Portion Of Binding Research Articles

The Role of Structural Flexibility in Hydrocarbon‐Stapled Peptides Designed to Block Viral Infection via Human ACE2 Mimicry

ABSTRACTThe COVID‐19 pandemic drove a uniquely fervent pursuit to explore the potential of peptide, antibody, protein, and small‐molecule‐based antiviral agents against severe acute respiratory syndrome‐coronavirus 2 (SARS‐CoV‐2). The interaction between the SARS‐CoV2 spike protein with the angiotensin‐converting enzyme 2 (ACE2) receptor that mediates viral cell entry was a particularly interesting target given its well‐described protein–protein interaction (PPI). This PPI is mediated by an α‐helical portion of ACE2 binding to the receptor binding domain (RBD) of the spike protein and thought to be susceptible to blockade through molecular mimicry. Small numbers of hydrocarbon‐stapled synthetic peptides designed to disrupt or block this interaction were tested individually and were found to have variable efficacy despite having related or overlapping sequences and similarly increased α‐helicity. Reasons for these differences are unclear and reported preclinical successes have been limited. This study sought to better understand reasons for these differences through evaluation of a comprehensive collection of hydrocarbon‐stapled peptides, designed based on four distinct principles: stapling position, number of staples, amino acid sequence, and primary sequence length. Surprisingly, we observed that the helicity and amino acid sequence iterations of hydrocarbon‐stapled peptides did not correlate with their bioactivity. Our results highlight the importance of iterative and combinatorial testing of these compounds to determine a configuration that best mimics natural binding and allows for chain flexibility while sacrificing structural helicity.

Structural basis for preferential binding of human TCF4 to DNA containing 5-carboxylcytosine.

The psychiatric risk-associated transcription factor 4 (TCF4) is linked to schizophrenia. Rare TCF4 coding variants are found in individuals with Pitt-Hopkins syndrome—an intellectual disability and autism spectrum disorder. TCF4 contains a C-terminal basic-helix-loop-helix (bHLH) DNA binding domain which recognizes the enhancer-box (E-box) element 5′-CANNTG-3′ (where N = any nucleotide). A subset of the TCF4-occupancy sites have the expanded consensus binding specificity 5′-C(A/G)-CANNTG-3′, with an added outer Cp(A/G) dinucleotide; for example in the promoter for CNIH3, a gene involved in opioid dependence. In mammalian genomes, particularly brain, the CpG and CpA dinucleotides can be methylated at the 5-position of cytosine (5mC), and then may undergo successive oxidations to the 5-hydroxymethyl (5hmC), 5-formyl (5fC), and 5-carboxyl (5caC) forms. We find that, in the context of 5′-0CG-1CA-2CG-3TG-3′(where the numbers indicate successive dinucleotides), modification of the central E-box 2CG has very little effect on TCF4 binding, E-box 1CA modification has a negative influence on binding, while modification of the flanking 0CG, particularly carboxylation, has a strong positive impact on TCF4 binding to DNA. Crystallization of TCF4 in complex with unmodified or 5caC-modified oligonucleotides revealed that the basic region of bHLH domain adopts multiple conformations, including an extended loop going through the DNA minor groove, or the N-terminal portion of a long helix binding in the DNA major groove. The different protein conformations enable arginine 576 (R576) to interact, respectively, with a thymine in the minor groove, a phosphate group of DNA backbone, or 5caC in the major groove. The Pitt-Hopkins syndrome mutations affect five arginine residues in the basic region, two of them (R569 and R576) involved in 5caC recognition. Our analyses indicate, and suggest a structural basis for, the preferential recognition of 5caC by a transcription factor centrally important in brain development.

Chloride binding of cement pastes with fly ash exposed to CaCl2 solutions at 5 and 23 °C

In cementitious materials exposed to solutions containing chloride, chloride binding typically results from the chemical reactions between chloride ions and aluminate phases to form Friedel's salt, and the interaction between chloride ions and calcium silicate hydrates (CSH). Calcium oxychloride can also form when Ca(OH)2 in cementitious materials reacts with CaCl2 solutions. This paper examines the chloride binding of hydrated cement pastes containing fly ash exposed to CaCl2 solutions of varying concentrations at 5 and 23 °C. Thermogravimetric analysis was used to quantify the chloride binding associated with Friedel's salt and calcium oxychloride. The amount of bound chloride by Friedel's salt is relatively independent of the exposure temperature, and as the chloride concentration [Cl−] increases, it increases until a plateau is reached at [Cl−] greater than 2 M. The addition of fly ash results in an increase in the chloride binding due to Friedel's salt. A lower exposure temperature leads to a greater amount of bound chloride associated with calcium oxychloride. In this study, no chloride binding associated with calcium oxychloride was observed in the cement pastes with 40% and 60% fly ash. The temperature-dependent chloride binding associated with CSH is a significant portion of the total chloride binding (19.8 %–70.8%) when cement pastes are exposed to CaCl2 solutions. As the replacement level of fly ash increases, the chloride binding by CSH increases first and then decreases. The amount of bound chloride by CSH increases linearly as the pH of the exposure solution decreases.

Genomic meta-analysis of the interplay between 3D chromatin organization and gene expression programs under basal and stress conditions

BackgroundOur appreciation of the critical role of the genome’s 3D organization in gene regulation is steadily increasing. Recent 3C-based deep sequencing techniques elucidated a hierarchy of structures that underlie the spatial organization of the genome in the nucleus. At the top of this hierarchical organization are chromosomal territories and the megabase-scale A/B compartments that correlate with transcriptional activity within cells. Below them are the relatively cell-type-invariant topologically associated domains (TADs), characterized by high frequency of physical contacts between loci within the same TAD, and are assumed to function as regulatory units. Within TADs, chromatin loops bring enhancers and target promoters to close spatial proximity. Yet, we still have only rudimentary understanding how differences in chromatin organization between different cell types affect cell-type-specific gene expression programs that are executed under basal and challenged conditions.ResultsHere, we carried out a large-scale meta-analysis that integrated Hi–C data from thirteen different cell lines and dozens of ChIP-seq and RNA-seq datasets measured on these cells, either under basal conditions or after treatment. Pairwise comparisons between cell lines demonstrate a strong association between modulation of A/B compartmentalization, differential gene expression and transcription factor (TF) binding events. Furthermore, integrating the analysis of transcriptomes of different cell lines in response to various challenges, we show that A/B compartmentalization of cells under basal conditions significantly correlates not only with gene expression programs and TF binding profiles that are active under the basal condition but also with those induced in response to treatment. Yet, in pairwise comparisons between different cell lines, we find that a large portion of differential TF binding and gene induction events occur in genomic loci assigned to A compartment in both cell types, underscoring the role of additional critical factors in determining cell-type-specific transcriptional programs.ConclusionsOur results further indicate the role of dynamic genome organization in regulation of differential gene expression between different cell types and the impact of intra-TAD enhancer–promoter interactions that are established under basal conditions on both the basal and treatment-induced gene expression programs.

Genome-Wide Mapping Reveals BRD4 in Regulation of Tumor-Driver Genes in Cutaneous T-Cell Lymphoma

Cutaneous T-cell lymphoma (CTCL) is a non-Hodgkin's lymphoma of skin homing malignant CD4+ T cells. Early treatment of patients with skin-directed therapies often yields successful short-term outcomes; however, survival of patients with late-stage CTCL is extremely poor, highlighting the need to identify novel therapies to inhibit key oncogenic processes. Although the underlying factors driving CTCL pathogenesis are poorly understood, recent findings from our lab highlight the critical role of epigenetic dysregulation in its development (Mishra et al., Blood, 122:1826, 2013). While many studies have been carried out on the role of epigenetic writers (histone acetyltransferases) and erasers (histone deacetylases) in CTCL gene regulation, little is known about the epigenetic readers (bromodomains; tandem PHD fingers; Pleckstrin homology domains) that coordinate transcriptional reprogramming. Previous studies have identified bromodomain-containing protein-4 (BRD4) as a super-enhancer transcriptional regulator that interacts with pairs of acetylated lysine residues on histone H3 to upregulate oncogenic transcription in multiple myeloma and diffuse large B-cell lymphoma (Loven, J. et al. Cell 153, 2013; Chapuy, B. et al. Cancer Cell 24, 2013). We analyzed BRD4 occupancy patterns in the CTCL genome and characterized downstream oncogenic pathways. Additionally, we investigated the effect of treatment with a BRD4 inhibitor, JQ1, on CTCL in vitro and in vivo. In order to gain insights into the role of BRD4 in CTCL, we generated a genome-wide map of BRD4 occupancy in CD4+ T-cells from CTCL patients and normal donors using "ChIP-seq", which combines chromatin immunoprecipitation (ChIP) with sequencing of BRD4-associated DNA. The analysis revealed an increased occupancy of BRD4 in patient's CD4+ T-cells compared to normal donor CD4+ T-cells at putative active regions, promoter active regions, distal active regions, and super-enhancers (Figure 1). The abnormal binding patterns in patients were reversed within 24 hours of treatment with JQ1 (Figure 1). Patient's cells treated with 1.0 μM JQ1 showed reduced cell viability (mean ± SEM of relative % viability in JQ1 vs control treated cells: 39.67 ± 1.838 vs 100.0 ± 2.335, n=4 each). Furthermore, we saw a similar effect of JQ1 in all the patient-derived CTCL cell lines tested (HH, Hut78, Hut102, SeAx, and MyLa) with EC50 concentrations ranging from 0.167 to 21.0 μM. Since both primary CD4+ T-cells from CTCL patients and CTCL cell lines displayed sensitivity to the cytotoxic effect of JQ1, we performed subsequent functional assays on Hut78 cell line. Propidium iodide/Annexin staining of JQ1-treated Hut78 cells (1.0 μM for 48 hours) revealed an increased frequency of cells in the G1 phase, indicating significant disruption of cell cycle progression upon BRD4 inhibition. Furthermore, considering that a large portion of BRD4 binding was detected in promoter and super-enhancer regions, we speculated that BRD4 regulates tumor driver genes. When examining BRD4 occupancy, we found that BRD4 binding at NOTCH1, and MYC was higher in patients' vs normal donors' CD4+ T-cells. Similar differential increase in BRD4 occupancy was also noted at the DNA-binding factor gene RBPJ, a known NOTCH1 activator and T-cell oncogenic co-factor. In order to determine whether BRD4 occupancy at these genes exerts transcriptional regulation, we examined expression levels of RBPJ, NOTCH1, and MYC in JQ1 treated cells. Semi-quantitative RT-PCR analysis on HuT78 cells treated with JQ1 showed significant reduction in RBPJ (mean ± SEM of relative expression in Control vs JQ1 treatment= 100.5 ± 07.086 vs. 35.39 ± 0.7832, n=3, P =0.0008), NOTCH1 (mean ± SEM of relative expression in Control vs JQ1 treatment= 100.0 ± 0.95 vs 0.61.77 ± 02.844, n=3, P =0.0002), and MYC (mean ± SEM of relative expression in Control vs JQ1 treatment= 100.1 ± 03.019 vs. 57.97 ± 01.997, n=3, P =0.0003). Additionally, in vivo treatment of CTCL mice with JQ1 (50mg/kg, 5 days/week for 4-weeks) halted as well as regressed progression of CTCL compared to placebo treated controls. Together, these findings demonstrate that BRD4 regulates the expression of the oncogenic drivers in CTCL, and that bromodomain inhibitors offer the opportunity to interrogate the mechanisms of BRD4-mediated T-cell oncogenesis in the CTCL mouse model and design new anti-cancer therapies in CTCL patients. [Display omitted] DisclosuresPorcu:Cell Medica: Research Funding; Celgene: Research Funding; Shape: Research Funding; Seattle Genetics: Research Funding; Infinity: Research Funding.

Endomorphins and DAMGO Utilize Different Mechanisms to Produce Analgesia

There is significant variability among animals in their response to mu opioid receptor (MOR1) analgesics. The mu opioid receptor gene (OPRM1) undergoes extensive alternative splicing, producing several splice variants with 1, 6, and 7 transmembrane (TM) domains. Variants containing 6 TMs lack TM1 and are associated with the exon 11 promoter. We previously showed that mice lacking exon 11 variants respond normally to methadone and morphine, but fentanyl, heroin, and morphine‐6‐glucuronide potency is significantly reduced (Pan et al, 2009). Here, we report that in exon 11 KO mice the DAMGO response is intact both supraspinally and spinally but endomorphin analgesiais significantly reduced. [3H]DAMGO binding in exon 11 KO brain and spinal cord was not significantly altered, but binding of the endomorphin 2 analog 125I‐DAPP (dmt(125I)‐D‐Ala‐Phe‐Phe‐NH2) was reduced.No binding of [3H]DAMGO or 125I‐DAPP was detected in mice lacking both exon 1 7TM MOR variants and exon 11 6TM variants. In [35S]GTPγS studies of brain and spinal cord binding, max stimulation by DAMGO was equal in wild type and exon 11 KO mice whereas IDAPP stimulation was reduced in exon 11 KO mice. Overall, our results indicate that DAMGO does not recruit 6TM exon 11 variants for binding and analgesia, whereas endomorphins require a mechanism including 6TM exon 11 variants for analgesia and a significant portion of binding.

N‐linked glycan profiling of GGTA1/CMAH knockout pigs identifies new potential carbohydrate xenoantigens

The temporary or long-term xenotransplantation of pig organs into people would save thousands of lives each year if not for the robust human antibody response to pig carbohydrates. Genetically engineered pigs deficient in galactose α1,3 galactose (gene modified: GGTA1) and N-glycolylneuraminic acid (gene modified: CMAH) have significantly improved cell survival when challenged by human antibody and complement in vitro. There remains, however, a significant portion of human antibody binding. To uncover additional xenoantigens, we compared the asparagine-linked (N-linked) glycome from serum proteins of humans, domestic pigs, GGTA1 knockout pigs, and GGTA1/CMAH knockout pigs using mass spectrometry. Carbohydrate structures were determined with assistance from GlycoWorkbench, Cartoonist, and SimGlycan software by comparison to existing database entries and collision-induced dissociation fragmentation data. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of reduced and solid-phase permethylated glycans resulted in the detection of high-mannose, hybrid, and complex type N-linked glycans in the 1000-4500m/z ion range. GGTA1/CMAH knockout pig samples had increased relative amounts of high-mannose, incomplete, and xylosylated N-linked glycans. All pig samples had significantly higher amounts of core and possibly antennae fucosylation. We provide for the first time a comparison of the serum protein glycomes of the human, domestic pig, and genetically modified pigs important to xenotransplantation.

THU0030 Neutralization of ACPA in Rheumatoid Arthritis - A Novel Principle of Treatment

BackgroundIn a previous study, we have identified endogeneously citrullinated sites in fibrinogen from RA synovial tissue1. Within the alpha chain, Arg573 and Arg591 were found citrullinated with an occupancy rate...

Comparison of nerve growth factor receptor binding models using heterodimeric muteins

Nerve growth factor (NGF) is a homodimer that binds to two distinct receptor types, TrkA and p75, to support survival and differentiation of neurons. The high-affinity binding on the cell surface is believed to involve a heteroreceptor complex, but its exact nature is unclear. We developed a heterodimer (heteromutein) of two NGF muteins that can bind p75 and TrkA on opposite sides of the heterodimer, but not two TrkA receptors. Previously described muteins are Δ9/13 that is TrkA negative and 7-84-103 that is signal selective through TrkA. The heteromutein (Htm1) was used to study the heteroreceptor complex formation and function, in the putative absence of NGF-induced TrkA dimerization. Cellular binding assays indicated that Htm1 does not bind TrkA as efficiently as wild-type (wt) NGF but has better affinity than either homodimeric mutein. Htm1, 7-84-103, and Δ9/13 were each able to compete for cold-temperature, cold-chase stable binding on PC12 cells, indicating that binding to p75 was required for a portion of this high-affinity binding. Survival, neurite outgrowth, and MAPK signaling in PC12 cells also showed a reduced response for Htm1, compared with wtNGF, but was better than the parent muteins in the order wtNGF > Htm1 > 7-84-103 >> Δ9/13. Htm1 and 7-84-103 demonstrated similar levels of survival on cells expressing only TrkA. In the longstanding debate on the NGF receptor binding mechanism, our data support the ligand passing of NGF from p75 to TrkA involving a transient heteroreceptor complex of p75-NGF-TrkA.

Junctional adhesion molecule-A is abnormally expressed in diffuse cutaneous systemic sclerosis skin and mediates myeloid cell adhesion

Objective:To investigate the role of junctional adhesion molecule-A (JAM-A) in the pathogenesis of systemic sclerosis (SSc).Methods:Biopsy specimens from proximal and distal arm skin and serum were obtained from patients with...

Unattached Kinetochores Catalyze Production of an Anaphase Inhibitor that Requires a Mad2 Template to Prime Cdc20 for BubR1 Binding

Premature anaphase onset is prevented by the mitotic checkpoint through production of a "wait anaphase" inhibitor(s) that blocks recognition of cyclin B and securin by Cdc20-activated APC/C, an E3 ubiquitin ligase that targets them for destruction. Using physiologically relevant levels of Mad2, Bub3, BubR1, and Cdc20, we demonstrate that unattached kinetochores on purified chromosomes catalytically generate a diffusible Cdc20 inhibitor or inhibit Cdc20 already bound to APC/C. Furthermore, the chromosome-produced inhibitor requires both recruitment of Mad2 by Mad1 that is stably bound at unattached kinetochores and dimerization-competent Mad2. We show that purified chromosomes promote BubR1 binding to APC/C-Cdc20 by acting directly on Mad2, but not BubR1. Our results support a model in which immobilized Mad1/Mad2 at kinetochores provides a template for initial assembly of Mad2 bound to Cdc20 that is then converted to a final mitotic checkpoint inhibitor with Cdc20 bound to BubR1.

Comparative antagonist pharmacology at the native mouse bradykinin B2 receptor: radioligand binding and smooth muscle contractility studies

The aim was to characterize the recently discovered non-peptide antagonist MEN16132 at the mouse B2 receptor, relative to other antagonists. [3H]-BK binding experiments used mouse lung and ileum tissue membranes and antagonist potency was measured in the isolated ileum contractility assay. Two BK binding sites resulted from saturation and homologous competition experiments. A role for the B1 receptor was excluded because of the poor affinity of B1 receptor ligands (pIC50<5). MEN16132, and the other reference antagonists, inhibited only one portion of BK specific binding, and the rank order of potency was (pIC50): Icatibant (lung 10.7; ileum 10.2)=MEN11270 (lung 10.4; ileum 9.9)=MEN16132 (lung 10.5; ileum 9.9).>LF16-0687 (lung 8.9; ileum 8.8)>FR173657 (lung 8.6; ileum 8.2). BK homologous curves performed with lung membranes after treatment with the antagonist MEN16132 or Icatibant (10 nM) displayed only the low affinity site. The functional antagonism by MEN16132 (pA2 9.4) and Icatibant (pA2 9.1), towards BK (control EC50 6.1 nM) induced ileum contractions, was concentration-dependent and surmountable, but the Schild plot slope was less than unity. In mouse tissue, radiolabelled BK recognizes two binding sites and B2 receptor antagonists can compete only for the higher affinity one. The pharmacological profile of the novel non-peptide antagonist MEN16132 indicates that it exhibits subnanomolar affinity and potency for the mouse B2 receptor and is suitable for further characterization in in vivo pathophysiological models.

Two Structurally Atypical HEAT Domains in the C-Terminal Portion of Human eIF4G Support Binding to eIF4A and Mnk1

The X-ray structure of the C-terminal region of human eukaryotic translation initiation factor 4G (eIF4G) has been determined at 2.2 A resolution, revealing two atypical HEAT-repeat domains. eIF4G recruits various translation factors and the 40S ribosomal subunit to the mRNA 5' end. In higher eukaryotes, the C terminus of eIF4G (4G/C) supports translational regulation by recruiting eIF4A, an RNA helicase, and Mnk1, the kinase responsible for phosphorylating eIF4E. Structure-guided surface mutagenesis and protein-protein interaction assays were used to identify binding sites for eIF4A and Mnk1 within the HEAT-repeats of 4G/C. p97/DAP5, a translational modulator homologous to eIF4G, lacks an eIF4A binding site in the corresponding region. The second atypical HEAT domain of the 4G/C binds Mnk1 using two conserved aromatic/acidic-box (AA-box) motifs. Within the first AA-box, the aromatic residues contribute to the hydrophobic core of the domain, while the acidic residues form a negatively charged surface feature suitable for electrostatic interactions with basic residues in Mnk1.

Chemometric Models of TemplateMolecularly Imprinted Polymer Binding

This report provides the first example of the use of chemometrics to describe and predict the extent of template binding to molecularly imprinted polymers. The binding of bupivacaine to imprinted and reference polymers was examined in different solvent mixtures and at various temperatures using equilibrium binding studies. Data were fitted to third-degree equations using partial least-squares regression, resulting in chemometric models describing template binding in this system. The mathematical models demonstrated good correlation (R = 0.72-0.98) and predictive ability (Q = 0.54-0.99), and binding could be described in terms of temperature and dielectric constant. Binding in a nonpolar, aprotic solvent was unaffected by temperature whereas in more polar solvent mixtures temperature had a greater influence. This was explained by changes in the balance between electrostatic and hydrophobic interactions. Moreover, the results demonstrate that temperature has a greater influence on the nonspecific portion of binding, particularly in water-containing solvent mixtures.

Anisotropy of Fluctuation Dynamics of Proteins with an Elastic Network Model

Fluctuations about the native conformation of proteins have proven to be suitably reproduced with a simple elastic network model, which has shown excellent agreement with a number of different properties for a wide variety of proteins. This scalar model simply investigates the magnitudes of motion of individual residues in the structure. To use the elastic model approach further for developing the details of protein mechanisms, it becomes essential to expand this model to include the added details of the directions of individual residue fluctuations. In this paper a new tool is presented for this purpose and applied to the retinol-binding protein, which indicates enhanced flexibility in the region of entry to the ligand binding site and for the portion of the protein binding to its carrier protein.

A novel Sp1-related cis element involved in intestinal alkaline phosphatase gene transcription.

We have used sodium butyrate-treated HT-29 cells as an in vitro model system to study the molecular mechanisms underlying intestinal alkaline phosphatase (IAP) gene activation. Transient transfection assays using human IAP-CAT reporter genes along with DNase I footprinting were used to localize a critical cis element (IF-III) corresponding to the sequence 5'-GACTGGGCGGGGTCAAGATGGA-3'. Deletion of the IF-III element resulted in a dramatic reduction in reporter gene activity, and IF-III was shown to function in the context of a heterologous (SV40) promoter in a cell type-specific manner, further supporting its functional role in IAP transactivation. Electrophoretic mobility shift assays revealed that IF-III binds Sp1 and Sp3, but these factors comprise only a portion of the total nuclear binding and appear to mediate only a small portion of its transcriptional activity. IF-III does not correspond to any previously characterized regulatory region from other intestine-specific genes. We have thus identified a novel, Sp1-related cis-regulatory element in the human IAP gene that appears to play a role in its transcriptional activation during differentiation in vitro.

Identification and pharmacological characterization of somatostatin receptors in rat lung.

1. [125I]-[LTT]SRIF-28 and [125I]-SMS 201-995 were used to identify and characterize somatostatin (SRIF) receptors localized in rat lung tissue. In vitro autoradiography of rat lung tissue sections showed the existence of specific, high affinity binding sites for [125I]-[LTT]SRIF-28 without any significant specific binding of the sst2/sst5-receptor selective ligand [125I]-SMS 201-995. 2. In radioligand binding studies, specific binding of [125I]-[LTT]SRIF-28 to membranes of rat lung was linearly related to the concentration of membrane protein used with only a small portion of nonspecific binding. With [125I]-SMS 201-995 no specific binding could be observed up to a membrane concentration of 0.1 mg of protein/assay tube. 3. [125I]-[LTT]SRIF-28 bound rapidly to rat lung membranes with an apparent association rate constant (kapp) of 1.8 +/- 0.1 h-1 (n = 3). The equilibrium of specific binding was reached after an incubation period of approximately 90 min at room temperature and remained constant for the next 3 h. The association rate constant (k1) was calculated to be 3.7 x 10(10) M-1 h-1. The dissociation reaction followed first order kinetics with a dissociation rate constant (k-1) = 0.44 +/- 0.07 h-1 corresponding to a half-time of 95 +/- 15 min (n = 3). From these kinetic experiments an equilibrium dissociation constant (KD) for the binding of [125I]-[LTT]SRIF-28 was calculated to be 11.9 pM. 4. Saturation binding of [125I]-[LTT]SRIF-28 revealed an equilibrium dissociation constant (KD) of 50.1 pM (pKD = 10.3 +/- 0.1; n = 3) and a receptor density (Bmax) of 78 +/- 3 fmol mg-1 protein. A Hill coefficient not significantly different from 1 indicated saturable binding to a single class of high affinity binding sites. 5. Specific binding of [125I]-[LTT]SRIF-28 to rat lung membranes was inhibited by SRIF-14, SRIF-28 and different SRIF analogues. SRIF and different synthetic short chain SRIF analogues exhibited the following rank order of potency: SRIF-28 > SRIF-14 > CGP 23996 >> RC 160 > BIM 23014 > SMS 201- 995 > BIM 23056 > MK 678. 6. The binding affinities for SRIF and the various SRIF analogues determined using rat lung tissue were in close correlation to those obtained with Chinese hamster ovary (CHO) cells stably expressing sst, (r = 0.92) and sst4 (r = 0.95) receptors, respectively. 7. Reverse transcriptase--polymerase chain reaction (RT-PCR) showed the predominant expression of mRNA specific for sst4 receptors as well as some weak sst1 mRNA expression. 8. The findings suggest that sst4 receptor expression is the predominant form of the somatostatin receptors identified in rat lung tissue. In this study we demonstrated for the first time the existence of sst4 receptors in mammalian tissue.

Quantitative Dynamic Multicompartmental Analysis of Cholecystokinin Receptor Movement in a Living Cell Using Dual Fluorophores and Reconstruction of Confocal Images

Receptor regulation is a key component of the phenomenon of desensitization in response to agonist stimulation which protects cells from overstimulation. Receptor internalization is one part of this response, often quantified by the portion of saturable ligand binding which becomes resistant to acidic washes. It is now clear that this can include receptor in multiple distinct cellular compartments. We have developed a morphological technique involving dual fluorescent probes to delineate the plasmalemma and the ligand-occupied receptor using confocal microscopy, with analysis involving three-dimensional reconstruction and quantitation of receptor movement through each compartment. When a radioiodinated cholecystokinin (CCK) analogue occupied its receptor on the CHO–CCKR cell line, it became progressively more resistant to dissociation with acidic medium. Quantitation of receptor internalization in these cells over time using this dynamic morphological technique correlated with the acid-resistant receptor fraction, and provided the additional information of the cellular compartments traversed. This technique will have multiple applications to explore the cell-specific handling of this and other ligand-occupied receptors.

Specific [3H]glutamate binding in the cerebral cortex and hippocampus of rats during development: effect of homocysteine-induced seizures.

Specific [3H]glutamate binding to synaptic membranes from the cerebral cortex and hippocampus of 7-, 12- and 18-day-old rats was examined, both in control animals and during seizures induced by homocysteine. In the cerebral cortex a transient peak of glutamate binding was observed in 7-day-old group, whereas in the hippocampus it occurred in 12-day-old animals. Total specific [3H]glutamate binding was not influenced by preceding seizure activity in either of the age groups and both the studied regions. NMDA- and QA-sensitive glutamate bindings represent the highest portion of the total binding. Moreover, NMDA-sensitive binding in the cerebral cortex of 7-day-old rats is significantly higher as compared to the two more mature groups. The proportion of individual receptor subtypes on total binding in each age group was not influenced by preceding seizure activity. However, NMDA-sensitive binding in the hippocampus of 12-day-old rats, sacrificed during homocysteine-induced seizures, was significantly increased as compared to corresponding controls. In contrast to the effect of NMDA, AMPA, kainate and quisqualate which displaced to a different extent [3H]glutamate binding, homocysteine had no effect when added to membrane preparations. Similarly, [3H]CPP and [3H]AMPA bindings were not affected in the presence of homocysteine. It thus seems unlikely that homocysteine is an effective agonist for conventional ionotropic glutamate receptors. Its potential activity at some of the modulatory sites at the NMDA receptor channel complex or at metabotropic receptors has to be clarified in further experiments.

3H]Dihydrorotenone Binding to NADH: Ubiquinone Reductase (Complex I) of the Electron Transport Chain: An Autoradiographic Study

Abnormalities of mitochondrial energy metabolism may play a role in normal aging and certain neurodegenerative disorders. In this regard, complex I of the electron transport chain has received substantial attention, especially in Parkinson's disease. The conventional method for studying complex I has been quantitation of enzyme activity in homogenized tissue samples. To enhance the anatomic precision with which complex I can be examined, we developed an autoradiographic assay for the rotenone site of this enzyme. [3H]dihydrorotenone ([3H]DHR) binding is saturable (KD = 15-55 nM) and specific, and Hill slopes of 1 suggest a single population of binding sites. Nicotinamide adenine dinucleotide (NADH) enhances binding 4- to 80-fold in different brain regions (EC50 = 20-40 microM) by increasing the density of recognition sites (Bmax). Nicotinamide adenine dinucleotide phosphate also increases binding, but NAD+ does not. In skeletal muscle, heart, and kidney, binding was less affected by NADH. [3H]DHR binding is inhibited by rotenone (IC50 = 8-20 nM), meperidine (IC50 = 34-57 microM), amobarbitol (IC50 = 375-425 microM), and MPP+ (IC50 = 4-5 mM), consistent with the potencies of these compounds in inhibiting complex I activity. Binding is heterogeneously distributed in brain with the density in gray matter structures varying more than 10-fold. Lesion studies suggest that a substantial portion of binding is associated with nerve terminals. [3H]DHR autoradiography is the first quantitative method to examine complex I with a high degree of anatomic precision. This technique may help to clarify the potential role of complex I dysfunction in normal aging and disease.