Prolonged cultured human hepatocytes as an in vitro experimental system for the evaluation of potency and duration of activity of RNA therapeutics: Demonstration of prolonged duration of gene silencing effects of a GalNAc-conjugated human hypoxanthine phosphoribosyl transferase (HPRT1) siRNA

2020 ◽  
pp. 114374
Author(s):  
Qian Yang ◽  
Sara C. Humphreys ◽  
Julie M. Lade ◽  
Albert P. Li
Keyword(s):  
Gene Silencing ◽  
Experimental System ◽  
Human Hepatocytes ◽  
Hypoxanthine Phosphoribosyl Transferase ◽  
Phosphoribosyl Transferase ◽  
Prolonged Duration

EVALUATION OF TISSUE STEROID BINDING IN VITRO

1971 ◽  
Vol 68 (1_Suppl) ◽  
pp. S223-S246 ◽  
Author(s):  
C. R. Wira ◽  
H. Rochefort ◽  
E. E. Baulieu
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Experimental System ◽  
Specific Protein ◽  
Coupling Mechanism ◽  
Target Tissue ◽  
Protein Binding Sites ◽  
Definition Of ◽  
Hormone Specificity

ABSTRACT The definition of a RECEPTOR* in terms of a receptive site, an executive site and a coupling mechanism, is followed by a general consideration of four binding criteria, which include hormone specificity, tissue specificity, high affinity and saturation, essential for distinguishing between specific and nonspecific binding. Experimental approaches are proposed for choosing an experimental system (either organized or soluble) and detecting the presence of protein binding sites. Techniques are then presented for evaluating the specific protein binding sites (receptors) in terms of the four criteria. This is followed by a brief consideration of how receptors may be located in cells and characterized when extracted. Finally various examples of oestrogen, androgen, progestagen, glucocorticoid and mineralocorticoid binding to their respective target tissues are presented, to illustrate how researchers have identified specific corticoid and mineralocorticoid binding in their respective target tissue receptors.

scholarly journals Coculture with hiPS-derived intestinal cells enhanced human hepatocyte functions in a pneumatic-pressure-driven two-organ microphysiological system

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marie Shinohara ◽  
Hiroshi Arakawa ◽  
Yuuichi Oda ◽  
Nobuaki Shiraki ◽  
Shinji Sugiura ◽  
...  
Keyword(s):  
Normal Cell ◽  
Human Hepatocytes ◽  
New Drugs ◽  
Intestinal Cells ◽  
Specific Gene ◽  
Gene Expressions ◽  
Normal Cells ◽  
Pneumatic Pressure ◽  
Pressure Driven

AbstractExamining intestine–liver interactions is important for achieving the desired physiological drug absorption and metabolism response in in vitro drug tests. Multi-organ microphysiological systems (MPSs) constitute promising tools for evaluating inter-organ interactions in vitro. For coculture on MPSs, normal cells are challenging to use because they require complex maintenance and careful handling. Herein, we demonstrated the potential of coculturing normal cells on MPSs in the evaluation of intestine–liver interactions. To this end, we cocultured human-induced pluripotent stem cell-derived intestinal cells and fresh human hepatocytes which were isolated from PXB mice with medium circulation in a pneumatic-pressure-driven MPS with pipette-friendly liquid-handling options. The cytochrome activity, albumin production, and liver-specific gene expressions in human hepatocytes freshly isolated from a PXB mouse were significantly upregulated via coculture with hiPS-intestinal cells. Our normal cell coculture shows the effects of the interactions between the intestine and liver that may occur in vivo. This study is the first to demonstrate the coculturing of hiPS-intestinal cells and fresh human hepatocytes on an MPS for examining pure inter-organ interactions. Normal-cell coculture using the multi-organ MPS could be pursued to explore unknown physiological mechanisms of inter-organ interactions in vitro and investigate the physiological response of new drugs.

scholarly journals Alcohol-and-HIV-Induced Lysosomal Dysfunction Regulates Extracellular Vesicles Secretion in Vitro and in Liver-Humanized Mice

Biology ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 29
Author(s):  
Raghubendra Singh Dagur ◽  
Moses New-Aaron ◽  
Murali Ganesan ◽  
Weimin Wang ◽  
Svetlana Romanova ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Extracellular Vesicles ◽  
Treated Group ◽  
Human Hepatocytes ◽  
In Vivo Studies ◽  
People Living With Hiv ◽  
Humanized Mouse Model ◽  
And Function

Background: Alcohol abuse is common in people living with HIV-1 and dramaticallyenhances the severity of HIV-induced liver damage by inducing oxidative stress and lysosomaldysfunction in the liver cells. We hypothesize that the increased release of extracellular vesicles(EVs) in hepatocytes and liver humanized mouse model is linked to lysosome dysfunction. Methods:The study was performed on primary human hepatocytes and human hepatoma RLWXP-GFP (Huh7.5 cells stably transfected with CYP2E1 and XPack-GFP) cells and validated on ethanol-fed liverhumanizedfumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chainknockout (FRG-KO) mice. Cells and mice were infected with HIV-1ADA virus. Results: We observedan increase in the secretion of EVs associated with a decrease in lysosomal activity and expressionof lysosomal-associated membrane protein 1. Next-generation RNA sequencing of primary humanhepatocytes revealed 63 differentially expressed genes, with 13 downregulated and 50 upregulatedgenes in the alcohol–HIV-treated group. Upstream regulator analysis of differentially expressedgenes through Ingenuity Pathway Analysis identified transcriptional regulators affecting downstreamgenes associated with increased oxidative stress, lysosomal associated disease, and function andEVs biogenesis. Our in vitro findings were corroborated by in vivo studies on human hepatocytetransplantedhumanized mice, indicating that intensive EVs’ generation by human hepatocytes andtheir secretion to serum was associated with increased oxidative stress and reduction in lysosomalactivities triggered by HIV infection and ethanol diet. Conclusion: HIV-and-ethanol-metabolisminducedEVs release is tightly controlled by lysosome status in hepatocytes and participates in thedevelopment of double-insult-induced liver injury.

scholarly journals Rapamycin Inhibits ALDH Activity, Resistance to Oxidative Stress, and Metastatic Potential in Murine Osteosarcoma Cells

Sarcoma ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Xiaodong Mu ◽  
Christian Isaac ◽  
Trevor Schott ◽  
Johnny Huard ◽  
Kurt Weiss
Keyword(s):  
Oxidative Stress ◽  
Experimental System ◽  
Metastatic Potential ◽  
Bone Morphogenetic Protein 2 ◽  
Stem Cell Marker ◽  
Migration And Invasion ◽  
Aldh Activity ◽  
Primary Malignancy ◽  
Rapamycin Treatment

Osteosarcoma (OS) is the most common primary malignancy of bone. Mortality is determined by the presence of metastatic disease, but little is known regarding the biochemical events that drive metastases. Two murine OS cell lines, K7M2 and K12, are related but differ significantly in their metastatic potentials: K7M2 is highly metastatic whereas K12 displays much less metastatic potential. Using this experimental system, the mammalian target of rapamycin (mTOR) pathway has been implicated in OS metastasis. We also discovered that aldehyde dehydrogenase (ALDH, a stem cell marker) activity is higher in K7M2 cells than K12 cells. Rapamycin treatment reduces the expression and enzymatic activity of ALDH in K7M2 cells. ALDH inhibition renders these cells more susceptible to apoptotic death when exposed to oxidative stress. Furthermore, rapamycin treatment reduces bone morphogenetic protein-2 (BMP2) and vascular endothelial growth factor (VEGF) gene expression and inhibits K7M2 proliferation, migration, and invasionin vitro. Inhibition of ALDH with disulfiram correlated with decreased mTOR expression and activity. In conclusion, we provide evidence for interaction between mTOR activity, ALDH activity, and metastatic potential in murine OS cells. Our work suggests that mTOR and ALDH are therapeutic targets for the treatment and prevention of OS metastasis.

In Vitro Metabolism of the Glucagon-Like Peptide-1 (GLP-1)–Derived Metabolites GLP-1(9-36)amide and GLP-1(28-36)amide in Mouse and Human Hepatocytes

2013 ◽  
Vol 41 (12) ◽  
pp. 2148-2157 ◽  
Author(s):  
Raman Sharma ◽  
Thomas S. McDonald ◽  
Heather Eng ◽  
Chris Limberakis ◽  
Benjamin D. Stevens ◽  
...  
Keyword(s):  
Human Hepatocytes ◽  
In Vitro Metabolism ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1

scholarly journals Molecular biology of potexviruses: recent advances

2007 ◽  
Vol 88 (6) ◽  
pp. 1643-1655 ◽  
Author(s):  
Jeanmarie Verchot-Lubicz ◽  
Chang-Ming Ye ◽  
Devinka Bamunusinghe
Keyword(s):  
Gene Silencing ◽  
Rna Synthesis ◽  
Triple Gene Block ◽  
Potato Virus X ◽  
Future Research ◽  
Virus Movement ◽  
Suppressor Activity ◽  
Gene Block ◽  
Recent Advances

Recent advances in potexvirus research have produced new models describing virus replication, cell-to-cell movement, encapsidation, R gene-mediated resistance and gene silencing. Interactions between distant RNA elements are a central theme in potexvirus replication. The 5′ non-translated region (NTR) regulates genomic and subgenomic RNA synthesis and encapsidation, as well as virus plasmodesmal transport. The 3′ NTR regulates both plus- and minus-strand RNA synthesis. How the triple gene-block proteins interact for virus movement is still elusive. As the potato virus X (PVX) TGBp1 protein gates plasmodesmata, regulates virus translation and is a suppressor of RNA silencing, further research is needed to determine how these properties contribute to propelling virus through the plasmodesmata. Specifically, TGBp1 suppressor activity is required for virus movement, but how the silencing machinery relates to plasmodesmata is not known. The TGBp2 and TGBp3 proteins are endoplasmic reticulum (ER)-associated proteins required for virus movement. TGBp2 associates with ER-derived vesicles that traffic along the actin network. Future research will determine whether the virus-induced vesicles are cytopathic structures regulating events along the ER or are vehicles carrying virus to the plasmodesmata for transfer into neighbouring cells. Efforts to assemble virions in vitro identified a single-tailed particle (STP) comprising RNA, coat protein (CP) and TGBp1. It has been proposed that TGBp1 aids in transport of virions or STP between cells and ensures translation of RNA in the receiving cells. PVX is also a tool for studying Avr–R gene interactions and gene silencing in plants. The PVX CP is the elicitor for the Rx gene. Recent reports of the PVX CP reveal how CP interacts with the Rx gene product.

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