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The impact of yogurts made with starter culture bacteria (L. bulgaricus and S. thermophilus) and supplemented with ingredients (maitake mushrooms, quercetin, L-glutamine, slippery elm bark, licorice root, N-acetyl-D-glucosamine, zinc orotate, and marshmallow root) that can help treat leaky gut were investigated using the Caco-2 cell monolayer as a measure of intestinal barrier dysfunction. Milk from the same source was equally dispersed into nine pails, and the eight ingredients were randomly allocated to the eight pails. The control had no ingredients. The Caco-2 cells were treated with isoflavone genistein (negative control) and growth media (positive control). Inflammation was stimulated using an inflammatory cocktail of cytokines (interferon-γ, tumor necrosis factor-α, and interleukin-1β) and lipopolysaccharide. The yogurt without ingredients (control yogurt) was compared to the yogurt treatments (yogurts with ingredients) that help treat leaky gut. Transepithelial electrical resistance (TEER) and paracellular permeability were measured to evaluate the integrity of the Caco-2 monolayer. Transmission electron microscopy (TEM), immunofluorescence microscopy (IM), and real-time quantitative polymerase chain reaction (RTQPCR) were applied to measure the integrity of tight junction proteins. The yogurts were subjected to gastric and intestinal digestion, and TEER was recorded. Ferrous ion chelating activity, ferric reducing potential, and DPPH radical scavenging were also examined to determine the yogurts’ antioxidant capacity. Yogurt with quercetin and marshmallow root improved the antioxidant activity and TEER and had the lowest permeability in fluorescein isothiocyanate (FITC)–dextran and Lucifer yellow flux among the yogurt samples. TEM, IM, and RTQPCR revealed that yogurt enhanced tight junction proteins’ localization and gene expression. Intestinal digestion of the yogurt negatively impacted inflammation-induced Caco-2 barrier dysfunction, while yogurt with quercetin, marshmallow root, maitake mushroom, and licorice root had the highest TEER values compared to the control yogurt. Yogurt fortification with quercetin, marshmallow root, maitake mushroom, and licorice root may improve functionality when dealing with intestinal barrier dysfunction. Full article

In situ hybridization (ISH) at the electron microscopic (EM) level is essential for elucidating the intracellular distribution and role of mRNA in protein synthesis. EM-ISH is considered to be an important tool for clarifying the intracellular localization of mRNA and the exact site of pituitary hormone synthesis on the rough endoplasmic reticulum. A combined ISH and immunohistochemistry (IHC) under EM (EM-ISH&IHC) approach has sufficient ultrastructural resolution, and provides two-dimensional images of the subcellular localization of pituitary hormone and its mRNA in a pituitary cell. The advantages of semiconductor nanocrystals (quantum dots, Qdots) and confocal laser scanning microscopy (CLSM) enable us to obtain three-dimensional images of the subcellular localization of pituitary hormone and its mRNA. Both EM-ISH&IHC and ISH & IHC using Qdots and CLSM are useful for understanding the relationships between protein and mRNA simultaneously in two or three dimensions. CLSM observation of rab3B and SNARE proteins such as SNAP-25 and syntaxin has revealed that both rab3B and SNARE system proteins play important roles and work together as the exocytotic machinery in anterior pituitary cells. Another important issue is the intracellular transport and secretion of pituitary hormone. We have developed an experimental pituitary cell line, GH3 cell, which has growth hormone (GH) linked to enhanced yellow fluorescein protein (EYFP). This stable GH3 cell secretes GH linked to EYFP upon stimulation by Ca²⁺ influx or Ca²⁺ release from storage. This GH3 cell line is useful for the real-time visualization of the intracellular transport and secretion of GH. These three methods from conventional immunohistochemistry and fluorescein imaging allow us to consecutively visualize the process of transcription, translation, transport and secretion of anterior pituitary hormone. Full article