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.

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.

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.

Steady-state fluorescence studies on lipase-vesicle interactions.

The interaction of lipase from Candida cylindracea (CCL) with positively charged polymerizable surfactant vesicles was studied by the use of steady-state fluorescence techniques. The phase transition of vesicles composed of nonpolymerized and polymerized N-allylbis[2-(hexadecanoyloxy)ethyl]methylammonium bromide (ABHEMA Br) was determined in the absence of lipase, by measuring the change in fluorescence anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The phase transition temperature for nonpolymerized vesicles is 49 degrees C and for the polymerized analogues 45 degrees C. Fluorescence anisotropy and resonance energy transfer measurements were used to illustrate the incorporation of the lipase in the vesicle membrane. These studies demonstrated that CCL is incorporated into the hydrophobic bilayer of the vesicle. By using an interfacial membrane probe 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene p-toluene sulphonate, TMA-DPH) and an internal membrane probe (DPH), it could be determined that the enzyme is incorporated more efficiently into nonpolymerized vesicles, and that the penetration of the enzyme into the bilayer is less deep in the case of the polymerized vesicles.

Integration of the classical and molecular linkage maps of tomato chromosome 6.

In the past, a classical map of the tomato genome has been established that is based on linkage data from intraspecific Lycopersicon esculentum crosses. In addition, a high density molecular linkage map has recently been constructed using a L. esculentum x L. pennellii cross. As the respective maps only partially match, they provide limited information about the relative positions of classical and molecular markers. In this paper we describe the construction of an integrated linkage map of tomato chromosome 6 that shows the position of cDNA-, genomic DNA- and RAPD markers relative to 10 classical markers. Integration was achieved by using a L. esculentum line containing an introgressed chromosome 6 from L. pennellii in crosses to a variety of L. esculentum marker lines. In addition, an improved version of the classical linkage map is presented that is based on a combined analysis of new linkage data for 16 morphological markers and literature data. Unlike the classical map currently in use, the revised map reveals clustering of markers into three major groups around the yv, m-2 and c loci, respectively. Although crossing-over rates are clearly different when comparing intraspecific L. esculentum crosses with L. esculentum x L. pennellii crosses, the clusters of morphological markers on the classical map coincide with clusters of genomic- and cDNA-markers on the molecular map constructed by Tanksley and coworkers.

Erythrocyte band 3 protein strongly interacts with phosphoinositides.

85% of the phosphorus coisolated with band 3 protein during separation of the intrinsic proteins of the human erythrocyte membrane by zonal electrophoresis in high concentrations of acetic acid was found to be derived from phosphoinositides, mainly phosphatidylinositol 4,5-bisphosphate. When native band 3 protein and pyrene-labelled phospholipids were present in micelles of the nonionic detergent nonaethyleneglycol lauryl ether, strong resonance energy transfer was observed between the tryptophan residues and phosphatidylinositol 4,5-bisphosphate and, to a smaller degree, phosphatidylinositol 4-phosphate. We conclude that band 3 protein strongly interacts with phosphoinositides, in particular with phosphatidylinositol 4,5-bisphosphate.

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.

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.

Fluorescence dynamics of diphenyl-1,3,5-hexatriene-labeled phospholipids in bilayer membranes.

A comparative study of the dynamical fluorescence properties of three phosphatidylcholines having a diphenyl-1,3,5-hexatriene (DPH) group attached at different depths from the head group incorporated into membrane vesicles has been carried out. The probes were covalently attached to the sn-2 position of the glycerol part of the phosphatidylcholine via either carboxyl, ethyl or propanoyl links. The vesicles were composed of either dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine. The experimental time-resolved polarized fluorescence data of the probes were analysed by two different methods: maximum entropy and global analysis. Distributed fluorescence lifetimes and correlation times of the DPH derivatives were obtained with the maximum entropy method. All DPH derivatives exhibited a bimodal distribution of fluorescence lifetimes with a dependence of the lifetime peak positions on the lipid phase, confirming previous data in the literature. The anisotropic rotational dynamics of the DPH moieties in the membranes could be described by several distributed correlation times. In the fluid phase of the membrane the residual anisotropy of free DPH became very small in contrast with those of the other probes, indicating that restriction of probe rotation is mainly imposed by the molecular geometry of the lipid probes. A two-dimensional analysis using the maximum entropy method demonstrated that both rotational correlation times were associated with the same set of fluorescence lifetimes. Global analysis of the data sets according to the general rotational diffusion model yielded weighted orientational distributions. Unexpectedly, a component of the DPH moiety oriented parallel to the membrane surface was obtained in the orientational distributions of the DPH lipids (as was reported earlier for DPH and TMA-DPH), which seems at variance with the geometric constraints imposed by the headgroups.

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.

The interaction of pyrene labeled diacylglycerol with protein kinase C in mixed micelles.

The binding of protein kinase C (PKC) to pyrene-labeled diacylglycerol (pDG) has been studied in a mixed micellar system by monitoring resonance energy transfer from excited tryptophans to pyrene with time-correlated single photon counting. The average lifetime of the excited state of the tryptophans in PKC showed a clear dependence on the mole percentage pDG in micelles in contrast with pyrene-labeled phosphatidylcholine (pPC). The binding data has been analyzed to a simple model which encompasses the size of the micelles and the binding constant of the pDG-PKC complex. From our data, though, these quantities cannot be determined independently. If we have no size information on the micelles we can determine a lower boundary of this quantity compatible with the data. When the micellar size is known, a binding constant for the DG-PKC complex can be extracted. The presented analytical approach can be applied to other systems in which lipid-protein interactions must be quantified.

Determination of panomifene in human plasma by high-performance liquid chromatography.

An ion-pair HPLC method was developed for the determination of the antiestrogenic drug panomifene, (E)-1,2-diphenyl-1-(4-[2-(2-hydroxyethylamino)ethoxy]phenyl)-3,3,3 - trifluoropropene, in human plasma. Tamoxifen, 20 ng in 1 ml of plasma, was used as an internal standard. The compounds were isolated from plasma by liquid-solid extraction. Fluorescence detection was achieved by on-line photochemical conversion of the compounds into highly fluorescent phenanthrene derivatives. The sensitivity of the method was 1 ng/ml. The within-day and between-day precision, linearity, extraction recovery and stability of panomifene in plasma and in deproteinized plasma were determined for validation of the method. The method is suitable for measuring plasma levels of panomifene and tamoxifen and for pharmacokinetic studies.

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.

Effect of neonatal allylestrenol treatment (hormonal imprinting) on the serum testosterone and progesterone concentration in adult rat.

Serum testosterone concentration was significantly elevated in adult male rats by a single perinatal allylestrenol administration. One week after a second allylestrenol treatment in adulthood the hormone concentration dropped below the control values. Serum progesterone concentration was significantly lowered in adult female rats by a single perinatal allylestrenol administration. Following a second allylestrenol treatment in adulthood the hormone concentration reached the control values. These experiments demonstrate that the hormonal imprinting caused by allylestrenol (a steroid used in the treatment of endangered pregnancy) not only acts at receptor level and produces changes in sexual behaviour, but also induces modifications in serum hormone concentrations.

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.

[Pharmacokinetic study of dibromodulcitol in children with brain tumors]. / A dibrómdulcit farmakokinetikai vizsgálata agytumoros gyermekekben.

Systemic pharmacokinetics of high-dose (500 mg/m2), orally administered dibromodulcitol (Elobromol) were studied in 16 chemotherapeutic courses administered to 5 patients. Cerebrospinal fluid dibromodulcitol levels were also analysed in two patients. Bromoepoxydulcitol, dianhydrodulcitol are cytotoxic, whereas bromoanhydrodulcitol, andhydroepoxydulcitol are inactive metabolites detectable during the biotransformation of dibromodulcitol. The HPLC method, developed by our team, is suitable for the determination of both dibromodulcitol and its main metabolites (dianhydrodulcitol and bromoanhydrodulcitol). Our publication is the first in the literature to describe the pharmacokinetic properties of these three hexite-derivatives in pediatric patients. With the exception of one patient, concentration-time curves were analysed by the one-compartment model. From the 30th minute following administration, dibromodulcitol was detectable in all plasma samples for at least 12 hours, its concentration however was usually undetectable by the 24th hour. Though highly variable in value, dianhydrodulcitol concentrations were detectable during all but one therapeutic courses. The following peak concentrations were observed: dibromodulcitol: 3.46-30.63 microM; dianhydroldulcitol: 1.70-6.17 microM; bromoanhydrodulcitol: 0-5.63 microM. The correlation of area under the curve for bromoanhydrodulcitol and dibromodulcitol was exponential up to 200 microMxh with no additional increase detectable above this limit; the distribution of dianhydrodulcitol values were described by a maximum-curve. The possibility of enterohepatic recirculation could not be excluded for any of the compounds studied. Each of the three hexitol derivatives were detectable in the cerebrospinal fluid even if the concentration of the individual metabolite remained undetectable in plasma. The cerebrospinal fluid concentrations of dibromodulcitol were almost constant in the period from 2.5 to 8 hours following administration.(ABSTRACT TRUNCATED AT 250 WORDS)