Identification of putative genetic modifying factors that influence the development of Papillon-Lefévre or Haim-Munk syndrome phenotypes.

BACKGROUND: Papillon-Lefévre syndrome (PLS; OMIM 245000) and Haim-Munk syndrome (HMS; OMIM 245010), which are both characterized by palmoplantar hyperkeratosis and periodontitis, are phenotypic variants of the same disease caused by mutations of the cathepsin C (CTSC) gene. AIM: To identify putative genetic modifying factors responsible for the differential development of the PLS or HMS phenotypes, we investigated two Hungarian patients with different phenotypic variants (PLS and HMS) but carrying the same homozygous nonsense CTSC mutation (c.748C/T; p.Arg250X). METHODS: To gain insights into phenotype-modifying associations, whole exome sequencing (WES) was performed for both patients, and the results were compared to identify potentially relevant genetic modifying factors. RESULTS: WES revealed two putative phenotype-modifying variants: (i) a missense mutation (rs34608771) of the SH2 domain containing 4A (SH2D4A) gene encoding an adaptor protein involved in intracellular signalling of cystatin F, a known inhibitor of the cathepsin protein, and (ii) a missense variant (rs55695858) of the odorant binding protein 2A (OBP2A) gene, influencing the function of the cathepsin protein through the glycosyltransferase 6 domain containing 1 (GLT6D1) protein. CONCLUSION: Our study contributes to the accumulating evidence supporting the clinical importance of phenotype-modifying genetic factors, which have high potential to aid the elucidation of genotype-phenotype correlations and disease prognosis.

Characterisation of bioenergetic pathways and related regulators by multiple assays in human tumour cells.

BACKGROUND: Alterations in cellular metabolism are considered as hallmarks of cancers, however, to recognize these alterations and understand their mechanisms appropriate techniques are required. Our hypothesis was to determine whether dominant bioenergetic mechanism may be estimated by comparing the substrate utilisation with different methods to detect the labelled carbon incorporation and their application in tumour cells. METHODS: To define the bioenergetic pathways different metabolic tests were applied: (a) measuring CO2 production from [1-(14)C]-glucose and [1-(14)C]-acetate; (b) studying the effect of glucose and acetate on adenylate energy charge; (c) analysing glycolytic and TCA cycle metabolites and the number of incorporated (13)C atoms after [U-(13)C]-glucose/[2-(13)C]-acetate labelling. Based on [1-(14)C]-substrate oxidation two selected cell lines out of seven were analysed in details, in which the highest difference was detected at their substrate utilization. To elucidate the relevance of metabolic characterisation the expression of certain regulatory factors, bioenergetic enzymes, mammalian target of rapamycin (mTOR) complexes (C1/C2) and related targets as important elements at the crossroad of cellular signalling network were also investigated. RESULTS: Both [U-(13)C]-glucose and [1-(14)C]-substrate labelling indicated high glycolytic capacity of tumour cells. However, the ratio of certain (13)C-labelled metabolites showed detailed metabolic differences in the two selected cell lines in further characterisation. The detected differences of GAPDH, ß-F1-ATP-ase expression and adenylate energy charge in HT-1080 and ZR-75.1 tumour cells also confirmed the altered metabolism. Moreover, the highly limited labelling of citrate by [2-(13)C]-acetate-representing a novel functional test in malignant cells-confirmed the defect of TCA cycle of HT-1080 in contrast to ZR-75.1 cells. Noteworthy, the impaired TCA cycle in HT-1080 cells were associated with high mTORC1 activity, negligible protein level and activity of mTORC2, high expression of interleukin-1ß, interleukin-6 and heme oxygenase-1 which may contribute to the compensatory mechanism of TCA deficiency. CONCLUSIONS: The applied methods of energy substrate utilisation and other measurements represent simple assay system using (13)C-acetate and glucose to recognize dominant bioenergetic pathways in tumour cells. These may offer a possibility to characterise metabolic subtypes of human tumours and provide guidelines to find biomarkers for prediction and development of new metabolism related targets in personalized therapy.

Highlights of a new type of intercellular communication: microvesicle-based information transfer.

Microvesicles (MVs) are membrane-covered cell fragments released by most cell types during apoptosis or activation. They are increasingly considered to play a pivotal role in information transfer between cells. Their presence and role have been proven in several physiological and pathological processes, such as immune modulation in inflammation and pregnancy, or blood coagulation and cancer. MVs represent a newly recognized system of intercellular communications. They not only may serve as prognostic markers in different diseases, but could also hold the potential to be new therapeutic targets or drug delivery systems. The present overview aims to highlight some aspects of this new means of cellular communication: "microvesicular communication".

T lymphocytes are targets for platelet- and trophoblast-derived microvesicles during pregnancy.

Microvesicles (MVs) can derive from several cell types and their membranes contain cell surface elements. Their role is increasingly recognized in cell-to-cell communication, as they act as both paracrine and remote messengers, occurring in circulating form as well as in plasma. Successful pregnancy requires a series of interactions between the maternal immune system and the implanted fetus, such that the semi-allograft will not be rejected. These interactions occur at the materno-placental interface and/or at a systemic level. In the present study we identified for the first time the in vivo plasma pattern of the MVs of third-trimester, healthy pregnant women, their cellular origin, and their target cells using flow cytometry and confocal laser microscopy. We searched for the cellular target molecules of thrombocyte-derived MVs with the help of neutralizing antibodies. We examined the in vitro effects of MVs on STAT3 phosphorylation of primary lymphocytes and Jurkat cells. We found that both placental trophoblast-derived and maternal thrombocyte-derived MVs bind to circulating peripheral T lymphocytes, but not to B lymphocytes or NK cells. We were able to show that the P-selectin (CD62P)-PSGL-1 (CD162) interaction is one mechanism binding platelet-derived MVs to T cells. We were also able to demonstrate that MV-lymphocyte interactions induce STAT3 phosphorylation in T cells. Our findings indicate that both thrombocyte- and trophoblast-derived MVs may play an important role in the immunomodulation of pregnancy. We suggest that the transfer of different signals via MVs represents a novel form of communication between the placenta and the maternal immune system, and that MVs contribute to the establishment of stable immune tolerance to the semi-allograft fetus.

Histamine regulates placental cytokine expression--in vivo study on HDC knockout mice.

Successful pregnancy is closely related to polarization toward a Th2 type immune response. As histamine is known to initiate Th2 dominance during inflammatory processes we raised the question whether histamine has any effect on the actual tuning of proper cytokine balance for the proceeding of the gestation. Histamine has multiple functions in the process of pregnancy, different studies have shown the direct and/or indirect presence of histamine action in the placenta as well. As HDC is the unique histamine producing enzyme in eukaryotes, we used HDC (so endogenous histamine)-deficient knockout mice as reliable model for studying histamine-related processes in vivo. We examined the placental histamine content and the expression of histamine receptors and Th1/Th2/Th3 type cytokines in the placenta. We showed for the first time the influence of histamine on the orchestrated regulation of placental cytokine expression. In the absence of local histamine the cytokine balance is shifted toward Th1 types at the maternal-placental interface, threatening pregnancy. We also measured splenic lymphocyte subpopulation ratios in pregnant and non-pregnant mice and found that in pregnancy they are independent of the presence of histamine.

Histidine decarboxylase deficiency in gene knockout mice elevates male sex steroid production.

Histamine is synthesized in cells by histidine decarboxylase (HDC). HDC-deficient knockout (KO) mice lack functional HDC and histamine in the tissues. In the present study we used this in vivo model for studying the role of HDC deficiency in the regulation of male steroid hormone metabolism. In agreement with earlier studies showing the lack of effects of central histamine on the basal secretion of gonadotrope hormones, we found no difference with in situ hybridization in the expression of GnRH in the hypothalamus of wild type and KO mice. The tissue concentrations of testosterone and several androgenic steroids were significantly elevated in the testes but not in the adrenal glands of HDC-KO mice. In contrast, serum estradiol levels failed to show a significant difference between the two groups. The weight of the testes was significantly smaller in both 7-day-old and adult KO mice. The ultrastructure of the adult testis indicated elevated steroid synthesis with more tightly coiled membranous whorls in Leydig cells. The present results suggest that changes in reproductive functions and sex steroid secretion in male HDC-KO mice are not due to altered hypothalamic GnRH expression but are probably related to definite modifications during fetal development of KO mice reinforced later by the lack of the effect of peripheral histamine. This may provide in vivo evidence that peripheral histamine is an important regulatory factor of male gonadal development during embryogenesis and of sex steroid metabolism later in adulthood.

Detection of protein oxidation in endothelial cells by fluorescently labelled tyramine.

BACKGROUND: Endothelial cell injury mediated by activated polymorphonuclear leucocytes (PMN) occurs during inflammation or reperfusion after brain ischemia. Protein oxidation caused by activated PMN may lead to functional disturbances, degeneration and death of the endothelial cells. The aim of this study was to detect protein oxidation in endothelial cells induced by activated neutrophils by using a novel fluorescent probe. MATERIAL AND METHODS: Protein oxidation of Human Umbilical Vein Endothelial Cells (HUVEC) in culture was investigated by a 15-min incubation with human neutrophils activated by phorbol myristate acetate (PMA) in the presence of tyramine coupled to the succinimidyl ester of (fluorescein -5 (and-6)-carboxamido) hexanoic acid. Dityrosine bond formation as reflected by the linkage of the fluorescent tyramine to proteins was determined by Western-blotting. RESULTS: The oxidative burst generated by activated neutrophils induced dityrosine formation in the extracellular proteins (ECP) of HUVEC. Similar results were obtained, when horseradish peroxidase (HRP) was used for the induction of oxidative stress. However, when hydrogen peroxide (0.1 mM) was used, dityrosine formation was not detected. CONCLUSIONS: Fluorescently labelled tyramine is a powerful tool for the detection of ECP oxidation in endothelial cells. As long as the oxidation by the activated neutrophils is limited to ECP, the endothelial cells may be protected by antioxidants.

Detection of protein oxidation in rat-1 fibroblasts by fluorescently labeled tyramine.

Oxidative damage to proteins has been postulated as a major cause of various degenerative diseases including the loss of functional capacity during aging. A prominent target for oxidation by reactive oxygen species (ROS) is the tyrosine residue. Here we present a highly sensitive method for the detection of tyrosyl radical formation in cells. The method is based on the fluorescein-labeled tyrosine analogue, tyramine, which upon oxidation may couple to proteins carrying a tyrosyl radical. Coupling of the probe (denoted TyrFluo) to standard proteins could be induced by generating ROS with horseradish peroxidase/hydrogen peroxide, SIN-1 or with peroxides (cumene or hydrogen peroxide) in combination with a transition metal. TyrFluo added to rat-1 fibroblasts remained outside the cell, whereas the acetylated form (acetylTyrFluo) was membrane-permeable and accumulated in the cell. Exposure of the cells to oxidative stress in the presence of either TyrFluo or acetylTyrFluo gave a cellular labeling characteristic for each probe. Western blot analysis confirmed that each probe labeled a specific set of proteins. This new method for the detection of ROS-induced oxidation of proteins may mimic the tendency of oxidized proteins to form dityrosine bonds.

Peptide-based targeting of fluorophores to organelles in living cells.

Peptides carrying organelle-specific import or retention sequences can target the fluorophore BODIPY(581/591) to the nucleus, peroxisomes, endoplasmic reticulum (ER), or the trans-Golgi network (TGN). The peroxisomal peptide contains the PTS1 sequence AKL. For targeting to the ER or TGN, the peptides carry the retention sequences KDEL and SDYQRL, respectively. A peptide carrying the nuclear leader sequence of the simian virus SV40 large tumor antigen, KKKRK, was used to direct the fluorophore to the nucleus. The fluorescent peptides for peroxisomes, ER, and the TGN spontaneously incorporate into living fibroblasts at 37 degrees C and accumulate in their target organelles within minutes. The uptake is still significant at 4 degrees C, indicating that endocytosis is not required for internalization. The highly charged nuclear peptide (net charge +4) does not spontaneously internalize. However, by transient permeabilization of the plasma membrane, this fluorescent peptide was found to rapidly accumulate in the nucleus. These fluorescent peptides open new opportunities to follow various aspects of specific organelles such as their morphology, biogenesis, dynamics, degradation, and their internal parameters (pH, redox).

Peptide-based targeting of fluorophores to peroxisomes in living cells.

Peptides carrying different fluorophores can be designed to incorporate spontaneously into living cells when added to the medium. By incorporating the peroxisome-targeting sequence PTS1, the peptide is recognized by the protein-import machinery of peroxisomes and, as a result, can accumulate in these organelles. Depending on the cell type, an inhibitor of the multidrug-resistance protein might be required to ensure strong accumulation. In this update, we discuss the potential of these peptide-linked fluorophores in solving issues related to organelle function and dynamics.

Ratio-fluorescence microscopy of lipid oxidation in living cells using C11-BODIPY(581/591).

A ratio-fluorescence assay was developed for on-line localization and quantification of lipid oxidation in living cells. The assay explores the oxidative sensitivity of C11-BODIPY(581/591). Upon oxidation, the fluorescence of this fluorophore shifts from red to green. The probe incorporates readily into cellular membranes and is about twice as sensitive to oxidation as arachidonic acid. Using confocal microscopy, the cumene hydroperoxide-induced oxidation of C11-BODIPY(581/591) was visualized at the sub-cellular level in rat-1 fibroblasts. Preloading of the cells with tocopherol retarded this oxidation. The data demonstrate that C11-BODIPY(581/591) is a valuable tool to quantify lipid oxidation and anti-oxidant efficacy in single cells.

Outer membrane phospholipase A is dimeric in phospholipid bilayers: a cross-linking and fluorescence resonance energy transfer study.

In the cell, the activity of outer membrane phospholipase A (OMPLA) is strictly regulated to prevent uncontrolled breakdown of the membrane lipids. Previously, it has been shown that the enzymatic activity is modulated by reversible dimerization. The current studies were carried out to define the oligomeric state of OMPLA in a membrane and to investigate the activation process. Three single-cysteine variant proteins H26C, H234C, and S144C were produced and purified to homogeneity. Using maleimido-based homo-bifunctional cross-linking reagents, H26C could be efficiently cross-linked as assessed by SDS-PAGE, whereas S144C and H234C could not be cross-linked. These data suggest that residue 26 is located close to the dimer symmetry axis. H26C was specifically labeled with 5-({[(2-iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid and N,N'-dimethyl-N-(iodoacetyl)-N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)ethylenediamine as the fluorescent energy donor and acceptor, respectively, and dimerization was investigated using fluorescence resonance energy transfer (FRET). Quenching of the donor in the presence of the acceptor demonstrated the dimeric nature of OMPLA, in agreement with cross-linking data. The observed FRET effect was dependent on the cofactor calcium, and the presence of substrate, indicating the specificity of the dimerization process. The labeled protein was reconstituted in phospholipid vesicles. In bilayers, OMPLA exhibited low activity and was dimeric as assessed by FRET. Addition of detergent resulted in a 70-fold increase in activity, while the protein remained dimeric. The results are discussed in terms of the activation of dimeric OMPLA due to changes in the physical state of the bilayer which occur upon perturbation of the membrane integrity.

Investigation on the formation of the hormone reception mechanism.

The formation of the hormone receptor mechanism is part of the embryonic development. The perinatal age seems to be critical for the maturation of this system, i.e. the development of the proper hormone selectivity, the number of the receptors, their sensitivity to the adequate hormone. Our team tried to get a closer view on this developmental period examining different parameters in different model systems. We were able to show that during the maturation of the hormonal system similar responses could be elicited by the later specific hormones or by the non-specific ones but structurally similar compounds. Also it became evident that one single hormonal treatment, applied during in the perinatal age, could influence the responsiveness to hormones of the adult animals.

Pharmacokinetics of panomifene in healthy volunteers at phase I/a study.

Panomifene (PAN) /E/-1,2,-diphenyl-1-[4-[2-(2-hydroxyethylamino)-ethoxy]-phenyl]-3, 3,3-trifluoropropene is a new original Hungarian compound and is a tamoxifen (TMX) analog. In the phase I/a study presented here the human tolerance, pharmacokinetics and endocrine effects of a single oral dose of panomifene were evaluated in healthy, post-menopausal, female volunteers. As to the dose escalation, pharmacokinetic studies were carried out at doses of 24, 48 and 96 mg in two volunteers, and 120 mg in one volunteer. To find a suitable dose or dose range, for further evaluation of the drug detailed pharmacokinetics were performed at a selected dose level (24 mg) in 10 volunteers. The pharmacokinetic study showed considerable interindividual variability of the parameters, and only a medium correlation between dose and AUC (r=0.876). At the selected dose level (24 mg p.o.) the peak concentration of the plasma was 67.7 +/- 17.4 ng/ml (Cmax(meas)), the time to peak was 3.6 +/- 1.8 h (t[max(meas)]). The mean of the terminal half-life was 70.0 +/- 23.1 h (t(1/2beta)). The area under the plasma concentration time curve (AUC) calculated by the kinetic equation (AUCcalc) was 4814 +/- 1172 and by the trapezoidal rule (AUCtrap) was 4612 +/- 1357 (ng/ml) h.

Reorientational properties of fluorescent analogues of the protein kinase C cofactors diacylglycerol and phorbol ester.

The reorientational properties of the fluorescently labelled protein kinase C (PKC) cofactors diacylglycerol (DG) and phorbol ester (PMA) in vesicles and mixed micelles have been investigated using time-resolved polarised fluorescence. The sn-2 acyl chain of DG was replaced by diphenylhexatriene- (DPH) propionic acid, while a dansyl labelled analogue of phorbol ester was used. The extent of ordering of DPH-DG in vesicles turned out to be slightly different from that of the control choline lipid DPH-PC. Addition of PKC to vesicles containing 30 mole% brain PS considerably slowed down the DPH-DG anisotropy decay. This was not observed when DPH-DG was replaced by DPH-PC. Analysis of the fluorescence anisotropy decays of these DPH-lipids in micelles polyoxyethylene-9-laurylether mixed with 10 mole% of the essential phosphatidylserine allowed estimation of their lateral diffusion, orientation distribution and reorientational dynamics within the micelles. Addition of PKC resulted in a significantly slower decay of the fluorescence anisotropy of both DPH-DG and DPH-PC even in the absence of calcium, indicating a calcium independent complexation of PKC with the PS containing micelles. Addition of calcium resulted in a further reduction of the decay of anisotropy of DPH-DG but not of DPH-PC indicating that the Ca2+ dependent immobilisation is cofactor-specific. Similar specific interactions with PKC resulted in a slower decay of dansylated PMA when calcium and PS were present.

Hormonal imprinting: neonatal treatment of rats with the peroxysome proliferator clofibrate irreversibly affects sexual behaviour.

In the rat, the peroxysome proliferator activated receptor (PPAR) inducer clofibrate can moderately influence the hormone (testosterone) level and, after single perinatal treatment, irreversibly affects sexual behavior through the mechanism of hormonal imprinting. The thymic glucocorticoid and estrogen receptors weren't significantly influenced. The experiments call the attention to the universality of false imprinting by molecules able to bind to the steroid/thyroid receptor superfamily, and point to the different sensitivity to different ligands.

Monoclonal antibodies against two electron reduced riboflavin and a quantification of affinity constants for this oxygen-sensitive molecule.

In order to create a protein environment that binds preferentially to the two-electron reduced form of flavin, monoclonal antibodies have been raised against a reduced flavin derivative. Due to the low fluorescence quantum yield and visible light absorption and to the instability of reduced flavin in an aerobic environment, it is not possible to determine the affinities of these antibodies for two-electron-reduced flavin using standard techniques. Because of its sensitivity, time-resolved fluorescence can be used to overcome this problem. This technique has been applied to study the binding of two antibodies, an IgG1 and an IgM, to reduced riboflavin (1,5-dihydroriboflavin) and oxidized riboflavin (riboflavin). The affinity of the IgG1 is more than 80 times larger for 1,5-dihydroriboflavin than for riboflavin. From analysis of the dynamical parameters of the system it is apparent that the internal motion of 1,5-dihydroriboflavin bound to IgG1 is much more restricted than that of riboflavin. In contrast, the affinity of the IgM is only slightly higher for 1,5-dihydroriboflavin than for riboflavin and the flexibility of binding of both flavin redox states in the antigen binding site is almost similar.

Quantitative analysis of lipid-lipid and lipid-protein interactions in membranes by use of pyrene-labeled phosphoinositides.

The lateral and rotational dynamics of phosphoinositides and their interactions with proteins were characterized using pyrene-labeled lipid analogues. In these systems, the collision frequency of pyrene-labeled lipids was studied by monitoring the monomeric pyrene fluorescence yield as a function of their mole fraction in the membranes. From this dependence, the lateral diffusion coefficient and a repulsion factor between two pyrene phosphoinositides could be estimated by applying an extended form of the Milling Crowd model [Eisinger, J., Flores, J., & Petersen, W. P. (1986) Biophys. J. 49, 987-1001]. The repulsion appeared to be highly dependent on the amount of negative charge of the lipid headgroups. From experiments with dioleoylphosphatidylcholine vesicles containing band 3 protein, the fraction of lipid molecules bound to this protein and the minimum number of sites possessing affinity for phosphatidylinositol-4-phosphate could be approximately estimated. The results of this study indicate that phosphoinositides are located preferentially adjacent to band 3. Intramolecular excimer formation of dipyrene-labeled phosphatidylcholine, phosphatidylinositol, and phosphatidylinositol-4-phosphate yielded information about the acyl chain dynamics of lipids surrounding the protein and of lipids in the bulk membrane. Time-resolved measurements of the pyrene fluorescence anisotropy showed that in membranes of resealed erythrocyte ghost cells the rotational freedom of pyrene-labeled phosphatidylinositol-4,5-bisphosphate is smaller than that of pyrene-labeled phosphatidylcholine. In contrast, no significant differences could be detected when these pyrene lipids were dispersed in dioleoylphosphatidylcholine membranes. It is proposed that the nonrandom distribution of the phosphoinositides induced by lipid-lipid repulsion and protein-lipid attraction will have a profound effect on the phospholipase C-catalyzed hydrolysis of the phosphoinositides into second-messenger molecules.