Mechanism of collagen biosynthesis up-regulation in cultured leiomyoma cells.

Uterine leiomyoma is the most common tumour in women with a reported incidence of 25-30%. The tumors are benign, composed of smooth muscle cells with variable amount of collagen - rich fibrous tissue. It is well established that accumulation of extracellular matrix in leiomyoma is key feature of tissue fibrosis. However, the pathogenesis of leiomyoma is still unclear. The aim of this study was to evaluate the metabolism of collagen in cultured leiomyoma cells and in control myometrium cells. The effect of estradiol, selective modulators of estrogen receptors (raloxifene, tamoxifen) and estrogen receptor down regulator (ICI 182.780) on collagen biosynthesis (measured by 5-[3H]-proline incorporation assay and measurement of prolidase activity) and collagen degradation (measured by metalloproteinase activity assay) was studied. It was found that collagen biosynthesis is strongly stimulated by low doses of estradiol (5 nM) in leiomyoma cells while it is not changed in control myometrium cells. An increase in estradiol concentration to 10 nM results in drastic decrease of this process both in leiomyoma as well as control cells. Although raloxifene and tamoxifen only slightly affected collagen biosynthesis in control myometrium cells, they significantly inhibited the process in leiomyoma cells. There was no coordinate correlation between collagen biosysignificantly inhibited the process in leiomyoma cells. There was no coordinate correlation between collagen biosynthesis and prolidase activity suggesting that regulation of this process may take place at transcriptional level. Both estrogen and SERMs were found to inhibit MMP-2 in leiomyoma as well as in control myometrium cells. The data suggest that stimulatory action of estrogen on collagen biosynthesis and inhibitory effect on MMP-2 activity in uterine leiomyoma may contribute to accumulation of this protein in ECM of this tissue

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

Prototype of an opto-capacitive probe for non-invasive sensing cerebrospinal fluid circulation

In brain studies, the function of the cerebrospinal fluid (CSF) awakes growing interest, particularly related to studies of the glymphatic system in the brain, which is connected with the complex system of lymphatic vessels responsible for cleaning the tissues. The CSF is a clear, colourless liquid including water (H2O) approximately with a concentration of 99 %. In addition, it contains electrolytes, amino acids, glucose, and other small molecules found in plasma. The CSF acts as a cushion behind the skull, providing basic mechanical as well as immunological protection to the brain. Disturbances of the CSF circulation have been linked to several brain related medical disorders, such as dementia. Our goal is to develop an in vivo method for the non-invasive measurement of cerebral blood flow and CSF circulation by exploiting optical and capacitive sensing techniques simultaneously. We introduce a prototype of a wearable probe that is aimed to be used for long-term brain monitoring purposes, especially focusing on studies of the glymphatic system. In this method, changes in cerebral blood flow, particularly oxy- and deoxyhaemoglobin, are measured simultaneously and analysed with the response gathered by the capacitive sensor in order to distinct the dynamics of the CSF circulation behind the skull. Presented prototype probe is tested by measuring liquid flows inside phantoms mimicking the CSF circulation

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

Nonsteroidal Anti-Inflammatory Drugs as PPAR&gamma; Agonists Can Induce PRODH/POX-Dependent Apoptosis in Breast Cancer Cells: New Alternative Pathway in NSAID-Induced Apoptosis

Nonsteroidal anti-inflammatory drugs (NSAIDs) are considered to be therapeutics in cancer prevention because of their inhibitory effect on cyclooxygenases (COX), which are frequently overexpressed in many types of cancer. However, it was also demonstrated that NSAIDs provoked a proapoptotic effect in COX knocked-out cancer cells. Here, we suggest that this group of drugs may provoke antineoplastic activity through the activation of PPAR&gamma;, which induces proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis. PRODH/POX is a mitochondrial enzyme that catalyzes proline degradation, during which ATP or reactive oxygen species (ROS) are generated. We have found that NSAIDs induced PRODH/POX and PPAR&gamma; expressions (as demonstrated by Western Blot or immunofluorescence analysis) and cytotoxicity (as demonstrated by MTT, cytometric assay, and DNA biosynthesis assay) in breast cancer MCF7 cells. Simultaneously, the NSAIDs inhibited collagen biosynthesis, supporting proline for PRODH/POX-induced ROS-dependent apoptosis (as demonstrated by an increase in the expression of apoptosis markers). The data suggest that targeting proline metabolism and the PRODH/POX&ndash;PPAR&gamma; axis can be considered a novel approach for breast cancer treatment