Functional Consequences of Intracellular Proline Levels Manipulation Affecting PRODH/POX-Dependent Pro-Apoptotic Pathways in a Novel in Vitro Cell Culture Model

Background/Aims: The effect of impaired intracellular proline availability for proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis was studied. Methods: We generated a constitutively knocked-down PRODH/POX MCF-7 breast cancer cell line (MCF-7(shPRODH/POX)) as a model to analyze the functional consequences of impaired intracellular proline levels. We have used inhibitor of proline utilization in collagen biosynthesis, 2-metoxyestradiol (MOE), inhibitor of prolidase that generate proline, rapamycin (Rap) and glycyl-proline (GlyPro), substrate for prolidase. Collagen and DNA biosynthesis were evaluated by radiometric assays. Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. Results: PRODH/POX knockdown decreased DNA and collagen biosynthesis, whereas increased prolidase activity and intracellular proline level in MCF-7(shPRODH/POX) cells. All studied compounds decreased cell viability in MCF-7 and MCF-7(shPRODH/POX) cells. DNA biosynthesis was similarly inhibited by Rap and MOE in both cell lines, but GlyPro inhibited the process only in MCF-7(shPRODH/POX) and MOE+GlyPro only in MCF-7 cells. All the compounds inhibited collagen biosynthesis, increased prolidase activity and cytoplasmic proline level in MCF-7(shPRODH/POX) cells and contributed to the induction of pro-survival mode only in MCF-7(shPRODH/POX) cells. In contrast, all studied compounds upregulated expression of pro-apoptotic protein only in MCF-7 cells. Conclusion: PRODH/POX was confirmed as a driver of apoptosis and proved the eligibility of MCF-7(shPRODH/POX) cell line as a highly effective model to elucidate the different mechanisms underlying proline utilization or generation in PRODH/POX-dependent pro-apoptotic pathways. (C) 2017 The Author(s) Published by S. Karger AG, Base

Analysis of positional candidate genes in the AAA1 susceptibility locus for abdominal aortic aneurysms on chromosome 19

ABSTRACT: BACKGROUND: Abdominal aortic aneurysm (AAA) is a complex disorder with multiple genetic risk factors. Using affected relative pair linkage analysis, we previously identified an AAA susceptibility locus on chromosome 19q13. This locus has been designated as the AAA1 susceptibility locus in the Online Mendelian Inheritance in Man (OMIM) database. METHODS: Nine candidate genes were selected from the AAA1 locus based on their function, as well as mRNA expression levels in the aorta. A sample of 394 cases and 419 controls was genotyped for 41 SNPs located in or around the selected nine candidate genes using the Illumina GoldenGate platform. Single marker and haplotype analyses were performed. Three genes (CEBPG, PEPD and CD22) were selected for DNA sequencing based on the association study results, and exonic regions were analyzed. Immunohistochemical staining of aortic tissue sections from AAA and control individuals was carried out for the CD22 and PEPD proteins with specific antibodies. RESULTS: Several SNPs were nominally associated with AAA (p < 0.05). The SNPs with most significant p-values were located near the CCAAT enhancer binding protein (CEBPG), peptidase D (PEPD), and CD22. Haplotype analysis found a nominally associated 5-SNP haplotype in the CEBPG/PEPD locus, as well as a nominally associated 2-SNP haplotype in the CD22 locus. DNA sequencing of the coding regions revealed no variation in CEBPG. Seven sequence variants were identified in PEPD, including three not present in the NCBI SNP (dbSNP) database. Sequencing of all 14 exons of CD22 identified 20 sequence variants, five of which were in the coding region and six were in the 3'-untranslated region. Five variants were not present in dbSNP. Immunohistochemical staining for CD22 revealed protein expression in lymphocytes present in the aneurysmal aortic wall only and no detectable expression in control aorta. PEPD protein was expressed in fibroblasts and myofibroblasts in the media-adventitia border in both aneurysmal and non-aneurysmal tissue samples. CONCLUSIONS: Association testing of the functional positional candidate genes on the AAA1 locus on chromosome 19q13 demonstrated nominal association in three genes. PEPD and CD22 were considered the most promising candidate genes for altering AAA risk, based on gene function, association evidence, gene expression, and protein expression

Biologiczna dostepnosc wapnia w serach typu Ricotta otrzymanych metoda termiczno-kwasowej koagulacji mleka

The calcium bioavailability from unripening cheeses produced by high heat-acid coagulation of milk was the major purpose of this work. The cheeses may supply a significant portion of the calcium needs for humans because of their total calcium content (from 747 to 605.6 mg/100 g cheese) and satisfactory Ca/P ratio (from 1.55 to 1.85). High bioavailability of calcium from the cheeses, expressed as the apparent absorption (from 57.4 ±0.6 to 60.5 ±1.1%) and retention (from 52.0±1.1 to 52.2±0.6%) make these dairy products to be an important source of available calcium in the human diet.Oznaczono zawartość białka, tłuszczu, wapnia, fosforu, magnezu, laktozy (tab.2) w serach typu Ricotta I i II otrzymanych doświadczalnie w skali półtechnicznej w hali technologicznej Instytutu Technologii Mleczarskiej AR-T w Olsztynie. Sery typu Ricotta I i II wyprodukowano z mleka pasteryzowanego (95 °C/15 s), poddanego następnie koagulacji kwasowej w temperaturze 90°C (ser typu Ricotta I) i 80°C (ser typu Ricotta II). Ponadto na rosnących szczurach szczepu Wistar określono biodostępność wapnia metodą bilansową i wyrażono ją za pomocą wskaźników absorpcji pozornej (A), retencji pozornej (R) oraz kumulacją tego składnika mineralnego w kościach (tab.3). Badania wykazały wysoką koncentrację wapnia i korzystny stosunek Ca : P w serach typu Ricotta I i II (tab.1). Uzyskano wysoką biodostępnośc wapnia z badanych produktów mlecznych (tab.3), które mogą stanowić istotne źródło przyswajalnego wapnia w racji pokarmowej człowieka

Hyaluronic acid abrogates ethanol-dependent inhibition of collagen biosynthesis in cultured human fibroblasts

Magdalena Donejko,1 Andrzej Przylipiak,1 Edyta Rysiak,2 Wojciech Miltyk,3 Elżbieta Galicka,1 Jerzy Przylipiak,4 Ilona Zaręba,2 Arkadiusz Surazynski21Department of Esthetic Medicine, 2Department of Medicinal Chemistry, 3Department of Pharmaceutical Analysis, Faculty of Pharmacy, Medical University of Białystok, 4Medical Office, Białystok, PolandIntroduction: The aim of the study was to evaluate the effect of ethanol on collagen biosynthesis in cultured human skin fibroblasts, and the role of hyaluronic acid (HA) in this process. Regarding the mechanism of ethanol action on human skin fibroblasts we investigated: expression of &beta;1 integrin and insulin-like growth factor 1 receptor (IGF-IR), signaling pathway protein expression: mitogen-activated protein kinases (MAPKs), protein kinase B (Akt), nuclear factor kappa B (NF-&kappa;B) transcription factor, cytotoxicity assay and apoptosis, metalloproteinase activity, as well as the influence of HA on these processes.Materials and methods: Collagen biosynthesis, activity of prolidase, DNA biosynthesis, and cytotoxicity were measured in confluent human skin fibroblast cultures that have been treated with 25, 50, and 100 mM ethanol and with ethanol and 500 &micro;g/mL HA. Western blot analysis and zymography were performed to evaluate expression of collagen type I, &beta;1 integrin receptor, IGF-IR, NF-&kappa;B protein, phospho-Akt protein, kinase MAPK, caspase 9 activity, and matrix metalloproteinases (MMP-9 and MMP-2).Results: Ethanol in a dose-dependent manner lead to the impairment of collagen biosynthesis in fibroblast cultures through decreasing prolidase activity and expression of &beta;1 integrin and IGF-IR. This was accompanied by an increased cytotoxicity, apoptosis and lowered expression of the signaling pathway proteins induced by &beta;1 integrin and IGF-IR, that is, MAPK (ERK1/2) kinases. The lowered amount of synthesized collagen and prolidase activity disturbance may also be due to the activation of NF-&kappa;B transcription factor, which inhibits collagen gene expression. It suggests that the decrease in fibroblast collagen production may be caused by the disturbance in its biosynthesis but not degradation. The application of HA has a protective effect on disturbances caused by the examined substances. It seems that regulatory mechanism of ethanol-induced collagen aberration take place at the level of collagen biosynthesis, since no effect of ethanol and HA was found on process of collagen degradation by MMP-2 and MMP-9.Conclusion: This study provides evidence that ethanol impairs collagen metabolism in human skin fibroblasts, leading to a significant decrease in the amount of produced protein. This mechanism probably is due to downregulation of prolidase activity, expression of &beta;1 integrin and IGF-IR receptors, and the signaling pathway proteins induced by these receptors.Keywords: collagen, ethanol, hyaluronic acid, fibroblas