Detection of cellular damage by hydrogen peroxide using SV40-T2 cells on shear horizontal surface acoustic wave (SH-SAW) sensor.

The rat lung epithelial cell line SV40-T2 was used to develop a cellular biosensing system to assay for environmental toxicants. The novel approach on which this system is based involves direct attachment of cultured rat or human cells onto a cell-adhesive matrix on the device through which shear horizontal surface acoustic waves (SH-SAW) are transmitted using 50 MHz SAW resonator. This novel design enables sensitive monitoring of changes of the electrophysical characteristics of cells, such as their conductivity and relative permittivity.

Synthesized basement membranes direct the differentiation of mouse embryonic stem cells into pancreatic lineages.

We previously reported that embryonic stem (ES) cells cultured on M15 cells, a mesoderm-derived supportive cell line, were efficiently differentiated towards an endodermal fate, finally adopting the specific lineages of various digestive organs such as the pancreas and liver. We show here that the endoderm-inducing activity of M15 cells is in part mediated through the extracellular matrices, and that laminin alpha5 is one of the crucial components. In an attempt to establish a feeder-free ES-cell procedure for pancreatic differentiation, we used a synthesized basement membrane (sBM) substratum using an HEK293 cell line stably expressing laminin-511.

Interleukin-1beta and tumor necrosis factor-alpha have opposite effects on fibroblasts and epithelial cells during basement membrane formation.

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are typical proinflammatory cytokines that influence various cellular functions, including metabolism of the extracellular matrix. We examined the roles of IL-1beta and TNF-alpha in basement membrane formation in an in vitro model of alveolar epithelial tissue composed of alveolar epithelial cells and pulmonary fibroblasts. Formation of the basement membrane by immortalized rat alveolar type II epithelial (SV40-T2) cells, which ordinarily do not form a continuous basement membrane, was dose-dependently upregulated in the presence of 2 ng/ml IL-1beta or 5 ng/ml TNF-alpha.

Role of basement membrane in EMMPRIN/CD147 induction in rat tracheal epithelial cells.

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a glycosylated transmembrane protein known to induce matrix metalloproteinases (MMPs). Although the expression of EMMPRIN is physiologically limited to fetal lung epithelium, the transcriptional regulation of this protein remains to be elucidated. We hypothesized that the interaction of epithelial cells with the basement membrane regulates EMMPRIN expression.

Seamless signal transduction from live cells to an NO sensor via a cell-adhesive sensing matrix.

A smart live-cell assay was developed as a cellular biosensing system. This system is based on novel tactics: the direct assembly of human cultured cells onto a cell-adhesive sensing matrix. This novel design provides considerable advantages, among them the possibility of capturing molecular signals immediately after they are secreted from living cells.

Keratinocyte apoptosis on type I collagen fibrils is prevented by Erk1/2 activation under high calcium condition.

Keratinocytes adhere and proliferate well on collagen-coated surfaces, but they undergo apoptosis without differentiation on collagen gels according to our past research. In the current studies, we investigated the necessary conditions for keratinocyte survival on fibrous collagen gels. We found that keratinocytes survived on collagen gels when the medium contains elevated levels (1.

Differentiation of tracheal basal cells to ciliated cells and tissue reconstruction on the synthesized basement membrane substratum in vitro.

Although lung epithelial cells directly attach to the basement membrane underneath in vivo, harvested epithelial cells are typically cultured on type I collagen gel (Col I-gel) in vitro. Recently we developed new culture substratum, designated as "synthesized Basement Membrane" (sBM), that has bared lamina densa on fibrillar collagen. To validate the usefulness of sBM substratum in airway tissue reconstitution in vitro, we cultured rat tracheal epithelial cells on sBM substratum and Col I-gel.

Homeobox B3, FoxA1 and FoxA2 interactions in epithelial lung cell differentiation of the multipotent M3E3/C3 cell line.

HOM/C homeobox (Hox) and forkhead box (Fox) factors are reported to be expressed in the foregut endoderm and are subsequently detected in a spatio-temporal pattern during lung development. Some of these factors were reported to influence the expression of lung marker proteins or to modulate lung development. To clarify the molecular mechanisms for generating functional lung cells from progenitor cell populations, we introduced the forkhead box factors, FoxA1 and FoxA2, and the homeobox factor, HoxB3, into the differentiation process in a multipotent hamster lung epithelial M3E3/C3 cell line.

Hepatocyte growth factor inhibits the formation of the basement membrane of alveolar epithelial cells in vitro.

Hepatocyte growth factor (HGF) is a pulmotrophic factor for the regeneration of injured pulmonary tissue. We investigated the role of HGF in basement membrane formation during wound healing by immortalized alveolar type II epithelial cells that could form a continuous basement membrane when they were cultured on collagen fibrils in the presence of entactin-contaminated laminin-1. Cells cultured with 5.

Assembly of the exogenous extracellular matrix during basement membrane formation by alveolar epithelial cells in vitro.

We found that immortalized alveolar type II epithelial cells (SV40-T2 cells) that were cultured on dense fibrillar collagen supplemented with Matrigel gel formed a thin and continuous lamina densa beneath them. Immunohistochemical analysis of laminin-1, type IV collagen, entactin (nidogen) and perlecan in the culture indicated that all these components were integrated into a sheet structure of basement membrane beneath the cells. Analysis of the temporal and spatial distribution of the basement membrane macromolecules revealed that the initial deposits of laminin-1 and entactin were significantly greater in area in the presence of Matrigel.

Transforming growth factor-beta1 regulates basement membrane formation by alveolar epithelial cells in vitro.

Immortalized alveolar type II epithelial (SV40-T2) cells formed a continuous, thin lamina densa when they were cultured on collagen fibrils with the supplement of 1.0 ng/ml TGF-beta1. Corresponding to lamina densa formation, immunohistochemical analysis of laminin, type IV collagen, perlecan, and entactin (nidogen) indicated integration of these components in a linear array beneath the SV40-T2 cells.

Effect of aging on nitric oxide production by rat alveolar macrophages.

Nitric oxide (NO) plays an important role in alveolar macrophages (AM)-mediated defense against infection. The elderly become highly susceptible to respiratory tract infection. Inhibition of NO production significantly suppresses defense against infections.

Assembly of basement membrane in vitro by cooperation between alveolar epithelial cells and pulmonary fibroblasts.

To investigate basement membrane formation by cooperation between pneumocytes and pulmonary fibroblasts, we cultured type II alveolar epithelial cells obtained from rats transfected with SV40-large T antigen gene (SV40-T2 cells) on type I collagen matrices. On fibroblasts-embedded gel (T2-Fgel), SV40-T2 cells ultrastructurally formed a continuous and thin layer of lamina densa, while on collagen gel without fibroblasts (T2-gel) SV40-T2 cells produced only discontinuous and diffuse deposits. Stripping SV40-T2 cells off the tissues by H2O2 treatment revealed a continuous and plane surface of lamina densa assembled on the T2-Fgel tissue, whereas only amorphous deposits appeared on the T2-gel tissue.

Increases in the mRNA levels of gamma-glutamyltransferase and heme oxygenase-1 in the rat lung after ozone exposure.

gamma-Glutamyltransferase (GGT) and heme oxygenase-1 (HO-1) are induced by chemical and physical stresses producing an oxidative burden on tissues and cells. Both enzymes are proposed to have an antioxidant role in protecting cells and tissues from oxidative burden. To explore the effects of ozone (O3), the major oxidant in photochemical smog, on the expression of GGT and HO-1 genes in the lung, we exposed rats to 0.

Stress-relaxation of fibroblasts in collagen matrices triggers ectocytosis of plasma membrane vesicles containing actin, annexins II and VI, and beta 1 integrin receptors.

To learn about the effects of tension on fibroblast function, we have been studying initial cellular responses to stress-relaxation. Human foreskin fibroblasts were cultured in anchored collagen matrices for 2 days, during which time mechanical stress developed. Subsequently, the matrices were dislodged; thereby allowing stress to dissipate.

Long-term effects of ozone and nitrogen dioxide on the metabolism and population of alveolar macrophages.

To investigate how alveolar macrophages adapt themselves to oxidative pollutants in the long term, rats were exposed to a strong oxidant, ozone (O3), or a weak oxidant, nitrogen dioxide (NO2), for a maximum duration of 12 wk. After exposures, alveolar macrophages were collected by pulmonary lavage. Throughout 11 wk of exposure to 0.

Stress relaxation of contracted collagen gels: disruption of actin filament bundles, release of cell surface fibronectin, and down-regulation of DNA and protein synthesis.

Relaxation of stressed collagen gels provides a model system uniquely suited to studying the regulation of cell morphology and biosynthetic function by tissue organization. Stress relaxation results in rapid, synchronous changes in cell morphology without enzymatic or other drug treatments, and makes possible an analysis of the initial cellular events associated with changes in tissue organization. During the first hour after stress relaxation, we observed transient hypercontraction of collagen gels and loss of collagen fibril organization as stress in the system dissipated.

Metabolic enhancement and increase of alveolar macrophages induced by ozone.

Male Wistar rats were exposed to 0.2 ppm ozone (O3) for 14 days and at intervals alveolar macrophages were collected by bronchoalveolar lavage to examine the effects of O3. The specific activities of glucose-6-phosphate dehydrogenase and glutathione peroxidase of alveolar macrophages increased to 1.

Subacute effects of nitrogen dioxide on membrane constituents of lung, liver, and kidney of rats.

Male Wistar rats were exposed to 0.4, 1.2, and 4.

Activation and increment of alveolar macrophages induced by nitrogen dioxide.

Male Wistar rats were exposed to 4 ppm nitrogen dioxide (NO2) for 10 d, and at intervals alveolar macrophages were collected by pulmonary lavage. A metabolic enhancement of alveolar macrophages was observed on d 4 of exposure. The specific activities of glucose-6-phosphate dehydrogenase and glutathione peroxidase of the peroxidative metabolic pathway increased to 1.

An increase in the activities of glycolytic enzymes in rat lungs produced by nitrogen dioxide.

Male Jcl: Wistar rats were exposed to 2, 4, and 10 ppm NO2 for 14, 10, and 7 d, respectively, to examine the effect of NO2 on the lung glycolytic pathway, a major energy-generating system in the lung. A highly significant increase in the activities of hexokinase, phosphofructokinase, 3-phosphoglycerate kinase, pyruvate kinase (PK), and lactate dehydrogenase was observed after 5 d exposure to 10 ppm NO2, and a significantly higher value was maintained until d 7. Similarly, the activities of all enzymes examined increased significantly by exposure to 4 ppm NO2, reaching the maximum between 4 and 7 d of exposure, and then approached to near the control levels.

In vivo effect of nitrogen dioxide on the activities of glycolytic enzymes in red blood cells of rats.

The effects of short-term exposure to NO2 on the glycolytic enzymes of rat red cells were examined. Exposure to 10 ppm NO2 resulted in decrease in activities of pyruvate kinase (PK) and phosphofructokinase (PFK) at day 1 and then increased progressively. Exposure to 4 ppm NO2 caused progressive increases in these enzyme activities from the first day.

Effects of nitrate and nitrite, chemical intermediates of inhaled nitrogen dioxide, on membrane components of red blood cells of rats.

Rat blood was incubated at 37 degrees C for 60 min with either NaNO3 or NaNO2 to examine the relationship between the decrease in the hexose content and Ca2+,Mg2+-ATPase activity of red cell membranes, and NO3- and NO2-. The hexose content decreased depending on the NaNO2 concentration up to 100 microM reaching 76% (p less than 0.05) of the control value.