Landscape of activating cancer mutations in FGFR kinases and their differential responses to inhibitors in clinical use.

Frequent genetic alterations discovered in FGFRs and evidence implicating some as drivers in diverse tumors has been accompanied by rapid progress in targeting FGFRs for anticancer treatments. Wider assessment of the impact of genetic changes on the activation state and drug responses is needed to better link the genomic data and treatment options. We here apply a direct comparative and comprehensive analysis of FGFR3 kinase domain variants representing the diversity of point-mutations reported in this domain.

Isolation and Culture of Mouse Hepatocytes: Gender-Specific Gene Expression Responses to Chemical Treatments.

In this chapter, the isolation of primary mouse hepatocytes and their response to chemical treatment are described. We show that it is important to consider, in the experimental design, the sex of the animals to be used. We demonstrate this by measuring the effect of sex hormones or xenobiotics on the expression of flavin-containing monooxygenase 5 in cultures of primary hepatocytes isolated from male and female mice.

Evaluation of phosphopeptide enrichment strategies for quantitative TMT analysis of complex network dynamics in cancer-associated cell signalling.

Defining alterations in signalling pathways in normal and malignant cells is becoming a major field in proteomics. A number of different approaches have been established to isolate, identify and quantify phosphorylated proteins and peptides. In the current report, a comparison between SCX prefractionation an antibody based approach, both coupled to TiO enrichment and applied to TMT labelled cellular lysates, is described.

Isolation of mouse hepatocytes.

Primary hepatocyte cultures better reflect the properties of the liver in vivo than do cell lines derived from the liver. Here we describe a method for the isolation and culture of mouse primary hepatocytes. The cells are viable, can be transfected by DNA, and retain key properties of liver cells such as the induction of cytochrome P450 gene expression by drugs such as phenobarbital.

Alternative promoters and repetitive DNA elements define the species-dependent tissue-specific expression of the FMO1 genes of human and mouse.

In humans, expression of the FMO1 (flavin-containing mono-oxygenase 1) gene is silenced postnatally in liver, but not kidney. In adult mouse, however, the gene is active in both tissues. We investigated the basis of this species-dependent tissue-specific transcription of FMO1.

Transfection of primary cultures of rat hepatocytes.

Five different transfection reagents-calcium phosphate, TransFast Transfection Reagent, Superfect Transfection Reagent, Effectene Transfection Reagent, and Tfx-20--were compared for their ability to effectively transfect primary cultures of male rat hepatocytes. Hepatocytes were isolated by the collagenase perfusion method and then cultured on Matrigel-coated plates for 24 h before transfection. The cells were transfected with either pGL3-Control or pGL3-Basic plasmids.

Transfection of Primary Cultures of Rat Hepatocytes.

Five different transfection reagents-calcium phosphate, TransFast™ Transfection Reagent, Superfect™ Transfection Reagent, Effectene™ Transfection Reagent, and Tfx™-20-were compared for their ability to effectively transfect primary cultures of male rat hepatocytes. Hepatocytes were isolated by the collagenase perfusion method and then cultured on Matrigel-coated plates for 24 h before transfection. The cells were transfected with either pGL3-Control or pGL3-Basic plasmids.

Relocalization of human chromatin remodeling cofactor TIP48 in mitosis.

TIP48 is a highly conserved eukaryotic AAA+ protein which is an essential cofactor for several complexes involved in chromatin acetylation and remodeling, transcriptional and developmental regulation and nucleolar organization and trafficking. We show that TIP48 abundance in HeLa cells did not change during the cell cycle, nor did its distribution in various biochemical fractions. However, we observed distinct changes in the subcellular localization of TIP48 during M phase using immunofluorescence microscopy.

Cyclophilin-D promotes the mitochondrial permeability transition but has opposite effects on apoptosis and necrosis.

Cyclophilin-D is a peptidylprolyl cis-trans isomerase of the mitochondrial matrix. It is involved in mitochondrial permeability transition, in which the adenine nucleotide translocase of the inner membrane is transformed from an antiporter to a non-selective pore. The permeability transition has been widely considered as a mechanism in both apoptosis and necrosis.