Analysis Of Saturation Curves Research Articles

Oxygen Saturation Curve Analysis in 2298 Hypoxia Awareness Training Tests of Military Aircrew Members in a Hypobaric Chamber.

We aim to provide reference values for military aircrews participating in hypoxia awareness training (HAT). We describe several parameters with potential biomedical interest based on selected segments and slopes of the changes in oxygen saturation (SatO2) during a standard HAT. A retrospective analysis of 2298 records of the SatO2 curve was performed, including 1526 military men aged 30.48 ± 6.47 years during HAT in a hypobaric chamber. HAT consisted of pre-oxygenation at 100% and an ascent to 7620 m, followed by O2 disconnection starting the phase of descent of SatO2 until reaching the time of useful consciousness (TUC), and finally reconnection to 100% O2 in the recovery phase. Using an ad hoc computational procedure, the time taken to reach several defined critical values was computed. These key parameters were the time until desaturation of 97% and 90% (hypoxia) after oxygen mask disconnection (D97/D90) and reconnection (R97/R90) phases, the time of desaturation (TUC-D97) and hypoxia (TUC-D90) during disconnection, the total time in desaturation (L97) or hypoxia (L90), and the slopes of SatO2 drop (SDSAT97 and SDSAT90) and recovery (SRSAT97). The mean of the quartiles according to TUC were compared by ANOVA. The correlations between the different parameters were studied using Pearson's test and the effect size was estimated with ω2. Potentially useful parameters for the HAT study were those with statistical significance (p < 0.05) and a large effect size. D97, D90, R97, and R90 showed significant differences with small effect sizes, while TUC-D97, TUC-D90, L97, L90, and SDSAT97 showed significant differences and large effect sizes. SDSAT97 correlated with TUC (R = 0.79), TUC-D97 (R = 0.81), and TUC-D90 (R = 0.81). In conclusion, several parameters of the SatO2 curve are useful for the study and monitoring of HAT. The SDSAT97 measured during the test can estimate the TUC and thus contribute to taking measures to characterize and protect the aircrew members.

Sulfur-Containing Persistent Free Radicals and Reactive Species on Photoaged Microplastics: Identification and the Formation Mechanism.

The elemental composition may affect the persistent free radical (PFR) and reactive species (RS) formation associated with photoaging microplastics; however, a relevant study is still lacking. This study systematically investigated the formation, evolution, and types of PFRs and RS on sulfur-containing microplastics (S-MPs) under simulated sunlight. Electron paramagnetic resonance detection and power saturation curve analysis isolated three different PFRs on each photoaging poly(phenylene sulfide) (PPS) and polysulfone (PSF). Combining the results of characterization and density functional theory calculation, these observed PFRs on the irradiated S-MPs were classified as oxygen-centered radicals with an adjacent S atom (namely, thio-oxygen radicals), oxygen-centered and sulfur-centered radicals, where the thio-oxygen radicals on PPS were benzenethiol-like radicals, and oxygen-centered radicals and sulfur-centered radicals on PSF that were identified as benzenesulfonic-like radicals and phenyl sulfonyl-like radicals, respectively. Moreover, potential precursor molecule fragments of PFRs on the photoaging S-MPs, including p-toluenesulfinic acid and benzenesulfonic acid, were detected by pyrolysis-gas chromatography/mass spectrometry and liquid chromatography-mass spectrometry. Interestingly, reactive sulfur species (SO3•-) was also observed on irradiated S-MPs in addition to reactive oxygen species, which was mainly derived from the reaction of •OH and sulfonyl radicals. These results have implications for assessing the potential risks of atmospheric S-MPs.

Peaksat: an R package for ChIP-seq peak saturation analysis

BackgroundEpigenomic profiling assays such as ChIP-seq have been widely used to map the genome-wide enrichment profiles of chromatin-associated proteins and posttranslational histone modifications. Sequencing depth is a key parameter in experimental design and quality control. However, due to variable sequencing depth requirements across experimental conditions, it can be challenging to determine optimal sequencing depth, particularly for projects involving multiple targets or cell types.ResultsWe developed the peaksat R package to provide target read depth estimates for epigenomic experiments based on the analysis of peak saturation curves. We applied peaksat to establish the distinctive read depth requirements for ChIP-seq studies of histone modifications in different cell lines. Using peaksat, we were able to estimate the target read depth required per library to obtain high-quality peak calls for downstream analysis. In addition, peaksat was applied to other sequence-enrichment methods including CUT&RUN and ATAC-seq.Conclusionpeaksat addresses a need for researchers to make informed decisions about whether their sequencing data has been generated to an adequate depth and subsequently sufficient meaningful peaks, and failing that, how many more reads would be required per library. peaksat is applicable to other sequence-based methods that include calling peaks in their analysis.

Fouling Potential Determination of a UASB Effluent Using Different Assessment Methods

The fouling potential of a pilot-scale UASB effluent was determined using different assessment methods (SDI and MFI tests and a mathematical analysis based on a saturation curve model). The UASB reactor was operated at HRT of 4, 8 and 12 h. Although the three methods employed were adequate as a predictive tool for UF membrane fouling, the saturation curve analysis revealed a clearer relationship between the fouling potential of the UASB effluent and the HRT applied. It was found that operating a UASB reactor under short HRT, such as 4 h, could increase membrane fouling in a post-treatment UF module.

Portfolios: Patterns in brand penetration, market share, and hero product variants

This research investigates the contribution of each stock-keeping unit (SKU) within a brand portfolio towards total brand penetration and market share, by adapting a method called Saturation Curve Analysis. The study utilises UK and US data on 90,000+ SKUs across 15 packaged goods categories. The results show that while the optimal number of SKUs in a portfolio is category specific, the top-selling SKU contributes around 50% of the brand penetration and 40% of sales. This establishes a benchmark for monitoring brand performance. These results emphasise the importance of having top-selling SKUs readily available to consumers, rather than sacrificing them over new product launches.

Using the MWC model to describe heterotropic interactions in hemoglobin.

Hemoglobin is a classical model allosteric protein. Research on hemoglobin parallels the development of key cooperativity and allostery concepts, such as the ‘all-or-none’ Hill formalism, the stepwise Adair binding formulation and the concerted Monod-Wymann-Changuex (MWC) allosteric model. While it is clear that the MWC model adequately describes the cooperative binding of oxygen to hemoglobin, rationalizing the effects of H+, CO2 or organophosphate ligands on hemoglobin-oxygen saturation using the same model remains controversial. According to the MWC model, allosteric ligands exert their effect on protein function by modulating the quaternary conformational transition of the protein. However, data fitting analysis of hemoglobin oxygen saturation curves in the presence or absence of inhibitory ligands persistently revealed effects on both relative oxygen affinity (c) and conformational changes (L), elementary MWC parameters. The recent realization that data fitting analysis using the traditional MWC model equation may not provide reliable estimates for L and c thus calls for a re-examination of previous data using alternative fitting strategies. In the current manuscript, we present two simple strategies for obtaining reliable estimates for MWC mechanistic parameters of hemoglobin steady-state saturation curves in cases of both evolutionary and physiological variations. Our results suggest that the simple MWC model provides a reasonable description that can also account for heterotropic interactions in hemoglobin. The results, moreover, offer a general roadmap for successful data fitting analysis using the MWC model.

Cooperative Binding of Metal Cations to a Spiropyran‐Conjugated Calix[4]arene

AbstractCooperative binding occurs frequently in biological systems, but is less exploited in synthetic receptor molecules. We synthesized a bis(spiropyran)‐conjugated calix[4]arene (bSPTC) through a 1,2,3‐triazole linker and found cooperativity in bindings of a series of divalent metal cations (Ca2+, Mg2+, Cd2+, Co2+, Fe2+, Hg2+, Ni2+, and Zn2+). UV‐vis absorption spectroscopy and principle component analysis were used to differentiate the recognized metal cations. The binding stoichiometry was determined by various continuation methods, which indicated that bSPTC has two binding ratios, 1:1 for Zn2+ and 1:2 for all other metal cations. Importantly, positive cooperativity was suggested for the 1:2 stoichiometry of bSPTC to Mg2+ ions from the sigmoidal saturation curve and Hill equation analysis. Density functional theory (DFT) calculations imply that the first binding of Mg2+ to one spiropyran destabilizes the second spiropyran, which facilitates binding of a second Mg2+.

Multiple AMPK activators inhibit l-carnitine uptake in C2C12 skeletal muscle myotubes.

Mutations in the gene that encodes the principal l-carnitine transporter, OCTN2, can lead to a reduced intracellular l-carnitine pool and the disease Primary Carnitine Deficiency. l-Carnitine supplementation is used therapeutically to increase intracellular l-carnitine. As AMPK and insulin regulate fat metabolism and substrate uptake, we hypothesized that AMPK-activating compounds and insulin would increase l-carnitine uptake in C2C12 myotubes. The cells express all three OCTN transporters at the mRNA level, and immunohistochemistry confirmed expression at the protein level. Contrary to our hypothesis, despite significant activation of PKB and 2DG uptake, insulin did not increase l-carnitine uptake at 100 nM. However, l-carnitine uptake was modestly increased at a dose of 150 nM insulin. A range of AMPK activators that increase intracellular calcium content [caffeine (10 mM, 5 mM, 1 mM, 0.5 mM), A23187 (10 μM)], inhibit mitochondrial function [sodium azide (75 μM), rotenone (1 μM), berberine (100 μM), DNP (500 μM)], or directly activate AMPK [AICAR (250 μM)] were assessed for their ability to regulate l-carnitine uptake. All compounds tested significantly inhibited l-carnitine uptake. Inhibition by caffeine was not dantrolene (10 μM) sensitive despite dantrolene inhibiting caffeine-mediated calcium release. Saturation curve analysis suggested that caffeine did not competitively inhibit l-carnitine transport. To assess the potential role of AMPK in this process, we assessed the ability of the AMPK inhibitor Compound C (10 μM) to rescue the effect of caffeine. Compound C offered a partial rescue of l-carnitine uptake with 0.5 mM caffeine, suggesting that AMPK may play a role in the inhibitory effects of caffeine. However, caffeine likely inhibits l-carnitine uptake by alternative mechanisms independently of calcium release. PKA activation or direct interference with transporter function may play a role.

Probing the Interfacial Chemistry of Ultrathin ALD-Grown TiO2 Films: An In-Line XPS Study

Ultrathin TiO2 films received renewed attention in the field of photoelectrochemical water splitting as corrosion protection layers for unstable, small-bandgap semiconductors. Because nucleation on the substrate can differ from steady-state growth of the film itself, it is important to understand the nucleation behavior on a specific surface. In this work, we studied the nucleation mechanism of atomic layer deposition-grown TiO2 from TiCl4 and H2O on as-received silicon by means of in-line X-ray photoelectron spectroscopy. Within a region of ∼0.4 nm of the SiO2/TiO2 interface, the presence of Ti3+ states are detected. In this region, the Ti, O, and Cl species are found to be more strongly bonded. At the initial stages of film growth, prolonged TiCl4 exposure is necessary to reach a saturated surface chemistry, which is in contrast to the outcome of growth per cycle saturation curve analysis. A prolonged water exposure experiment suggests that residual chlorine impurities can be prevented by using a suffic...

Mechanism of 3,3′-Disulfopropyl-5,5′-Dichlorothiacyanine Anion Interaction With Citrate-Capped Silver Nanoparticles: Adsorption and J-Aggregation

The paper presents the results of the experimental and theoretical study of 3,3′-disulfopropyl-5,5′-dichlorothiacyanine sodium salt (TC) adsorption and J-aggregation on the surface of citrate-capped silver nanoparticles (AgNPs) with an average particle size 10 nm. Various nanospectroscopy techniques such as UV–vis, TEM, AFM, DLS, ζ potential, and fluorescence measurements were carried out in order to characterize the hybrid system. Besides this, a set of simple density functional theory calculations (DFT) was performed and this suggested that TC dye, from the thermodynamical point of view, most likely interacted with AgNPs via oxygen atom from SO3– groups. The methods, which considered AgNPs as the macromolecule with several binding sites and TC dye as the ligand, were applied for the analysis of saturation curves constructed from the fluorescence data to obtain the binding constant (Ka = 1.5 × 106 M–1) and the average number of TC dye molecules bonded per AgNP (∼330). Moreover, TC fluorescence was quench...

Inhibitory effects of eppididymal protease inhibitor on the activity of prostate specific antigen

Objective To investigate the inhibitory effects of the epididymal protease inhibitor (Eppin) on the activity of prostate specific antigen (PSA).Methods Recombinant Eppin and recombinant PSA were produced by molecular cloning technique in vitro.The enzymatical analysis of Eppin inhibiting PSA was done in the reaction buffer 0.1 mol/L Tris-HC1,pH 8.3,1.0 mol/L NaCl.The hydrolysis of velocity of PSA to the chromogenic substrate S-2586 was detected by spectrometer.Results Recombinant Eppin and PSA with high purity were produced by molecular cloning technique.The recombinant PSA had the enzymatical activity by hydrolyzing its substrate S-2586.0.3μmol/L PSA and the substate S-2586 reaction to the saturation curve analysis showed that Km( response rate of half the maximum reaction rate when the substrate concentration) value was 0.5μmol,and substrate saturated concentration was 0.2 mmol/L.With the increases of Eppin (0,0.56,1.16,2.32μmol/L),the hydrolysis velocity of PSA to S-2586 was slowed down.Conclusion Eppin,as a new inhibitor,specifically inhibits the activity of PSA. Key words: Epididymal; Protease inhibitor; Prostate specific antigen

Evaluation of weathering-resistance classes in clastic rocks on the example of Polish sandstones

The scope of the paper is an attempt at the identification of the weathering-resistance classes within clastic rocks by means of analysis of capillary pressure saturation curves. The porosimetric parameters corresponding to the cementing character and the grains’ mineralogical content are very important features of stone building materials, because of the weathering processes. The analysed rocks were Polish sandstones and muddy sandstones used for building purposes, collected from different geological units of Poland (i.e. Sudety Mts. Carpathian Mts. and Holy Cross Mts.) The results indicate the usefulness of sandstone materials for building purposes. They could also be used in conservation procedures and for the reconstruction of existing buildings and monuments. Basing on the parameterisation, with the van Genuchten function, of cumulative capillary pressure saturation curves, it was possible to distinguish four groups of the sampled rocks. The lithological features and weathering sustainability within the groups are quite uniform, what allow identifying the weathering resistance classes. Taking into account the complicated nature of all the factors influencing weathering processes, it is supposed that the presented parameterisation could be a useful tool for weathering-resistance classification of clastic rocks. The classification could be useful in building industry and in conservation of historical stone monuments.

Critical Active-Site Residues Identified by Site-Directed Mutagenesis in Pseudomonas aeruginosa Phosphorylcholine Phosphatase, A New Member of the Haloacid Dehalogenases Hydrolase Superfamily

Pseudomonas aeruginosa phosphorylcholine phosphatase (PChP), the product of the PA5292 gene, is synthesized when the bacteria are grown with choline, betaine, dimethylglycine, or carnitine. In the presence of Mg(2+), PChP catalyzes the hydrolysis of both phosphorylcholine (PCh) and p-nitrophenylphosphate (p-NPP). PCh saturation curve analysis of the enzyme with or without the signal peptide indicated that the peptide was the fundamental factor responsible for decreasing the affinity of the second site of PChP for PCh, either at pH 5.0 or pH 7.4. PChP contained three conserved motifs characteristic of the haloacid dehalogenases superfamily. In the PChP without the signal peptide, motifs I, II, and III correspond to the residues (31)DMDNT(35), (166)SAA(168), and K(242)/(261)GDTPDSD(267), respectively. To determine the catalytic importance of the D31, D33, T35, S166, K242, D262, D265, and D267 on the enzyme activity, site-directed mutagenesis was performed. D31, D33, D262, and D267 were identified as the more important residues for catalysis. D265 and D267 may be involved in the stabilization of motif III, or might contribute to substrate specificity. The substitution of T35 by S35 resulted in an enzyme with a low PChP activity, but conserves the catalytic sites involved in the hydrolysis of PCh (K(m1) 0.03 mM: , K(m2) 0.5 mM: ) or p-NPP (K(m) 2.1 mM: ). Mutating either S166 or K242 revealed that these residues are also important to catalyze the hydrolysis of both substrates. The substitution of lysine by arginine or by glutamine revealed the importance of the positive charged group, either from the amino or guanidinium groups, because K242Q was inactive, whereas K242R was a functional enzyme.

Quantitative Analysis of Muscarinic Acetylcholine Receptor Homo- and Heterodimerization in Live Cells: REGULATION OF RECEPTOR DOWN-REGULATION BY HETERODIMERIZATION

Although previous pharmacological and biochemical data support the notion that muscarinic acetylcholine receptors (mAChR) form homo- and heterodimers, the existence of mAChR oligomers in live cells is still a matter of controversy. Here we used bioluminescence resonance energy transfer to demonstrate that M1, M2, and M3 mAChR can form constitutive homo- and heterodimers in living HEK 293 cells. Quantitative bioluminescence resonance energy transfer analysis has revealed that the cell receptor population in cells expressing a single subtype of M1, M2, or M3 mAChR is predominantly composed of high affinity homodimers. Saturation curve analysis of cells expressing two receptor subtypes demonstrates the existence of high affinity M1/M2, M2/M3, and M1/M3 mAChR heterodimers, although the relative affinity values were slightly lower than those for mAChR homodimers. Short term agonist treatment did not modify the oligomeric status of homo- and heterodimers. When expressed in JEG-3 cells, the M2 receptor exhibits much higher susceptibility than the M3 receptor to agonist-induced down-regulation. Coexpression of M3 mAChR with increasing amounts of the M2 subtype in JEG-3 cells resulted in an increased agonist-induced down-regulation of M3, suggesting a novel role of heterodimerization in the mechanism of mAChR long term regulation.

Determination of Equilibrium Dissociation Constants in Fluorescence Polarization

A simple mathematical model (the FP K(d) model) is used to generate the dissociation equilibrium constant (K(d)) for G protein-coupled receptor-ligand binding measured using fluorescence polarization (FP) saturation curve analysis. The model generates data that may be analyzed by the method of Scatchard. The validity of the FP K(d) model is proven in six model systems in which the modeled K(d) values are within a factor of 5 of inhibitory equilibrium constant values obtained from radioligand competition assays.

Analysis of low-density lipoprotein catabolism by primary cultures of hepatic cells from normal and low-density lipoprotein receptor knockout mice

Low-density lipoproteins (LDL) are taken up by LDL receptor (LDLr)-dependent and -independent pathways; the role and importance of the latest being less well defined. We analyzed the importance of these pathways in the mouse by comparing LDL binding to primary cultures of hepatocytes from LDLr knockout (LDLr KO) and normal C57BL/6J mice. Saturation curve analysis shows that 125I-LDL bind specifically to normal and LDLr KO mouse hepatocytes with similar dissociation constants ( K d) (31.2 and 22.9 μg LDL-protein/ml, respectively). The maximal binding capacity ( B max) is, however, reduced by 48% in LDLr KO mouse hepatocytes in comparison to normal hepatocytes. Conducting the assay in the presence of a 200-fold excess of high-density lipoprotein-3 (HDL3) reduced by 39% the binding of 125I-LDL to normal hepatocytes and abolished the binding to the LDLr KO mouse hepatocytes. These data indicate that in normal mouse hepatocytes, the LDLr is responsible for approximately half of the LDL binding while a lipoprotein binding site (LBS), interacting with both LDL and HDL3, is responsible for the other half. It can also be deduced that both receptors/sites have a similar affinity for LDL. The metabolism of LDL-protein and cholesteryl esters (CE) was analyzed in both types of cells. 125I-LDL-protein degradation was reduced by 95% in LDLr KO hepatocytes compared to normal hepatocytes. Comparing the association of 125I-LDL and 3H-CE-LDL revealed a CE-selective uptake of 35.6- and 22-fold for normal and LDLr KO mouse hepatocytes, respectively. Adding a 200-fold excess of HDL3 in the assay reduced by 71% the CE-selective uptake in LDLr KO hepatocytes and by 96% in normal hepatocytes. This indicates that mouse hepatocytes are able to selectively take up CE from LDL by the LBS. The comparison of LDL-CE association also showed that the LBS pathway provides 5-fold more LDL-CE to the cell than the LDLr. Overall, our results indicate that in mouse hepatocytes, LDLr is almost completely responsible for LDL-protein degradation while the LBS is responsible for the major part of LDL-CE entry by a CE-selective uptake pathway.

Calcitonin modifies ligand binding to muscarinic receptor in CNS membranes

Calcitonin (CT) is a peptide produced by the thyroid gland, whose best described role is to prevent bone reabsorption, though it also participates in other biological functions through both central and peripheral mechanisms. CT is able to inhibit brain Na +, K +-ATPase activity (Rodrı́guez de Lores Arnaiz, López Ordieres, Peptides 1997;18:613–5) and a relationship between such enzyme activity and cholinergic function has been suggested. Accordingly, we tested CT effect on [ 3H]-quinuclidinyl benzilate ([ 3H]-QNB) binding to rat CNS membranes to determine whether the peptide is able to modify the cholinergic muscarinic receptor as well. It was found that 1×10 −7–1×10 −5 M CT decreased 20–70% ligand binding to hippocampal, cerebellar, cortical and striatal membranes. Scatchard analysis of saturation curves showed that 5×10 −6 M CT significantly modified binding kinetic constants, thus it increased roughly 220% K d values and decreased 20–36% B max values in cerebral cortical and cerebellar membranes. Since the peptide decreases affinity ligand binding and reduces the number of binding sites, CT may well be acting as a cholinergic modulator through a decrease in muscarinic receptor functionality.

Effects of training on the concentration of Na+, K+-ATPase in foal muscle.

We investigated the effect of training on the Na+, K+-ATPase concentration in foal skeletal muscle by measurement of [3H]ouabain binding. From the 7th day after birth, 12 foals were divided in 3 groups: a) staying in a box stall (Box); b) staying in a box stall with a training regimen of an increasing number of sprints per day (Training); and c) staying on pasture (Pasture). Euthanasia was performed after 5 months. In semitendinosus muscle, the concentration of [3H]ouabain binding sites (pmol/g wet wt) was 181 +/- 6 in the Box, 220 +/- 15 in the Training, and 197 +/- 8 in the Pasture group (all n = 6; Box vs. Training, P<0.05). In gluteus medius, the concentration of [3H]ouabain binding sites was 168 +/- 9 in the Box, 219 +/- 12 in the Training, and 175 +/- 4 in the Pasture group (all n = 6; Box or Pasture vs. Training, P<0.02). Scatchard analysis of saturation curves showed that the difference in [3H]ouabain binding sites between the 3 groups could not be ascribed to differences in the Kd for ouabain. Both for semitendinosus and gluteus medius muscle, the concentration of [3H]ouabain binding sites increased in the order Box < Pasture < Training (a total increase of around 20%). This suggests a specific effect of the amount and intensity of exercise on the Na+, K+-ATPase concentration in horse skeletal muscle, and may lead to a better performance during exercise.

Low density lipoprotein-receptor plays a major role in the binding of very low density lipoproteins and their remnants on HepG2 cells

The binding to HepG2 cells of very low density lipoproteins (VLDL) and their remnants (IDL) was alternatively, in the past, attributed to the low density lipoprotein receptor (LDLr) or to an apoE-specific receptor. In order to resolve this issue, we have compared the binding of those lipoproteins labelled with iodine-125 to normal and LDLr deficient HepG2 cells. Those deficient cells were obtained by a constitutive antisense strategy and their LDLr level is 14% the level of normal HepG2 cells. By saturation curve analysis, we show that VLDL and IDL bind to high and low affinity sites on cells. The low affinity binding was eliminated by conducting the assay in presence of a 200-fold excess of HDL3 respective to the concentrations of 125 I -labelled VLDL and IDL. For 125 I -VLDL high affinity binding to normal HepG2 cells, we found a dissociation constant ( K d) of 21.2±3.7 μg prot./ml (S.E., N=5) and a maximal binding capacity ( B max) of 0.0312±0.0063 μg prot./mg cell prot, while we have measured a K d of 5.3±0.8 and a B max of 0.0081±0.0014 with LDLr deficient cells. This indicates that LDLr is responsible for 74% of VLDL binding to HepG2 cells and that the non-LDLr high affinity receptor has a higher affinity for VLDL than LDLr. A 53% loss of 125 I -IDL binding capacity was measured with LDLr deficient cells compared with normal cells ( B max:0.028±0.005 versus 0.059±0.006), while no significant statistical difference was found between affinities. The study shows that the LDLr is almost the only contributor in VLDL binding, while it shares IDL binding capacity with another high affinity receptor. The physiological importance of LDLr is confirmed by an almost equivalent loss of IDL and VLDL degradation in LDLr deficient cells.

Dopamine D2-like receptors on human peripheral blood lymphocytes: a radioligand binding assay and immunocytochemical study.

1. Peripheral blood lymphocytes express dopamine D1-like and D2-like receptors which were investigated using radioligand binding assay and molecular biology techniques. Analysis of dopamine D2-like receptors expressed by human peripheral blood lymphocytes with radioligand binding assay may offer a rapid technique for assessing receptor changes in disorders characterized by involvement of the dopaminergic system. However, the suitability of radioligand binding assay techniques to measure dopamine D2-like receptors is questioned. 2. In view of the discrepancy between data of dopamine D2-like receptor determination with molecular biology and radioligand binding assay techniques, we have assayed dopamine D2-like receptors expressed by human peripheral blood lymphocytes using as radioligands the dopamine receptor agonist 7-[3H]-hydroxy-N,N-di-n-propyl-2-aminotetraline ([3H]-7-OH-DPAT) and two antagonists ([3H]-spiperone and [3H]-nemonapride). 3. Analysis of saturation curves revealed a concentration-dependent binding of all compounds to human peripheral blood lymphocytes. Dissociation constant (Kd) values averaged between 0.15 and 0.40 nM for different radioligands. The maximum density of binding sites (Bmax) was low, ranging from 4.15 +/- 0.05 fmol/10(6) cells with [3H]-spiperone and 8.66 +/- 0.04 fmol/10(6) cells with [3H]-7-OH-DPAT. 4. Displacement curves of [3H]-7-OH-DPAT, [3H]-spiperone and [3H]-nemonapride binding to human peripheral blood lymphocytes revealed, using radioligand concentrations giving the highest specific:non-specific binding ratio, a pharmacological profile consistent with the labelling of dopamine D2-like receptors. The use of higher radioligand concentrations resulted in a poorly displaceable and characterizable binding. 5. Detection of dopamine D2, D3 and D4 receptor immunoreactivity in cytospin centrifuged peripheral blood lymphocytes revealed dopamine D3 and D4 but not D2 receptor immunostaining. 6. The above findings indicate in agreement with molecular biology studies, that dopamine D2-like receptors expressed by human peripheral blood lymphocytes belong to the D3 and D4 receptor subtypes. These receptors are detectable using either dopamine D2-like receptor agonists and antagonists as radioligands if controlled experimental conditions are followed. The standardisation of immunocytochemical techniques for detecting human peripheral blood lymphocyte dopamine receptors may contribute to clarify their role in lymphocyte function or as a peripheral marker of the status of the dopaminergic system.