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PubMed1992

Photoaffinity labeling of avermectin binding sites from Caenorhabditis elegans and Drosophila melanogaster.

An azido-avermectin analog [4'' alpha-(4-azidosalicylamido-epsilon-caproylamido-beta-alan ylamido)-4''-deoxyavermectin B1a; azido-AVM] was synthesized and used to photoaffinity label avermectin binding sites present in the membranes of Caenorhabditis elegans and Drosophila melanogaster. Azido-AVM was biologically active and behaved like a competitive inhibitor of [3H]ivermectin binding to C. elegans membranes (Ki = 0.2 nM). Radiolabeled azido-AVM bound specifically and with high affinity to C. elegans membranes (Kd = 0.14 nM) and, upon photoactivation, became covalently linked to three C. elegans polypeptides of 53, 47, and 8 kDa. Photoaffinity labeling of a membrane preparation from D. melanogaster heads resulted in labeling of a single major polypeptide of approximately 47 kDa. The proteins that were covalently tagged in these experiments are believed to be associated with avermectin-sensitive chloride channels present in the neuromuscular systems of C. elegans and D. melanogaster. Azido-AVM did not bind to rat brain membranes and therefore was selective for the nematode and insect receptors.

Affinity Labels↗
PubMed1986

High yield photoaffinity labeling of angiotensin II receptors.

The angiotensin II analogues, [Sar1, (4'-N3)Phe8]All and [Sar1, (4'-N3)D-Phe8]All were synthetized and prepared in their iodinated and radioiodinated forms. On rat liver membranes 125I-SarN3PheAll and 125I-SarN3DPheAll labeled a single population of sites (maximal binding capacity, 1.13 +/- 0.16 pmol/mg of protein) with high affinity (dissociation constants of 0.18 +/- 0.05 and 0.37 +/- 0.16 nM, respectively) and high specificity (nonspecific binding less than 10% of total binding for an 0.8 fractional receptor occupancy). 125I-SarN3PheAll and 125I-SarN3DPheAll allowed photoaffinity labeling of liver angiotensin receptors with high efficiency. The yield of photoaffinity labeling was independent of fractional receptor occupancy. 125I-SarN3PheAll and 125I-SarN3DPHeAll allow recovery in a soluble and covalently labeled form of about 40% of the total number of angiotensin receptors from rat liver membranes. For this reason they can be considered as potentially very useful tools for the purification of angiotensin receptors. The material covalently labeled with the antagonist 125I-SarN3DPHeAll was eluted from an Ultrogel ACA34 column as a homogeneous peak (Stoke's radius: 5.5 +/- 0.2 nm). The material covalently labeled with the agonist 125I-SarN3PHeAll was more heterogeneous. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of 125I-SarN3PheAll- and 125I-SarN3DPHeAll-labeled material revealed a single component of 63,000 molecular weight.

Adenylyl Cyclases↗
PubMed1997

Photoaffinity labeling of the ligand-interacting helix of the retinoic acid receptor alpha.

Two photoaffinity-labeling probes for retinoic acid receptor (RAR) alpha, 4-[(3-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl)carboxamido]ben zoic acid (3DIAM) and its para-isomer (4DIAM), were designed and synthesized. Both compounds possess high affinity for recombinant RAR alpha (MBP-RAR alpha/E) and bind covalently to its cognate ligand-binding site. The labeled site of MBP-RAR alpha/E with 3DIAM was determined, by the endoproteinase combination method, to be located in helix 11 of the ligand-binding domain of RAR alpha, which is the position at which the ligand is considered to bind, on the basis of the reported crystal structure of the retinoic acid/RAR gamma complex.

Azirines↗
PubMed1992

Photoaffinity labelling of steroid-hormone-binding glutathione S-transferases with [3H]methyltrienolone. Inhibition of steroid-binding activity by the anticarcinogen indole-3-carbinol.

The identification and characterization of steroid-hormone-binding glutathione S-transferases (GST) were undertaken using photoaffinity-labelling techniques. Irradiation of mouse liver cytosol, in the presence of 50 nM-[3H]methyltrienolone, resulted in the specific affinity labelling of five proteins. One of these proteins, designated MBP27, had an approximate molecular mass of 27 kDa under denaturing conditions and was induced by treatment of mice with either 2(3)-t-butyl-4-hydroxyanisole (BHA) or phenobarbital (PB). An additional affinity-labelled protein, MBP25, which was not detected in untreated mouse cytosol, was induced in the liver cytosols from BHA- and PB-treated mice. The molecular masses of these proteins and their induction by BHA and PB suggested that they may be steroid-hormone-binding GST subunits. Irradiation of mouse liver cytosol in the presence of [3H]methyltrienolone, followed by immunoprecipitation using GST-specific antibodies established that both GST mu and GST alpha bind [3H]methyltrienolone and both contribute to the affinity-labelled protein designated MBP27. GST Ya1 Ya1, an alpha class GST that is not expressed in untreated mouse liver but is induced by BHA and PB, was also found to bind [3H]methyltrienolone and is identical with the affinity-labelled protein designated MBP25. Experiments were undertaken next to assess the effects of the anticarcinogenic plant compound indole-3-carbinol (I3C) on GST-mediated steroid hormone-binding using the photoaffinity labelling techniques. Treatment of mice with I3C resulted in the induction of immunoreactive GST mu and GST Ya1 Ya1. However, the steroid-binding activity of these proteins in vitro was severely inhibited by the acid-condensation products of I3C that are generated in the stomach after ingestion. These results suggest that I3C may inhibit GST-mediated steroid-binding activity which could contribute to the anticarcinogenic activity of this compound.

Affinity Labels↗
PubMed2005

Fluorescent photoaffinity labeling of cytochrome P450 3A4 by lapachenole: identification of modification sites by mass spectrometry.

While photoaffinity ligands (PALs) have been widely used to probe the structures of many receptors and transporters, their effective use in the study of membrane-bound cytochrome P450s is less established. Here, lapachenole has been used as an effective photoaffinity ligand of human P450 3A4, and mass spectrometry data demonstrating the efficient and specific photoaffinity labeling of CYP3A4 by this naturally occurring benzochromene compound is presented. Without photolysis, lapachenole is a substrate of CYP3A4 and can be metabolized to hydroxylated products by this enzyme. A high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) procedure was developed to analyze small amounts of intact purified CYP3A4, and analysis of the labeled protein showed the presence of one molecule of lapachenole bound per monomer of protein. Photolabeled CYP3A4 peptide adducts were further characterized by mass spectrometric analysis after proteolytic digestion and isolation of fluorescent photolabeled peptides. Two peptide adducts accounting for >95% of the labeled peptides were isolated by HPLC, and both peptides, ECYSVFTNR (positions 97-105) and VLQNFSFKPCK (positions 459-469), were identified by nano-LC/ESI quadrupole time-of-flight (QTOF) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. The sites of modification were further localized to positions Cys-98 and Cys-468 for each peptide by nano-LC/ESI QTOF tandem mass spectrometry (MS/MS). The results provided the first direct evidence for interaction between the PAL and the putative B-B' loop region, which may serve as a substrate access channel or as a part of the CYP3A4 active site. In conclusion, benzochromene analogues are effective PALs, which may be used in the study of other cytochrome P450 structures.

Benzopyrans↗
PubMed1983

Inhibition of bile salt transport in brush-border membrane vesicles from rat small intestine by photoaffinity labeling.

The uptake of photolabile bile acid derivative, (7,7-azo-3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24 oyl)-2-aminoethanesulfonate (7,7-azo-TC), was investigated in rat ileal brush-border membrane vesicles. The uptake of 7,7-azo-TC showed a transient vesicle to medium ratio greater than one in the presence of a Na+ gradient. The Na+-dependent uptake of 7,7-azo-TC was inhibited by taurocholate and vice versa. 130 microM 7,7-azo-TC inhibited Na+-dependent taurocholate uptake by 50%. The degree of inhibitory power on taurocholate uptake was taurodeoxycholate greater than cholate greater than 7,7-azo-TC. Photoaffinity labeling of membrane vesicles with 7,7-azo-TC irreversibly inhibited Na+-dependent taurocholate and D-glucose transport but not Na+-dependent L-alanine transport. Kinetic and photoaffinity labeling experiments indicate that this representative photoaffinity probe interacts with the ileal Na+, bile salt cotransporter and may be used to identify polypeptide components of this transport system.

Affinity Labels↗
PubMed1993

Intrinsic photoaffinity labeling of native and recombinant rat pancreatic secretin receptors.

BACKGROUND: Structural characterization of pancreatic secretin receptors has been limited by difficulties in generating suitable radioligands and obtaining sufficient substrate. The aims of this study were to design, synthesize, and characterize high affinity radiolabeled analogues of secretin suitable for "intrinsic" photoaffinity labeling and to clone, express, and characterize the recombinant rat pancreatic secretin receptor. METHODS: The ability of synthetic analogues to stimulate amylase secretion by pancreatic acini was studied. Receptor complementary DNA (cDNA) was cloned by screening a rat pancreatic library with a probe based on the sequence of a neural cell secretin-binding protein. Competition binding and affinity labeling were performed with membranes prepared from rat pancreas and transfected cells. RESULTS: Two probes were fully efficacious secretagogues, which bound in a specific, high-affinity, rapid, and temperature-dependent manner. Only ([125I]Tyr10, pNO2-Phe22) rat secretin 27 covalently labeled a 50,000-62,000-molecular weight pancreatic membrane protein, with labeling inhibited in a concentration-dependent manner by secretin but not vasoactive intestinal polypeptide. Hybridization screening yielded a full-length cDNA identical to the neural clone. Photoaffinity labeling of this recombinant receptor identified a 57,000-62,000-molecular weight protein with specificity similar to that of native pancreas. Both native and recombinant receptors migrated at a molecular weight of 42,000 after endoglycosidase F deglycosylation. CONCLUSIONS: This study provides evidence for the molecular identity of the pancreatic secretin receptor and presents a novel probe important in structural characterization of its agonist-binding domain.

Affinity Labels↗
PubMed1983

Photoaffinity labelling of the benzodiazepine receptor cannot be used to predict ligand efficacy.

Irreversible attachment of flunitrazepam to brain membranes occurs on exposure to UV light, resulting in a loss of the number of binding sites detected by saturation binding of [3H]flunitrazepam, but not of [3H]ethyl-beta-carboline-3-carboxylate (beta CCE). Changes in the affinity of ligands for the benzodiazepine receptor produced by photoaffinity labelling, as indicated by the relative IC50 values for the displacement of [3H]beta CCE in photoaffinity labelled versus control membranes, appear to be related to their chemical structure rather than to their pharmacological profile.

Animals↗
PubMed1988

Analysis of photoaffinity label derivatives to probe thyroid hormone receptor in human fibroblasts, GH1 cells, and soluble receptor preparations.

The regulation of growth hormone gene expression by thyroid hormone in cultured GH1 cells is mediated by a chromatin-associated receptor. We have previously described a photoaffinity label derivative of 3,5,3'-triiodo-L-thyronine (L-T3) in which the alanine side chain was modified to form N-2-diazo-3,3,3-trifluoropropionyl-L-T3 (L-[125I]T3-PAL). On exposure to 254 nm UV light, L-[125I]T3-PAL generates a carbene which covalently modifies two thyroid hormone receptor forms in intact GH1 cells; an abundant 47,000 Mr species and a less abundant 57,000 Mr form. We have now synthesized similar photoaffinity label derivatives of 3,5,3',5'-tetraiodo-L-thyronine (L-T4) and 3,3',5'-triiodo-L-thyronine (L-rT3). Both compounds identify the same receptor forms in intact cells and in nuclear extracts in vitro as L-[125I]T3-PAL. Labeling by L-[125I]rT3-PAL was low and consistent with the very low occupancy of receptor by L-rT3. Underivatized L-[125I]T3 and L-[125I]T4 labeled the same receptor forms at 254 nm but at a markedly lower efficiency than their PAL derivatives. In contrast, N-bromoacetyl-L-[125I]T3, a chemical affinity labeling agent, did not derivatize either receptor form in vitro. The relative efficiency of coupling to receptor at 254 nm was L-[125I]T4-PAL greater than L-[125I]T3-PAL greater than L-[125I]T4 greater than L-[125I]T3. Although L-[125I]T4-PAL has a lower affinity for receptor than L-[125I]T3-PAL, its coupling efficiency was 5-10-fold higher. This suggests that the alanine side chain of L-[125I]T4-PAL is positioned in the ligand binding region near a residue which is efficiently modified by photoactivation. With L-[125I]T4-PAL we were able to identify three different molecular weight receptor species in human fibroblast nuclei.

Affinity Labels↗
PubMed1984

Photoaffinity labeling of the ouabain binding site in Na, K-ATPase in developing brine shrimp.

Analysis of purified Na,K-ATPase from brine shrimp nauplii by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals two large (alpha) subunits [G.L. Peterson, R.D. Ewing, S.R. Hootman, and F.P. Conte (1978) J. Biol. Chem. 253:4762]. The band with lower mobility in a neutral or alkaline gel is designated alpha 1 and the band with higher mobility alpha 2. Ouabain prevents dephosphorylation of both alpha 1 and alpha 2 as documented by gel analysis, but a higher concentration of ouabain is required to prevent dephosphorylation of alpha 2. The photoaffinity label, [3H]4'(2-ethyldiazomalonyl) digitoxigenin monodigitoxiside, specifically labels alpha in a ouabain-protectable manner without labeling other contaminating proteins in the preparation. Greater than 93% of the total ouabain-protectable labeling of the alpha subunits is associated with alpha 1. The photoaffinity label, [3H]4"' (2-ethyldiazomalonyl) digitoxin, specifically labels alpha 1 and beta in a ouabain-protectable manner without labeling other contaminating proteins. These data show that in the brine shrimp the third digitoxose residue of digitoxin binds in a region in which the alpha 1 and beta chains are in close proximity. Less than 5% of the specific ouabain-protectable labeling of total alpha is associated with alpha 2. These studies indicate that cardioactive steroids have higher affinity for the alpha 1 subunit.

Affinity Labels↗
PubMed2003

Photoaffinity labeling under non-energized conditions of a specific drug-binding site of the ABC multidrug transporter LmrA from Lactococcus lactis.

The Lactococcus lactis multidrug resistance ABC transporter protein LmrA has been shown to confer resistance to structurally and functionally diverse antibiotics and anti-cancer drugs. Using a previously characterized photoreactive drug analogue of Rhodamine 123 (iodo-aryl azido-Rhodamine 123 or IAARh123), direct and specific photoaffinity labeling of LmrA in enriched membrane vesicles could be achieved under non-energized conditions. This photoaffinity labeling of LmrA occurs at a physiologically relevant site as it was inhibited by molar excess of ethidium bromide>Rhodamine 6G>vinblastine>doxorubicin>MK571 (a quinoline-based drug) while colchicine had no effect. The MDR-reversing agents PSC 833 and cyclosporin A were similarly effective in inhibiting IAARh123 photolabeling of LmrA and P-glycoprotein. In-gel digestion with Staphyloccocus aureus V8 protease of IAARh123-photolabeled LmrA revealed several IAARh123 labeled polypeptides, in addition to a 6.8kDa polypeptide that comprises the last two transmembrane domains of LmrA.

ATP-Binding Cassette Transporters↗
PubMed1996

Structural studies of a human pi class glutathione S-transferase. Photoaffinity labeling of the active site and target size analysis.

The glutathione S-transferases (GSTs; EC 2.5.1.18) are a family of dimeric proteins that catalyze reactions between glutathione (GSH) and various electrophiles. A partial cDNA for human GST pi was obtained and the open reading frame completed. The completed cDNA was cloned, and GST pi protein was expressed in bacteria. Cloned enzyme was purified and had the same kinetic constants, molecular mass, pI value, and N-terminal sequence as placental GST pi except that some of the polypeptides had N-terminal methionines. A radiolabeled azido derivative of GSH, S-(p-azidophenacyl)-[3H]glutathione, was used to photoaffinity-label the active site of the cloned enzyme. Labeled enzyme did not bind to a GSH-agarose affinity column. Labeling was prevented in the presence of S-hexylglutathione, and noncovalently-bound azido affinity label was a competitive inhibitor towards 1-chloro-2,4-dinitrobenzene and GSH. These results suggest that the azido label was binding at the active site of the enzyme. Photoaffinity-labeled enzyme was trypsinized, and two labeled peptides were purified and sequenced. One peptide corresponded to residues 183-188, whereas the other corresponded to residues 183-186. These residues appear to form part of the hydrophobic (H-site) binding region of human GST pi that has not been shown previously. Cloned enzyme was subjected to radiation inactivation to assess the importance of subunit interactions in the maintenance of catalytic activity. The target size of enzymatic activity (23 kDa) was not significantly different from that of the protein monomer (24 kDa). Therefore, each subunit of human GST pi appears to be capable of independent catalytic activity.

Affinity Labels↗
PubMedDate not supplied

Evaluation of a highly efficient aryl azide photoaffinity labeling reagent for the progesterone receptor.

16 alpha,17 alpha-[(R)-1'-(4-Azidophenyl)ethylidenedioxy]pregn-4-ene- 3,20-dione (7) was prepared in high specific activity tritium-labeled form (20 Ci/mmol) and shown to bind to the progesterone receptor with an affinity (Kd = 0.80 nM) that is 47% of that of [3H]-R 5020 (Kd = 0.38 nM). [3H]Progestin aryl azide 7 exhibits high photoattachment efficiency (60% at 1 h) compared to the commonly used progesterone receptor photoaffinity labeling reagent [3H]-R 5020 (2.2% at 1 h) and is the most efficient progesterone receptor photoaffinity labeling reagent prepared to date. The photoattachment observed with 7 proceeds in a time-dependent fashion, with most of the attachment occurring within the first 10 min of photolysis. Characterization of the photolabeled proteins by SDS-polyacrylamide gel electrophoresis shows specific labeling of two adducts of molecular weight 108,500 +/- 800 and 87,000 +/- 1,500 (n = 3), the same species as labeled by [3H]-R 5020. The ratio of progesterone receptor subunits A:B was determined to be 3.3:1 with both [3H]progestin azide 7 and [3H]-R 5020. Information on the specific amino acid(s) that attach to the ligand during photolysis awaits further analysis of the covalently bound ligand-protein adduct.

Affinity Labels↗
PubMed1988

125I-BW-A844U, an antagonist radioligand with high affinity and selectivity for adenosine A1 receptors, and 125I-azido-BW-A844U, a photoaffinity label.

3-(4-Amino)phenethyl-1-propyl-8-cyclopentylxanthine (BW-A844U) has been synthesized and shown to bind with high affinity to adenosine A1 receptors of bovine brain membranes (KD = 0.23 nM). This compound is highly selective for A1 receptors; the KI for binding to A2 receptors of human platelet membranes is 2.0 microM (A2/A1 ratio = 8700). Radioiodination of the 3-aminophenethyl group resulted in 125I-BW-A844U, a radioligand that retains high affinity for A1 receptors in bovine brain membranes (KD = 0.14 nM) and to 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate-solubilized receptors (KD = 0.34 nM). Specific binding of 125I-BW-A844U represented greater than 90% of the total binding at the KD. From the association constant (K1 = 5.0 X 10(8) M-1min-1) and the dissociation constant (K-1 = 0.064 min-1), the kinetic KD (K-1/K1) in membranes was calculated to be 0.13 nM. NaCl (1 M) had little effect on the binding affinity of 125I-BW-A844U, in contrast to the large effect of salt on the binding affinity of acidic antagonist radioligands. 8-Sulfophenyltheophylline inhibited radioligand binding with a Hill coefficient of 1.0, indicative of a single affinity binding state for the antagonist. By comparison, two distinct agonist affinity states of A1 receptors for the agonist (R)-phenylisopropyladenosine could be resolved, a high affinity state (62%, KH = 74 pM) and a low affinity state (KL = 3.83 nM). The addition of 0.1 mM guanylylimidodiphosphate converted all receptors to the low affinity state. Addition of NaCl (0.5 M) decreased the fraction of receptors in the high affinity state and increased both KH and KL, suggesting that NaCl alters coupling of receptors to G proteins and influences the conformation of the receptor polypeptide, whether or not the receptor is coupled to a G protein. Conversion of the arylamine on the 3-position of 125I-BW-A844U to an aryl azide resulted in a photoaffinity label, 125I-azido-BW-A844U. Upon photoactivation, the photoaffinity label was specifically photoincorporated into the 34,000-dalton polypeptide of the adenosine A1 receptor.

Adenosine↗
PubMed1987

Cytoplasmic androgen binding protein of rat liver: molecular characterization after photoaffinity labeling and functional correlation with the age-dependent synthesis of alpha 2u-globulin.

The liver of the mature male rat contains a moderate affinity (Kd = 10(-8)M), low-capacity, cytoplasmic androgen binding protein (CAB) whose appearance during puberty and disappearance during senescence correlate with the androgen-dependent synthesis of alpha 2u-globulin. Molecular properties of CAB were examined by photoaffinity labeling with tritiated methyltrienolone (R-1881), a synthetic androgen, and by its localization within the hepatocytes which are competent to produce alpha 2u-globulin. Photoaffinity labeling of the liver cytosol derived from postpubertal male rats, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, showed a predominant androgen binding band corresponding to Mr 31,000. This 31-kilodalton (kDa) binding component was conspicuously absent in the liver of androgen-insensitive prepubertal and senescent male rats and in adult male rats treated with estradiol-17 beta. In addition, unlike the cytoplasmic extract, the nuclear lysate of the male rat hepatocytes did not contain the 31-kDa androgen binder. Disappearance of the 31-kDa androgen binding band from the cytosolic fraction of androgen-insensitive animals was associated with a concomitant appearance of a minor androgen binding component of apparent Mr 29,000. The livers of postpubertal male rats normally contain two subpopulations of hepatocytes, only one of which is highly active (competent) in alpha 2u-globulin synthesis. Separation of these two subpopulations through a fluorescence-activated cell sorter followed by whole cell labeling showed more than a 2-fold higher uptake of R-1881 by the competent cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1982

Direct photoaffinity labeling of an allosteric site on subunit protein M1 of mouse ribonucleotide reductase by dTTP.

The protein M1 subunit of ribonucleotide reductase contains at least two allosteric nucleotide binding sites that control the capacity of the enzyme to reduce ribonucleotides to the deoxyribonucleotides required for DNA synthesis. Direct photoaffinity labeling of partially purified protein M1 from mouse T-lymphoma (S49) cells was observed after UV irradiation in the presence of dTTP at 0 degrees C. The relative molar incorporation of nucleotide per subunit was 4-8%. Competition experiments showed that the dTTP was bound to an allosteric domain genetically and kinetically defined as the substrate specificity site of the enzyme. An altered protein M1 isolated from a thymidine-resistant mutant cell line showed significantly decreased photoincorporation of dTTP, consistent with the fact that its CDP reductase activity is resistant to feedback inhibition by dTTP. Specific photolabeling of several other proteins with pyrimidine and purine nucleotides was also found, indicating the general usefulness of direct photoaffinity labeling in the study of enzymes involved in nucleotide and nucleic acid metabolism.

Affinity Labels↗
PubMed1986

Photoaffinity labeling of neurotensin binding sites on rat brain sections.

The photoaffinity labeling of neurotensin (NT) binding sites was carried out on rat midbrain sections using a monoiodo analogue of NT (125I-azidobenzoyl [Trp11] NT; 125IAB-NT). Autoradiographic data showed that the 125IAB-NT binding site localization was quite similar to that obtained with 125I-NT, with high densities in both substantia nigra and ventral tegmental area. Covalent specific binding was only observed when sections were irradiated with UV after the incubation, followed by various histological treatments necessary for light and electron microscopy.

Affinity Labels↗
PubMed1987

Photoaffinity labeling of a novel benzodiazepine binding protein in rat brain.

Photoaffinity labeling experiments were conducted with [3H]clonazepam to investigate the possible existence of a benzodiazepine binding site that is distinct from the central- and peripheral-type benzodiazepine binding sites. These studies demonstrate the presence of a novel benzodiazepine binding protein in rat brain with a relative mobility of 65,000 daltons that binds benzodiazepines stereoselectivity in the high nanomolar-low micromolar concentration range.

Affinity Labels↗