-Ser(2)] and [Gly(2)]glucagon retained high affinity binding and biological potency, similar to native glucagon in vitro. [D-Ser(2)]Glucagon exhibited enhanced hyperglycemic activity in a bioassay, whereas [Gly(2)]glucagon was not completely resistant to DPIV degradation.

Animals↗
PubMed2000

The first selective agonist for the neuropeptide YY5 receptor increases food intake in rats.

The first Y(5) receptor-selective analog of neuropeptide Y (NPY), [Ala(31),Aib(32)]NPY, has been developed and biologically characterized. Using competition binding assays on cell lines that express different Y receptors, we determined the affinity of this analog to be 6 nm at the human Y(5) receptor, >500 nm at the Y(1) and Y(2) receptors, and >1000 nm at the Y(4) receptor. Activity studies performed in vitro using a cAMP enzyme immunoassay, and in vivo using food intake studies in rats, showed that the peptide acted as an agonist. Further peptides obtained by the combination of the Ala(31)-Aib(32) motif with chimeric peptides containing segments of NPY and pancreatic polypeptide displayed the same selectivity and even higher affinity (up to 0.2 nm) for the Y(5) receptor. In vivo administration of the new Y(5) receptor-selective agonists significantly stimulated feeding in rats. The NMR solution structures of NPY and [Ala(31),Aib(32)]NPY showed a different conformation in the C-terminal region, where the alpha-helix of NPY was substituted by a more flexible, 3(10)-helical turn structure.

Amino Acid Motifs↗
PubMed2000

Identification and characterization of a potent, selective, and orally active antagonist of the CC chemokine receptor-1.

The CC chemokine receptor-1 (CCR1) is a prime therapeutic target for treating autoimmune diseases. Through high capacity screening followed by chemical optimization, we identified a novel non-peptide CCR1 antagonist, R-N-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl ]-2-oxoethoxy]phenyl]urea hydrochloric acid salt (BX 471). Competition binding studies revealed that BX 471 was able to displace the CCR1 ligands macrophage inflammatory protein-1alpha (MIP-1alpha), RANTES, and monocyte chemotactic protein-3 (MCP-3) with high affinity (K(i) ranged from 1 nm to 5.5 nm). BX 471 was a potent functional antagonist based on its ability to inhibit a number of CCR1-mediated effects including Ca(2+) mobilization, increase in extracellular acidification rate, CD11b expression, and leukocyte migration. BX 471 demonstrated a greater than 10,000-fold selectivity for CCR1 compared with 28 G-protein-coupled receptors. Pharmacokinetic studies demonstrated that BX 471 was orally active with a bioavailability of 60% in dogs. Furthermore, BX 471 effectively reduces disease in a rat experimental allergic encephalomyelitis model of multiple sclerosis. This study is the first to demonstrate that a non-peptide chemokine receptor antagonist is efficacious in an animal model of an autoimmune disease. In summary, we have identified a potent, selective, and orally available CCR1 antagonist that may be useful in the treatment of chronic inflammatory diseases.

Administration, Oral↗
PubMed2000

Characterization of type XI collagen-glycosaminoglycan interactions.

Using competitive binding experiments, it was found that native type XI collagen binds heparin, heparan sulfate, and dermatan sulfate. However, interactions were not evident with hyaluronic acid, keratan sulfate, or chondroitin sulfate chains over the concentration range studied. Chondrocyte-matrix interactions were investigated using cell attachment to solid phase type XI collagen. Pretreatment of chondrocytes with either heparin or heparinase significantly reduced attachment to type XI collagen. Incubation of denatured and cyanogen bromide-cleaved type XI collagen with radiolabeled heparin identified sites of interaction on the alpha1(XI) and alpha2(XI) chains. NH(2)-terminal sequence data confirmed that the predominant heparin-binding peptide contained the sequence GKPGPRGQRGPTGPRGSRGAR from the alpha1(XI) chain. Using rotary shadowing electron microscopy of native type XI collagen molecules and heparin-bovine serum albumin conjugate, an additional binding site was identified at one end of the triple helical region of the collagen molecule. This coincides with consensus heparin binding motifs present at the amino-terminal ends of both the alpha1(XI) and the alpha2(XI) chains. The contribution of glycosaminoglycan-type XI collagen interactions to cartilage matrix stabilization is discussed.

Amino Acid Sequence↗
PubMed2006

Molecular properties of Oep21, an ATP-regulated anion-selective solute channel from the outer chloroplast membrane.

The flux of phosphorylated carbohydrates, the major export products of chloroplasts, is regulated at the level of the inner and presumably also at the level of the outer membrane. This is achieved through modulation of the outer membrane Oep21 channel currents and tuning of its ion selectivity. Refined analysis of the Oep21 channel properties by biochemical and electrophysiological methods revealed a channel formed by eight beta-strands with a wider pore vestibule of dvest approximately 2.4 nm at the intermembrane site and a narrower filter pore of drestr approximately 1 nm. The Oep21 pore contains two high affinity sites for ATP, one located at a relative transmembrane electrical distance delta = 0.56 and the second close to the vestibule at the intermembrane site. The ATP-dependent current block and reduction in anion selectivity of the Oep21 channel is relieved by the competitive binding of phosphorylated metabolic intermediates like 3-phosphoglycerate and glycerinaldehyde 3-phosphate. Deletion of a C-terminal putative FX4K binding motif in Oep21 decreased the capability of the channel to tune its ion selectivity by about 50%, whereas current block remained unchanged.

Adenosine Triphosphate↗
PubMed2002

Estrogenicity of organophosphorus and pyrethroid pesticides.

Although organophosphorus and pyrethroid pesticides are considered environmental contaminants, their estrogenic potentials are still ubiquitous and unclear. The present study was undertaken to evaluate the estrogenic activities of nine pesticides (phoxim, malathion, monocrotophos, dimethoate, opunal, fenvalerate, cypermethrin, permethrin, and deltamethrin) using three in vitro methods [E-Screen assay, estrogen receptor (ER) competitive binding assay, and pS2 expression assay]. All the pyrethroid pesticides tested induced MCF-7 cell proliferation significantly, while organophosphorus pesticides did not. The estrogenic potency were ranked as permethrin > fenvalerate > cypermethrin > deltamethrin. The proliferation induced by cypermethrin, permethrin, and deltamethrin was blocked by ICI 182.780, while fenvalerate only partly inhibited it. In addition, pyrethroid pesticides inhibited the binding of [3H]estradiol to ER, while the organophosphorus failed to do so. Fenvalerate, permethrin, and cypermethrin induced pS2 mRNA expression with varying potency, while there were no significant effects in deltamethrin-treated groups. Our findings provide evidence to support the idea that pyrethroid pesticides tested produce an ER-specific, agonist response. Fenvalerate induced MCF-7 cell proliferation by a mechanism not involving ER-mediated pathway. Organophosphorus pesticides tested showed no estrogenic potential. Compared with the pS2 expression assay, E-Screen was a more sensitive and useful assay for screening of the xenoestrogenic chemicals.

Animals↗
PubMed2001

Estrogen receptors in rat bone: their interaction with estrogen receptor modulators.

Estrogen receptors (ER) were studied in rat bone cytosol using immunoprecipitation, and Western blot technique. Ligand specificity of bone ER was studied using various known modulators of ER. Competitive experiments were performed under exchange conditions in bone tissue obtained from one day old rats. ER alpha and beta subtypes were identified using immunoblotting experiments compared with that of ovarian and uterine tissues. In competitive binding assay, maximum inhibition in specific 3H-E2 binding was shown by E2 followed by tamoxifen and diethylstilbestrol. 7-Hydroxycentchroman and 85/287 also inhibited specific 3H-E2 binding but were less potent as compared to tamoxifen and diethylstilbestrol. However, 85/287 was less effective (81%) as compared to 7-hydroxycentchroman. Polyacrylamide gel electrophoresis of cytosol and Western blot analysis revealed the presence of 55 kD and 66 kD ER immunoreactive bands corresponding to alpha and beta subtypes, respectively, in bone as well as in uterus. Interestingly, the concentration of 55 kD ER was 3-fold higher than that of 66 kD ER. Ovarian cytosol revealed the presence of a 55 kD band only in Western blot analysis. These studies suggest the action of estrogens/ER modulators on osteoblasts which contain a limited number of classical alpha as well as beta sub types of ER that are known to be structurally different in their hormone-binding domains.

Animals↗
PubMedDate not supplied

Homologous regulation of melanocortin-1 receptor (MC1R) expression in melanoma tumor cells in vivo.

As G protein-coupled receptors (GPCRs) are the target of numerous signaling molecules, including about half of the therapeutic drugs currently used, it is important to understand the consequences of homologous (ligand-induced) receptor regulation. Continuous exposure of GPCRs to agonist in vitro most frequently results in receptor down-regulation, but receptor up-regulation may occur as well. These phenomena are expected to play a role in the physiological adaptation to endogenous ligands and also in the response to repetitive administration of drugs in the clinic. However, there is little information on homologous regulation of GPCRs in vivo. Here, we report on the regulation of melanocortin-1 receptor (MC1R) expression in melanoma cells implanted into mice. Two melanoma cell lines were investigated, D10 and B16F1, which in vitro had previously been shown to undergo homologous receptor up- and down-regulation, respectively. After implantation into mice and exposure to the natural MC1R agonist alpha-melanocyte-stimulating hormone (alpha-MSH), cell-surface MC1R expression was evaluated by competition binding experiments in tumor membrane preparations. In B 16F1 cells, a single injection of 50 to 500 microg alpha-MSH induced a rapid but moderate dose-dependent MC1R down-regulation which could be totally reverted within 16-24 h. By continuous administration of alpha-MSH via osmotic minipumps, MC1R down-regulation was considerably amplified and reached the level observed in vitro, demonstrating that prolonged receptor interaction was necessary to induce a maximal effect in vivo. Similar results were obtained in vitro, which demonstrates that homologous MC1R regulation in B16F1 cells is essentially independent of the physiological environment. In D10 cells, however, up-regulation could not be reproduced in vivo, suggesting that MC1R up-regulation is more dependent on the physiological environment. These results demonstrate the importance of in vivo receptor regulation studies, in particular in view of the potential use of MC1R as a target for melanoma therapy.

Animals↗