Detection of metabolic fluxes of O and H atoms into intracellular water in mammalian cells.

Metabolic processes result in the release and exchange of H and O atoms from organic material as well as some inorganic salts and gases. These fluxes of H and O atoms into intracellular water result in an isotopic gradient that can be measured experimentally. Using isotope ratio mass spectroscopy, we revealed that slightly over 50% of the H and O atoms in the intracellular water of exponentially-growing cultured Rat-1 fibroblasts were isotopically distinct from growth medium water.

Reactive oxygen species alter autocrine and paracrine signaling.

Cytochrome P450 (P450) 3A4 (CYP3A4) is the most abundant P450 protein in human liver and intestine and is highly inducible by a variety of drugs and other compounds. The P450 catalytic cycle is known to uncouple and release reactive oxygen species (ROS), but the effects of ROS from P450 and other enzymes in the endoplasmic reticulum have been poorly studied from the perspective of effects on cell biology. In this study, we expressed low levels of CYP3A4 in HepG2 cells, a human hepatocarcinoma cell line, and examined effects on intracellular levels of ROS and on the secretion of a variety of growth factors that are important in extracellular communication.

Structure of the EGF receptor transactivation circuit integrates multiple signals with cell context.

Transactivation of the epidermal growth factor receptor (EGFR) is thought to be a process by which a variety of cellular inputs can be integrated into a single signaling pathway through either stimulated proteolysis (shedding) of membrane-anchored EGFR ligands or by modification of the activity of the EGFR. As a first step towards building a predictive model of the EGFR transactivation circuit, we quantitatively defined how signals from multiple agonists were integrated both upstream and downstream of the EGFR to regulate extracellular signal regulated kinase (ERK) activity in human mammary epithelial cells. By using a "non-binding" reporter of ligand shedding, we found that transactivation triggers a positive feedback loop from ERK back to the EGFR such that ligand shedding drives EGFR-stimulated ERK that in turn drives further ligand shedding.

Rapid and sustained nuclear-cytoplasmic ERK oscillations induced by epidermal growth factor.

Although the ERK pathway has a central role in the response of cells to growth factors, its regulatory structure and dynamics are incompletely understood. To investigate ERK activation in real time, we expressed an ERK-GFP fusion protein in human mammary epithelial cells. On EGF stimulation, we observed sustained oscillations of the ERK-GFP fusion protein between the nucleus and cytoplasm with a periodicity of approximately 15 min.

Regulation of the low-dose radiation paracrine-specific anchorage-independent growth response by annexin A2.

Here we identify the release of annexin A2 into the culture medium in response to low-dose X-radiation exposure and establish functional linkages to an established paracrine factor-mediated anchorage-independent growth response. Using a standard bicameral coculture model, we demonstrate that annexin A2 is secreted into the medium by irradiated cells (seeded in upper chamber) and is capable of binding to nonirradiated neighboring cells (seeded in lower chamber). The paracrine factor-mediated anchorage-independent growth response to low-dose X irradiation is reduced when irradiated annexin A2-silenced (shRNA) JB6 cells are co-cultured with nonirradiated cells relative to co-culture with irradiated annexin A2-competent vector control cells.

Lysophosphatidic acid-induced ERK activation and chemotaxis in MC3T3-E1 preosteoblasts are independent of EGF receptor transactivation.

Bone-forming osteoblasts and their progenitors are target cells for the lipid growth factor lysophosphatidic acid (LPA) which is produced by degranulating platelets at sites of tissue injury. LPA is a potent inducer of bone cell chemotaxis, proliferation and survival in vitro, and this lipid factor is an attractive candidate to facilitate preosteoblast migration during skeletal regeneration in vivo. In this study we sought to more clearly define the intracellular signaling pathways mediating the effects of LPA on bone cells.

Multiple mechanisms are responsible for transactivation of the epidermal growth factor receptor in mammary epithelial cells.

The number of distinct signaling pathways that can transactivate the epidermal growth factor receptor (EGFR) in a single cell type is unclear. Using a single strain of human mammary epithelial cells, we found that a wide variety of agonists, such as lysophosphatidic acid (LPA), uridine triphosphate, growth hormone, vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), and tumor necrosis factor-alpha, require EGFR activity to induce ERK phosphorylation. In contrast, hepatocyte growth factor can stimulate ERK phosphorylation independent of the EGFR.

ProMAT: protein microarray analysis tool.

Summary: ProMAT is a software tool for statistically analyzing data from enzyme-linked immunosorbent assay microarray experiments. The software estimates standard curves, sample protein concentrations and their uncertainties for multiple assays. ProMAT generates a set of comprehensive figures for assessing results and diagnosing process quality.

Studying cellular processes and detecting disease with protein microarrays.

Protein microarrays are a rapidly developing analytic tool with diverse applications in biomedical research. These applications include profiling of disease markers or autoimmune responses, understanding molecular pathways, protein modifications, and protein activities. One factor that is driving this expanding usage is the wide variety of experimental formats that protein microarrays can take.

Suppression of cytochrome P450 3A protein levels by proteasome inhibitors.

We have previously reported that CYP3A cross-links with polyubiquitinated proteins in microsomes from nicardipine-treated rats in a process that is distinct from classical polyubiquitination. To further examine the role of the proteasome in CYP3A degradation, we investigated the effects of proteasome inhibitors lactacystin, MG132, proteasome inhibitor 1, and hemin in primary cultures of rat and human hepatocytes. With the exception of hemin, these agents increased the total pool of ubiquitinated proteins in microsomes isolated from rat hepatocytes, indicating that lactacystin, MG132, and proteasome inhibitor 1 effectively inhibited the proteasome in these cells.