High-affinity Recognition Sites Research Articles

Nongenomic roles of thyroid hormones and their derivatives in adult brain: are these compounds putative neurotransmitters?

We review the evidence regarding the nongenomic (or non-canonical) actions ofthyroid hormones (thyronines) and their derivatives (including thyronamines and thyroacetic acids) in the adult brain. The paper seeks to evaluate these compounds for consideration as candidate neurotransmitters. Neurotransmitters are defined by their (a) presence in the neural tissue, (b) release from neural tissue or cell, (c) binding to high-affinity and saturable recognition sites, (d) triggering of a specific effector mechanism and (e) inactivation mechanism. Thyronines and thyronamines are concentrated in brain tissue and show distinctive patterns of distribution within the brain. Nerve terminals accumulate a large amount of thyroid hormones in mature brain, suggesting a synaptic function. However, surprisingly little is known about the potential release of thyroid hormones at synapses. There are specific binding sites for thyroid hormones in nerve-terminal fractions (synaptosomes). A notable cell-membrane binding site for thyroid hormones is integrin αvβ3. Furthermore, thyronines bind specifically to other defined neurotransmitter receptors, including GABAergic, catecholaminergic, glutamatergic, serotonergic and cholinergic systems. Here, the thyronines tend to bind to sites other than the primary sites and have allosteric effects. Thyronamines also bind to specific membrane receptors, including the trace amine associated receptors (TAARs), especially TAAR1. The thyronines and thyronamines activate specific effector mechanisms that are short in latency and often occur in subcellular fractions lacking nuclei, suggesting nongenomic actions. Some of the effector mechanisms for thyronines include effects on protein phosphorylation, Na+/K+ ATPase, and behavioral measures such as sleep regulation and measures of memory retention. Thyronamines promptly regulate body temperature. Lastly, there are numerous inactivation mechanisms for the hormones, including decarboxylation, deiodination, oxidative deamination, glucuronidation, sulfation and acetylation. Therefore, at the current state of the research field, thyroid hormones and their derivatives satisfy most, but not all, of the criteria for definition as neurotransmitters.

Facilely self-assembled and dual-molecule calibration aptasensor based on SERS for ultra-sensitive detection of tetrodotoxin in pufferfish

Tetrodotoxin (TTX) is one of the most lethal neurotoxins, so the reliable quantitative analysis of TTX is crucial for food and environmental safety monitoring. Herein, a novel dual-molecule calibration aptasensor was developed for detection of TTX based on Surface-enhanced Raman scattering (SERS). The adaptive surface has high affinity recognition sites for the target of interest, which ensures the high specificity and stability of the aptasensor. In addition, the uniquely labeled signal molecules located in the Raman silent region (1800–2400 cm−1) can avoid the interference of other exogenous biological signal molecules. Meanwhile, in quantitative analysis, the SERS signal generated by the reporter is calibrate in real time using the second-order peak of silicon molecule (Si). The detection linear range of the aptasensor was 0.0319 ng/mL-319.27 ng/mL, with a limit of detection (LOD) of 0.024 ng/mL and the excellent uniformity (RSD = 8.8%) for TTX detection. As a promising and versatile detection candidate, the ultra-sensitive quantitative detection aptasensor of TTX had important practical significance, which can offer more favorable persuasion for TTX analysis in real seafood samples.

Botulinum Neurotoxin Injection as a Therapeutic Option in Thoracic Outlet Syndrome Caused by a Supernumerary Scalenus Muscle: A Case Report

Thoracic Outlet Syndrome (TOS) is a neurovascular syndrome and may occur as a result of compression of brachial plexus elements and/or subclavian vessels as they traverse the cervicoaxillary canal. One of the causes of TOS is the existence of a supernumerary scalene muscle, usually denominated as scalenus minimus. Botulinum Toxin Type A (BoNT-A) acts by binding presynaptically to high-affinity recognition sites on the cholinergic nerve terminals and decreases the release of acetylcholine, suppressing muscle overactivity. BoNT-A reduces contraction in injected muscles, causing focal chemodenervation and has been described as a non surgical effective treatment in selected cases of TOS. Here, is the case of a 44-year-old woman diagnosed with refractory TOS, who presented with paresthesia in the fourth and fifth fingers of her right hand, associated with a feeling of lack of coordination in the same hand. On Magnetic Resonance Imaging (MRI), a supernumerary scalene muscle was identified as a probable cause of TOS. She was treated with chemodenervation of the scalenus anterior muscle (30 Units) and scalenus medius muscle (30 Units), using incobotulinumtoxin A. The patient had a very significant clinical improvement after Anterior Scalene (AS) and Middle Scalene (MS) muscle BoNT-A injections. Incobotulinumtoxin A neurotoxin injection is a therapeutic option in TOS.

Blocking, Bending, and Binding: Regulation of Initiation of Chromosome Replication During the Escherichia coli Cell Cycle by Transcriptional Modulators That Interact With Origin DNA.

Genome duplication is a critical event in the reproduction cycle of every cell. Because all daughter cells must inherit a complete genome, chromosome replication is tightly regulated, with multiple mechanisms focused on controlling when chromosome replication begins during the cell cycle. In bacteria, chromosome duplication starts when nucleoprotein complexes, termed orisomes, unwind replication origin (oriC) DNA and recruit proteins needed to build new replication forks. Functional orisomes comprise the conserved initiator protein, DnaA, bound to a set of high and low affinity recognition sites in oriC. Orisomes must be assembled each cell cycle. In Escherichia coli, the organism in which orisome assembly has been most thoroughly examined, the process starts with DnaA binding to high affinity sites after chromosome duplication is initiated, and orisome assembly is completed immediately before the next initiation event, when DnaA interacts with oriC’s lower affinity sites, coincident with origin unwinding. A host of regulators, including several transcriptional modulators, targets low affinity DnaA-oriC interactions, exerting their effects by DNA bending, blocking access to recognition sites, and/or facilitating binding of DnaA to both DNA and itself. In this review, we focus on orisome assembly in E. coli. We identify three known transcriptional modulators, SeqA, Fis (factor for inversion stimulation), and IHF (integration host factor), that are not essential for initiation, but which interact directly with E. coli oriC to regulate orisome assembly and replication initiation timing. These regulators function by blocking sites (SeqA) and bending oriC DNA (Fis and IHF) to inhibit or facilitate cooperative low affinity DnaA binding. We also examine how the growth rate regulation of Fis levels might modulate IHF and DnaA binding to oriC under a variety of nutritional conditions. Combined, the regulatory mechanisms mediated by transcriptional modulators help ensure that at all growth rates, bacterial chromosome replication begins once, and only once, per cell cycle.

Molecularly Imprinted Silk Fibroin Nanoparticles.

Nanosized biomimetics prepared by the strategy of molecular imprinting, that is, the stamping of recognition sites by means of a template-assisted synthesis, are demonstrating potential as plastic antibodies in medicine, proving effective for cell imaging and targeted therapies. Most molecularly imprinted nanoparticles (MIP-NPs) are currently made of soft matter, such as polyacrylamide and derivatives. Yet, MIP-NPs biocompatibility is crucial for their effective translation into clinical uses. Here, we propose the original idea to synthesize fully biocompatible molecularly imprinted nanoparticles starting from the natural polymer silk fibroin (MIP SF-NPs), which is nontoxic and highly biocompatible. The conditions to produce MIP SF-NPs of different sizes (dmean ∼ 50 nm; dmean ∼ 100 nm) were set using the response surface method. The stamping of a single, high affinity (KD = 57 × 10–9 M), and selective recognition site per silk fibroin nanoparticle was demonstrated, together with the confirmation of nontoxicity. Additionally, MIP SF-NPs were used to decorate silk microfibers and silk nanofibers, providing a general means to add entailed biofunctionalities to materials.

Building the bacterial orisome: high‐affinity DnaA recognition plays a role in setting the conformation of oriC DNA

During assembly of the E. coli pre-replicative complex (pre-RC), initiator DnaA oligomers are nucleated from three widely separated high-affinity DnaA recognition sites in oriC. Oligomer assembly is then guided by low-affinity DnaA recognition sites, but is also regulated by a switch-like conformational change in oriC mediated by sequential binding of two DNA bending proteins, Fis and IHF, serving as inhibitor and activator respectively. Although their recognition sites are separated by up to 90 bp, Fis represses IHF binding and weak DnaA interactions until accumulating DnaA displaces Fis from oriC. It remains unclear whether high-affinity DnaA binding plays any role in Fis repression at a distance and it is also not known whether all high-affinity DnaA recognition sites play an equivalent role in oligomer formation. To examine these issues, we developed origin-selective recombineering methods to mutate E. coli chromosomal oriC. We found that, although oligomers were assembled in the absence of any individual high-affinity DnaA binding site, loss of DnaA binding at peripheral sites eliminated Fis repression, and made binding of both Fis and IHF essential. We propose a model in which interaction of DnaA molecules at high-affinity sites regulates oriC DNA conformation.

Bacterial origin recognition complexes direct assembly of higher-order DnaA oligomeric structures

Eukaryotic initiator proteins form origin recognition complexes (ORCs) that bind to replication origins during most of the cell cycle and direct assembly of prereplication complexes (pre-RCs) before the onset of S phase. In the eubacterium Escherichia coli, there is a temporally similar nucleoprotein complex comprising the initiator protein DnaA bound to three high-affinity recognition sites in the unique origin of replication, oriC. At the time of initiation, this high-affinity DnaA-oriC complex (the bacterial ORC) accumulates additional DnaA that interacts with lower-affinity sites in oriC, forming a pre-RC. In this paper, we investigate the functional role of the bacterial ORC and examine whether it mediates low-affinity DnaA-oriC interactions during pre-RC assembly. We report that E. coli ORC is essential for DnaA occupation of low-affinity sites. The assistance given by ORC is directed primarily to proximal weak sites and requires oligomerization-proficient DnaA. We propose that in bacteria, DnaA oligomers of limited length and stability emerge from single high-affinity sites and extend toward weak sites to facilitate their loading as a key stage of prokaryotic pre-RC assembly.

Dextromethorphan/quinidine: a novel dextromethorphan product for the treatment of emotional lability

Dextromethorphan (DM) is among the most widely used, non-narcotic antitussives, with a predictable safety profile. In 1981, a non-opioid, high-affinity brain recognition site for DM was discovered, and since then a unique neuropharmacological profile has emerged for this ‘old’ drug, suggesting novel applications. However, an extensive body of research for DM alone in treating various neurological conditions has been inconsistent. This may be largely due to its rapid first-pass metabolism. DM is currently being reintroduced as the active ingredient in a novel combination product in which low-dose quinidine is added to inhibit its breakdown, elevating blood levels of DM and increasing its likelihood of reaching neuronal targets. This has opened new possibilities for therapeutic use; the best evidence at present being for neurological disorders affecting emotional control.

3H]A-317491, a novel high-affinity non-nucleotide antagonist that specifically labels human P2X2/3 and P2X3 receptors.

A-317491 is a potent and selective antagonist of P2X3 and P2X(2/3) receptors. In the present studies, the ability of [3H]A-317491 to label recombinant human P2X(2/3) and P2X(3) receptors was characterized. Using membranes prepared from 1321N1 cells expressing P2X(2/3) receptors, [3H]A-317491 specifically labeled high-affinity (Kd = 0.9 nM) recognition sites. High-affinity [3H]A-317491 binding was not detected in membrane preparations from native 1321N1 cells or cells expressing homomeric P2X1, P2X2, or P2X3 receptors. Specific [3H]A-317491 P2X3 receptors could only be reliably detected following treatment of intact P2X3 receptor-expressing cells with apyrase (1 U/ml) both before and during membrane preparation. Under these conditions, [3H]A-317491 also labeled high-affinity (Kd = 9 nM) binding sites. Lower affinity binding components (Kd values of 87-790 nM) were detected in both assays using higher ligand concentrations that likely represent nonfunctional recognition sites. [3H]A-317491 binding to both P2X(2/3) and P2X3 receptors was reversible, and ligand kinetic studies provided similar estimates of the high-affinity binding constants. Potent P2X3 receptor agonists 2-methylthio-ATP, 2,3-O-(4-benzoylbenzoyl)-ATP, and alpha,beta-methylene adenosine triphosphate also potently inhibited specific [3H]A-317491 binding to both P2X(2/3) and P2X3 receptors. The pharmacological profile for P2X receptor antagonists to inhibit [3H]A-317491 binding to P2X(2/3) and P2X3 receptors was highly correlated (r = 0.98, P < 0.05), and a similar rank order of potency was observed for blockade of P2X(2/3) receptor-mediated calcium influx. These data demonstrate that [3H]A-317491 is the first useful radioligand for the specific labeling of P2X3-containing channels.

Characteristics and composition of the vitamin K-dependent gamma-glutamyl carboxylase-binding domain on osteocalcin.

Two different sites on vitamin K-dependent gamma-glutamyl carboxylase (VKC) are involved in enzyme-substrate interaction: the propeptide-binding site required for high-affinity substrate binding and the active site for glutamate carboxylation. Synthetic descarboxy osteocalcin (d-OC) is a low-K(m) substrate for the VKC, but unique since it possesses a high-affinity recognition site for the VKC, distinct from the propeptide which is essential as a binding site for VKC. However, the exact location and composition of this VKC-recognition domain on d-OC has remained unclear until now. Using a stereospecific substrate analogue [t-butyloxycarbonyl-(2S,4S)-4-methylglutamic acid-Glu-Val (S-MeTPT)] we demonstrate in this paper that the high affinity of d-OC for VKC cannot be explained by a direct interaction with either the active site or with the propeptide-binding site on VKC. It is shown using various synthetic peptides derived from d-OC that there are two domains on d-OC necessary for recognition: one located between residues 1 and 12 and a second between residues 26 and 39, i.e. at the C-terminal side of the gamma-carboxyglutamate (Gla) domain. Both internal sequences contribute substantially to the efficiency of carboxylation. On the basis of these data we postulate the presence of a second high-affinity substrate-binding site on VKC capable of specifically binding d-OC, which is the first vitamin K-dependent substrate of which the VKC binding domain is interrupted by the Gla domain.

Characterisation of agonist binding on human 5-HT 2C receptor isoforms

The 5-HT 2C receptor is expressed in different isoforms as a result of mRNA editing. Both INI (unedited) and VSV (a fully edited version) isoforms are abundant in rat brain. The VSV isoform lacks the high affinity recognition site for 5-HT, which may be caused by low efficiency coupling to G-proteins. In this study we have investigated the pharmacology of the agonist binding site of these two isoforms of the 5-HT 2C receptor. The VSV isoform was expressed in Chinese hamster ovary cells (CHO) and the INI isoform in both Chinese hamster ovary cells and human embryonic kidney cells (HEK-293). Saturation analysis using [ 3H]5-HT revealed high and low affinity recognition sites on the INI isoform in both cell types whilst the VSV isoform did not have the high affinity binding site for [ 3H]5-HT. Displacement studies were undertaken using [ 3H]5-HT to label the receptors. In these studies the affinity of agonists (5-HT, Ro600175 (( S)-2-(6-Chloro-5-fluoroindol-1-yl)-1-methylethylamine), MK212 (6-Chloro-2-(piperazinyl) pyrazine), mCPP (1-( m-chlorophenyl)-piperazine), TfMPP ( N-( m-trifluoromethylphenyl)piperazine), DOI (1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane), DOB (1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane) and 8OH-DPAT (8-hydroxy-2-(di- N-propylamino)tetralin) was higher at the INI isoform, whilst antagonist affinity (ketanserin and mesulergine) did not change between the two receptor isoforms. There were no differences between the INI isoform expressed in the CHO and HEK-293. This suggests that the INI isoform of the 5-HT 2C receptor is pharmacologically similar to the VSV form of the 5-HT 2C receptor but that it couples more efficiently to G-proteins.

Characterization of subclones of the epithelial cell line from Chironomus tentans resistant to the insecticide RH 5992, a non-steroidal moulting hormone agonist

Selection of hormone resistant subclones in the continuous presence of the insecticide and ecdysteroid mimick RH 5992 (tefubenozide) resulted preferentially in clones with defects in ecdysteroid receptor function. RH 5992 is already degraded to polar products in wild-type cells; no increase in metabolism of tefubenozide is observed in resistant clones. According to Western blots, ecdysteroid receptor (EcR) and its heterodimerization partner ultraspiracle (USP) are present in all resistant clones. The concentrations are comparable to wild-type cells, but in three clones the extent of phosphorylation of USP is diminished. With regard to hormone binding several types of hormone resistance are distinguished: (1) The same two high-affinity hormone recognition sites are present as in wild-type cells (K D1=0.31±0.28 nM, K D2=6.5±2.4 nM) but the number of binding sites is reduced. (2) The binding site with the lower affinity (K D2) is missing. (3) The binding site with the higher affinity (K D1) is missing. (4) No specific binding is observed. Ponasterone A binding can be rescued by addition of EcR but not by USP. (5) Ligand specificity is altered. RH 5992 can not compete [ 3H]-ponasterone A as efficient as in wild-type cells.

A Metal-Chelating Microscopy Tip as a New Toolbox for Single-Molecule Experiments by Atomic Force Microscopy

In recent years, the atomic force microscope (AFM) has contributed much to our understanding of the molecular forces involved in various high-affinity receptor-ligand systems. However, a universal anchor system for such measurements is still required. This would open up new possibilities for the study of biological recognition processes and for the establishment of high-throughput screening applications. One such candidate is the N-nitrilo-triacetic acid (NTA)/His-tag system, which is widely used in molecular biology to isolate and purify histidine-tagged fusion proteins. Here the histidine tag acts as a high-affinity recognition site for the NTA chelator. Accordingly, we have investigated the possibility of using this approach in single-molecule force measurements. Using a histidine-peptide as a model system, we have determined the binding force for various metal ions. At a loading rate of 0.5 μm/s, the determined forces varied from 22 ± 4 to 58 ± 5 pN. Most importantly, no interaction was detected for Ca 2+ and Mg 2+ up to concentrations of 10 mM. Furthermore, EDTA and a metal ion reloading step demonstrated the reversibility of the approach. Here the molecular interactions were turned off (EDTA) and on (metal reloading) in a switch-like fashion. Our results show that the NTA/His-tag system will expand the “molecular toolboxes” with which receptor-ligand systems can be investigated at the single-molecule level.

Purification of DNA‐Binding Proteins Using Biotin/Streptavidin Affinity Systems

This unit presents purification protocols that exploit the tight and essentially irreversible complex that biotin forms with streptavidin. A DNA fragment containing a high-affinity binding site for the protein of interest is prepared and a molecule of biotinylated nucleotide is incorporated into one of the ends of the DNA fragment. The protein of interest is allowed to bind to the high-affinity recognition site present in the biotinylated fragment. The tetrameric protein streptavidin is then bound to the biotinylated end of the DNA fragment. Next, the protein/biotinylated fragment/streptavidin ternary complex is efficiently removed by adsorption onto a biotin-containing resin. Since streptavidin is multivalent, it is able to serve as a bridge between the biotinylated DNA fragment and the biotin-containing resin. Proteins remaining in the supernatant are washed away under conditions that maximize the stability of the DNA-protein complex. Finally, the protein of interest is eluted from the resin with a high-salt buffer. Both batch and column formats are presented, as is a protocol for the use of streptavidin-agarose. A support protocol describes a mobility shift assay for detecting sequence-specific DNA-binding proteins.

Spectroscopic Evaluation of Molecular Imprinting Polymerization Systems

An evaluation of the extent of self-assembly of functional monomer–template complexes in molecularly imprinted polymer prepolymerization mixtures has been performed. The results provide general insights into the nature of the prepolymerisation self-assembly phase. Furthermore, the method allows for the estimation of the number of medium- to high-affinity recognition sites in the product polymer and a means for the rapid evaluation of molecular imprinting systems.

Modification of the photodynamic action of δ-aminolaevulinic acid (ALA) on rat pancreatoma cells by mitochondrial benzodiazepine receptor ligands

We have shown that addition of exogenous delta-aminolaevulinic acid (ALA) to rat pancreatoma AR4-2J cells in culture leads to the increased production of porphobilinogen (PBG) and the accumulation of photoactive protoporphyrin IX (PPix) in these cells. Exposure to light (lambda > 400 nm) at an intensity of 0.2 mW cm-2 for 8 min resulted in an ALA dose-dependent cytolysis of the cells, with an EC50 of 6.6 +/- 0.7 microM. This cytolytic effect was light intensity dependent, with greater cell destruction after exposure to light at an intensity of 0.47 mW cm-2 than at 0.2 mW cm-2; it was also dependent on the duration of illumination, cell survival decreasing with increasing illumination times. The photodestruction of the AR4-2J cells following exposure to ALA can be attributed to the production of endogenous PPix, a photoactive porphyrin that we have shown to generate singlet oxygen upon illumination, whereas ALA itself does not. Further investigation of the molecular mechanisms underlying the photodynamic action of ALA demonstrated the involvement of the mitochondrial (peripheral) benzodiazepine receptor (MBR), a high-affinity recognition site for dicarboxylic porphyrins, and especially PPix. The centrally acting benzodiazepine compounds clonazepam and flumazenil, which have negligible affinities for the MBR, had no effect on ALA-mediated phototoxicity. In contrast, both the isoquinoline carboxamide PK11195 and the benzodiazepine Ro 5-4864 ligands, displaying a high affinity for the MBR, did affect ALA-mediated phototoxicity, each markedly increasing the EC50 for cell photodestruction and thus exerting a photoprotective effect. It is concluded that the MBR may play an important role in the expression of ALA-mediated PPix phototoxicity and that MBR ligands, by diminishing the actions of endogenous PPix, have the potential to rescue cells from porphyrin-induced photolysis.

Heterodimerization and functional interaction between EGF receptor family members: a new signaling paradigm with implications for breast cancer research.

The EGF receptor (EGFR) and HER2 are members of a growth factor receptor family. Overexpression of either protein in advanced breast cancer correlates with poor prognosis. EGF stimulates growth by binding to EGFR, activating the receptor's intracellular tyrosine kinase. The initial consequence is phosphorylation of specific tyrosine-containing sequences in the receptor's carboxyl terminus. These phosphotyrosines serves as high affinity recognition sites for proteins that, in turn, transmit the growth signal inside the cell. Mechanistic studies suggest that EGF binds to a single EGFR, triggering dimerization with another like receptor molecule. This dimerization is thought to initiate the tyrosine kinase activation. The EGF receptor family was recently expanded with the sequencing of HER3 and HER4. Each of the four family members was postulated to regulate a unique growth or differentiation signaling repertoire when activated by a receptor-specific ligand. However, new data from numerous laboratories suggest that EGFR family members may play a complex and ultimately more flexible role in signaling by forming heterodimers between family members, e.g. EGFR:HER2 or HER4:HER2. These heterodimers may form even when only one member of the pair binds its ligand. This review summarizes current work on heterodimerization and attempts to predict the consequences for downstream signaling. In brief, when compared to ligand-dependent receptor homodimers comprised of two proteins with the same internalization sequence and phosphorylated tyrosine residues, heterodimers are likely to: i) expand substrate selection and downstream signaling pathway activation; ii) promote interaction between sets of substrates in the mixed receptor complexes that would not ordinarily be physically juxtaposed; iii) alter the duration of receptor signaling by changing rates of receptor internalization, ligand loss, kinase inactivation, recycling, etc.; and iv) alter rates of receptor and substrate dephosphorylation. In addition to understanding interactions of heterodimers with the internalization machinery, identification of receptor-specific substrates and binding proteins for each EGFR family member will be necessary to explicate the role of heterodimers in growth and differentiation.

Conformational changes induced by DNA binding of NF-kappa B.

The transcription factor NF-kappa B makes extensive contacts with its recognition site over one complete turn of the double helix. Structural transitions, in both protein and DNA, that accompany formation of the DNA-protein complex were analysed by proteinase sensitivity and circular dichroism (CD) spectroscopy. In the absence of DNA chymotrypsin cleaved p50 after residues Y60 and N78, while proteinase K cleaved p50 after residues S74 and Q180. Previous experiments had indicated that trypsin cleaved p50 after K77. Cleavages after Y60, S74, K77 and N78 were blocked in the presence of bound DNA, whereas cleavage after Q180 was enhanced. Y60, S74, K77 and N78 are all located in the p50 N-terminal domain AB loop, whereas Q180 is located in the mainly alpha-helical region between p50 N-terminal domain beta-strands G' and H. As only Y60 makes direct contact with the DNA it is likely that the AB loop is highly unstructured in the absence of DNA, but is held in a rigid, proteinase-resistant structure by bound DNA. These conclusions were supported by CD spectroscopic studies of recombinant p50 and p65 homodimers, which indicated that both species changed conformation when binding DNA. Examination of the near UV CD spectra revealed that with some DNA sequences the bound and free forms of the DNA assumed different conformations. While this was evident for a fully symmetrical, high affinity recognition site DNA, it was not apparent with less tightly bound DNA.

A nonconsensus octamer‐recognition sequence (TAATGARAT‐motif) identifies a novel DNA binding protein in human merkel cell carcinoma cell lines

We have established a number of cell lines from Merkel cell carcinoma (MCC) of the skin. In many respects these cell lines resemble those established from small cell lung cancer (SCLC) and it is difficult to differentiate between metastatic MCC and SCLC on morphological or histochemical criteria. Both are thought to be neuroendocrine tumours and may express a number of neuroendocrine markers. SCLC cell lines express the octamer-DNA binding transcription factor brn-2 gene products N-Oct3 and N-Oct5, which are restricted to the neuroectodermal cell lineage. In DNA binding studies using a consensus octamer recognition site we have found that 4 of 8 MCC cell lines examined expressed brn-2 in at least trace amounts compared with 3 SCLC cell lines which all expressed brn-2 proteins at high levels. Moreover, these DNA binding studies were extended by using a high-affinity brn-2 recognition site related to the degenerate octamer TAATGARAT-motif. This identified a novel DNA binding protein in a subset of MCC cell lines. The protein was absent from the three SCLC cell lines, melanoma cells and brain tissue. This binding activity, which we term Merkel cell DNA nuclear (MNF), was shown to be specific by competitive inhibition with oligonucleotide binding sites and was not inhibited by polyclonal antisera against the Oct-1, Oct-2 or Brn-2 proteins. This protein may serve as a unique marker for MCC compared with SCLC cells and may be involved in regulating the Merkel cell phenotype.

DNA sequences that act as high affinity targets for the vitamin D3 receptor in the absence of the retinoid X receptor.

DNA binding site discrimination within a subgroup of nuclear receptors, including the human vitamin D3 receptor (hVDR), appears to be influenced primarily by spacing and orientation differences of response element half-sites, since many receptors recognize and bind to the same hexameric half-site sequence, AGGTCA. Small sequence differences within half-sites, however, may also play an important role in distinguishing between different receptor complexes. Several laboratories have reported that the AGGTCA element in a direct repeat (DR) configuration appears to be a high affinity recognition site for only nuclear receptor-9 retinoid X receptor (RXR) heterodimers. However, we have previously shown that a closely related, but distinct, element (AGTTCA; essentially the mouse osteopontin [Spp-1] vitamin D response element) acts as a high affinity target for purified hVDR in the absence of RXR. This suggests that some half-site sequences could be targets for hVDR alone while others serve as recognition elements for hVDR-RXR complexes. In this report, we test this hypothesis by selecting, using purified hVDR only, for high affinity receptor binding sites in a complex DNA mixture which should by chance contain such sequences. We find that the purified receptor selects a heptameric sequence resembling a half-site of the osteopontin vitamin D response element, consistent with osteopontin-like sequences acting as high affinity targets for hVDR in the absence of RXR. We directly test this by comparing the in vitro DNA binding activity of purified hVDR to DR+3 elements comprised of osteopontin-like AGTTCA or AGGTCA half-sites.(ABSTRACT TRUNCATED AT 250 WORDS)