Biology

Aquaporin-9 is expressed in rat Sertoli cells and interacts with the cystic fibrosis transmembrane conductance regulator
Men with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are usually subfertile/infertile. Besides playing a role in Cl−/HCO3− transport, it has been proposed that CFTR interacts with water membrane transport systems, particularly aquaporins, to control seminiferous tubular secretion, which is regulated by the somatic Sertoli cells (SCs). As aquaporin-9 (AQP...


Bicarbonate transport in health and disease
Bicarbonate (HCO3−) has a central place in human physiology as the waste product of mitochondrial energy production and for its role in pH buffering throughout the body. Because bicarbonate is impermeable to membranes, bicarbonate transport proteins are necessary to enable control of bicarbonate levels across membranes. In humans, 14 bicarbonate transport proteins, members of the SLC4 and SLC...
Electrocautery-induced localized colonic injury elicits increased levels of pro-inflammatory cytokines in small bowel and decreases jejunal alanine absorption
Colitis is associated with functional abnormalities in proximal non-inflamed gut areas, but animal models to study small bowel dysfunction in colitis have limitations. This study aims to determine small intestinal alanine absorption and cytokine expression in a novel model of colonic ulceration induced by electro-cautery. A descending colon ulcer was induced in rats by a bipolar electro-cautery...


The elastin peptide (VGVAPG) 3 induces the 3D reorganisation of PML-NBs and SC35 speckles architecture, and accelerates proliferation of fibroblasts and melanoma cells
During melanoma tumour growth, cancerous cells are exposed to the immediate surrounding the micro- and macro environment, which is largely modified through the degradation of the extracellular matrix by fibroblast-derived metalloproteinases. Among the degradation products, (VGVAPG)3, an elastin peptide is known to stimulate the proliferation of both fibroblasts and cancerous cells by binding to...
Cell-Flow Technique
Various devices have been used to flow neurotransmitter solutions over cells containing receptors (e.g., ligand-gated ion channels) for whole-cell current recordings. With many of the devices, the orientation between the porthole of the flow device and the cell is not maintained absolutely constant. Orientation is critical for reproducibility in kinetic experiments. To be able to change the com...


Caged Neurotransmitters and Other Caged Compounds: Design and Application
The approaches using caged neurotransmitters described here enable transient kinetic investigations to be made with membrane-bound proteins (receptors) on a cell surface with the same time resolution as was previously possible only with proteins in solution.
In Vivo Calcium Recordings and Channelrhodopsin-2 Activation through an Optical Fiber
We describe here an approach for the fluorometric monitoring of population activity in neurons in live mice combined with the activation of optogenetic actuators in vivo. In this protocol, a thin multimode fiber, which is used for both delivering excitation light and collecting emitted fluorescence signals, is inserted into the skull of a mouse. When combined with multicell bolus loading of Ca2...

Basic Quantitative Polymerase Chain Reaction Using Real-Time Fluorescence Measurements
This protocol uses quantitative polymerase chain reaction (qPCR) to measure the number of DNA molecules containing a specific contiguous sequence in a sample of interest (e.g., genomic DNA or cDNA generated by reverse transcription). The sample is subjected to fluorescence-based PCR amplification and, theoretically, during each cycle, two new duplex DNA molecules are produced for each duplex DN...
Purification of Mitochondria by Sucrose Step Density Gradient Centrifugation
Mitochondrial fractions isolated from tissue culture cells or tissue such as liver after differential centrifugation can be purified further by density gradient centrifugation. Here we describe the use of sucrose for this purpose because it is commonly used and inexpensive and the resulting mitochondria preparations are useful for many purposes.

Measurement of Luminal pH of Acidic Stores as a Readout for NAADP Action
In addition to mobilizing Ca2+, NAADP plays a role in modulating the luminal pH (pHL) of acidic stores of the endolysosomal system. The effects of NAADP on pHL have been most extensively studied in the sea urchin egg, both in the intact egg and in egg homogenates. Related observations have also been made in mammalian systems (e.g., guinea pig atrial myocytes and pancreatic acinar cells). Althou...
Myelinating Cocultures of Rat Retinal Ganglion Cell Reaggregates and Optic Nerve Oligodendrocyte Precursor Cells
This protocol describes the generation of a rapidly myelinating central nervous system coculture for the study of complex neuronal-glial interactions in vitro. Postnatal rat retinal ganglion cells (RGCs) purified by immunopanning are promoted to cluster into reaggregates and then allowed to extend dense beds of radial axons for 10–14 d. Subsequently, rodent oligodendrocyte precursor cells...

Fluorescence Microscopy
Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal ...
Myelinating Cocultures of Purified Oligodendrocyte Lineage Cells and Retinal Ganglion Cells
In this article, we introduce methods for generating rapidly myelinating cocultures with reaggregates of purified retinal ganglion cells and optic nerve oligodendrocyte precursor cells. This coculture system facilitates the study of complex central nervous system neuronal–glial interactions and myelination. It enables control of the extracellular environment and allows the use of transfec...
Tetracycline-Regulated Mouse Models of Cancer
Genetically engineered mouse models (GEMMs) have proven essential to the study of mammalian gene function in both development and disease. However, traditional constitutive transgenic mouse model systems are limited by the temporal and spatial characteristics of the experimental promoter used to drive transgene expression. To address this limitation, considerable effort has been dedicated to de...
Transcriptional Regulation by Trithorax-Group Proteins
The trithorax group of genes (trxG) was identified in mutational screens that examined developmental phenotypes and suppression of Polycomb mutant phenotypes. The protein products of these genes are primarily involved in gene activation, although some can also have repressive effects. There is no central function for these proteins. Some move nucleosomes about on the genome in an ATP-dependent ...