Affinity Ones Research Articles

Impact of SARS-CoV-2 vaccination on FcγRIIIA/CD16 dynamics in Natural Killer cells: relevance for antibody-dependent functions.

Natural Killer (NK) cells contribute to the protective effects of vaccine-induced antibodies thanks to the low affinity receptor for IgG, FcγRIIIA/CD16, whose aggregation leads to the killing of infected cells and IFNγ release, through which they potentiate adaptive immune responses. Forty-seven healthy young individuals undergoing either homologous (ChAdOx1-S/ChAdOx1-S) or heterologous (ChAdOx1-S/BNT162B2) SARS-CoV-2 vaccination settings were recruited. Peripheral blood samples were collected immediately prior to vaccination and 8 weeks after the booster dose. The phenotypic and functional profile of NK cells was evaluated by flow cytometry at both time points. Serum samples were tested to evaluate circulating anti-Spike IgG levels and cytomegalovirus serostatus. CD16 F158V polymorphism was assessed by sequencing analysis. The downregulation of CD16 and the selective impairment of antibody-dependent cytotoxicity and IFNγ production in CD56dim NK population, persisting 8 weeks after boosting, were observed in heterologous, but not in homologous SARS-CoV-2 vaccination scheme. While the magnitude of CD16-dependent functions of the global CD56dim pool correlated with receptor levels before and after vaccination, the responsivity of NKG2C+ subset, that displays amplified size and functionality in HCMV+ individuals, resulted intrinsically insensitive to CD16 levels. Individual CD16 responsiveness was also affected by CD16F158V polymorphism; F/F low affinity individuals, characterized by reduced CD16 levels and functions independently of vaccination, did not show post-vaccinal functional impairment with respect to intermediate and high affinity ones, despite a comparable CD16 downregulation. Further, CD16 high affinity ligation conditions by means of afucosylated mAb overcame vaccine-induced and genotype-dependent functional defects. Finally, the preservation of CD16 expression directly correlated with anti-Spike IgG titer, hinting that the individual magnitude of receptor-dependent functions may contribute to the amplification of the vaccinal response. This study demonstrates a durable downmodulation of CD16 levels and Ab-dependent NK functions after SARS-CoV-2 heterologous vaccination, and highlights the impact of genetic and environmental host-related factors in modulating NK cell susceptibility to post-vaccinal Fc-dependent functional impairment.

Key features of inhibitor binding to the human mitochondrial pyruvate carrier hetero-dimer

ObjectiveThe mitochondrial pyruvate carrier (MPC) has emerged as a promising drug target for metabolic disorders, including non-alcoholic steatohepatitis and diabetes, metabolically dependent cancers and neurodegenerative diseases. A range of structurally diverse small molecule inhibitors have been proposed, but the nature of their interaction with MPC is not understood, and the composition of the functional human MPC is still debated. The goal of this study was to characterise the human MPC protein in vitro, to understand the chemical features that determine binding of structurally diverse inhibitors and to develop novel higher affinity ones. MethodsWe recombinantly expressed and purified human MPC hetero-complexes and studied their composition, transport and inhibitor binding properties by establishing in vitro transport assays, high throughput thermostability shift assays and pharmacophore modeling. ResultsWe determined that the functional unit of human MPC is a hetero-dimer. We compared all different classes of MPC inhibitors to find that three closely arranged hydrogen bond acceptors followed by an aromatic ring are shared characteristics of all inhibitors and represent the minimal requirement for high potency. We also demonstrated that high affinity binding is not attributed to covalent bond formation with MPC cysteines, as previously proposed. Following the basic pharmacophore properties, we identified 14 new inhibitors of MPC, one outperforming compound UK5099 by tenfold. Two are the commonly prescribed drugs entacapone and nitrofurantoin, suggesting an off-target mechanism associated with their adverse effects. ConclusionsThis work defines the composition of human MPC and the essential MPC inhibitor characteristics. In combination with the functional assays we describe, this new understanding will accelerate the development of clinically relevant MPC modulators.

Regulatory regions of the C elegans genome contain more low-affinity REF-1 transcription-factor binding sites than high-affinity sites

Regulatory regions that control transcription generally contain multiple binding sites for sequence-specific, DNA-binding proteins. Such regions are characterized by clusters of cis-acting sites that are more evolutionarily conserved than neighboring nonfunctional sequences. Here, we present evidence that the regulatory regions of the Caenorhabditis elegans genome contain more low-affinity binding sites for REF-1, a Hairy/Enhancer of split family transcription factor that contains two basic Helix-Loop-Helix DNA-binding domains, than high affinity ones. We modified the systematic evolution of ligands by exponential enrichment-serial analysis of gene expression (SELEX-SAGE) procedure to obtain detailed information on the low- and high-affinity DNA-binding sequences for REF-1. We then investigated whether the REF-1 binding sites are involved in any characteristic features of the C elegans genome. Statistics clearly showed that the actual frequencies of both low- and high-affinity binding sites within the genome are significantly higher than the expected frequencies. Computational analysis also shows that both low- and high-affinity binding sites for REF-1 are enriched within the regions of the genome that contain more regulatory motifs, suggesting that both types of sites are involved in regulatory events. The low-affinity sites are enriched more than the high-affinity sites within the evolutionarily conserved regions of the genome, which suggests the possibility that a single high-affinity site can compose a regulatory element, but low-affinity sites must be clustered to become an active regulatory element.

P1‐253: Research and development of anti‐Aß oligomers antibodies for the treatment of Alzheimer's disease

Amyloid beta oligomers (AbOs) are promising target for the treatment of Alzheimer's disease (AD) because they are believed as major pathogenic factor of AD. One of the hopeful therapeutic approaches is passive immunotherapy using anti- AbOs specific antibody. This approach has two merits, which are efficient therapeutic effect and decrease of side effects, because the antibody does not bind to amyloid precursor protein (APP) nor naturally existed Abeta. At AAIC2011 last year, we reported the screening results of the best anti-AbOs antibody by in vitro tests and in vivo test using AD model mouse. Here, the progress will be presented. Anti-AbOs antibodies were generated by immunization of antigens prepared by our original method to mouse. The initial screening was performed by ELISA and dotblot assay. The amino acid sequences of all positive clones were analyzed and sequence-redundant clones were eliminated. Then, antibodies that have higher selectivity to AbOs were selected by competitive ELISA. Next, antibodies that displayed non-binding property to normal human tissue array were chosen. Then, their affinities to AbOs were measured by SPR (Surface Plasmon Resonance) analysis, following that the antibodies were divided into high or low affinity groups. Next, passive immunization test using Tg2576 mouse was performed in several antibodies from each group. 184 antibodies were obtained in the results of first screening and subsequent determination of amino acid sequence. Then, 49 antibodies which have higher selectivity to AbOs were selected by the competitive ELISA. Moreover, dozens of antibodies were chosen from 49 antibodies by their non-binding feature to normal human tissue array. Next, these antibodies were divided into high and low affinity groups by SPR analysis. Then in vivo efficacy in several antibodies in each groups were examined by passive immunization test by using Tg2576 mouse. The results showed that high affinity antibodies have better efficacy of improvement of learning memory than low affinity ones, and also there were difference in clearance of Abeta in the brains among the high affinity antibodies. We have obtained the best antibody which has high specificity and affinity to AbOs and also has high efficacy in in vivo experiments.

Research and development of anti-Abeta oligomers antibodies for the treatment of Alzheimer's disease

Amyloid beta oligomers (AbOs) are a promising target for treatment of Alzheimer's disease (AD) because they are considered as major pathogenic factor of AD. One of the hopeful therapeutic approaches targeted to AbOs is passive immunotherapy using anti-AbOs specific antibody. This approach has two merits, which are efficient therapeutic effect and decrease of side effects, because the antibody does not bind to amyloid precursor protein (APP) nor naturally existed Abeta. We generated a number of anti-AbOs antibodies and performed screening to select the best one by in vitro tests and in vivo test using AD model mouse. These results will be presented. Anti-AbOs antibodies were generated by immunization of antigens prepared by our original method to mouse. The initial screening was performed by ELISA and dotblot assay. The amino acid sequences of all positive clones were analyzed and sequence-redundant clones were eliminated. Then, antibodies that have higher selectivity to AbOs were selected by competitive ELISA using Abeta monomer or AbOs as competitors. Next, antibodies that displayed non-binding property to normal human tissue array were chosen. Then, their affinities to AbOs were measured by SPR (Surface Plasmon Resonance) analysis, following that the antibodies were divided into high or low affinity groups. Finally, passive immunization test using Tg2576 mouse was performed in several antibodies from each group. Behavioral tests and biochemical analysis were used for the evaluation. 184 antibodies were obtained in the results of first screening and subsequent determination of amino acid sequence. Then, 49 antibodies which have higher selectivity to AbOs were selected by the competitive ELISA. Moreover, dozens of antibodies were chosen from 49 antibodies by their non-binding feature to normal human tissue array. Next, these antibodies were divided into high and low affinity groups by SPR analysis. Finally, in vivo efficacy in several antibodies in each groups were examined by passive immunization test by using Tg2576 mouse. The results showed that high affinity antibodies have better efficacy of improvement of learning memory than low affinity ones. And also there were difference in clearance of Abeta in the brains among the high affinity antibodies. The screening to select the best antibody, which has high specificity and affinity to AbOs and also has in vivo efficacy, was finished.

Interaction of bovine serum albumi with two alkylimidazolium-based ionic liquids investigated by microcalorimetry and circular dichroism

The interactions of bovine serum albumin (BSA) with two alkylimidazolium-based ionic liquids, 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim]BF4) and 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim]PF6), in buffer solutions at pH 7.0 were investigated by isothermal titration calorimetry (ITC) and circular dichroism (CD). CD spectra showed that the two ionic liquids changed the secondary structure of BSA. Data process was based on the supposition that there were several independent types of binding sites on each BSA molecule for the two ligand molecules. The results obtained by using this supposition combined with Langmuir adsorption model showed that there were two types of such binding sites. One was the high affinity binding site, and the other was the low affinity binding site. The binding constants, changes in enthalpy, entropy and Gibbs free energy for the two types of binding were obtained, which showed that the two types of binding were driven by a favorable entropy increase. Furthermore, for either the ionic liquids, the number of the high affinity binding sites is much smaller than that of the low affinity ones. These results were interpreted with the molecular structure of BSA and the different substituent groups on imidazole ring of the two ionic liquid molecules.

The oxygen-binding modulation of hemocyanin from the Southern spiny lobster Palinurus gilchristi

Arthropod hemocyanins transport and store oxygen and are composed of six subunits, or multiples thereof depending on the species. Palinurus gilchristi hemocyanin is found only as 1 x 6-mers, as normally occurs in spiny lobsters. An alkaline pH and removal of calcium ions induce a wholly reversible dissociation into monomers. The oxygen-binding properties of 1 x 6-meric hemocyanin from P. gilchristi were investigated with respect to pH and modulating effect exerted by calcium, lactate and urate. The oxygen affinity was highly affected by pH in the presence of calcium ions, while in its absence the Bohr coefficient became 60% lower. The protein is insensitive to lactate, but affected by urate which markedly increased hemocyanin-oxygen affinity, acting as the physiological major positive effector. Calcium ions decrease oxygen affinity at low concentration range (0-1 mM), while as concentration becomes higher than 100 mM, the oxygen affinity increases, indicating the presence of two independent types of calcium-binding sites with high and low affinity, respectively. The previous hypothesis, that the presence of high-affinity binding sites in addition to low affinity ones could be a characteristic feature of Palinuran hemocyanins, has been tested by analyzing, with respect to calcium-hemocyanin interaction, three other species belonging to Palinura.

Existence of high and low affinity dinucleotides pentaphosphate-induced calcium responses in individual synaptic terminals and lack of correlation with the distribution of P2X 1–7 subunits

Individual analysis of synaptic terminals calcium responses, induced by dinucleotides pentaphosphate, Ap 5A or Gp 5G, demonstrates the presence of two main groups considering the concentration required for stimulation. The first group corresponds to those responding to Ap 5A or Gp 5G at nanomolar concentration, representing 16% and 12%, respectively, and the second one responds to micromolar concentration and represents, respectively, 17% and 14%, of the total functional synaptosomal population in rat midbrain. Dose–response curves in single terminals showed an Ap 5A EC 50 values of 0.9 ± 0.2 nM and 11.8 ± 0.9 μM, being the maximal intrasynaptosomal calcium increase of 200 ± 0.3 and 125 ± 0.2 nM for the high and low affinity responding terminals, respectively. Combination of microfluorimetric and immunocytochemical studies showed lack of correlation between dinucleotides pentaphosphate responses and P2X receptor subunits expression, in spite of the abundance of P2X 2, P2X 3 and P2X 7 at the presynaptic level in rat midbrain synaptosomes. Pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), a P2X receptors antagonist, showed no effect on low affinity dinucleotides receptors population, and partial inhibition on the high affinity one. On the other hand, diinosine pentaphosphate (Ip 5I) completely abolished the low affinity dinucleotides responses, and 60% inhibition of the high affinity ones.

The evolutionary and structural ‘logic’ of antigen receptor diversity

Most vertebrate species utilize antibody and T-cell receptor (TCR) genes to create a vast repertoire of antigen sensor molecules on their B and T lymphocytes, respectively. While the organization of these genes exhibits substantial variation between species, one common theme is that, in almost every case, there is at least one variable region with a highly diverse CDR3 region and often much less diversity elsewhere in the binding site. Whereas with αβ TCRs this skewing of diversity correlates well with the need to recognize diverse peptides bound to MHC molecules, this cannot explain why this same pattern is evident in immunoglobulins (Igs) or γδ TCRs. Instead we have postulated that in the primary repertoire, all or most antigen receptors have a bipartite binding site, in which diverse CDR3 loops act as a highly antigen specific ‘core’ whereas other CDRs bind in a largely opportunistic fashion. In the case of antibodies, somatic hypermutation then acts to improve the complementarity to a given antigen and increase antibody affinity. A test of this model in mice engineered to have a very limited V region repertoire shows that primary antibodies can be generated that are highly specific for distinct antigens, yet identical in sequence except for their V H CDR3. Furthermore, very high affinity antibodies can be raised by repeated immunizations, showing that somatic hypermutation can mold these low affinity antibodies into high affinity ones. Thus, the wide variations seen in V region repertoires amongst vertebrates is likely to be of lesser importance than the preservation of one or more diverse CDR3 regions.

Interaction of bovine (BSA) and human (HSA) serum albumins with ionic surfactants: spectroscopy and modelling

The binding of several different categories of small molecules to bovine (BSA) and human (HSA) serum albumins has been studied for many years through different spectroscopic techniques to elucidate details of the protein structure and binding mechanism. In this work we present the results of the study of the interactions of BSA and HSA with the anionic sodium dodecyl sulfate (SDS), cationic cethyltrimethylammonium chloride (CTAC) and zwitterionic N-hexadecyl- N, N-dimethyl-3-ammonium-1-propanesulfonate (HPS) monitored by fluorescence spectroscopy of the intrinsic tryptophans at pH 5.0. Similarly to pH 7.0 and 9.0, at low concentrations, the interaction of BSA with these surfactants shows a quenching of fluorescence with Stern–Volmer quenching constants of (1.1±0.1)×10 4 M −1, (3.2±0.1)×10 3 M −1 and (2.1±0.1)×10 3 M −1 for SDS, HPS and CTAC, respectively, which are associated to the ‘effective’ association constants to the protein. On the interaction of these surfactants with HSA, an opposite effect was observed as compared to BSA, i.e., an enhancement of fluorescence takes place. For both proteins, at low surfactant concentrations, a positive cooperativity was observed and the Hill plot model was used to estimate the number of surfactant binding sites, as well as the association constants of the surfactants to the proteins. It is worthy of notice that the binding constants for the surfactants at pH 5.0 are lower as compared to pH 7.0 and 9.0. This is probably due to fact that the protein at this acid pH is quite compact reducing the accessibility of the surfactants to the hydrophobic cavities in the binding sites. The interaction of myristic acid with both proteins shows a similar fluorescence behaviour, suggesting that the mechanism of the interaction is the same. Recently published crystallographic studies of HSA–myristate complex were used to perform a modelling study with the aim to explain the fluorescence results. The crystallographic structure reveals that a total of five myristic acid molecules are asymmetrically bound in the macromolecule. Three of these sites correspond to higher affinity ones and correlate with high association constants described in the literature. Our models for BSA and HSA with bound SDS suggest that the surfactant could be bound at the same sites as those reported in the crystal structure for the fatty acid. The differences in tryptophan vicinity upon surfactant binding are explored in the models in order to explain the observed spectroscopic changes. For BSA the quenching is due to a direct contact of a surfactant molecule with the indole of W 131 residue. It is clear that the binding site in BSA which is very close, in contact with tryptophan W 131, corresponds to a lower affinity site, explaining the lower binding constants obtained from fluorescence studies. In the case of HSA the enhancement of fluorescence is due to the removal of static quenching of W 214 residue in the intact protein caused by nearby residues in the vicinity of this tryptophan.

Regulation of the expression of low affinity GABAA receptors in rat cerebellar granule cells.

GABAA receptors of cerebellar granule cells obtained from neonatal rats and kept in culture were studied by labelled muscimol binding. The data show that, according to the maturational state of those cells in vivo, one or two binding components appear. The low affinity component seems to be the one appearing later. The expression of this component seems to be regulated by protein tyrosine phosphorylation. In fact, its expression is down regulated by the protein tyrosine kinase (PTK) inhibitor, genistein. Viceversa, its expression is upregulated by insulin like growth factor I (IGF-I), most probably via PTK activation. A possible interpretation of the data is that in vivo IGF-I is one of the endogenous messages leading to the expression of this component during development. Another endogenous factor involved may be GABA itself. Low affinity GABAA receptors appear to be the ones involved in inhibitory synaptic transmission at glomeruli. Whereas the high affinity ones probably correspond to extrasynaptic GABAA receptors mediating the tonic form of inhibition in cerebellar granules.

Adsorption of metal ions onto goethite: single-adsorbate and competitive systems

Adsorption of anthropogenically released toxic metals such as Ni and Zn to goethite effects their mobility and bioavailability in aquatic environments. In this research sorption studies were conducted to understand competitive adsorption of environmentally important metals such as Ni, Zn, and Ca onto the goethite surface. Adsorption edges conducted as a function of ionic strength suggest that these metals are chemisorbed to the goethite surface. Furthermore, the adsorption affinity follows the order of the inverse of the hydrated radii multiplied by the number of waters in the primary solvation shell: Zn>Ni>Ca. Isotherm studies revealed a linear relation between the amount of metal adsorbed and the aqueous bulk phase concentrations, where site saturation was obtained by reducing the goethite concentration to 0.1 g l −1. Accordingly the single-site Langmuir model provided a good fit; equilibrium constants were found to be independent of pH indicative of one type of adsorption reaction. The equilibrium constants for both transition metals (Ni and Zn) were greater than that of Ca, suggesting that transition metals have a greater affinity for the surface. Analyses of site densities revealed two types of sites on the surface of goethite: high affinity ones to which transition metals bind, and low affinity sites that comprise 100× that of the high affinity ones. From the isotherm studies, it appears that only the alkaline earth metals such as Ca adsorb to this lower affinity site. The single-site Langmuir model was able to accurately describe adsorption competition between Ni and Zn for the goethite surface. In contrast, no competitive effects were observed in Ni–Ca and Zn–Ca binary systems.

Calcium Binding of Transglutaminases: A 43Ca NMR Study Combined with Surface Polarity Analysis

Transglutaminases (TGases) form cross-links between glutamine and lysine side-chains of polypeptides in a Ca2+-dependent reaction. The structural basis of the Ca2+-effect is poorly defined. 43Ca NMR, surface polarity analysis combined with multiple sequence alignment and the construction of a new homology model of human tissue transglutaminase (tTGase) were used to obtain structural information about Ca2+ binding properties of factor XIII-A2, tTGase and TGase 3 (each of human origin). 43Ca NMR provided higher average dissociation constants titrating on a wide Ca2+-concentration scale than previous studies with equilibrium dialysis performed in shorter ranges. These results suggest the existence of low affinity Ca2+ binding sites on both FXIII-A and tTGase in addition to high affinity ones in accordance with our surface polarity analysis identifying high numbers of negatively charged clusters. Upon increasing the salt concentration or activating with thrombin, FXIII-A2 partially lost its original Ca2+ affinity; the NMR data suggested different mechanisms for the two activation processes. The NMR provided structural evidence of GTP-induced conformational changes on the tTGase molecule diminishing all of its Ca2+ binding sites. NMR data on the Ca2+ binding properties of the TGase 3 are presented here; it binds Ca2+ the most tightly, which is weakened after its proteolytic activation. The investigated TGases seem to have very symmetric Ca2+ binding sites and no EF-hand motifs.

Expression of sodium channels with different saxitoxin affinity during rat forebrain development

This work characterizes the development of the saxitoxin (STX)-sensitive Na + channels from rat whole forebrain between embryonic day 15 (E15) and postnatal day 90 (P90), both with binding studies and with single channel studies. The Na + channel total mRNA and the individual mRNAs encoding Na + channels I, II and III were also determined. The total STX binding rose about 40-fold from E15 to reach a plateau at P30 and its temporal course correlated with the expression of Na + channel total mRNA. Low affinity and high-affinity STX binding sites, predominant in embryonic and postnatal forebrains, respectively, were found. The single channel studies of batrachotoxin-modified channels also revealed two main populations. In E15 only low-affinity channels ( K D = 32.7 nM; 200 mM NaCl) and in P30 only high affinity ones ( K D = 1.6 nM) were present. At P0 channels with intermediate affinity ( K D range 3–34 nM) were observed. The increase in affinity was due to a gradual increase in the STX association rate.

Heterogeneity of Guinea-Pig Lung Muscarinic Receptors Revealed by [3H]4-DAMP-binding

Summary: Muscarinic receptors present in guinea-pig lung were characterized using the M3 selective radioligand [3H]4-diphenylacetoxy-N-methyl-piperidine methiodide ([3H]4-DAMP). In saturation studies, [3H]4-DAMP identified two populations of binding sites with approximately 4% of the sites displaying high affinity (Kd=0.21 nM and Bmax= 10 fmol/mg prot.) while the remaining sites were low affinity ones (Kd=18.11 nM and Bmax=269 fmol/mg prot.). In competition studies with [3H]4-DAMP (0.35 nM), methoctramine and hexahydro-siladifenidol (HHSiD) identified 50 and 70% of high affinity binding sites displaying the pharmacological profile of the M2 and the M3 receptors, respectively. No evidence was found for high affinity [3H]pirenzepine binding sites in guinea-pig lung. However, pirenzepine/[3H]4-DAMP competition experiments suggested that pirenzepine recognized an equal proportion of [3H]4-DAMP binding sites with intermediate and low affinity binding constants. The intermediate affinity binding constant was inconsistent with the presence of M1 receptors and reflected more the presence of M4 or a mixture of M3 and M4 receptors. The low affinity pirenzepine binding sites may represent M2 receptors. These results provide further evidence for the occurrence of M2 and M3 receptors and suggest the presence of the M4 muscarinic receptor subtype in guinea-pig lung.

Exposure to particle-bound polyaromatic hydrocarbons in the Al-Mansoria residential area during the Kuwait oil fires: A qualitative appraisal of the adsorption role

High ambient levels of inhalable particulate matter (PM 10) were detected in residential areas during the oil well burning in Kuwait (February–November 1991). Because inhalation exposures to PM 10 were significant (data on PAH quantification are scarce), it became possible to describe the exposure to PM 10-associated PAHs of alternative courses of events, such as PAH-particle interaction mechanisms. Depending on particle adsorption characteristics (affinity and site availability), it is concluded that, contrary to what is currently believed, low levels of ambient PM 10 levels did not indicate low PAH exposures in Al-Mansoria residential area during May 10–31, 1991. Due to the frequent presence of dust particles in the ambient air caused by the heavy dust fallout in Al-Mansoria (average > 65 tons/km 2) during May, 1991, the predicted patterns can be explained by two hypothesized mechanisms. The first is a two-step process: loss of PAHs from low affinity sites and reabsorption onto stronger affinity ones leading to low surface coverage at high PM 10 concentrations. The second involves dilution of PAH-containing soot with aeolian particles. Both events can lead to low ambient PAHs at high PM 10 levels or high ambient PAHs at low PM 10 levels.

Uptake of Proteins Modified with 3-Deoxyglucosone, a Maillard Reaction Intermediate, by the Type I Macrophage Scavenger Receptor

We investigated the mechanism of recognition by murine peritoneal macrophages of bovine serum albumin (BSA) modified with glucose and 3-deoxyglucosone (3DG), the main intermediate compound in the Maillard reaction of proteins with glucose. A scatchard analysis of the binding data of BSA incubated with 3DG for 17 days (3DG-BSA 17d) and of glycated BSA indicated two binding sites, each with a different binding affinity, in both cases. The apparent Kd values for the higher affinity sites of 3DG-BSA 17d and of glycated BSA were 8.5 nM and 4.6 nM, and those for the lower affinity ones were 0.95 μM and 1.3 μM, respectively. 3DG-BSA 17d and glycated BSA competed with each other in the specific binding to macrophages, this binding being most effectively inhibited by maleylated BSA and polyinosinic acid. The binding of 3DG-BSA 17d and glycated BSA to the type I macrophage scavenger receptor [Kodama et al. Nature, 343, 531–535 (1990)] was investigated, because these modified BSAs share binding sites with maleylated...

Regulation of beta-endorphin receptor expression in mouse spleen cells with Con A and rIL-2.

The expression of the beta-endorphin receptor on both activated and unstimulated mouse spleen cells was studied. Results showed that unstimulated cells have only one type of beta-endorphin receptor with a specific low affinity (Kd = 1.034 +/- 0.0237 x 10(-7) M, 25,000 sites/cell). After Con A stimulation, cells express two types of receptors, one with a low affinity (Kd = 1.034 +/- 0.024 x 10(-7) M, 320,000 sites/cell) and the other with a high affinity (Kd = 1.052 +/- 0.033 x 10(-9) M, 49,000 sites/cell). The kinetic experiments during 4 days after Con A activation indicated that the receptor of high affinity emerged from 24 to 72 h, while the low affinity one increased in number after stimulation. The receptor numbers of both high and low affinity ones reached a maximum peak at 72 h, then began to decline. The addition of exogenous rIL-2 depressed the Con A-induced increment of the receptor numbers of both the high and low affinity ones, but enhanced the proliferative response of the cells. It is suggested that the degree of the expression of the receptors does not simply depend on the mitogenic degree of the cells. In addition, our experiment demonstrated that splenocytes cultured in medium with or without Con A or Con A + rIL-2 for 96 h did not secrete any detectable amount of beta-endorphin with use of the RIA assay, which is sensitive enough to detect the much lower levels of beta-endorphin than that necessary for biological effects. We suggest that the expression of the high affinity beta-endorphin receptor on the activated T-lymphocytes may have to precede the production of IL-2 to potentiate the T-cell proliferative response. The mechanisms and modes of interaction between the neuroendocrine system and the immune system were discussed.

Effect of diazepam on the low affinity GABA binding sites at different developmental stages of the chick optic lobe

The developmental profile of GABA receptor sites was studied under experimental conditions where the low affinity binding site is mainly measured. The in vitro effects of nanomolar concentrations of diazepam upon low affinity GABA binding sites during development were also examined. The addition of 50 nM diazepam enhanced specific [3H]GABA binding at all stages of development, this stimulation being greater at early stages (10th–14th embryonic day). Scatchard analysis performed at the 14th embryonic day revealed only the binding sites of low affinity with a Kd of 520 Nm and a maximal number of binding sites (Bmax) of 7.60 pmol/mg protein. At hatching two populations of GABA binding sites were present: one with high affinity (Kd = 10.47 nM and Bmax = 0.22 pmol/mg protein) and the other with low affinity (Kd = 686.51 nM and Bmax = 13.28 pmol/mg protein). The addition of 50 nM diazepam increased the affinity of the low affinity GABA binding sites in both stages of development, while that of the high affinity ones remained unchanged. The apparent Kds of [H3]GABA binding at the 14th embryonic day and at hatching were 120.50 and 452.06 nM, respectively, while the densities of receptor sites were unchanged. Our results show that diazepam increases low affinity GABA binding in an age-dependent manner, this effect being higher in tissue obtained from embryonic chicken optic lobe as compared to the adult one. The binding data obtained in this study suggest a tighter coupling of the GABA-benzodiazepine complex at early stages of development.

3H]tyrosine binding to plasma membrane preparation of rat corpora lutea.

Plasma membrane preparations of rat corpora lutea have been incubated with [3H]tyrosine. [3H]tyrosine binding sites are demonstrated and Scatchard analysis shows that there exist two types of binding sites, one with high affinity and low capacity, the other with low affinity and high capacity. The kinetics studies demonstrate that the [3H]tyrosine binding to the two types of binding sites is reversible and the speed of binding to the high affinity type is faster than that to the low affinity type. The analysis of the chemical structure of tyrosine analogues and related compounds with respect to the specificity of the binding sites reveal that both types of binding sites show specificity, but the specificity of the high affinity sites is higher than that of the low affinity sites. The relations of tyrosine structure to binding processing and to tyrosine inhibitory action on hCG-induced progesterone production are discussed. It is suggested that the high affinity binding sites might be regarded as "tyrosine receptors" and the low affinity ones glycoprotein carriers for tyrosine across the plasma membrane.