Cytotoxic effects in transformed and non-transformed human breast cell lines after exposure to silver nanoparticles in combination with selected aluminium compounds, parabens or phthalates.

This study was undertaken to assess cytotoxic effects of selected aluminium compounds, parabens and phthalates in combination with silver nanoparticles (AgNP, 15 and 45 nm by STEM, Ag15 and Ag45, respectively) on cell lines of the human breast epithelium, normal (MCF-10A) and transformed (MDA-MB-231 and MCF-7). Combination indices were the most spectacular at effective concentrations (ED) inducing 25 % decrease in viability for the combinations of Ag15 with AlCl3 for MDA-MB-231 cells or aluminium zirconium tetrachlorohydrex Gly (AlZr) for MCF-10A and MCF-7 cells, where rather strong antagonism was revealed. As the ED values increased, those effects were enhanced (e.g. Ag15+AlCl3 for MDA-MB-231) or reversed into synergism (e.g. Ag15+AlZr for MCF-7). Another strong effect was observed for aluminium chloride hydroxide, which increasing ED, induced synergistic effect with both Ag15 and Ag45 on MCF-10A cells. Another interesting synergistic effect was observed for DBPh, but only in combination with Ag45 on MCF-10A and MCF-7. The results on cytotoxicity, cell cycle and oxidative stress induction indicate complex response of the cell lines to combined treatment with silver nanoparticles and the chemicals, which were influenced by diverse factors, such as physico-chemical characteristics of AgNP, method of their synthesis, concentrations used, and finally cell type.

Synthesis and anti-tumour, immunomodulating activity of diosgenin and tigogenin conjugates.

A series of novel diosgenin (DSG) and tigogenin (TGG) derivatives with diosgenin or tigogenin steroid aglycons linked to levulinic and 3,4-dihydroxycinnamic acids, dipeptides and various amino acids by an ester bond at the C3-oxygen atom of the steroid skeleton has been synthesized. Diosgenyl esters have been prepared by an esterification reaction (DCC/DMAP) of diosgenin with the corresponding acids. All analogues have been evaluated in vitro for their antiproliferative profile against cancer cell lines (MCF-7, MDA-MB-231, PC-3) and human umbilical vein endothelial cells (HUVEC). Analogue2c (l-serine derivative of TGG), the best representative of the series showed IC50 of 1.5 µM (MCF-7), and induced apoptosis in MCF-7 by activating caspase-3/7. The immunomodulatory properties of six synthesized analogues have been determined by examining their effects on the expression of cytokine genes essential for the functioning of the human immune system (IL-1, IL-4, IL-10, IL-12 and TNF-α). Biological evaluation has revealed that new compounds 4c and 16a do not induce the expression of pro-inflammatory cytokines in THP-1 cells after the lipopolysaccharide (LPS) stimulation. They also stimulate the expression of anti-inflammatory IL-10 that acts stronger than diosgenin itself. An in silico ADME properties(absorption, distribution, metabolism, excretion) study was also performed to predict the pharmacokinetic profile of the synthesized compounds. To shed light on the molecular interactions between the synthesized compounds and the glucocorticoid receptor and the estrogen receptor, 2c, 4c and 16a compounds were docked into the active binding sites of these receptors. The in silico and in vitro data suggested that this new group of compounds might be considered as a promising scaffold for further modification of more potent and selective anticancer and immunomodulatory agents.

Purinergic implication in amyotrophic lateral sclerosis-from pathological mechanisms to therapeutic perspectives.

Amyotrophic lateral sclerosis (ALS) is a clinically heterogeneous disorder characterized by degeneration of upper motor neurons in the brainstem and lower motor neurons in the spinal cord. Multiple mechanisms of motor neuron injury have been implicated, including more than 20 different genetic factors. The pathogenesis of ALS consists of two stages: an early neuroprotective stage and a later neurotoxic. During early phases of disease progression, the immune system through glial and T cell activities provides anti-inflammatory factors that sustain motor neuron viability. As the disease progresses and motor neuron injury accelerates, a rapidly succeeding neurotoxic phase develops. A well-orchestrated purine-mediated dialog among motor neurons, surrounding glia and immune cells control the beneficial and detrimental activities occurring in the nervous system. In general, low adenosine triphosphate (ATP) concentrations protect cells against excitotoxic stimuli through purinergic P2X4 receptor, whereas high concentrations of ATP trigger toxic P2X7 receptor activation. Finally, adenosine is also involved in ALS progression since A2A receptor antagonists prevent motor neuron death. Given the complex cellular cross-talk occurring in ALS and the recognized function of extracellular nucleotides and adenosine in neuroglia communication, the comprehensive understanding of purinome dynamics might provide new research perspectives to decipher ALS and help to design more efficient and targeted drugs. This review will focus on the purinergic players involved in ALS etiology and disease progression and current therapeutic strategies to enhance neuroprotection and suppress neurotoxicity.

A probabilistic atlas of fiber crossings for variability reduction of anisotropy measures.

Diffusion imaging enables assessment of human brain white matter (WM) in vivo. WM microstructural integrity is routinely quantified via fractional anisotropy (FA). However, FA is also influenced by the number of differentially oriented fiber populations per voxel. To date, the precise statistical relationship between FA and fiber populations has not been characterized, complicating microstructural integrity assessment. Here, we used 630 state-of-the-art diffusion datasets from the Human Connectome Project, which allowed us to infer the number of fiber populations per voxel in a model-free fashion. Beyond the known impact on mean FA, variance of anisotropy distributions was drastically impacted, not only for FA, but also the more recent anisotropy indices generalized FA and multidimensional anisotropy. To ameliorate this bias, we introduce a probabilistic WM atlas delineating the number of distinctly oriented fiber populations per voxel. Our atlas shows that the majority of WM voxels features two differentially directed fiber populations (44.7%) rather than unidirectional fibers (32.9%) and identified WM regions with high numbers of crossing fibers, referred to as crossing pockets. Compartmentalizing anisotropy drastically reduced variance in group comparisons ranging from the whole brain to a few voxels in a single slice. In summary, we demonstrate a systematic effect of intra-voxel diffusion inhomogeneity on anisotropy. Moreover, we introduce a potential solution: The provided probabilistic WM atlas can easily be used with any given diffusion dataset to enhance the statistical robustness of anisotropy measures and increase their neurobiological utility.

Genotoxic effects in transformed and non-transformed human breast cell lines after exposure to silver nanoparticles in combination with aluminium chloride, butylparaben or di-n-butylphthalate.

In the present study genotoxic effects after combined exposure of human breast cell lines (MCF-10A, MCF-7 and MDB-MB-231) to silver nanoparticles (AgNP, citrate stabilized, 15 and 45nm by STEM, Ag15 and Ag45, respectively) with aluminium chloride, butylparaben, or di-n-butylphthalate were studied. In MCF-10A cells exposed for 24h to Ag15 at the concentration of 23.5µg/mL a statistically significant increase in DNA damage in comet assay (SSB) was observed. In the presence of the test chemicals the genotoxic effect was decreased to a level comparable to control values. In MCF-7 cells a significant increase in SSB level was observed after exposure to Ag15 at 16.3µg/mL. The effect was also diminished in the presence of the three test chemicals. In MDA-MB-231 cells no significant increase in SSB was observed, however increased level of oxidative DNA damage (incubation with Fpg enzyme) was observed after exposure to combinations of both AgNP with aluminium chloride. No increase in micronuclei formation was observed in neither cell line after the single nor combined treatments. Our results point to a low risk of increased genotoxic effects of AgNP when used in combination with aluminium salts, butylparaben or di-n-butylphthalate in consumer products.

Interactive effects of dietary lipids and vitamin E level on performance, blood eicosanoids, and response to mitogen stimulation in broiler chickens of different ages.

The effects of the dietary polyunsaturated fatty acids (PUFA) n-6:n-3 ratio and vitamin E (vE) on the levels of pro-inflammatory eicosanoids, the incorporation of docosahexaenoic acid (DHA) and arachidonic acid (AA) into immune tissues, and changes in leukocyte population after phytohemagglutinin (PHA) challenge were investigated in broiler chickens of different ages. One-day-old female broilers (48 per treatment) were fed 4 different wheat-soybean-corn-based diets containing corn oil with a high PUFA n-6:n-3 ratio (HR) or a mixture of linseed and fish oils with a low PUFA n-6:n-3 ratio (LR). Diets contained either 50 mg vE kg-1 of diet (basal vE) or 300 mg vE kg-1 of diet (increased vE). At d 14 and d 34, 8 chickens per treatment were challenged with PHA, and wing web swelling (WWS) was measured. The blood concentration of leukotriene (LTB4), prostaglandin (PGE2), and thromboxane (TBX2) in 17-day-old and 43-day-old chickens was determined. The pattern of AA and DHA incorporation into bursa, spleen, and brain lipids reflected the level of their precursors in the diet. WWS was the highest in chickens fed a LR diet and in 14-day-old chickens (P < 0.01). Leukocyte proportions varied with dietary PUFA n-6:n-3 ratio and with age. The heterophil:lymphocyte ratio was the highest at 6 h post PHA challenge, and was higher in 34-day-old chickens (P < 0.001). TBX2 and PGE2 concentrations were higher in chickens fed HR diet, whereas TBX2 and LTB4 concentrations were lower at high vE level. Lower PGE2 and LTB4, but higher TBX2 concentrations were measured in younger birds (P < 0.001). The results indicated that LR increased the phagocytic cell proportion in the blood; HR promoted the incorporation of AA into the immune tissues, which increased the levels of more pro-inflammatory eicosanoids in the blood; and vE counteracts these effects to some extent. Owing to the immaturity of the immune system, dietary interventions might be promising at the early stage of chicken growth.

First evidence of infectious hematopoietic necrosis virus (IHNV) in the Netherlands.

In spring 2008, infectious hematopoietic necrosis virus (IHNV) was detected for the first time in the Netherlands. The virus was isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), from a put-and-take fishery with angling ponds. IHNV is the causative agent of a serious fish disease, infectious hematopoietic necrosis (IHN). From 2008 to 2011, we diagnosed eight IHNV infections in rainbow trout originating from six put-and-take fisheries (symptomatic and asymptomatic fish), and four IHNV infections from three rainbow trout farms (of which two were co-infected by infectious pancreatic necrosis virus, IPNV), at water temperatures between 5 and 15 °C. At least one farm delivered trout to four of these eight IHNV-positive farms. Mortalities related to IHNV were mostly <40%, but increased to nearly 100% in case of IHNV and IPNV co-infection. Subsequent phylogenetic analysis revealed that these 12 isolates clustered into two different monophyletic groups within the European IHNV genogroup E. One of these two groups indicates a virus-introduction event by a German trout import, whereas the second group indicates that IHNV was already (several years) in the Netherlands before its discovery in 2008.

Sex-dependent differences in the effect of early weaning on the chosen hormones secretion in sheep during the postnatal transition to puberty--preliminary results.

The influence of early weaning on the cortisol, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and growth hormone (GH) secretion in lambs of both sexes and testosterone (T4) level in male lambs during the postnatal transition to puberty was investigated by radioimmunoassay. It was hypothesized that this influence is long-term and sexually dimorphic. Hence, the effect of weaning at 5 weeks of age in comparison with the weaning at 9 weeks of age on hormone concentra- tions in peripheral blood plasma of 5-, 9-, 12-, and 16-week-old lambs of both sexes was investigated. The cortisol concentrations were greater (P < 0.05) in control and early weaned female lambs than in male lambs at investigated stages. Weaning at 5 weeks of age resulted in the lover (P < 0.05) cortisol secretion in male lambs in contrast to the greater (P < 0.05) cortisol secretion in female lambs at 16 weeks of age. Weaning at 5 weeks of age stimulated (P < 0.001) the FSH secretion, but reduced (P < 0.001) the LH, GH and T4 secretion in 16-week-old male lambs. In female lambs early weaning inhibited (P < 0.05) the FSH secretion at 9 weeks of age, LH secretion after 9 weeks of age and GH secretion after 12 weeks of age. Thus, early weaning results in the sexually dimorphic stress reaction that is more potent and long-lasting in female in contrast to male lambs. This maternal deprivation stress contributes to the inhibition of LH and GH secretion in lambs of both sexes and T4 secretion in male lambs during the postnatal transition to puberty.

In vivo quantitative NMR imaging of fruit tissues during growth using Spoiled Gradient Echo sequence.

Nondestructive studies of physiological processes in agronomic products require increasingly higher spatial and temporal resolutions. Nuclear Magnetic Resonance (NMR) imaging is a non-invasive technique providing physiological and morphological information on biological tissues. The aim of this study was to design a robust and accurate quantitative measurement method based on NMR imaging combined with contrast agent (CA) for mapping and quantifying water transport in growing cherry tomato fruits. A multiple flip-angle Spoiled Gradient Echo (SGE) imaging sequence was used to evaluate the intrinsic parameters maps M0 and T1 of the fruit tissues. Water transport and paths flow were monitored using Gd(3+)/[Fe(CN)6](3-)/D-mannitol nanoparticles as a tracer. This dynamic study was carried out using a compartmental modeling. The CA was preferentially accumulated in the surrounding tissues of columella and in the seed envelopes. The total quantities and the average volume flow of water estimated are: 198 mg, 1.76 mm(3)/h for the columella and 326 mg, 2.91 mm(3)/h for the seed envelopes. We demonstrate in this paper that the NMR imaging technique coupled with efficient and biocompatible CA in physiological medium has the potential to become a major tool in plant physiology research.

Local termination pattern analysis: a tool for comparing white matter morphology.

Disconnections between structures in the brain have long been hypothesized to be the mechanism behind numerous disease states and pathological behavioral phenotypes. Advances in diffusion weighted imaging (DWI) provide an opportunity to study white matter, and therefore brain connectivity, in great detail. DWI-based research assesses white matter at two different scales: voxelwise indexes of anisotropy such as fractional anisotropy (FA) are used to compare small units of tissue and network-based methods compare tractography-based models of whole-brain connectivity. We propose a method called local termination pattern analysis (LTPA) that considers information about both local and global brain connectivity simultaneously. LTPA itemizes the subset of streamlines that pass through a small set of white matter voxels. The "local termination pattern" is a vector defined by counts of these streamlines terminating in pairs of cortical regions. To assess the reliability of our method we applied LTPA exhaustively over white matter voxels to produce complete maps of local termination pattern similarity, based on diffusion spectrum imaging (DSI) data from 11 individuals in triplicate. Here we show that local termination patterns from an individual are highly reproducible across the entire brain. We discuss how LTPA can be deployed into a clinical database and used to characterize white matter morphology differences due to disease, developmental or genetic factors.

Clinical and immunological determinants of severe/refractory asthma (SRA): association with Staphylococcal superantigen-specific IgE antibodies.

BACKGROUND: Demographic and immunological determinants of severe refractory asthma (SRA) are not well characterized. Because Staphylococcus aureus enterotoxins with superantigenic activity have been associated with upper and lower airway inflammation, we aimed to evaluate the association of sensitization to Staphylococcal enterotoxins with asthma severity and various asthma phenotypes. METHODS: The study included 109 patients with SRA diagnosed according to the American Thoracic Society Workshop 2000, and 101 patients with nonsevere asthma, followed for at least 12 months. Specific IgE to Staphylococcus enterotoxins and total IgE and eosinophil cationic protein concentrations were measured in serum with immunoassays. FINDINGS: A significant risk for severe asthma was associated with female gender [Odds Ratio (OR) = 2.04], history of wheezing in childhood (OR = 2.47), presence of hypersensitivity to aspirin (OR = 1.96) and with body mass index (OR = 3.08). The mean level of enterotoxin-specific IgE was 3-fold higher in patients with severe asthma when compared to patients with nonsevere asthma (P = 0.01). Serum-specific IgE to enterotoxins was significantly associated with low respiratory function parameters (FEV1, FEV1/FVC and MEF 25/75) and increased airway reversibility in response to albuterol. The presence of specific IgE to enterotoxin carried a significant risk for patients to have serum total IgE level above 100 kU/l (OR = 7.84). INTERPRETATION: Specific immunological response to enterotoxins is associated with clinical and immunological parameters of asthma severity, suggesting a role for Staphylococcal enterotoxins in the asthma pathogenesis.

Concrete with carpet recyclates: suitability assessment by surface energy evaluation.

Worn out textile floor coverings are burdensome wastes that are degraded in landfill sites after a very long period of time. One of the ways to manage this kind of waste may be the use of carpet recyclate (CR) as an additive for concrete reinforcement. Therefore, an attempt was made to predict the effects of recyclate additives on the durability a concrete-carpet mixture by employing the method of assessing surface properties of components in the concrete-carpet recyclates composite. Testing was performed on carpet wastes, containing polyamide (PA) and polypropylene (PP) piles and butadiene-styrene resin with chalk filler (BSC) as back coating, to assess the suitability of CR additive for concrete reinforcement by surface energy evaluation. Based on the measurements of contact angles, the free surface energy of recyclate components was determined. The reversible work of adhesion at the interface between these components in dry and wet states was also calculated. The results show that CR with both PA and PP fibers form a strong and water-resistant bond with concrete.

85-kDa cytosolic phospholipase A2 group IValpha gene promoter polymorphisms in patients with severe asthma: a gene expression and case-control study.

Cytosolic phospholipase A(2) (cPLA(2)) group IValpha is a critical enzyme involved in the liberation of arachidonic acid from cellular membranes. cPLA(2)(-/-) mice have reduced allergen-induced bronchoconstriction and bronchial hyperresponsiveness. The goal of this study was to investigate polymorphisms of the (CA)(n) and (T)(n) microsatellites and surrounding regions in the cPLA(2)alpha gene promoter. We analysed the cPLA(2) promoter regions containing (CA)(n) and (T)(n) repeats in 87 patients with severe asthma and in 48 control subjects by bidirectional sequencing. Functional studies were performed utilizing reporter genes derived from subjects with varying numbers of these repeats, and on constructs with a series of deletions. We found that the (CA)(n) and (T)(n) regions are polymorphic and that constructs with CA or T repeats or CA and T repeats deleted revealed, respectively, a 41.8 +/- 7%, 22.3 +/- 5% and 100 +/- 20% increase in reporter gene activity. A lower number of CA or T repeats caused higher cPLA(2) promoter luciferase activity. The group of shorter alleles of the (CA)(n) microsatellite region (n = 12-18) (P(cor) = 0.00006), and the group of shorter alleles of (T)(n) repeats region (n = 17-38) (P(cor) = 0.0039) occurred significantly more often in patients with severe asthma. We also found novel SNPs in positions -292 C > G, -185 A > C, -180 T > C and -165 A > C. Two of them were associated with the severe asthma phenotype: -180T allele (P(cor) = 0.03996) and -185 A allele (P(cor) = 0.03966). These results demonstrate that (CA)(n) and (T)(n) repeats may have an influence on cPLA(2) transcription which might play a role in severe asthma pathogenesis.

Aspirin-triggered 15-HETE generation in peripheral blood leukocytes is a specific and sensitive Aspirin-Sensitive Patients Identification Test (ASPITest).

BACKGROUND: We have previously demonstrated that aspirin triggers specific generation of 15-hydroxyeicosateraenoic acid (15-HETE) from nasal polyp epithelial cells and peripheral blood leukocytes (PBL) from aspirin-sensitive (AS) but not aspirin-tolerant (AT) patients with asthma/rhinosinusitis. The goal of this study was to assess the diagnostic value of ASA-induced 15-HETE generation measurement to identify AS patients. METHODS: PBL were obtained from 43 AS patients with asthma and rhinosinusitis, 35 AT asthmatics and 17 healthy control (HC) subjects. PBL were incubated with 2-200 muM aspirin (ASA) and 15-HETE release was measured in cell supernatants with competitive ELISA. RESULTS: Unstimulated PBL from all three groups of patients generated similar amount of 15-HETE. Incubation with 200 microM ASA resulted in an increase in an 15-HETE generation (mean increase +421%) in AS-asthmatics but small and nonsignificant response in AT-asthmatics or control subjects. Receiver operating curve (ROC) analysis revealed that the sensitivity of the test for confirmation of ASA-sensitivity was 83% and the specificity 82%. Positive predictive value was 0.79 and negative predictive value was 0.86. Naproxen induced a significant increase in 15-HETE only in some AS-asthmatics, but not in AT-asthmatics. CONCLUSION: Our data demonstrate that ASA-induced 15-HETE generation by PBL is a specific and sensitive aspirin-sensitive patients identification test (ASPITest).

[Spinal cord anchored at S2 level: diagnosis difficulties and risks]. / Zakotwiczenie rdzenia kregowego do S2--trudnosci diagnostyczne i niebezpieczenstwa.

Anchoring of spinal cord at S2 level is described in a female patient aged 46, associated with bilateral equinovarus deformity and spina bifida in lumbar segment. The onset of the disease was sudden due to prolapse of intervertebral disc at L2-L3 and L3-L4 levels followed by bilateral flaccid-spastic paraparesis. In view of congenital spinal deformity lumbar tap was abandoned and MRI was carried out which showed spinal cord reaching down to S2 level. Diagnostic and therapeutic management is described and literature review is presented.

Emission pattern of high-energy pions: A new probe for the early phase of heavy-Ion collisions

The emission pattern of charged pions has been measured in Au+Au collisions at 1 GeV/nucleon incident energy. In peripheral collisions and at target rapidities, high-energy pions are emitted preferentially towards the target spectator matter. In contrast, low-energy pions are emitted predominantly in the opposite direction. The corresponding azimuthal anisotropy is explained by the interaction of pions with projectile and target spectator matter. This interaction with the spectator matter causes an effective shadowing which varies with time during the reaction. Our observations show that high-energy pions stem from the early stage of the collision whereas low-energy pions freeze out later.

[Pseudomonas aerogunosa pneumonia in patients treated at the Hospital for Chest Diseases]. / Zapalenia pluc wywolane przez paleczke ropy blekitnej u chorych leczonych w Szpitalu Chorób Pluc.

Retrospective analysis of pneumonia caused by Pseudomonas aeruginosa was made in 66 patients, treated in hospital. Nosocomial pneumonia was diagnosed in 11 (17%) patients. In 51 patients coexisting lung diseases were present: mainly COPD and bronchiectasis. Strains of Pseudomonas aeruginosa were susceptible mostly to imipenem, meropenem, aztreonam, ticarcillin-clavulanic acid, ceftazidime, ciprofloxacin, amikacin, piperacillin-tazobactam, netilmicin. Duration of treatment in hospital was very long--59% were treated over 30 days. Combined antibacterial therapy was applied in 35 (53%) patients and monotherapy, often with different antibiotics--in 31 (47%) patients. Treatment was successful in 45 (68%) patients. In 9 patients the results of treatment was not successful: mainly because of empyema in 7 pts. Twelve (18%) patients (with coexisting COPD--6 and lung cancer--6) died. We can support current recommendations for treatment of Pseudomonas aeruginosa infection with combination of aminoglycosides or fluoroquinolones plus one of remaining antipseudomonal antibiotics. Treatment failures occurred mainly in patients with severe coexisting diseases and/or empyema.

[Pet allergens in a house-dust and elimination procedures against them]. / Alergeny zwierzece zawarte w kurzu domowym oraz sposoby ich eliminacji.

The paper presents a group of allergens carried by the most popular home-kept animals (furred animals). Ubiquitous presence of these allergens (at schools and in public places) is caused by their specific characteristics different from those of house-dust-mites. It is stressed that the serum albumines coming from such animals can be the cause of allergic symptoms in the respiratory tract of patients suffering from bronchial asthma. The trials carried out so far have confirmed the reduction of the animal allergens' amount in different indoor environments when several ways of their elimination were applied. However only few clinical trials have been made to estimate the improvement of ventilation parameters, bronchodilatators requirement and the symptom intensification in bronchial asthma cases, after employing some of the recommended methods often in the continuing presence of the animal at home. Education and encouraging bronchial asthma patients and their families to get rid of animals from their homes and acquiting them with various reservoirs of animal allergens as well as the allergens' distribution and translocation should become the basis of therapeutic management in that group of patients.

Peptide ligands can bind to distinct sites in integrin alphaIIbbeta3 and elicit different functional responses.

The spatial relationship between the binding sites for two cyclic peptides, cyclo(S,S)KYGCRGDWPC (cRGD) and cyclo(S,S)KYGCHarGDWPC (cHarGD), high affinity analogs for the RGD and HLGGAKQAGDV peptide ligands, in integrin alphaIIbbeta3 (GPIIb-IIIa) has been characterized. For this purpose, cRGD and cHarGD were labeled with fluorescein isothiocyanate and tetramethylrhodamine 5-isothiocyanate, respectively. Both cyclic peptides were potent inhibitors of fibrinogen binding to alphaIIbbeta3, particularly in the presence of Mn2+; IC50 values for cRGD and cHarGD were 1 and <0.1 nM in the presence of Mn2+. Direct binding experiments and fluorescence resonance energy transfer analysis using the purified receptor showed that both peptides interacted simultaneously with distinct sites in alphaIIbbeta3. The distance between these sites was estimated to be 6.1 +/- 0.5 nm. Although cRGD bound preferentially to one site and cHarGD to the other, the sites were not fully specific, and each cyclic peptide or its linear counterpart could displace the other to some extent. The binding affinity of the cHarGD site was dramatically affected by Mn2+. cRGD, but not cHarGD, bound to recombinant beta3-(95-373) in a cation-dependent manner, indicating that the cRGD site is located entirely within this fragment. With intact platelets, binding of c-RGD and cHarGD to alphaIIbbeta3 resulted in distinct conformational alterations in the receptor as indicated by the differential exposure of ligand-induced binding site epitopes and also induced the opposite on membrane fluidity as shown by electron paramagnetic resonance analyses using 5-doxylstearic acid as a spin probe. These data support the concept the two peptide ligands bind to distinct sites in alphaIIbbeta3 and initiate different functional consequences within the receptor itself and within platelets.