This study examined the localization and the effect of circulating peptides on the expression of aminopeptidase N (EC 3.4.11.2) in caprine mammary gland. Four lactating goats in mid to late lactation ...were used in a crossover design and were subjected to 2 dietary treatments. Abomasal infusion of casein hydrolysate was used to increase the concentration of peptide-bound amino acid in the circulation. Samples of mammary gland tissue from each goat were taken by biopsy at the end of each treatment period to measure gene and protein expression of aminopeptidase N in the tissue. There were no measurable effects on feed intake and milk production for any of the treatments. Western blot analysis showed that aminopeptidase N is located on the basolateral side of parenchymal cells and not on the apical membranes. Abomasal infusion of casein hydrolysate caused a marked change in the profile of arterial blood free amino acids and peptide-bound amino acids smaller than 1500Da. Abundance of aminopeptidase N mRNA and protein increased by 51 and 58%, respectively, in casein hydrolysate-infused goats compared with the control treatment. It was concluded that aminopeptidase N is one candidate actively involved in the mammary gland to support protein synthesis and milk production. In accordance with the nutritional conditions in the current experiment, it is suggested that aminopeptidase N expression is partly controlled by the metabolic requirements of the gland and postabsorptive forms of amino acids in the circulation.
A 970 bp cDNA Na+/glucose cotransporter (SGLT1) was isolated and sequenced from chicken jejunum by reverse transcriptase polymerase chain reaction (RT-PCR) using primers based on conserved regions. ...Using the 970 bp PCR product as a specific probe, Northern Blot hybridization indicated a transcript of ca. 4 kb. The isolated chicken intestinal SGLT1 cDNA was used to quantitate mRNA expression. Glucose uptake activity and kinetics were determined in brush border membrane vesicles (BBMV) from jejunum tissue of chickens which were either fed, food-deprived or refed following food deprivation. Net glucose uptake to BBMV was higher (P < 0.02) in the control and refed chicks (149 ± 11.9, 139.6 ± 7.43 pmol · mg protein−1 · s−1) than in food-deprived chicks (107 ± 4.23 pmol · mg protein−1 · s−1). The km (150 μmol/L) and Vmax (1111.1 pmol · mg protein−1 · s−1) were higher in the food-deprived chicks compared to control and refed birds (25, 24 μmol/L and 227, 142 pmol · mg protein−1 · s−1, respectively). Expression of SGLT1 mRNA was significantly enhanced in the food-deprived and refed birds. In food-deprived chicks the lower affinity and higher activity of the SGLT1 transporter for glucose were accompanied by higher expression of mRNA which might indicate that the transporter was upregulated by low substrate concentration. Quantification of expression of intestinal mRNA of SGLT1 provides important information concerning control of nutrient uptake. J. Nutr. 130: 2174–2179, 2000.
Abstract
Background
Inflammatory Bowel Diseases (IBD) result from abnormal interactions between the immune system, epithelial barrier, and gut microbiota. However, understanding the abnormal ...interaction between aerobic epithelia and mainly anaerobic microbiota is complicated and is limited in part by the lack of human-relevant models.
Methods
We developed a model system that is based on whole human fecal samples and human-derived epithelia to study the interactions between the gut bacteria and the epithelia. To capture patient heterogeneity, we prioritized and pooled fecal material from 10 Crohn disease (CD), 10 ulcerative colitis (UC), and 10 healthy subjects. Prioritization of samples was based on our published gut microbial health index (PMID: 35197084), matching gender and age. Monolayered epithelial cells (Caco2 cells and patient-derived colonoids) were used for the co-culturing. Measurable outputs included cell viability, epithelial integrity, and secretion of CXCL1.
Results
Fecal samples were processed by separating the supernatant, heat-killing the bacteria, and rejoining the samples (Fig. 1A). 16S sequencing showed that bacterial composition and alpha diversity (Faith) were overall preserved after the heat-killing (Fig. 1B), as seen in the original samples. The gut microbial health index was higher among the control samples and pool, compared to the IBD samples (Fig. 1C), preserving the IBD pathogenic signals. Because previous studies indicated the enrichment of salivary bacteria in IBD feces, salivary samples were also processed for comparison. We confirmed that similar amounts of bacterial DNA (as a proxy of bacterial mass) were used for coculturing as indicated by qPCR of the 16S-V4 (Fig. 1D). Co-culturing the fecal and saliva pools with Caco-2 cells revealed a significant increase in CXCL1 proinflammatory chemokine secretion to the media in comparison to no-treatment (Fig. 2A). Moreover, the UC and CD pools significantly decreased epithelial integrity transepithelial electrical resistance (TEER), without affecting cellular viability (Fig. 2B-D). Finally, incubation of the fecal content with colon-derived organoids (from 2 controls and 1 UC) resulted in a substantial reduction of cell viability, which was more pronounced with fecal content derived from UC patients (Fig. 2E), and this effect was noted most with the UC fecal supernatants.
Conclusion
Using a novel co-culturing system, we demonstrated the effects of fecal contents from IBD and control subjects on CXCL1 secretion and epithelial integrity in Caco2 cells, and on colonoids viability. These models will be used to identify fecal factors and interventions relevant to IBD epithelial functions.
Development of a portable gamma camera with coded aperture Gal, Olivier; Gmar, Mehdi; Ivanov, Oleg P. ...
Nuclear instruments & methods in physics research. Section A, Accelerators, spectrometers, detectors and associated equipment,
07/2006, Volume:
563, Issue:
1
Journal Article
Peer reviewed
Since the beginning of the 1990s, the CEA is being involved in the development of compact gamma cameras based on a pinhole collimator, a scintillator and an intensified CCD. In collaboration with the ...Kurchatov Institute, the CEA has recently developed a miniature coded mask, which significantly improves sensitivity and angular resolution. Laboratory tests have demonstrated excellent performance. They are summarized, accompanied with on-site test results. We also present preliminary results of Medipix2/CdTe, a semiconductor pixel detector.
The small intestine of the chicken undergoes intensive changes in the immediate posthatch period, increasing in size and developing crypts, villi and mature enterocytes. During this time, chicks are ...also transferring from nutrition based on the lipid-rich yolk to exogenous carbohydrate-rich feeds. The cdx homeobox genes participate in axial patterning and in definition of cell identity in embryos, and some cdx genes remain active postpartum in organs such as the intestine. In this study, the transcription patterns of two of these genes, cdxA and cdxB, were examined in the small intestine of the embryo and posthatch chick; in addition, the effects on these genes of starving for 48 h at hatch were examined. Both cdx transcription factors were upregulated toward the time of hatch and were observed in proliferating enterocytes; this enhanced expression continued posthatch. Distribution of cdxA changed with age and was found at higher concentrations in mature enterocytes. Starving from 0 to 48 h posthatch retarded growth and decreased enterocyte proliferation and expression of cdxA and cdxB. After access to feed, expression of cdx genes was enhanced. Chicken homeobox genes cdxA and cdxB are expressed in all enterocytes during embryonic and posthatch development; however, cdxA may have a role in enterocyte maturation posthatch. CdxB was expressed later in development then previously reported.
Abstract
Background
Ulcerative colitis (UC) patients show high variations in disease phenotype and response to therapy. Intestinal epithelia play a key role in UC pathogenesis, and therefore ...patient-derived epithelial model to study this variation are warranted. Few studies suggest that colonoids derived from UC patients retain some disease-related transcriptional and epigenetic changes, but they also raise questions regarding their persistence in culture. Here, we aimed to characterize if UC epithelial dysfunctions and response to inflammatory signals retained in colonoid culture.
Methods
Rectal biopsies were used to generate epithelial organoids (colonoids) that were grown through 5–10 passages using L-WRN conditioned medium. Media and total RNA were collected for measuring CXCL1 secretion using ELISA and qPCR, respectively.
Results
3 control and 5 UC patients-derived colonoids were generated. Median age was 17 years and 75% were females. Control patients were evaluated for loose stool, abdominal pain, and anemia, but had normal rectal mucosa endoscopically and histologically. UC included newly diagnosed and established patients. Colonoids were allowed to differentiate for 48 hours before applying different inflammatory cocktails to induce inflammation for 24 h with IFNγ+LPS, IFNγ+TNFα, and TNFα+LPS (20 ng/ml each trigger). UC organoids secreted significant higher levels of CXCL1 as detected in the media at baseline (p=0.02) and with IFNy+TNFa (p=0.01), but no difference was noted with TNFa+LPS (Fig. 1). In addition, CXCL1 mRNA showed significant higher levels after inflammatory triggering in UC vs. controls (Fig. 1) with IFNγ +LPS, (p=0.02), IFNγ+TNFα (p=0.003), and TNFα+LPS (p=0.016). Similarly, several other genes showed significant and substantial induction in UC organoids vs. controls, in some or all the inflammatory triggers (Fig. 2). The tight junction-associated protein, ZO1 showed substantially higher induction in UC compared to controls with the 3 inflammatory cocktails IFNγ +LPS, (p=0.03), IFNγ+TNFα (p=0.02), and TNFα+LPS (p=0.005). Significantly higher expression in UC organoid was further noted in comparison to control in IL8 levels upon IFNγ +LPS (p=0.002), in interferon inducible IDO1 with IFNγ+TNFα (p=0.001), and in goblet associated MUC2 (p=0.04) and bacterial sensing DUOX2 (p=0.001) with TNFα+LPS.
Conclusion
Our data suggest that UC colonoids retain some inflammatory response activity in-vitro even after several passages. We show that UC organoids secrete higher level of CXCL1. Additionally, we note higher transcriptional response to different inflammatory signals in UC organoids potentially implying an “immune” epithelial transcriptional memory that may contribute to UC chronicity.
Aminopeptidases are members of a membrane-bound metallopeptidase family that are expressed at a high level on the brush-border membrane of enterocytes. Because the rapid growth of meat-type chickens ...depends on the dietary supply of amino acids, a study of intestinal aminopeptidases, which play a central role in protein digestion, is important. This study is the first reported isolation of the partial cDNA of chicken intestinal aminopeptidase and sequencing of a 1.7-kb cDNA fragment. The gene was isolated by reverse transcriptase polymerase chain reaction using six primers chosen from conserved regions of the aminopeptidase genes. Amplified fragments were extracted from the gel, purified, and sequenced. By using this chicken cDNA as a probe, northern blot analysis revealed a transcript of approximately 3.5 kb in the chicken duodenum, jejunum, and ileum tissues. Higher RNA expression and activity of aminopeptidase were found in the ileum tissue compared with the duodenum and jejunum segments.
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