Binding of IgG-opsonized particles to Fc gamma R is an active stage of phagocytosis that involves receptor clustering and phosphorylation

Fc gammaR mediate the phagocytosis of IgG-coated particles and the clearance of IgG immune complexes. By dissecting binding from internalization of the particles, we found that the binding stage, rather than particle internalization, triggered tyrosine phosphorylation of Fc gammaR and accompanying proteins. High amounts of Lyn kinase were found to associate with particles isolated at the binding stage from J774 cells. PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), an Src kinase inhibitor, but not piceatannol, an inhibitor of Syk kinase, reduced the amount of Lyn associated with the bound particles and simultaneously diminished the binding of IgG-coated particles. Studies of baby hamster kidney cells transfected with wild-type and mutant Fc gammaRIIA revealed that the ability of the receptor to bind particles was significantly reduced when phosphorylation of the receptor was abrogated by Y298F substitution in the receptor signaling motif. Under these conditions, binding of immune complexes of aggregated IgG was depressed to a lesser extent. A similar effect was exerted on the binding ability of wild-type Fc gammaRIIA by PP2. Moreover, expression of mutant kinase-inactive Lyn K275R inhibited both Fc gammaRIIA phosphorylation and IgG-opsonized particle binding. To gain insight into the mechanism by which protein tyrosine phosphorylation can control Fc gammaR-mediated binding, we investigated the efficiency of clustering of wild-type and Y298F-substituted Fc gammaRIIA upon binding of immune complexes. We found that a lack of Fc gammaRIIA phosphorylation led to an impairment of receptor clustering. The results indicate that phosphorylation of Fc gammaR and accompanying proteins, dependent on Src kinase activity, facilitates the clustering of activated receptors that is required for efficient particle binding.     

Genetic mapping of ovary colour and quantitative trait loci for carotenoid content in the fruit of <Emphasis Type="Italic">Cucurbita maxima</Emphasis> Duchesne

The high content of carotenoids, sugars, dry matter, vitamins and minerals makes the fruit of winter squash (Cucurbita maxima Duchesne) a valuable fresh-market vegetable and an interesting material for the food industry. Due to their nutritional value, long shelf-life and health protective properties, winter squash fruits have gained increased interest from researchers in recent years. Despite these advantages, the genetic and genomic resources available for C. maxima are still limited. The aim of this study was to use the genetic mapping approach to map the ovary colour locus and to identify the quantitative trait loci (QTLs) for high carotenoid content and flesh colour. An F₆ recombinant inbred line (RIL) mapping population was developed and used for evaluations of ovary colour, carotenoid content and fruit flesh colour. SSR markers and DArTseq genotyping-by-sequencing were used to construct an advanced genetic map that consisted of 1824 molecular markers distributed across linkage groups corresponding to 20 chromosomes of C. maxima. Total map length was 2208 cM and the average distance between markers was 1.21 cM. The locus affecting ovary colour was mapped at the end of chromosome 14. The identified QTLs for carotenoid content in the fruit and fruit flesh colour shared locations on chromosomes 2, 4 and 14. QTLs on chromosomes 2 and 4 were the most meaningful. A correlation was clearly confirmed between fruit flesh colour as described by the chroma value and carotenoid content in the fruit. A high-density genetic map of C. maxima with mapped loci for important fruit quality traits is a valuable resource for winter squash improvement programmes.     

Association between single nucleotide polymorphisms of TPH1 and TPH2 genes,and depressive disorders

Tryptophan catabolites pathway disorders are observed in patients with depression. Moreover, single nucleotide polymorphisms of tryptophan hydroxylase genes may modulate the risk of depression occurrence. The objective of our study was to confirm the association between the presence of polymorphic variants of TPH1 and TPH2 genes, and the development of depressive disorders. Six polymorphisms were selected: c.804‐7C>A (rs10488682), c.‐1668T>A (rs623580), c.803+221C>A (rs1800532), c.‐173A>T (rs1799913)—TPH1, c.‐1449C>A (rs7963803), and c.‐844G>T (rs4570625)—TPH2. A total of 510 DNA samples (230 controls and 280 patients) were genotyped using TaqMan probes. Among the studied polymoorphisms, the G/G genotype and G allele of c.804‐7C>A—TPH1, the T/T homozygote of c.803+221C>A—TPH1, the A/A genotype and A allele of c.1668T>A—TPH1, the G/G homozygote and G allele of c.‐844G>T—TPH2, and the C/A heterozygote and A allele of c.‐1449C>A—TPH2 were associated with the occurrence of depression. However, the T/T homozygote of c.‐1668T>A—TPH1, the G/T heterozygote and T allele of c.‐844G>T—TPH2, and the C/C homozygote and C allele of c.‐1449C>A—TPH2 decreased the risk of development of depressive disorders . Each of the studied polymorphisms modulated the risk of depression for selected genotypes and alleles. These results support the hypothesis regarding the involvement of the pathway in the pathogenesis of depression.     

Tuning of Hemes b Equilibrium Redox Potential Is Not Required for Cross-Membrane Electron Transfer

In biological energy conversion, cross-membrane electron transfer often involves an assembly of two hemes b. The hemes display a large difference in redox midpoint potentials (ΔE_{m_}b), which in several proteins is assumed to facilitate cross-membrane electron transfer and overcome a barrier of membrane potential. Here we challenge this assumption reporting on heme b ligand mutants of cytochrome bc₁ in which, for the first time in transmembrane cytochrome, one natural histidine has been replaced by lysine without loss of the native low spin type of heme iron. With these mutants we show that ΔE_{m_}b can be markedly increased, and the redox potential of one of the hemes can stay above the level of quinone pool, or ΔE_{m_}b can be markedly decreased to the point that two hemes are almost isopotential, yet the enzyme retains catalytically competent electron transfer between quinone binding sites and remains functional in vivo. This reveals that cytochrome bc₁ can accommodate large changes in ΔE_{m_}b without hampering catalysis, as long as these changes do not impose overly endergonic steps on downhill electron transfer from substrate to product. We propose that hemes b in this cytochrome and in other membranous cytochromes b act as electronic connectors for the catalytic sites with no fine tuning in ΔE_{m_}b required for efficient cross-membrane electron transfer. We link this concept with a natural flexibility in occurrence of several thermodynamic configurations of the direction of electron flow and the direction of the gradient of potential in relation to the vector of the electric membrane potential.     

Novel naphthyloxy derivatives – Potent histamine H3 receptor ligands. Synthesis and pharmacological evaluation

A series of 1- and 2-naphthyloxy derivatives were synthesized and evaluated for histamine H₃ receptor affinity. Most compounds showed high affinities with K_i values below 100?nM. The most potent ligand, 1-(5-(naphthalen-1-yloxy)pentyl)azepane (11) displayed high affinity for the histamine H₃ receptor with a K_i value of 21.9?nM. The antagonist behaviour of 11 was confirmed both in vitro in the cAMP assay (IC₅₀?=?312?nM) and in vivo in the rat dipsogenia model (ED₅₀?=?3.68?nM). Moreover, compound 11 showed positive effects on scopolamine induced-memory deficits in mice (at doses of 10 and 15?mg/kg) and an analgesic effect in the formalin test in mice with ED₅₀?=?30.6?mg/kg (early phase) and ED₅₀?=?20.8?mg/kg (late phase). Another interesting compound, 1-(5-(Naphthalen-1-yloxy)pentyl)piperidine (13; H₃R K_i?=?53.9?nM), was accepted for Anticonvulsant Screening Program at the National Institute of Neurological Disorders and Stroke/National Institute of Health (Rockville, USA). The screening was performed in the maximal electroshock seizure (MES), the subcutaneous pentylenetetrazole (scPTZ) and the 6-Hz psychomotor animal models of epilepsy. Neurologic deficit was evaluated by the rotarod test. Compound 13 inhibited convulsions induced by the MES with ED₅₀ of 19.2?mg/kg (mice, i.p.), 17.8 (rats, i.p.), and 78.1 (rats, p.o.). Moreover, 13 displayed protection against the 6-Hz psychomotor seizures (32?mA) in mice (i.p.) with ED₅₀ of 33.1?mg/kg and (44?mA) ED₅₀ of 57.2?mg/kg.Furthermore, compounds 11 and 13 showed in vitro weak influence on viability of tested cell lines (normal HEK293, neuroblastoma IMR-32, hepatoma HEPG2), weak inhibition of CYP3A4 activity, and no mutagenicity. Thus, these compounds may be used as leads in a further search for histamine H₃ receptor ligands with promising in vitro and in vivo activity.     

Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells

Epidermal and hepatocyte growth factors can stimulate invasive abilities of melanoma cells, while treatment with combination of their receptors' (EGFR and MET, respectively) inhibitors reduces viability of these cells, as we have previously shown. Proposed therapy has potential; however, used drugs block more than one goal effectively, what raises the question about the real target of analysed inhibitors. For this reason, we analysed direct involvement of these receptors in the invasion of melanoma cells inducing EGFR and MET up‐ and down‐regulations in examined cells. Results were acquired with assays evaluating cell migration and invasion (scratch wound assay, Transwell filter‐based method and single‐cell tracking). We revealed that cells' motile abilities are increased after EGFR overexpression and decreased following EGFR and MET silencing. This outcome correlates with elevated (EGFR up‐regulation) or reduced (EGFR/MET down‐regulation) number of formed invadopodia, visualized with immunofluorescence, and their rate of proteolytic abilities, evaluated by fluorescent gelatin degradation assay, and gelatin zymography, compared to control cells. Above‐mentioned data indicate that both—EGFR and MET signalling is directly connected with melanoma cells invasion, what establishes these receptors as promising targets for anti‐cancer treatment.