Continuous Flow Mucosal Cells for Measuring the in-Vitro Permeability of Small Tissue Samples

1997 ◽  
Vol 86 (1) ◽  
pp. 82-84 ◽  
Author(s):  
Christopher A. Squier ◽  
Mary Kremer ◽  
Philip W. Wertz
Keyword(s):  
Continuous Flow ◽  
Tissue Samples ◽  
Small Tissue ◽  
Mucosal Cells

PROSTAGLANDIN PRODUCTION BY INTRA-UTERINE TISSUES FROM PERIPARTURIENT SHEEP: USE OF A SUPERFUSION TECHNIQUE

1978 ◽  
Vol 76 (1) ◽  
pp. 111-121 ◽  
Author(s):  
M. D. MITCHELL ◽  
A. P. F. FLINT
Keyword(s):  
Arachidonic Acid ◽  
Relative Rate ◽  
Prostaglandin E ◽  
Tissue Samples ◽  
Prostaglandin Production ◽  
Small Tissue ◽  
Prostaglandin F

SUMMARY A technique for the continuous superfusion of small tissue samples in vitro has been applied to the study of prostaglandin production by ovine intra-uterine tissues. Basal and oxytocin-stimulated production of prostaglandins was studied at 120–125 days of pregnancy and after dexamethasone-induced delivery. In general, the relative rate of prostaglandin production by tissues was: foetal cotyledon = maternal cotyledon>myometrium and in quantitative order the prostaglandins produced were prostaglandin E (PGE) > prostaglandin F (PGF) = 13,14-dihydro-15-oxo-prostaglandin F (PGFM). Considerable variation was found between the rates of prostaglandin production in individual sheep. Oxytocin had no effect on the production of prostaglandins by tissues obtained before labour but myometrium and maternal cotyledon obtained at delivery exhibited a significant increase in production of PGE and PGF (though not PGFM) in response to oxytocin. Administration of arachidonic acid increased the production of PGE and PGF by the foetal cotyledon.

scholarly journals Trafficking of Full-Length and N-Terminally Truncated Cathepsin B in Human Colorectal Carcinoma Cells

2021 ◽  
Vol 11 (24) ◽  
pp. 11936
Author(s):  
Tripti Tamhane ◽  
Robin W. Njenga ◽  
Roberta E. Burden ◽  
Heiko Büth ◽  
Gunhild M. Maelandsmo ◽  
...  
Keyword(s):  
Colorectal Carcinoma ◽  
Cathepsin B ◽  
Carcinoma Cells ◽  
Full Length ◽  
Adenocarcinoma In Situ ◽  
Tissue Samples ◽  
Mucosal Cells ◽  
Lysosomal Cysteine Protease

Cathepsin B is an endo-lysosomal cysteine protease. However, its increased expression and altered localization to the extracellular space, to mitochondria, or to the nucleus has been linked to tumor progression. In the present study, we show enhanced levels of cathepsin B in adenocarcinoma tissue in comparison to adjacent normal colon. Additionally, cathepsin B was observed in the nuclear compartment of mucosal cells in adenocarcinoma tissue samples and in the nuclei of the colorectal carcinoma cell line HCT116. Accordingly, a distinct 40-kDa form of cathepsin B was detected in HCT116 cells, which is proposed to represent a specific form lacking the signal peptide and parts of the propeptide. Trafficking studies with an EGFP-tagged N-terminally truncated form, mimicking the 40-kDa form, demonstrated accumulation in aggresome-like inclusion bodies, while EGFP-tagged full-length cathepsin B revealed regular sorting to endo-lysosomes. We conclude that the identity of nuclear cathepsin B in colorectal adenocarcinoma (in situ) and in carcinoma cells (in vitro) cannot be attributed to either full-length or 40-kDa N-terminally truncated cathepsin B forms. Hence, future studies are needed to demonstrate which form/s of cathepsin B may be sorted to the nuclei of colorectal carcinoma cells, and whether redundant regulation of related cathepsin expression occurs.

scholarly journals Possible Role of IRS-4 in the Origin of Multifocal Hepatocellular Carcinoma

Cancers ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2560
Author(s):  
Luis G. Guijarro ◽  
Patricia Sanmartin-Salinas ◽  
Eva Pérez-Cuevas ◽  
M. Val Toledo-Lobo ◽  
Jorge Monserrat ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Liver Cancer ◽  
Hepg2 Cells ◽  
Cellular Polarity ◽  
Ki 67 ◽  
Tissue Samples ◽  
Vitro Analysis ◽  
Tumoral Cells

New evidence suggests that insulin receptor substrate 4 (IRS-4) may play an important role in the promotion of tumoral growth. In this investigation, we have evaluated the role of IRS-4 in a pilot study performed on patients with liver cancer. We used immunohistochemistry to examine IRS-4 expression in biopsies of tumoral tissue from a cohort of 31 patient suffering of hepatocellular carcinoma (HCC). We simultaneously analyzed the expression of the cancer biomarkers PCNA, Ki-67, and pH3 in the same tissue samples. The in vitro analysis was conducted by studying the behavior of HepG2 cells following IRS-4 overexpression/silencing. IRS-4 was expressed mainly in the nuclei of tumoral cells from HCC patients. In contrast, in healthy cells involved in portal triads, canaliculi, and parenchymal tissue, IRS-4 was observed in the cytosol and the membrane. Nuclear IRS-4 in the tumoral region was found in 69.9 ± 3.2%, whereas in the surrounding healthy hepatocytes, nuclear IRS-4 was rarely observed. The percentage of tumoral cells that exhibited nuclear PCNA and Ki-67 were 52.1 ± 7%, 6.1 ± 1.1% and 1.3 ± 0.2%, respectively. Furthermore, we observed a significant positive linear correlation between nuclear IRS-4 and PCNA (r = 0.989; p < 0.001). However, when we correlated the nuclear expression of IRS-4 and Ki-67, we observed a significant positive curvilinear correlation (r = 0.758; p < 0.010). This allowed us to define two populations, (IRS-4 + Ki-67 ≤ 69%) and (IRS-4 + Ki-67 > 70%). The population with lower levels of IRS-4 and Ki-67 had a higher risk of suffering from multifocal liver cancer (OR = 16.66; CI = 1.68–164.8 (95%); p < 0.05). Immunoblot analyses showed that IRS-4 in normal human liver biopsies was lower than in HepG2, Huh7, and Chang cells. Treatment of HepG2 with IGF-1 and EGF induced IRS-4 translocation to the nucleus. Regulation of IRS-4 levels via HepG2 transfection experiments revealed the protein’s role in proliferation, cell migration, and cell-collagen adhesion. Nuclear IRS-4 is increased in the tumoral region of HCC. IRS-4 and Ki-67 levels are significantly correlated with the presence of multifocal HCC. Moreover, upregulation of IRS-4 in HepG2 cells induced proliferation by a β-catenin/Rb/cyclin D mechanism, whereas downregulation of IRS-4 caused a loss in cellular polarity and in its adherence to collagen as well as a gain in migratory and invasive capacities, probably via an integrin α2 and focal adhesion cascade (FAK) mechanism.

scholarly journals EP4 as a Negative Prognostic Factor in Patients with Vulvar Cancer

Cancers ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1410
Author(s):  
Anna Buchholz ◽  
Aurelia Vattai ◽  
Sophie Fürst ◽  
Theresa Vilsmaier ◽  
Christina Kuhn ◽  
...  
Keyword(s):  
Overall Survival ◽  
Prognostic Factor ◽  
Vulvar Cancer ◽  
Cox Regression ◽  
Disease Free Survival ◽  
Common Finding ◽  
Cancer Tissue ◽  
Vulvar Carcinoma ◽  
Tissue Samples

New prognostic factors and targeted therapies are urgently needed to improve therapeutic outcomes in vulvar cancer patients and to reduce therapy related morbidity. Previous studies demonstrated the important role of prostaglandin receptors in inflammation and carcinogenesis in a variety of tumor entities. In this study, we aimed to investigate the expression of EP4 in vulvar cancer tissue and its association with clinicopathological data and its prognostic relevance on survival. Immunohistochemistry was performed on tumor specimens of 157 patients with vulvar cancer treated in the Department of Obstetrics and Gynecology, Ludwig-Maximilian-University of Munich, Germany, between 1990 and 2008. The expression of EP4 was analyzed using the well-established semiquantitative immunoreactivity score (IRS) and EP4 expression levels were correlated with clinicopathological data and patients’ survival. To specify the tumor-associated immune cells, immunofluorescence double staining was performed on tissue samples. In vitro experiments including 5-Bromo-2′-Deoxyuridine (BrdU) proliferation assay and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid (MTT) viability assay were conducted in order to examine the effect of EP4 antagonist L-161,982 on vulvar carcinoma cells. EP4 expression was a common finding in in the analyzed vulvar cancer tissue. EP4 expression correlated significantly with tumor size and FIGO classification and differed significantly between keratinizing vulvar carcinoma and nonkeratinizing carcinoma. Survival analysis showed a significant correlation of high EP4 expression with poorer overall survival (p = 0.001) and a trending correlation between high EP4 expression and shorter disease-free survival (p = 0.069). Cox regression revealed EP4 as an independent prognostic factor for overall survival when other factors were taken into account. We could show in vitro that EP4 antagonism attenuates both viability and proliferation of vulvar cancer cells. In order to evaluate EP4 as a prognostic marker and possible target for endocrinological therapy, more research is needed on the influence of EP4 in the tumor environment and its impact in vulvar carcinoma.

Myofibrillar degeneration with diphtheria toxin

2020 ◽  
Vol 45 (4) ◽  
pp. 351-357
Author(s):  
Bilge Özerman Edis ◽  
Muhammet Bektaş ◽  
Rüstem Nurten
Keyword(s):  
Protein Synthesis ◽  
Actin Filaments ◽  
Diphtheria Toxin ◽  
Cardiac Tissue ◽  
Culture Conditions ◽  
Bacterial Toxin ◽  
Filamentous Actin ◽  
Cardiac Damage ◽  
Tissue Samples

AbstractObjectivesCardiac damage in patient with diphtheritic myocarditis is reported as the leading cause of mortality. Diphtheria toxin (DTx) is a well-known bacterial toxin inducing various cytotoxic effects. Mainly, catalytic fragment inhibits protein synthesis, induces cytotoxicity, and depolymerizes actin filaments. In this study, we aimed to demonstrate the extent of myofibrillar damage under DTx treatment to porcine cardiac tissue samples.MethodsTissue samples were incubated with DTx for 1–3 h in culture conditions. To analyze whole toxin (both fragments) distribution, conjugation of DTx with FITC was performed. Measurements were carried out with fluorescence spectrophotometer before and after dialysis. Immunofluorescence microscopy was used to show localization of DTx-FITC (15 nM) on cardiac tissue incubated for 2 h. Ultrastructural characterization of cardiac tissue samples treated with DTx (15 or 150 nM) was performed with transmission electron microscopy.ResultsDTx exerts myofibrillar disorganization. Myofilament degeneration, mitochondrial damage, vacuolization, and abundant lipid droplets were determined with 150 nM of DTx treatment.ConclusionsThis finding is an addition to depolymerization of actin filaments as a result of the DTx-actin interactions in in vitro conditions, indicating that myofilament damage can occur with DTx directly besides protein synthesis inhibition. Ultrastructural results support the importance of filamentous actin degeneration at diphtheritic myocarditis.

scholarly journals Novel Identified Circular Transcript of RCAN2, circ-RCAN2, Shows Deviated Expression Pattern in Pig Reperfused Infarcted Myocardium and Hypoxic Porcine Cardiac Progenitor Cells In Vitro

2021 ◽  
Vol 22 (3) ◽  
pp. 1390
Author(s):  
Julia Mester-Tonczar ◽  
Patrick Einzinger ◽  
Johannes Winkler ◽  
Nina Kastner ◽  
Andreas Spannbauer ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Progenitor Cells ◽  
Regulatory Networks ◽  
Circular Rnas ◽  
Cardiac Progenitor Cells ◽  
Tissue Samples ◽  
Healthy Myocardium ◽  
Acute Mi

Circular RNAs (circRNAs) are crucial in gene regulatory networks and disease development, yet circRNA expression in myocardial infarction (MI) is poorly understood. Here, we harvested myocardium samples from domestic pigs 3 days after closed-chest reperfused MI or sham surgery. Cardiac circRNAs were identified by RNA-sequencing of rRNA-depleted RNA from infarcted and healthy myocardium tissue samples. Bioinformatics analysis was performed using the CIRIfull and KNIFE algorithms, and circRNAs identified with both algorithms were subjected to differential expression (DE) analysis and validation by qPCR. Circ-RCAN2 and circ-C12orf29 expressions were significantly downregulated in infarcted tissue compared to healthy pig heart. Sanger sequencing was performed to identify the backsplice junctions of circular transcripts. Finally, we compared the expressions of circ-C12orf29 and circ-RCAN2 between porcine cardiac progenitor cells (pCPCs) that were incubated in a hypoxia chamber for different time periods versus normoxic pCPCs. Circ-C12orf29 did not show significant DE in vitro, whereas circ-RCAN2 exhibited significant ischemia-time-dependent upregulation in hypoxic pCPCs. Overall, our results revealed novel cardiac circRNAs with DE patterns in pCPCs, and in infarcted and healthy myocardium. Circ-RCAN2 exhibited differential regulation by myocardial infarction in vivo and by hypoxia in vitro. These results will improve our understanding of circRNA regulation during acute MI.

scholarly journals Splicing factor SRSF1 promotes breast cancer progression via oncogenic splice switching of PTPMT1

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Jun-Xian Du ◽  
Yi-Hong Luo ◽  
Si-Jia Zhang ◽  
Biao Wang ◽  
Cong Chen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Progression ◽  
High Risk Group ◽  
Clinical Samples ◽  
Binding Motif ◽  
Ki 67 ◽  
Tissue Samples

Abstract Background Intensive evidence has highlighted the effect of aberrant alternative splicing (AS) events on cancer progression when triggered by dysregulation of the SR protein family. Nonetheless, the underlying mechanism in breast cancer (BRCA) remains elusive. Here we sought to explore the molecular function of SRSF1 and identify the key AS events regulated by SRSF1 in BRCA. Methods We conducted a comprehensive analysis of the expression and clinical correlation of SRSF1 in BRCA based on the TCGA dataset, Metabric database and clinical tissue samples. Functional analysis of SRSF1 in BRCA was conducted in vitro and in vivo. SRSF1-mediated AS events and their binding motifs were identified by RNA-seq, RNA immunoprecipitation-PCR (RIP-PCR) and in vivo crosslinking followed by immunoprecipitation (CLIP), which was further validated by the minigene reporter assay. PTPMT1 exon 3 (E3) AS was identified to partially mediate the oncogenic role of SRSF1 by the P-AKT/C-MYC axis. Finally, the expression and clinical significance of these AS events were validated in clinical samples and using the TCGA database. Results SRSF1 expression was consistently upregulated in BRCA samples, positively associated with tumor grade and the Ki-67 index, and correlated with poor prognosis in a hormone receptor-positive (HR+) cohort, which facilitated proliferation, cell migration and inhibited apoptosis in vitro and in vivo. We identified SRSF1-mediated AS events and discovered the SRSF1 binding motif in the regulation of splice switching of PTPMT1. Furthermore, PTPMT1 splice switching was regulated by SRSF1 by binding directly to its motif in E3 which partially mediated the oncogenic role of SRSF1 by the AKT/C-MYC axis. Additionally, PTPMT1 splice switching was validated in tissue samples of BRCA patients and using the TCGA database. The high-risk group, identified by AS of PTPMT1 and expression of SRSF1, possessed poorer prognosis in the stage I/II TCGA BRCA cohort. Conclusions SRSF1 exerts oncogenic roles in BRCA partially by regulating the AS of PTPMT1, which could be a therapeutic target candidate in BRCA and a prognostic factor in HR+ BRCA patient.

In vitro Action of Gastric Mucosal Lysosomal Enzymes on Intracellular Gastric Glycoproteins

1979 ◽  
Vol 34 (1-2) ◽  
pp. 90-95 ◽  
Author(s):  
Fouad M. Fouad ◽  
D. Waldron-Edward
Keyword(s):  
Cell Wall ◽  
Lysosomal Enzymes ◽  
Gastric Ulceration ◽  
Mucosal Cell ◽  
Gastric Lesions ◽  
Acid Hydrolases ◽  
Gastric Mucosal Cells ◽  
Mucosal Cells ◽  
Specific Increase

Abstract The results show that incubation of gastric mucosal cells from rat at pH ~4.5 or in the presence of aspirin is associated with a specific increase in the activity of some acid-hydrolases. Intracellular glycoproteins, isolated by non-degradative techniques from rat or dog fundic mucosal cells, were found to be potential bio-substrates for these acid-hydrolyses. This may suggest that cleavage of the carbohydrate moieties of the intracellular and mucosal cell wall glycoproteins is a fundamental step in the development of gastric ulceration. A model for gastric lesions is proposed and discussed in the light of the results obtained.

scholarly journals Impact of Chronic Tetracycline Exposure on Human Intestinal Microbiota in a Continuous Flow Bioreactor Model

Antibiotics ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 886
Author(s):  
Youngbeom Ahn ◽  
Ji Young Jung ◽  
Ohgew Kweon ◽  
Brian T. Veach ◽  
Sangeeta Khare ◽  
...  
Keyword(s):  
Molecular Analysis ◽  
Intestinal Microbiota ◽  
Continuous Flow ◽  
Antimicrobial Drug ◽  
Bioreactor Culture ◽  
Analysis Techniques ◽  
Human Intestinal Microbiota ◽  
Traditional Cultures ◽  
The Impact

Studying potential dietary exposure to antimicrobial drug residues via meat and dairy products is essential to ensure human health and consumer safety. When studying how antimicrobial residues in food impact the development of antimicrobial drug resistance and disrupt normal bacteria community structure in the intestine, there are diverse methodological challenges to overcome. In this study, traditional cultures and molecular analysis techniques were used to determine the effects of tetracycline at chronic subinhibitory exposure levels on human intestinal microbiota using an in vitro continuous flow bioreactor. Six bioreactor culture vessels containing human fecal suspensions were maintained at 37 °C for 7 days. After a steady state was achieved, the suspensions were dosed with 0, 0.015, 0.15, 1.5, 15, or 150 µg/mL tetracycline, respectively. Exposure to 150 µg/mL tetracycline resulted in a decrease of total anaerobic bacteria from 1.9 × 107 ± 0.3 × 107 down to 2 × 106 ± 0.8 × 106 CFU/mL. Dose-dependent effects of tetracycline were noted for perturbations of tetB and tetD gene expression and changes in acetate and propionate concentrations. Although no-observed-adverse-effect concentrations differed, depending on the traditional cultures and the molecular analysis techniques used, this in vitro continuous flow bioreactor study contributes to the knowledge base regarding the impact of chronic exposure of tetracycline on human intestinal microbiota.

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