Gene expression profiles in chondrosarcoma cells subjected to cyclic stretching and hydrostatic pressure. A cDNA array study

Mechanical forces have a profound effect on cartilage tissue and chondrocyte metabolism. Strenuous loading inhibits the cellular metabolism, while optimal level of loading at correct frequency raises an anabolic response in chondrocytes. In this study, we used Atlas Human Cancer cDNA array to investigate mRNA expression profiles in human chondrosarcoma cells stretched 8% for 6 hours at a frequency of 0.5 Hz. In addition, cultures were exposed to continuous and cyclic (0.5 Hz) 5 MPa hydrostatic pressure. Cyclic stretch had a more profound effect on the gene expression profiles than 5 MPa hydrostatic pressure. Several genes involved with the regulation of cell cycle were increased in stretched cells, as well as mRNAs for PDGF-B, glucose-1-phosphate uridylyltransferase, Tiam1, cdc37 homolog, Gem, integrin alpha6, and matrix metalloproteinase-3. Among down-regulated genes were plakoglobin, TGF-alpha, retinoic acid receptor-alpha and Wnt8b. A smaller number of changes was detected after pressure treatments. Plakoglobin was increased under cyclic and continuous 5 MPa hydrostatic pressure, while mitogen-activated protein kinase-9, proliferating cell nuclear antigen, Rad6, CD9 antigen, integrins alphaE and beta8, and vimentin were decreased. Cyclic and continuous pressurization induces a number of specific changes. In conclusion, a different set of genes were affected by three different types of mechanical stimuli applied on chondrosarcoma cells.     

Effect of high hydrostatic pressure on the bacterial mechanosensitive channel MscS

We have investigated the effect of high hydrostatic pressure on MscS, the bacterial mechanosensitive channel of small conductance. Pressure affected channel kinetics but not conductance. At negative pipette voltages (corresponding to membrane depolarization in the inside-out patch configuration used in our experiments) the channel exhibited a reversible reduction in activity with increasing hydrostatic pressure between 0 and 900 atm (90 MPa) at 23°C. The reduced activity was characterized by a significant reduction in the channel opening probability resulting from a shortening of the channel openings with increasing pressure. Thus high hydrostatic pressure generally favoured channel closing. Cooling the patch by approximately 10°C, intended to order the bilayer component of the patch by an amount similar to that caused by 50 MPa at 23°C, had relatively little effect. This implies that pressure does not affect channel kinetics via bilayer order. Accordingly we postulate that lateral compression of the bilayer, under high hydrostatic pressure, is responsible. These observations also have implications for our understanding of the adaptation of mechanosensitive channels in deep-sea bacteria.A Proceeding of the 28th Annual Meeting of the Australian Society for Biophysics.     

cDNA microarray reveals the alterations of cytoskeleton-related genes in osteoblast under high magneto-gravitational environment

The diamagnetic levitation as a novel ground-based model for simulating a reduced gravity environment has been widely applied in many fields. In this study, a special designed superconducting magnet, which can produce three apparent gravity levels （0, 1, and 2 g）, namely high magneto-gravitational environment （HMGE）, was used to simulate space gravity environment. The effects of HMGE on osteoblast gene expression profile were investigated by microarray. Genes sensitive to diamagnetic levitation environment （0g）, gravity changes, and high magnetic field changes were sorted on the basis of typical cell func- tions. Cytoskeleton, as an intracellular load-bearing struc- ture, plays an important role in gravity perception. Therefore, 13 cytoskeleton-related genes were chosen according to the results of microarray analysis, and the expressions of these genes were found to be altered under HMGE by real-time PCR. Based on the PCR results, the expressions of WASF2 （WAS protein family, member 2）, WIPF1 （WAS/WASL interacting protein family, member 1）, paxillin, and talin 1 were further identified by western blot assay. Results indicated that WASF2 and WIPF1 were more sensitive to altered gravity levels, and talin 1 and paxillin were sensitive to both magnetic field and gravity changes. Our findings demonstrated that HMGE can affect osteoblast gene expression profile and cytoskele- ton-related genes expression. The identification of mechanosensitive genes may enhance our understandings to the mechanism of bone loss induced by microgravity and may provide some potential targets for preventing and treating bone loss or osteoporosis.     

Profiling of differentially expressed genes in LRRC4 overexpressed glioblastoma cells by cDNA array

Our previous study has shown that LRRC4 is a novel member of the leucine-rich repeat （LRR） superfamily and has the potential to suppress brain tumor growth. In order to further analyze the functions of LRRC4 on the maintenance of normal function and suppression of tumorigenesis in the central nervous system, we investigated alterations in gene expression related to neurobiology by the Atlas array in two inducible dual-stable LRRC4-overexpressing cell lines. Seventeen of 588 genes spotted on the Atlas membrane showed altered expression levels in LRRC4 transfected U251MG Tet-on cells, which are involved in cell proliferation and cell cycle progression, tumor invasion and metastasis, and neurotransmitter synthesis and release. In addition, cell invasion assay results showed that LRRC4 can inhibit the U251MG cell migration. These studies represent the first cDNA array analysis of the effects of LRRC4 on the involvement of different neurobiological genes in U251MG glioblastoma cells and provide new insights into the function of LRRC4 in glioma.     

The protective effect of osmoprotectant TMAO on bacterial mechanosensitive channels of small conductance MscS/MscK under high hydrostatic pressure

Activity of the bacterial mechanosensitive channels of small conductance MscS/MscK of E. coli was investigated under high hydrostatic pressure (HHP) using the “flying-patch” patch-clamp technique. The channels were gated by negative pipette voltage and their open probability was measured at HHP of 0.1 to 80 MPa. The channel open probability decreased with increasing HHP. When the osmolyte methylamine N-oxide (TMAO) was applied to the cytoplasmic side of the inside-out excised membrane patches of E. coli giant spheroplasts the inhibitory effect of HHP on the channel activity was suppressed at pressures of up to 40 MPa. At 40 MPa and above the channel open probability decreased in a similar fashion with or without TMAO. Our study suggests that TMAO helps to counteract the effect of HHP up to 40 MPa on the MscS/MscK open state by “shielding” the cytoplasmic domain of the channels.     

The protective effect of osmoprotectant TMAO on bacterial mechanosensitive channels of small conductance MscS/MscK under high hydrostatic pressure

Activity of the bacterial mechanosensitive channels of small conductance MscS/MscK of E. coli was investigated under high hydrostatic pressure (HHP) using the “flying-patch” patch-clamp technique. The channels were gated by negative pipette voltage and their open probability was measured at HHP of 0.1 to 80 MPa. The channel open probability decreased with increasing HHP. When the osmolyte methylamine N-oxide (TMAO) was applied to the cytoplasmic side of the inside-out excised membrane patches of E. coli giant spheroplasts the inhibitory effect of HHP on the channel activity was suppressed at pressures of up to 40 MPa. At 40 MPa and above the channel open probability decreased in a similar fashion with or without TMAO. Our study suggests that TMAO helps to counteract the effect of HHP up to 40 MPa on the MscS/MscK open state by “shielding” the cytoplasmic domain of the channels.     

High pressure microscopy--a powerful tool for monitoring cells and macromolecules under high hydrostatic pressure.

A high pressure chamber, which withstands a pressure up to 300 MPa has been developed. The so-called HPDS (Hartmann, Pfeifer, Dornheim, Sommer) High Pressure Cell in combination with an inverted microscope and an analysis system allows brilliant microscopic colour pictures with an optical resolution better than 0.56 microm. The pressure chamber allows the in situ observation of dynamic changes of microscopic structures in bright field, phase contrast and fluorescence microscopy. This publication should demonstrate the capabilities of the system using results of experiments with two types of Spirogyra algae. The pictures have shown significant variations of the chloroplasma and the cell wall membrane at pressures of up to 120 MPa. The new system provides a simple way to perform microscopic analyses at pressures of up to 300 MPa.     

Life under pressure: hydrostatic pressure in cell growth and function

H(2)O is one of the most essential molecules for cellular life. Cell volume, osmolality and hydrostatic pressure are tightly controlled by multiple signaling cascades and they drive crucial cellular functions ranging from exocytosis and growth to apoptosis. Ion fluxes and cell shape restructuring induce asymmetries in osmotic potential across the plasma membrane and lead to localized hydrodynamic flow. Cells have evolved fascinating strategies to harness the potential of hydrodynamic flow to perform crucial functions. Plants exploit hydrodynamics to drive processes including gas exchange, leaf positioning, nutrient acquisition and growth. This paradigm is extended by recent work that reveals an important role for hydrodynamics in pollen tube growth.     

Analysis of cDNA transcripts from Coniothyrium minitans reveals a diverse array of genes involved in key processes during sclerotial mycoparasitism

Coniothyrium minitans colonises and destroys the sclerotia of Sclerotinia sclerotiorum in nature exhibiting ecologically obligate mycoparasitism as its spores remain dormant in soil and only grow actively in the presence of the sclerotia. Molecular mechanisms underlying sclerotial mycoparasitism are poorly defined. We identified 251 unisequences representing genes preferentially expressed by C. minitans during sclerotial mycoparasitism, substantially increasing the molecular knowledge of this commercially important biocontrol agent. Genes associated with signalling and cellular communication, degradation of host cell walls and energy reserves, nutrient utilisation, detoxification and stress response were identified suggesting that C. minitans employs a number of key processes during host colonisation. Several of these genes are novel to fungal-fungal interactions (e.g. PTH11-like GPCR and the ETP gene cluster). Secretin receptor-like GPCR and the TGF-beta signalling system have not yet been characterised in filamentous fungi. This study provides the basis for in-depth gene function analysis in sclerotial mycoparasitism.     

Molecular dynamics simulations of HPr under hydrostatic pressure

The histidine-containing protein (HPr) plays an important role in the phosphotransferase system (PTS). The deformations induced on the protein structure at high hydrostatic pressure values (4, 50, 100, 150, and 200 MPa) were previously (H. Kalbitzer, A. G?rler, H. Li, P. Dubovskii, A. Hengstenberg, C. Kowolik, H. Yamada, and K. Akasaka, Protein Science 2000, Vol. 9, pp. 693-703) analyzed by NMR experiments: the nonlinear variations of the amide chemical shifts at high pressure values were supposed to arise from induced shifts in the protein conformational equilibrium. Molecular dynamics (MD) simulations are here performed, to analyze the protein internal mobility at 0.1 MPa, and to relate the nonlinear variations of chemical shifts observed at high pressure, to variations in conformational equilibrium. The global features of the protein structure are only slightly modified along the pressure. Nevertheless, the values of the Voronoi residues volumes show that the residues of alpha-helices are more compressed that those belonging to the beta-sheet. The alpha-helices are also displaying the largest internal mobility and deformation in the simulations. The nonlinearity of the 1H chemical shifts, computed from the MD simulation snapshots, is in qualitative agreement with the nonlinearity of the experimentally observed chemical shifts.     

cDNA expression array analysis of DNA repair genes in human glioma cells that lack or express DNA-PK

M059J cells provide the only example of DNA-PKcs (now known as PRKDC) deficiency in a human cell line. M059K cells, derived from the same tumor specimen, express PRKDC protein and activity and, together with M059J, provide a useful model in which to study the role of DNA-PK in cellular responses to DNA-damaging agents. Because these cells are of tumor origin, we used Atlas human cancer cDNA expression arrays to investigate possible differential expression of other DNA repair genes in control and irradiated samples. cDNA array results indicated differential expression of 14 genes. Northern blotting confirmed relatively greater expression of replication factor C 37-kDa subunit mRNA in M059J cells compared to M059K cells and reduced expression of DNA ligase IV compared to ligase III in both cell lines independent of irradiation. These results suggest that other DNA repair proteins are altered in these cell lines and that repair mechanisms predicted from the study of normal tissues may be fundamentally altered in human cancer cells.     

Structural stability of human butyrylcholinesterase under high hydrostatic pressure

Human butyrylcholinesterase is a nonspecific enzyme of clinical, pharmacological and toxicological significance. Although the enzyme is relatively stable, its activity is affected by numerous factors, including pressure. In this work, hydrostatic pressure dependence of the intrinsic tryptophan fluorescence in native and salted human butyrylcholinesterase was studied up to the maximum pressure at ambient temperature of about 1200 MPa. A correlated large shift toward long wavelengths and broadening observed at pressures between 200 and 700 MPa was interpreted as due to high pressure-induced denaturation of the protein, leading to an enhanced exposure of tryptophan residues into polar solvent environment. This transient process in native butyrylcholinesterase presumably involves conformational changes of the enzyme at both tertiary and secondary structure levels. Pressure-induced mixing of emitting local indole electronic transitions with quenching charge transfer states likely describes the accompanying fluorescence quenching that reveals different course from spectral changes. All the pressure-induced changes turned irreversible after passing a mid-point pressure of about 400 ± 50 MPa. Addition of either 0.1 M ammonium sulphate (a kosmotropic salt) or 0.1 M lithium thiocyanate (a chaotropic salt) to native enzyme similarly destabilized its structure.     

Stability of subtilisin and lysozyme under high hydrostatic pressure

The stabilities of subtilisin and lysozyme under hydrostatic pressures up to 200 MPa were investigated for up to 7 days at 25 degrees C. Methods were chosen to assess changes in tertiary and secondary protein structure as well as aggregation state. Tertiary structure was monitored in situ with second derivative UV spectroscopy and after pressure treatment by dynamic light scattering and second derivative UV spectroscopy. Secondary structure and potential secondary structural changes were characterized by second derivative FTIR spectroscopy. Changes in aggregation state were assessed using dynamic light scattering. Additionally, protein concentration balances were carried out to detect any loss of protein as a function of pressure. For the conditions tested, neither protein shows measurable changes in tertiary or secondary structure or signs of aggregation. Lysozyme concentration balances show no dependence on pressure. Subtilisin concentration balances at high protein concentration (4 mg/mL and higher) do not show pressure dependence. However, the concentration balances carried out at 0.4 mg/mL show a clear sign of pressure dependence. These results may be explained by protein interaction with the vial surface and appear to be rate limited by the equilibrium between active and inactive protein on the surface. Pressure increases protein loss, and the estimated partial molar volume change between the two states is estimated to be -20 +/- 10 mL/mol.