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We demonstrated that the plasma membrane Ca2+ ATPase PMCA4b inhibits migration and metastatic activity of BRAF mutant melanoma cells. Actin dynamics are essential for cells to move, invade and metastasize, therefore, we hypothesized that PMCA4b affected cell migration through remodeling of the actin cytoskeleton. We found that expression of PMCA4b in A375 BRAF mutant melanoma cells induced a profound change in cell shape, cell culture morphology, and displayed a polarized migratory character. Along with these changes the cells became more rounded with increased cell–cell connections, lamellipodia and stress fiber formation. Silencing PMCA4b in MCF-7 breast cancer cells had a similar effect, resulting in a dramatic loss of stress fibers. In addition, the PMCA4b expressing A375 cells maintained front-to-rear Ca2+ concentration gradient with the actin severing protein cofilin localizing to the lamellipodia, and preserved the integrity of the actin cytoskeleton from a destructive Ca2+ overload. We showed that both PMCA4b activity and trafficking were essential for the observed morphology and motility changes. In conclusion, our data suggest that PMCA4b plays a critical role in adopting front-to-rear polarity in a normally spindle-shaped cell type through F-actin rearrangement resulting in a less aggressive melanoma cell phenotype.

Two related multisubunit tethering complexes promote endolysosomal trafficking in all eukaryotes: Rab5-binding CORVET that was suggested to transform into Rab7-binding HOPS. We have previously identified miniCORVET, containing Drosophila Vps8 and three shared core proteins, which are required for endosome maturation upstream of HOPS in highly endocytic cells (Lőrincz et al., 2016a). Here, we show that Vps8 overexpression inhibits HOPS-dependent trafficking routes including late endosome maturation, autophagosome-lysosome fusion, crinophagy and lysosome-related organelle formation. Mechanistically, Vps8 overexpression abolishes the late endosomal localization of HOPS-specific Vps41/Lt and prevents HOPS assembly. Proper ratio of Vps8 to Vps41 is thus critical because Vps8 negatively regulates HOPS by outcompeting Vps41. Endosomal recruitment of miniCORVET- or HOPS-specific subunits requires proper complex assembly, and Vps8/miniCORVET is dispensable for autophagy, crinophagy and lysosomal biogenesis. These data together indicate the recruitment of these complexes to target membranes independent of each other in Drosophila, rather than their transformation during vesicle maturation.

Loss of epithelial cell polarity and tissue disorganization are hallmarks of carcinogenesis, in which Ca 2+ signaling plays a significant role. Here we demonstrate that the plasma membrane Ca 2+ pump PMCA4 (ATP2B4) is downregulated in luminal breast cancer, and this is associated with shorter relapse-free survival in patients with luminal A and B1 subtype tumors. Using the MCF-7 breast cancer cell model we show that PMCA4 silencing results in the loss of cell polarity while a forced increase in PMCA4b expression induces cell polarization and promotes lumen formation. We identify Arf6 as a regulator of PMCA4b endocytic recycling essential for PMCA4-mediated lumen formation. Silencing of the single pmca gene in Drosophila melanogaster larval salivary gland destroys lumen morphology suggesting a conserved role of PMCAs in lumen morphogenesis. Our findings point to a role of PMCA4 in controlling epithelial cell polarity, and in the maintenance of normal glandular tissue architecture. PMCA4 promotes epithelial polarization and lumen formation, while its loss induces partial EMT in luminal breast cancer cells. Its downregulation in LUMA/B1 breast cancer correlates with poor survival and may therefore serve as a prognostic marker.

Metastatic melanoma is the most aggressive type of skin cancer. Previously, we identified the plasma membrane Ca2+ pump isoform 4b (PMCA4b or ATP2B4) as a putative metastasis suppressor in BRAF mutant melanoma cells. Metastasis suppressors are often downregulated in cancer, therefore, it is important to identify the pathways involved in their degradation. Here, we studied the role of p38 MAPK in PMCA4b degradation and its effect on melanoma metastasis. We found that activation of p38 MAPK induces internalization and subsequent degradation of PMCA4b through the endo/lysosomal system that contributes to the low PMCA4b steady-state protein level of BRAF mutant melanoma cells. Moreover, BRAF wild type cell models including a doxycycline-inducible HEK cell system revealed that p38 MAPK is a universal modulator of PMCA4b endocytosis. Inhibition of the p38 MAPK pathway markedly reduced migration, colony formation and metastatic activity of BRAF mutant cells in vitro partially through an increase in PMCA4b and a decrease in β4 integrin abundance. In conclusion, our data suggest that the p38 MAPK pathway plays a key role in PMCA4b degradation and inhibition of this pathway—by increasing the stability of PMCA4b—may provide a potential therapeutic target for inhibition of melanoma progression and metastasis.

Loss of epithelial cell polarity is a hallmark of carcinogenesis, in which Ca2+ signaling plays a role. Here we demonstrate that the plasma membrane Ca2+ pump PMCA4 is downregulated in luminal breast cancer, and this is associated with shorter relapse-free survival of patients with luminal A and B1 sub-type tumors. We find that in vitro silencing of PMCA4 in MCF-7 breast cancer cells induces internalization of E-cadherin and Dlg1 leading to a severe loss of cell polarity while re-expression of the b variant of PMCA4 restores cell polarity and promotes lumen formation mediated by the Arf6-trafficking pathway. We also demonstrate via an in vivo model using Drosophila melanogaster that silencing of the single pmca gene destroys lumen morphology in the larval salivary gland suggesting a conserved role of PMCAs in lumen morphogenesis. Additionally, enhanced secretory vesicle accumulation in PMCA(4)-deficient cells indicates a profound secretion defect both in vitro and in vivo. Our findings point to a novel role of PMCA4 in restoring epithelial cell polarity, and maintaining normal glandular tissue architecture.

We demonstrated that the plasma membrane Ca ATPase PMCA4b inhibits migration and metastatic activity of BRAF mutant melanoma cells. Actin dynamics are essential for cells to move, invade and metastasize, therefore, we hypothesized that PMCA4b affected cell migration through remodeling of the actin cytoskeleton. We found that expression of PMCA4b in A375 BRAF mutant melanoma cells induced a profound change in cell shape, cell culture morphology, and displayed a polarized migratory character. Along with these changes the cells became more rounded with increased cell-cell connections, lamellipodia and stress fiber formation. Silencing PMCA4b in MCF-7 breast cancer cells had a similar effect, resulting in a dramatic loss of stress fibers. In addition, the PMCA4b expressing A375 cells maintained front-to-rear Ca concentration gradient with the actin severing protein cofilin localizing to the lamellipodia, and preserved the integrity of the actin cytoskeleton from a destructive Ca overload. We showed that both PMCA4b activity and trafficking were essential for the observed morphology and motility changes. In conclusion, our data suggest that PMCA4b plays a critical role in adopting front-to-rear polarity in a normally spindle-shaped cell type through F-actin rearrangement resulting in a less aggressive melanoma cell phenotype.

I.István királyunk fiának, Inre hercegnek az éles téről csupán egyetlen hazsi legenda mersdt ránk. Ezt a legendát lengyel vonatkozásu részekkel több e- a magyar legendás tól gyökeresen különböző e. lengyel Inro-logonda egésztti ki. A megyar legendának, melyet több kódex megőrzött számunkra, háron különböző bevezetése van. Bartoniek véleménye s20- rint! az eredeti bevezetést később kétszer átdolgozták, még\" pedig először a Hartvik püspök által szerkesztett István-legenda alapján, majd egy teljesen ismeretlen forrásnak a fele jegyzéseiből. A magyar legenda alapszövege a ránk maradt kódexekben egy-két szavas, illetve egy-egy mondatnyi eltéréstől eltekintve teljesen azonoss Az utóbb keletkezett bevezetések , megszerkesztésének időpontjáról semmi biztosat nem tudunks A kódoxek legnagyobb része az első bevezetést tartalmazó l109- gendát őrizte mege. Est közli egy ősnyontatvány is,\" először adták ki a magyar legendákat nyomtatásban.A legrégibb és legjobb kódex, a reuni /Sstájerorszóg/ cisztercita apátság 69. szánu, 13. század eloji, /esetleg 124 század végi kódexe/, amely 173 kis folió alaku lapot tartalnaz.s Előszó az Imreslegenda szövegének kisadásás hoz Seríiptores Rerum Hungaricarum -- a továbbiakban; SRH -- ILBPes 19384/ öt.tegendas Sanctorum regni Hungariae in [Lombardica/Historia! non contentae, Argentinae, 1486., Strassburg, 1485. és 1498., továbbá még néhány kisdás a 15. században év és hely negje- lőlése nélkül /Ls [ains Repertoriun Bibliographivum II., £ le Téöö; 251. old, 92996-77998./ és Velence, 1512. - Vö. Bars tonieck : i.sn. 447. oldás.A 27\"e-38! oldalon István legendáját /uns Hartvik legends/; a Z8Y-stl\" oldalon az Inreslegendát közli. A kódex külsejéről és feltételezhetően magyar eredetéről Menyhért közöl adatokat,? A kódex kemény táblája leszekedt, Mai formás jában két --s később egybekötött e- Önálló részből áll. Közülük csak az első rész lehetett Magyarországons Ennek boritós lapja az egybekötés alkalmával kerülhetett az egész kötet vés gére. Ezon a hátsó két, 15. százsdi késtől származó bejegyzés olvasható: \"Capella sancti spiritus in Supronio\" és \"Martinus Odenburg\", Ugyanezen kéztől származik az Istvánslogonda e kódexbeli szövegében a 29\" oldalon a hibás \"hespremn\" kijavitása \"Wesprem\"eres.\" Ebből ugy látszik, hogy az említett Martinus magyar ember lehetett. A kódex magyar eredetét tánogatja az is, hogy az István-logenda pannonhalmi adonányáról szóló részlete feltünően egyesik a pannonhalmi oöklevéltár 1249.

A novel, validated, reversed-phase (RP), chiral high performance liquid chromatography (HPLC) method was developed for the enantiopurity control analysis of naproxen, a frequently used non-steroidal anti-inflammatory agent using polysaccharide-type chiral stationary phase (CSP). In the screening phase of method development, seven columns were tested in polar organic (PO) mode using mobile phases consisting of 0.1% acetic acid in methanol, ethanol, 2-propanol, and acetonitrile. Enantiorecognition was observed only in five cases. The best enantioseparation was observed on a Lux Amylose-1 column with 0.1% (v/v) acetic acid in ethanol with a resolution (Rs) of 1.24. The enantiomer elution order was unfavorable, as the distomer eluted after the eutomer. When the ethanolic mobile phase was supplemented with water, enantiomer elution order reversal was observed, indicating a difference in the enantiorecognition mechanism upon switching from PO to RP mode. Furthermore, by changing ethanol to methanol, not only lower backpressure, but also higher resolution was obtained. Subsequent method optimization was performed using a face-centered central composite design (FCCD) to achieve higher chiral resolution in a shorter analysis time. Optimized parameters offering baseline separation were as follows: Lux Amylose-1 stationary phase, thermostated at 40 °C, and a mobile phase consisting of methanol:water:acetic acid 85:15:0.1 (v/v/v), delivered with 0.65 mL/min flow rate. Using these optimized parameters, a Rs = 3.21 ± 0.03 was achieved within seven minutes. The optimized method was validated according to the ICH guidelines and successfully applied for the analysis of different pharmaceutical preparations, such as film-coated tablets and gel, as well as fixed-dose combination tablets, containing both naproxen and esomeprazole.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that plays significant regulatory roles in the differentiation of the central nervous system and peripheral organs. A lack of the neuropeptide can lead to abnormalities in long bone development. In callus formation, a possible signaling balance shift in PACAP KO mice has been demonstrated, but Notch signalization, with its potential connection with PACAP 1-38, has not been investigated in ossification. Our main goal was to show connections between PACAP and Notch signaling in osteogenesis. Notch signalization showed an elevation in the long bones of PACAP-gene-deficient mice, and it was also elevated during the PACAP 1-38 treatment of UMR-106 and MC3T3-E1 osteogenic cells. Moreover, the inhibition of Notch signaling was compensated by the addition of PACAP 1-38 in vitro. The inorganic and organic matrix production of UMR-106 cells was increased during PACAP 1-38 treatment under the inhibition of Notch signaling. As a possible common target, the expression and nuclear translocation of NFATc1 transcription factor was increased during the disturbance of PACAP and Notch signaling. Our results indicate a possible synergistic regulation during bone formation by PACAP and Notch signalization. The crosstalk between Notch and PACAP signaling pathways highlights the complexity of bone development and homeostasis.