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C3H10T1/2 mouse embryo fibroblasts differentiate into adipocytes when stimulated by a standard hormonal mixture (IDMB). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), via the aryl hydrocarbon receptor (AhR), inhibits induction of the key adipogenic gene peroxisome proliferator-activated receptor gamma (PPARgamma) and subsequent adipogenesis. This TCDD-mediated inhibition requires activation of the extracellular signal-regulated kinase (ERK) pathway, which can be accomplished by serum, epidermal growth factor (EGF), or fibroblast growth factor (FGF). In the absence of serum or growth factors, IDMB induced adipogenesis without mitosis. Microarray analysis identified 200 genes that exhibited expression changes of at least twofold after 24 h of IDMB treatment. This time precedes most PPARgamma stimulation but follows the period of TCDD/ERK cooperation and periods of increased cell contraction and DNA synthesis. Functionally related gene clusters include genes associated with cell structure, triglyceride and cholesterol metabolism, oxidative regulation, and secreted proteins. In the absence of growth factors TCDD inhibited 30% of these IDMB responses without inhibiting the process of differentiation. A combination of EGF and TCDD that blocks differentiation cooperatively blocked a further 44 IDMB-responsive genes, most of which have functional links to differentiation, including PPARgamma. Cell cycle regulators that are stimulated by EGF were substantially inhibited by IDMB but these responses were unaffected by TCDD. By contrast, TCDD and EGF cooperatively reversed IDMB-induced changes in cell adhesion complexes immediately prior to increases in PPARgamma1 expression. Changes in adhesion-linked signaling may play a key role in TCDD affects on differentiation.

The aryl hydrocarbon receptor (AhR) is activated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), but activation without an exogenous ligand also occurs when normal cell-cell contact is prevented. Suspension of several C3H10T1/2 fibroblast clonal sub-lines that contain an integrated AhR-responsive reporter produced a time course and level of reporter activation and CYP1B1 induction that paralleled TCDD stimulation in confluent monolayer culture. Suspension activation was, however, more transient. Loss of cell-cell contact at low density also activated these reporters independent of cell cycle changes to levels comparable to TCDD stimulation of confluent cells. Loss of cell-cell contact may, therefore, activate AhR. Suspension and TCDD activations exhibited comparable nuclear translocation of AhR and then AhR/ARNT complex formation. Each AhR activation process was equally attenuated by inhibition of, respectively, HSP90 ATPase, the 26S proteosome, and by depletion of intracellular Ca2+. By contrast, the AhR antagonist alpha-naphthoflavone (alphaNF) blocked ligand-stimulated AhR activity, but not activation through loss of cell-cell contact. Suspension-induced reporter activation was selectively enhanced by LiCl, which prevented GSK-3beta effects on the simultaneously released beta-catenin. The effects of suspension and LiCl on reporters were reversed by Ro-31-8220, which did not affect beta-catenin, TCDD-activation processes, or AhR turnover. Neither LiCl nor Ro-31-8220 altered suspension-induced AhR/ARNT complex formation. Loss of cell-cell contact permits nuclear translocation and AhR activation that is largely replicated after TCDD binding, but with activity differences due to contact-sensitive factors functioning after AhR/ARNT complex formation.

Adipogenesis is typically stimulated in mouse embryo fibroblast (MEF) lines by a standard hormonal combination of insulin (I), dexamethasone (D), and methylisobutylxanthine (M), administered with a fresh serum renewal. In C3H10T1/2 (10T1/2) cells, peroxisome proliferator-activated receptor gamma1 (PPARgamma1) expression, an early phase key adipogenic regulator, is optimal after 36 h of IDM stimulation. Although previous studies provide evidence that mitotic clonal expansion of 3T3-L1 cells is essential for adipogenesis, we show, here, that 10T1/2 cells do not require mitotic clonal expansion, but depend on cell cycle progression through S-phase to commit to adipocyte differentiation. Exclusion of two major mitogenic stimuli (DM without insulin and fresh serum renewal) from standard IDM protocol removed mitotic clonal expansion, but sustained equivalent PPARgamma1 synthesis and lipogenesis. Different S-phase inhibitors (aphidicolin, hydroxyurea, l-mimosine, and roscovitin) each arrested cells in S-phase, under hormonal stimulation, and completely blocked PPARgamma1 synthesis and lipogenesis. However, G2/M inhibitors effected G2/M accumulation of IDM stimulated cells and prevented mitosis, but fully sustained PPARgamma1 synthesis and lipogenesis. DM stimulation with or without fresh serum renewal elevated DNA synthesis in a proportion of cells (measured by BrdU labeling) and accumulation of cell cycle progression in G2/M-phase without complete mitosis. By contrast, standard IDM treatments with fresh serum renewal caused elevated DNA synthesis and mitotic clonal expansion while achieved equivalent level of adipogenesis. At most, one-half of the 10T1/2 mixed cell population differentiated to mature adipocytes, even when clonally isolated. PPARgamma was exclusively expressed in the cells that contained lipid droplets. IDM stimulated comparable PPARgamma1 synthesis and lipogenesis in isolated cells at low cell density (LD) culture, but in about half of the cells and with sensitivity to G1/S, but not G2/M inhibitors. Importantly, growth arrest occurred in all differentiating cells, while continuous mitotic clonal expansion occurred in non-differentiating cells. Irrespective of confluence level, 10T1/2 cells differentiate after progression through S-phase, where adipogenic commitment induced by IDM stimulation is a prerequisite for PPARgamma synthesis and subsequent adipocyte differentiation.

Activation of the aryl-hydrocarbon receptor (AhR) by pretreatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) blocks hormone (IDM/BRL)-induced adipocyte differentiation of C3H10T1/2 cells in proportion to the suppression of the elevation of the key mediator, peroxisome proliferator-activated receptor (PPARgamma1). Inhibition of MEK-induced ERK phosphorylation had no effect on adipogenesis but prevented this TCDD suppression. Initiation of MEK inhibition up to 6 h after IDM/BRL stimulation in combination with serum addition completely reversed the TCDD-mediated suppression but declined to ineffectiveness when delayed to 24 h after stimulation. This period occurs well after the decline of serum-induced ERK activation, at a time when ERK phosphorylation is low, and prior to the onset of IDM/BRL-stimulated PPARgamma1 expression. This temporal separation of ERK activation from the affected PPARgamma1 expression suggests that ERK does not act directly on either PPARgamma1 transcription or receptor function. Thus, ERK activation and TCDD/AhR stimulation work synergistically to inhibit adipocyte differentiation. Nonrenewal of serum at the time of IDM/BRL addition removed most of the ERK activation and also the TCDD-mediated suppressions of PPARgamma1 expression and adipocyte differentiation. Transfection of a vector expressing constitutively active MEK1 generated a constant, high level of phosphorylated ERK comparable to the peak serum-induced level and fully restored TCDD suppression without a TCDD-mediated effect on ERK phosphorylation. We conclude that low levels of activated MEK and ERK cooperate with AhR-induced factor(s) to generate a suppressor that prevents PPARgamma1 transcription and then differentiation.

The aryl hydrocarbon receptor (AhR) is activated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), but activation without an exogenous ligand also occurs when normal cell-cell contact is prevented. Suspension of several C3H10T1/2 fibroblast clonal sub-lines that contain an integrated AhR-responsive reporter produced a time course and level of reporter activation and CYP1B1 induction that paralleled TCDD stimulation in confluent monolayer culture. Suspension activation was, however, more transient. Loss of cell-cell contact at low density also activated these reporters independent of cell cycle changes to levels comparable to TCDD stimulation of confluent cells. Loss of cell-cell contact may, therefore, activate AhR. Suspension and TCDD activations exhibited comparable nuclear translocation of AhR and then AhR/ARNT complex formation. Each AhR activation process was equally attenuated by inhibition of, respectively, HSP90 ATPase, the 26S proteosome, and by depletion of intracellular Ca2+. By contrast, the AhR antagonist alpha-naphthoflavone (alphaNF) blocked ligand-stimulated AhR activity, but not activation through loss of cell-cell contact. Suspension-induced reporter activation was selectively enhanced by LiCl, which prevented GSK-3beta effects on the simultaneously released beta-catenin. The effects of suspension and LiCl on reporters were reversed by Ro-31-8220, which did not affect beta-catenin, TCDD-activation processes, or AhR turnover. Neither LiCl nor Ro-31-8220 altered suspension-induced AhR/ARNT complex formation. Loss of cell-cell contact permits nuclear translocation and AhR activation that is largely replicated after TCDD binding, but with activity differences due to contact-sensitive factors functioning after AhR/ARNT complex formation.

We report here progress on a NASA program to develop fiber optic interferometric sensors for aeroacoustic measurements. As reported earlier, NASA's first fiber-optic microphone was developed and fabricated. Preliminary anechoic chamber tests demonstrated successfully its feasibility as an aeroacoustic sensor. Improved performance of a newly designed sensor head is presented here in terms of frequency response function and noise floor.© (1994) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Advanced pressure sensors have been developed at NASA Ames Research Center, using fiber‐optic technology. The development includes: compact size microphone for aeroacoustic measurements in wind tunnels with minimized flow–sensor interaction, and compact size pressure sensors for real‐time measurements of flow transition over an airfoil. A prototype pressure sensor was designed and fabricated with a silicon nitride diaphragm mounted on a miniaturized ferrule. The sensor has a 3‐mm diameter. Preliminary tests demonstrated excellent performance. The frequency response is steady and uniform within the design frequency limit, 100 to 5000 Hz. Its sensitivity, measured in terms of signal‐to‐noise ratio, is almost 10 dB better than the best condenser microphone available in a similar size (0.125‐in diameter), and is better than any existing fiber‐optic pressure sensor by at least three orders of magnitude. The overall performance of this sensor exceeds the initial expectation. The compact size and light weight of these sensors provide several advantages. The small size could allow tens of hundreds of sensors to be used together for applications such as microphone arrays. The flow–sensor interaction is smaller, providing more accurate measurements of pressure fluctuation of air flows over flight surfaces.

A simple method is described for the determinatio n of α-tocopherol in fish liver by high performance liquid chromatography. This method does not involve saponification or thin layer chromatography and thus avoids the destruction of α-tocopherol during sample preparation. The homogenized liver sample is extracted twice with dioxane–isooctane (20+80, v/v), and the combined extracts are dried under vacuum. The residue is extracted 3 times with acetonitrile from which α-tocopherol is then extracted with isooctane. The residue from the dried isooctane solution is dissolved in methanol–water (90+10, v/v) which is injected directly onto a micro Bondapak C18 high performance liquid chromatograph. The detection response to α-tocopherol at 280 nm was measured by peak height which was linear from 1 to 10 μg/25 μL injection. The coefficients of variation for retention time and peak height for 5 replicate analyses of the standard during 2 weeks were 1.4 and 2.4%, respectively. Recovery of α-tocopherol added to the sample before homogenization was 80–92% with a mean of 86.2% and a coefficient of variation of 4.9%. The minimum amount of sample and the concentration of α-tocopherol that can be accurately determined by the method are 0.5 g liver and 1 μg α-tocopherol/g liver, respectively.