Structural changes in root and shoot ofBacopa monniera in response to salt stress

Various concentrations of salt (NaCI) were shown to have an influence on the differentiation of tissues in the root and stem ofBacopa monniera (L) Wettst. Higher concentrations induced drastic changes in roots grown on salt-supplemented media; epidermal and cortical cells experienced changes in shape, size, and orientation and/or were got disintegrated. A low concentration of salt induced a profuse development of root hairs which gradually disappeared at higher concentrations. Air spaces in the stem cortex were enlarged and xylem cell walls in the vascular ring were thickened.     

Metabolic response of biofilm to shear stress in fixed-film culture

AIMS: In a biofilm reactor, detachment force resulting from hydraulic shear is a major factor that determines the formation and structure of steady state biofilm. The metabolic response of biofilm to change in shear stress was therefore investigated. METHODS AND RESULTS: A conventional annular reactor made of PVC was used, in which shearing over the rotating disc surface was strictly defined. Results from the steady state aerobic biofilm reactor showed that the biofilm structure (density and thickness) and metabolic behaviour (growth yield and dehydrogenase activity) were closely related to the shear stress exerted on the biofilm. Smooth, dense and stable biofilm formed at relatively high shear stress. Higher dehydrogenase activity and lower growth yield were obtained when the shear stress was raised. Growth yield was inversely correlated with the catabolic activity of biofilm. The reduced growth yield, together with the enhanced catabolic activity, suggests that a dissociation of catabolism from anabolism would occur at high shear stress. CONCLUSION: Biofilms may respond to shear stress by regulating metabolic pathways associated with the substrate flux flowing between catabolism and anabolism. A biological phenomenon, besides a simple physical effect, is underlying the observed relation between the shear stress and resulting biofilm structure. SIGNIFICANCE AND IMPACT OF THE STUDY: A hypothesis is proposed that the shear-induced energy spilling would be associated with a stimulated proton translocation across the cell membrane, which favours formation of a stronger biofilm. This research may provide a basis for experimental data on biofilm obtained at different shear stresses to be interpreted in relation to energy.     

Effect of spatial gradient in fluid shear stress on morphological changes in endothelial cells in response to flow

Arterial bifurcations are common sites for development of cerebral aneurysms. Although this localization of aneurysms suggests that high shear stress (SS) and high spatial SS gradient (SSG) occurring at the bifurcations may be crucial factors for endothelial dysfunction involved in aneurysm formation, the details of the relationship between the hemodynamic environment and endothelial cell (EC) responses remain unclear. In the present study, we sought morphological responses of ECs under high-SS and high-SSG conditions using a T-shaped flow chamber. Confluent ECs were exposed to SS of 2-10 Pa with SSG of up to 34 Pa/mm for 24 and 72 h. ECs exposed to SS without spatial gradient elongated and oriented to the direction of flow at 72 h through different processes depending on the magnitude of SS. In contrast, cells did not exhibit preferred orientation and elongation under the combination of SS and SSG. Unlike cells aligned to the flow by exposure to only SS, development of actin stress fibers was not observed in ECs exposed to SS with SSG. These results indicate that SSG suppresses morphological changes of ECs in response to flow.     

Heterogeneous response of microvascular endothelial cells to shear stress

We investigated changes in calcium concentration in cultured bovine aortic endothelial cells (BAECs) and rat adrenomedulary endothelial cells (RAMECs, microvascular) in response to different levels of shear stress. In BAECs, the onset of shear stress elicited a transient increase in intracellular calcium concentration that was spatially uniform, synchronous, and dose dependent. In contrast, the response of RAMECs was heterogeneous in time and space. Shear stress induced calcium waves that originated from one or several cells and propagated to neighboring cells. The number and size of the responding groups of cells did not depend on the magnitude of shear stress or the magnitude of the calcium change in the responding cells. The initiation and the propagation of calcium waves in RAMECs were significantly suppressed under conditions in which either purinergic receptors were blocked by suramin or extracellular ATP was degraded by apyrase. Exogenously applied ATP produced similarly heterogeneous responses. The number of responding cells was dependent on ATP concentration, but the magnitude of the calcium change was not. Our data suggest that shear stress stimulates RAMECs to release ATP, causing the increase in intracellular calcium concentration via purinergic receptors in cells that are heterogeneously sensitive to ATP. The propagation of the calcium signal is also mediated by ATP, and the spatial pattern suggests a locally elevated ATP concentration in the vicinity of the initially responding cells.     

Cell morphological response to low shear stress in a two-dimensional culture microsystem with magnitudes comparable to interstitial shear stress

Slow interstitial flow can lead to large changes in cell morphology. Since conventional biological assays are adapted to two-dimensional culture protocols, there is a need to develop a microfluidic system that can generate physiological levels of interstitial flow. Here we developed a system that uses a passive osmotic pumping mechanism to generate sustained and steady interstitial flows for two-dimensional cultures. Two different cell types, fibroblasts and mesenchymal stem cells, were selected because they are generally exposed to interstitial flow. To quantify the cellular response to interstitial shear flow in terms of proliferation and alignment, 4 rates of flow were applied. We found that the proliferation rate of fibroblasts varied linearly with wall shear stress. In addition, alignment of fibroblast cells depended linearly on the magnitude of the shear stress, whereas mesenchymal stem cells were aligned regardless of the magnitude of shear stress. This suggested that mesenchymal stem cells are very sensitive to shear stresses, even at levels generated by interstitial flow. The results presented here emphasize the need to consider the mechanosensitivity and the natural role of different cell types when evaluating their responses to fluid flow.     

Respiratory plasticity in response to changes in oxygen supply and demand

Aerobic organisms maintain O₂ homeostasis by responding to changesin O₂ supply and demand in both short and long time domains.In this review, we introduce several specific examples of respiratoryplasticity induced by chronic changes in O₂ supply (environmentalhypoxia or hyperoxia) and demand (exercise-induced and temperature-inducedchanges in aerobic metabolism). These studies reveal that plasticityoccurs throughout the respiratory system, including modificationsto the gas exchanger, respiratory pigments, respiratory muscles,and the neural control systems responsible for ventilating thegas exchanger. While some of these responses appear appropriate(e.g., increases in lung surface area, blood O₂ capacity, andpulmonary ventilation in hypoxia), other responses are potentiallyharmful (e.g., increased muscle fatigability). Thus, it maybe difficult to predict whole-animal performance based on theplasticity of a single system. Moreover, plastic responses maydiffer quantitatively and qualitatively at different developmentalstages. Much of the current research in this field is focusedon identifying the cellular and molecular mechanisms underlyingrespiratory plasticity. These studies suggest that a few keymolecules, such as hypoxia inducible factor (HIF) and erythropoietin,may be involved in the expression of diverse forms of plasticitywithin and across species. Studying the various ways in whichanimals respond to respiratory challenges will enable a betterunderstanding of the integrative response to chronic changesin O₂ supply and demand.     

Modeling of mechanosensitive channel gating in response to wall shear stress

The accurate biological function of mechanosensitive (MS) channels is crucial for maintaining the viability of living cells. For instance, in vascular endothelial cells, calcium influx from the extracellular environment into cytoplasm is regulated by stretch-activated channels. However, the mechanism by which cells sense force remains unclear. For this study, we hypothesized that gating of ion channels is simply regulated by the direct mechanical stress induced in a membrane. We modeled a membrane channel using crystallographic data of the bacteria Mycobacterium tuberculosis (Tb-MscL) because MscL homologs are integral membrane proteins with sequence similarity to most known ion channels. Molecular dynamics (MD) simulations were performed to elucidate the gating mechanism of the channel protein in response to the fluid shear stress. Results suggest that the stretched membrane drives the interfacial part of the protein–membrane complex to expand and maintains the stability of the constricted part of the transmembrane pore. Moreover, structural similarities between Tb-MscL and the family of ligand-gated ion channels suggest that the conformational change of this model in response to fluid shear stress is useful for modeling the gating mechanism in a broad class of gated channels.     

Oscillatory structures of blood flow reflect the dynamics of information processes in microcirculatory networks

Laser Doppler flowmetry with wavelet analysis of oscillations of the blood flow in the glabrous skin of the second and fifth fingertips of 30 healthy individuals and 57 patients before and after median and ulnar nerve sutures (n = 29) and after hand sympathectomy (n = 28). Information processes in microvascular networks include both the stationary and oscillatory components. This study is the first to apply wavelet analysis of the blood flow oscillatory organization to studying the dynamics of information processes in microcirculatory networks. A methodological approach was proposed to estimate the total quantity of information, the value and semantic features of different information channels and the information regime (multichannel or resonance) in microcirculatory networks. The deficit of both general information and its content (particularly, of external information), a decrease in the accessibility to the information and the system of self-organization were typical during the denervation syndrome. The semantic information signs changes mainly after sympathectomy. The dynamics of the information process reflects the functional significance of microcirculation in the course of nerve regeneration and skin reinnervation. The increment in information quantity with trophic content occurred at the preimpulse stage of nerve regeneration; it corresponds to the quantitative development of skin microvascular networks for trophic support of reinnervation. The semantic information content was predominantly modulated at the impulse stage due to the increase in homeostatic control intensity. Information peculiarities in the transitional period (from the preimpulse to impulse stage) included a decrease in sporadic processes, an increase in determinism in the system control, the predomination of the trophic content assimilation with the increment in the myogenic activity proper, and the possibility of both multichannel and resonance regimes. The general biological law of the antientropic importance of information for decreasing stochastic processes and the system control support has been confirmed.     

RGD-dependent mechanotransduction of suspension cultured Taxus cell in response to shear stress

Plant cells cultured in bioreactors are strongly influenced by mechanical forces. However, the molecular mechanism of plant cell mechanoreception has maintained unclear. In animal cells, the Arg-Gly-Asp (RGD) motif can be found in proteins of the extracellular matrix. Integrins link the intracellular cytoskeleton of cells with the extracellular matrix by recognizing this RGD motif. Integrin has been demonstrated to function as an apparatus not only for adhesion but also for mechanotransduction. In plant cells, the molecules that mediate the structural continuity between wall and membrane are unknown. Here, we found that synthetic RGD peptide could dramatically reduce the level of phosphorylation of MAPK-like cascades that are activated by shear stress and reduce the alkalinization response, production of reactive oxygen species (ROS) and accumulation of phenolics by Taxus cuspidata cells during shear stress. These results implicate that a RGD recognition system may exist in Taxus cells and play an important role in signal transduction of shear stress. Although the Arabidopsis genome database shows that the plant seems to lack a homologue of animal integrin, plant cells may use other RGD-binding proteins to recognize the RGD motif. The correlative mechanism is discussed.     

Proteomic analysis of vascular endothelial cells in response to laminar shear stress

Isotope-coded affinity tags (cICAT) coupled with mass spectrometric analysis is one of the leading technologies for quantitative proteomic profiling and protein quantification. We performed proteomic analysis of bovine aortic endothelial cells (BAEC) in response to laminar shear stress using cICAT labeling coupled with LC-MS/MS. Protein expressions in BAEC under 15 dynes/cm2 of shear stress for 10 min, 3 h, and 6 h were compared with matched stationary controls. Analysis of each sample produced 1800-2400 proteins at >or=75% confidence level. We found 142, 213, and 186 candidate proteins that were up- or down-regulated by at least two-fold after 10 min, 3 h, and 6 h of shear stress, respectively. Some of these proteins have known cellular functions and they encompass many signaling pathways. The signaling pathways that respond to shear stress include those of integrins, G-protein-coupled receptors, glutamate receptors, PI3K/AKT, apoptosis, Notch and cAMP-mediated signaling pathways. The validity of the mass spectrometric analysis was also confirmed by Western blot and confocal immunofluorescence microscopy. The present quantitative proteomic analysis suggests novel potential regulatory mechanisms in vascular endothelial cells in response to shear stress. These results provide preliminary footprints for further studies on the signaling mechanisms induced by shear stress.     

An apparatus to study the response of cultured endothelium to shear stress

An apparatus which has been developed to study the response of cultured endothelial cells to a wide range of shear stress levels is described. Controlled laminar flow through a rectangular tube was used to generate fluid shear stress over a cell-lined coverslip comprising part of one wall of the tube. A finite element method was used to calculate shear stresses corresponding to cell position on the coverslip. Validity of the finite element analysis was demonstrated first by its ability to generate correctly velocity profiles and wall shear stresses for laminar flow in the entrance region between infinitely wide parallel plates (two-dimensional flow). The computer analysis also correctly predicted values for pressure difference between two points in the test region of the apparatus for the range of flow rates used in these experiments. These predictions thus supported the use of such an analysis for three-dimensional flow. This apparatus has been used in a series of experiments to confirm its utility for testing applications. In these studies, endothelial cells were exposed to shear stresses of 60 and 128 dynes/cm2. After 12 hr at 60 dynes/cm2, cells became aligned with their longitudinal axes parallel to the direction of flow. In contrast, cells exposed to 128 dynes/cm2 required 36 hr to achieve a similar reorientation. Interestingly, after 6 hr at 128 dynes/cm2, specimens passed through an intermediate phase in which cells were aligned perpendicular to flow direction. Because of its ease and use and the provided documentation of wall shear stress, this flow chamber should prove to be a valuable tool in endothelial research related to atherosclerosis.     

Cytoplasmic calcium response to fluid shear stress in cultured vascular endothelial cells

Summary Vascular endothelial cells modulate their structure and functions in response to changes in hemodynamic forces such as fluid shear stress. We have studied how endothelial cells perceive the shearing force generated by blood flow and the substance(s) that may mediate such a response. We identify cytoplasmic-free calcium ion (Ca⁺⁺), a major component of an internal signaling system, as a mediator of the cellular response to fluid shear stress. Cultured monolayers of bovine aortic endothelial cells loaded with the highly fluorescent Ca⁺⁺-sensitive dye Fura 2 were exposed to different levels of fluid shear stress in a specially designed flow chamber, and simultaneous changes in fluorescence intensity, reflecting the intracellular-free calcium concentration ([Ca⁺⁺] _i ), were monitored by photometric fluorescence microscopy. Application of shear stress to cells by fluid perfusion led to an immediate severalfold increase in fluorescence within 1 min, followed by a rapid decline for about 5 min, and finally a plateau somewhat higher than control levels during the entire period of the stress application. Repeated application of the stress induced similar peak and plateau levels of [Ca⁺⁺] _i but at reduced magnitudes of response. These responses were observed even in Ca⁺⁺-free medium. Thus, a shear stress transducer might exist in endothelial cells, which perceives the shearing force on the membrane as a stimulus and mediates the signal to increase cytosolic free Ca⁺⁺. This work was partly supported by a grant-in-aid, for Special Project Research no. 61132008, from the Japanese Ministry of Education, Science and Culture and a research fund from the Atherosclerosis Study Association.     

Colon cancer cell adhesion in response to Src kinase activation and actin-cytoskeleton by non-laminar shear stress

Malignant cells shed from tumors during surgical resection or spontaneous metastasis experience physical forces such as shear stress and turbulence within the peritoneal cavity during irrigation, laparoscopic air insufflation, or surgical manipulation, and within the venous or lymphatic system. Since physical forces can activate intracellular signals that modulate the biology of various cell types in vitro, we hypothesized that shear stress and turbulence might increase colon cancer cell adhesion to extracellular matrix, potentiating metastatic implantation. Primary human malignant colon cancer cells isolated from resected tumors and SW620 were subjected to shear stress and turbulence by stirring cells in suspension at 600 rpm for 10 min. Shear stress for 10 min increased subsequent SW620 colon cancer cell adhesion by 40.0 +/- 3.0% (n = 3; P < 0.001) and primary cancer cells by 41.0 +/- 3.0% to collagen I when compared to control cells. In vitro kinase assay (1.5 +/- 0.13 fold) and Western analysis (1.34 +/- 0.04 fold) demonstrated a significant increase in Src kinase activity in cells exposed shear stress. Src kinase inhibitors PP1 (0.1 microM), PP2 (20 microM), and actin-cytoskeleton stabilizer phalloidin (10 microM) prevented the shear stress stimulated cell adhesion to collagen I. Furthermore, PP2 inhibited basal (50.0 +/- 2.8%) and prevented shear stress induced src activation but phalloidin pretreatment did not. These results raise the possibility that shear stress and turbulence may stimulate the adhesion of malignant cells shed from colon cancers by a mechanism that requires both actin-cytoskeletal reorganization an independent physical force activation of Src kinase. Blocking this pathway might reduce tumor metastasis during surgical resection.     

Structural changes in mitochondria induced by uncoupling reagents. The response to proteolytic enzymes

Interaction of uncoupling reagents with bovine serum albumin markedly inhibited its hydrolysis by proteolytic enzymes. The inhibition presumably is due to conformational transitions in the protein substrate induced by the binding of the ligand-uncoupling reagents. The proteolysis of casein, a protein that does not bind these reagents, was not affected, indicating that the proteinases themselves were not inactivated. In contrast, interaction of uncoupling reagents with freshly isolated rat liver mitochondria enhanced their susceptibility to proteolytic enzymes. This was shown by an increase in the release of ninhydrin-reacting material, by an increase in free acid groups and by a decrease in the turbidity of the mitochondrial suspensions. These effects, although opposite in direction to those obtained with albumin, are also presumed to indicate structural changes in the mitochondrial proteins and a disorganization of the protein-phospholipid complex. It is suggested that such structural alterations are expressed functionally as the uncoupling of oxidative phosphorylation.     

Conservation of microRNA-mediated regulatory networks in response to copper stress in grapevine

Jatropha curcas L. as a bio energy plant belonging to the Euphorbiaceae family is gaining progressive importance over the last years. In 2012 and 2014 field experiments were carried out to assess the effects of cytokinins 6-Benzyladenine (BA) and Forchlorfenuron (CPPU) acting as plant growth regulators (PGRs). Number of flowers per inflorescence, female-to-male ratio of flowers, fruits per infructescence, fruiting rate, number of seeds per fruit, seed size and weight as well as seed oil content were determined. It was suggested that concerning effectiveness of exogenous application of PGRs the developmental stage of flower is very important. Due to that, special interest was laid on the age of inflorescences at the time of application. Our experiments revealed a strong dependence of cytokinin effectiveness on the developmental stage of flowering. So treatment of plants with 6-Benzyladenine at the beginning of flower formation (stage 1) significantly increased the number of male and female flowers per inflorescence, whereas treatment at an advanced flowering stage (stage 2) or at the stage of fully developed flowers (stage 3) had only slight or no effects. In contrast, fruit retention was progressively increased by treatment in stage 2 and 3. Application of Forchlorfenuron significantly increased female-to-male ratio in stage 1 flowers but showed no effects on stage 2 and 3. 6-Benzyladenine as well as Forchlorfenuron showed equal effects on number of fruit inflorescences treated in stage 1. Our results show a significant decrease in seed weight due to BA- and CPPU-application while kernel weight remained stable. Concerning fruits, clusters and oil yield per tree, BA-application showed significant increasing effects. This study indicates that 6-Benzyladenine and Forchlorfenuron can improve yield affecting parameters in J. curcas when the phenological stage of flowering at time of application is precisely considered.