Genome-Wide Gene Expression Analysis of NIH 3T3 Cell Line Under Mechanical Stimulation

Cyclic mechanical stretching induces biological and biomechanical response in cells. These responses are firstly determined by gene expression regulation in the cells of tissue. A method based on the CellDrum (R) Technology provided the environment for cyclic mechanical stimulation of NIH 3T3 cells in vitro. Cells were cultured on a silicone membrane. mRNA expression levels of the genes Egr1, Fgfr2, Tp53, Itgb3, and Itgb5 was evaluated by real-time PCR at stimulation times ranging from 5 min to 12 h with a cyclic strain of 0.25% at 0.25 Hz in order to decide which time period was most suitable for a subsequent detailed profiling. The genome-wide expression profile of NIH 3T3 cells was carried out by whole mouse genome microarrays. The mRNA expression levels of most genes tested were significantly changed after 1 h of mechanical stimulation. Subsequently, the mRNA samples of the 1-h stretched cells were hybridized to obtain a gene expression profile using microarrays. Real-time PCR results are shown to agree with the microarray results. The early response genes, such as Egr1, Egr2, Fos, Myc, Rela, Fas, Egfr1, and Fgfr2 playing a role in stretch activation of the signal transduction pathways were significantly up-regulated, whereas the only significantly down-regulated gene is Tfrc. Low level of mechanical stimulation was found to effect the expression of early responsive genes initiates alteration of NIH 3T3 behaviors to control the homeostasis of the fibroblasts

Hemoglobin senses body temperature

When aspirating human red blood cells (RBCs) into 1.3 mum pipettes (DeltaP = -2.3 kPa), a transition from blocking the pipette below a critical temperature T(c) = 36.3 +/- 0.3 degrees C to passing it above the T(c) occurred (micropipette passage transition). With a 1.1 mum pipette no passage was seen which enabled RBC volume measurements also above T(c). With increasing temperature RBCs lost volume significantly faster below than above a T(c) = 36.4 +/- 0.7 (volume transition). Colloid osmotic pressure (COP) measurements of RBCs in autologous plasma (25 degrees C < or = T < or = 39.5 degrees C) showed a T (c) at 37.1 +/- 0.2 degrees C above which the COP rapidly decreased (COP transition). In NMR T(1)-relaxation time measurements, the T(1) of RBCs in autologous plasma changed from a linear (r = 0.99) increment below T(c) = 37 +/- 1 degrees C at a rate of 0.023 s/K into zero slope above T(c) (RBC T(1) transition). In conclusion: An amorphous hemoglobin-water gel formed in the spherical trail, the residual partial sphere of the aspirated RBC. At T(c), a sudden fluidization of the gel occurs. All changes mentioned above happen at a distinct T(c) close to body temperature. The T(c) is moved +0.8 degrees C to higher temperatures when a D(2)O buffer is used. We suggest a mechanism similar to a "glass transition" or a "colloidal phase transition". At T(c), the stabilizing Hb bound water molecules reach a threshold number enabling a partial Hb unfolding. Thus, Hb senses body temperature which must be inscribed in the primary structure of hemoglobin and possibly other proteins

Adhesion of erythrocytes to endothelial cells after acute exercise: Differences in red blood cells from juvenile and adult rats

Erythrocytes (RBC) from untrained male Wistar rats and rat glomerular endothelial cells (EC) were used to investigate the effects of acute exercise (speed: 20 m/min, slope: 0, duration: 1 hour) on RBC membrane protein oxidation and adhesion to cultured EC. Experimental animals were divided into juvenile (age 10 weeks) and adult (age 30 weeks) groups for these studies. Immediately following exercise, juvenile rat RBC membrane protein oxidation was significantly enhanced. Adult rat RBC showed significantly higher basal protein oxidation than juvenile RBC; but the level of adult rat RBC membrane protein oxidation was unaffected by exercise. Prior to exercise, adult rat RBC showed significantly higher adhesion to EC than RBC of juvenile rat. There was no difference in plasma fibronectin or fibrinogen levels following exercise. Only juvenile rat RBC showed a significant decrease in sialic acid residue content following exercise. These experiments show that there are changes in RBC-EC interactions following exercise that are influenced by animal age

Does antioxidant supplementation alter the effects of acute exercise on erythrocyte aggregation, deformability and endothelium adhesion in untrained rats?

This study aimed to determine whether high-dose antioxidant supplementation had an impact on the acute exercise effects related to erythrocyte membrane mechanics. Experimental animals (n= 32) were divided into four groups as control, exercised, supplemented, and supplemented + exercise. Four-week antioxidant supplementation (vitamin C, vitamin E, and zinc) was applied to experimental animals. Following acute exercise on a motor-driven rodent treadmill, erythrocyte aggregation and deformability, erythrocyte adhesion to endothelial cells, superoxide dismutase (SOD), and glutathione peroxidase activities of the erythrocytes were analyzed. In both supplemented and non-supplemented exercised groups, there was a significant decrease in SOD activities and erythrocyte aggregation, and an increase in adhesion to endothelial cell although there was no change on erythrocyte deformability. There were no differences in the responses to the exercise of supplemented and non-supplemented rats. The data suggested that high-dose antioxidant supplementation did not alter the effects of acute exercise on erythrocyte membrane mechanics

Body Temperature-Related Structural Transitions of Monotremal and Human Hemoglobin

In this study, temperature-related structural changes were investigated in human, duck-billed platypus (Ornithorhynchus anatinus, body temperature T(b) = 31–33°C), and echidna (Tachyglossus aculeatus, body temperature T(b) = 32–33°C) hemoglobin using circular dichroism spectroscopy and dynamic light scattering. The average hydrodynamic radius (R(h)) and fractional (normalized) change in the ellipticity (F(obs)) at 222 ± 2 nm of hemoglobin were measured. The temperature was varied stepwise from 25°C to 45°C. The existence of a structural transition of human hemoglobin at the critical temperature T(c) between 36–37°C was previously shown by micropipette aspiration experiments, viscosimetry, and circular dichroism spectroscopy. Based on light-scattering measurements, this study proves the onset of molecular aggregation at T(c). In two different monotremal hemoglobins (echidna and platypus), the critical transition temperatures were found between 32–33°C, which are close to the species' body temperature T(b). The data suggest that the correlation of the structural transition's critical temperature T(c) and the species' body temperature T(b) is not mere coincidence but, instead, is a more widespread structural phenomenon possibly including many other proteins