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The small GTPase protein RhoA has two effectors, ROCK (Rho-associated protein kinase 1) and mDIA1 (protein diaphanous homolog 1), which cooperate reciprocally. However, temporal regulation of RhoA and its effectors in obesity-induced kidney damage remains unclear. Here, we investigated the role of RhoA activation in the proximal tubules at the early and late stages of obesity-induced kidney damage. In mice, a three-week high-fat-diet induced proximal tubule hypertrophy and damage without increased albuminuria, and RhoA/mDIA1 activation without ROCK activation. Conversely, a 12-week high-fat diet induced proximal tubule hypertrophy, proximal tubule damage, increased albuminuria, and RhoA/ROCK activation without mDIA1 elevation. Proximal tubule hypertrophy resulting from cell cycle arrest accompanied by downregulation of the multifunctional cyclin-dependent kinase inhibitor p27Kip1 was elicited by RhoA activation. Mice overexpressing proximal tubule-specific and dominant-negative RHOA display amelioration of high-fat diet-induced kidney hypertrophy, cell cycle abnormalities, inflammation, and renal impairment. In human proximal tubule cells, mechanical stretch mimicking hypertrophy activated ROCK, which triggered inflammation. In human kidney samples from normal individuals with a body mass index of about 25, proximal tubule cell size correlated with body mass index, proximal tubule cell damages, and mDIA1 expression. Thus, RhoA activation in proximal tubules is critical for the initiation and progression of obesity-induced kidney damage. Hence, the switch in the downstream RhoA effector in proximal tubule represents a transition from normal to pathogenic kidney adaptation and to body weight gain, leading to obesity-induced kidney damage.

We recently reported that fluorescent dye PB430, which consisted of a 2-phenyl-substituted benzophosphole P-oxide skeleton that was reinforced by a methylene bridge, showed pronounced photostability and, thus, high utility for applications in super-resolution stimulated emission depletion (STED) microscopy. Herein, we replaced the methylene bridge with another P=O group to 1) investigate the role of the bridging moieties; and 2) further modulate the fluorescence properties of this skeleton. We synthesized a series of phospholo[3,2-b]phosphole-based dyes-trans-PO-PB430, cis-PO-PB430, and trans-PO-PB460-all of which showed sufficient water solubility. Moreover, trans-PO-PB430 and trans-PO-PB460 exhibited intense green and orange fluorescence, respectively, and a high photostability that was comparable to that of PB430. In contrast, cis-PO-PB430 underwent rapid photobleaching upon continuous photoirradiation, which demonstrated the importance of steric shielding of the polycyclic skeleton by the substituents on the bridging moieties. The fluorescence properties of these dyes were insensitive to concentration, pH value, and polarity changes of the environment in solution. In addition, even in the solid state, these dyes showed strong green to orange emissions. These results demonstrate the potential utility of trans-PO-PB430 and trans-PO-PB460 as highly photostable fluorescent dyes.

The present study aimed to investigate the effects of olmesartan, an antagonist for angiotensin II receptor type 1(AT1), on the activation of extracellular signal-regulated kinases (ERK)1/2, tissue remodeling, and pro-inflammatory signals in the right ventricle and lung of mice during the early phase of hypobaric hypoxia. Phosphorylation of ERK1/2 in both tissue types in response to hypoxia peaked at 1-3 days, and declined rapidly in the right ventricle, whereas in the lung it was sustained for at least 8 days. Upregulation of angiotensinogen mRNA was observed in the hypoxic lung at 4-9 days, but not in the hypoxic right ventricle and pulmonary artery. Olmesartan inhibited the hypoxia-induced phosphorylation of ERK1/2 in the lung, but not in the right ventricle. Neither right ventricular hypertrophy nor the thickening of the intrapulmonary arterial wall was ameliorated by olmesartan. However, this drug inhibited the expression of the mRNA for angiotensinogen and several pro-inflammatory factors, including interleukin-6 and inducible nitric oxide synthase in the hypoxic lung. These results suggest that olmesartan blocks a potential positive feedback loop of the angiotensin II-AT1 receptor system, which may lead to attenuate pro-inflammatory signals in the mouse lung, that are associated with hypoxic pulmonary hypertension, without inducing any appreciable effects on the compensatory cardiopulmonary hypertrophy at an early phase of exposure to a hypobaric hypoxic environment.

We developed a method of hybrid particle/cubic interpolated propagation (CIP) to predict the motion of micro- and macrofree surfaces within gas-liquid flows. Microfree surfaces (smaller than the grid sizes) were simulated with the particle method, and macrofree surfaces (larger than the grid sizes) were simulated with the grid method (CIP is a kind of grid method). With the hybrid, velocities given by the advection part of the particle method were combined with those given by the advection part of CIP. Furthermore, the particles used with the particle method were assigned near the macrofree surfaces by using the volume fraction of liquid that was calculated with CIP. The method we developed was used to predict the collapse of a liquid column. Namely, it was simultaneously able to predict both large deformation in the liquid column and its fragmentation, and the predicted configurations for the liquid column agreed well with the experimentally measured ones. It was also used to predict the behavior of liquid films at the outlet of a fuel injector used for automobile engines. The particle method in the simulation was mainly used for liquid films in the air region and the grid method was used for the other regions to shorten the computational time. The predicted profile of the liquid film was very sharp in the air region where the liquid film became thinner than the grid sizes; there was no loss of liquid film with numerical diffusion.

Fine atomization of the liquid jet from a fuel injector in an automobile engine lowers engine emissions and improves fuel efficiency. Simulation of liquid-film breakup near the injector outlet is useful for predicting the atomization. However, grid methods and particle methods require a large amount of computation time in the practical engineering aspect because fine meshes smaller than liquid films or a large number of particles are needed. In this study, we have tried to simulate the breakup of liquid films by using groups of particles in the particle method. In the simulation, the particle method was applied only to the liquid film and the grid method was used in other areas to shorten the computation time. We then simulated the breakup of a liquid film near the outlet of a fuel injector used for automobile engines, and found that our hybrid method could simulate the breakup of the liquid film into ligaments.

Muscle contraction is accompanied by passive stretching or deformation of cells and tissues. The present study aims to clarify whether or not acute passive stretching evokes glucose transporter 4 (GLUT4) translocation and glucose uptake in skeletal muscles of mice. Passive stretching mainly induced GLUT4 translocation from an intracellular membrane-rich fraction (PF5) to a plasma membrane-rich fraction (F2) and accelerated glucose uptake in hindlimb muscles; whereas electrical stimulation, which mimics physical exercise in vivo, and insulin, each induced GLUT4 translocation from an intracellular membrane-rich fraction (PF5) to a fraction rich in plasma membrane (F2), and to one rich in transverse tubules (PF3), along with subsequent glucose uptake. Mechanical stretching increased phosphorylation of Akt and p38 mitogen-activated protein kinase (p38 MAPK), but it had no apparent effect on the activity of AMP-activated protein kinase (AMPK). Electrical stimulation augmented the activity of not only AMPK but also phosphorylation of Akt and p38 MAPK. Our results suggest that passive stretching produces translocation of GLUT4 mainly from the fraction rich in intracellular membrane to that rich in plasma membrane, and that the glucose uptake could be Akt- and p38 MAPK-dependent, but AMPK-independent manners.

Obesity frequently promotes a variety of cardiovascular diseases including atherosclerosis, hypertension, and type 2 diabetes. In a view of both the preventive and therapeutic aspects of the abovementioned diseases, most intensive clinical interventions have been primarily directed at decreasing excessive amounts of fat tissue by a change in the balance between intake and expenditure of energy; such changes are typically effected via daily exercise and diet control. Mechanical stimuli such as stretching and rubbing of fat tissues using gymnastic exercises or massage are believed to decrease obesity; however, there is no report concerning the direct effect of the mechanical stimulation on adipocytes. Here, we demonstrated that cyclic stretch inhibited adipocyte differentiation of mouse 3T3-L1 cells, which was attributable to a reduced expression of adipogenic transcription factor peroxisome proliferator-activated receptor (PPAR)gamma(2) via the activation of an extracellular signal-regulated protein kinase (ERK) pathway. The inhibitory effect of the cyclic stretching on the differentiation of 3T3-L1 cells could be restored by troglitazone, a synthetic ligand for PPARgamma. Our results provide a molecular basis for the physiological significance of the local application of mechanical stimuli to fat tissues, which is totally independent of a mechanism for systemic energy consumption.

Stretch-induced contraction of rabbit pulmonary artery depends on endothelium-derived vasoactive prostanoids. We investigated which prostanoid(s) was responsible for the stretch-induced contraction of the artery, and whether integrin was involved in this mechanotransduction process. Stretch increased productions of untransformed prostaglandin H(2), prostaglandin E(2), prostaglandin F(2alpha), and thromboxane A(2) in the pulmonary artery with intact endothelium. A blocking peptide for integrins (RGD peptide) significantly inhibited productions of thromboxane A(2) and prostaglandin F(2alpha), but the peptide did not affect productions of untransformed prostaglandin H(2) and prostaglandin E(2), as well as contraction in response to stretch. SQ29,548, a prostaglandin H(2)/thromboxane A(2) receptor antagonist, inhibited the contractile response to not only stretch but also exogenous prostaglandin H(2). Acetylcholine (up to 30 microM) also contracted the artery in an endothelium-dependent manner. Ozagrel (10 nM-1 microM), an inhibitor of thromboxane synthase, abolished the production of thromboxane A(2), in response to both stretch and acetylcholine, whereas the inhibitor mostly inhibited acetylcholine-induced contraction, but it did not suppress stretch-induced contraction. The results suggested that prostaglandin H(2) and thromboxane A(2), either released from endothelium by mechanical stretch or by acetylcholine, produced contraction of rabbit pulmonary artery in a RGD-independent manner.

The effects of protein-tyrosine kinase (PTK) and protein-tyrosine phosphatase (PTP) inhibitors on voltage-activated barium currents (I(Ba)) through L-type calcium channels increased by hypotonic solution were investigated in canine basilar arterial myocytes by the whole-cell patch-clamp technique. I(Ba) was elicited by depolarizing step from a holding potential of -80 to +10 mV and identified by using an L-type calcium channel agonist, Bay K 8644 (100 nM), and an L-type calcium channel blocker, nicardipine (1 microM). Hypotonic superfusate induced cell swelling and acted as a stretch stimulus, which reversibly increased peak I(Ba) amplitude at +10 mV. I(Ba) was also decreased by nicardipine (1 microM) under the hypotonic condition. PTK inhibitors such as herbimycin A (30 nM), genistein (10 microM), and lavendustin A (10 microM) decreased I(Ba) enhanced by hypotonic solution. Genistein also decreased I(Ba) in a concentration-dependent manner under the isotonic condition. The inactive genistein analogue daidzein (10 microM) had no effect on I(Ba) under either the isotonic or hypotonic condition. By contrast, herbimycin A did not decrease I(Ba) under the isotonic condition. Sodium orthovanadate (10 microM), a PTP inhibitor, increased I(Ba) under both conditions. The present results suggest that cell swelling by hypotonic solution increases the L-type calcium channel currents in canine basilar artery and that herbimycin-sensitive PTK activity is primarily involved in the enhancement of calcium channel currents.

The mechanism by which fasudil inhibits pressure-induced myogenic contraction was studied with regard to tyrosine phosphorylation in rat cerebral artery. Intracellular Ca2+ concentration ([Ca2+]i) and vessel diameter were simultaneously measured. Total tyrosine phosphorylation level and phosphorylation of tyrosine 419 on pp60src required for its full catalytic activity were immunocytochemically detected in situ. Fasudil (1–100 μM) partially suppressed the increase in [Ca2+]i, and totally attenuated contraction elicited by pressurization from 10 to 60 mmHg. Furthermore, fasudil (100 μM) significantly attenuated tyrosine phosphorylation and the activity of pp60src augmented in situ by pressure. Herbimycin A (1–100 nM) and genistein (3–30 μM), tyrosine kinase inhibitors, effectively attenuated the pressure-induced increase in [Ca2+]i, contraction, tyrosine phosphorylation, and activation of pp60src. Both fasudil and herbimycin A directly inhibited the pp60src activity in a cell free system. Orthovanadate (100 μM), a tyrosine phosphatase inhibitor, significantly potentiated the pressure-induced increase in [Ca2+]i and contraction. Nicardipine (100 nM), a Ca2+ antagonist, completely inhibited pressure-induced increase in [Ca2+]i and contraction, but affected neither tyrosine phosphorylation nor activity of pp60src in the pressurized arteries. Arginine-glycine-aspartic acid-serine peptide (1–100 μM) concentration-dependently reduced the pressure-induced contraction. In addition to the hitherto reported vasodilatory actions of fasudil, the present results suggest the inhibition by fasudil of pressure-induced tyrosine phosphorylation and pp60src activation. The wide spectrum of inhibitory actions of fasudil may contribute to the effective attenuation of the pressure-induced contraction in the cerebral artery. British Journal of Pharmacology (2000) 130, 219–230; doi:10.1038/sj.bjp.0703292

With regard to the mechanotransduction mechanisms of vasculature involved in hypertensive diseases, we aimed to identify tyrosine-phosphorylated proteins in pulmonary artery that responded to mechanical stress. Mechanical stretch simultaneously augmented protein-tyrosine phosphorylation in p55, p95, p105, p115, p130, p165, p180 in pulmonary artery tissue and pulmonary artery-derived smooth muscle cells (PASMC), whereas p115 and p55 were preferentially phosphorylated by the stretch in endothelial cells (PAEC). A series of experiments designed to characterize these proteins indicated that p115 and p180 were focal adhesion kinase (FAK) and platelet-derived growth factor receptor β (PDGF-Rβ), respectively, and that stretch augmented the surface-expression of PDGF-Rβ in PASMC but not in PAEC. Moreover, a significant increase in the steady-state mRNA level for PDGF-Rβ was observed in the pulmonary artery of rats with monocrotaline-induced pulmonary hypertension, where the artery should be overstretched due to increasing pulmonary arterial blood pressure. These results suggest that stretch-induced overexpression of cell-surface PDGF-Rβ as well as augmentation of tyrosine phosphorylation of proteins including FAK in PASMC might be involved in the mechanotransduction of pulmonary artery.

1 The mechanism of stretch-induced contraction of the intrapulmonary artery of rabbit was studied with special regard to the endothelium-dependence and production of prostanoids. 2 Isolated intrapulmonary artery of rabbits in ring form produced contraction when stretched slowly up to 180% of its initial muscle length (=100%) at a rate of 0.44 mm s−1, with a stimulus period of 5 min. 3 The stretch-induced contraction was attenuated by the mechanical removal of the endothelium, inhibitors of cyclo-oxygenase such as aspirin and indomethacin, [1S-[1α,2α(Z),3α,4α]]-7-[3-[[2-[(phenylamino) carbonyl] hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (SQ 29,548), which is a thromboxane A2/prostaglandin H2 receptor antagonist, or by ozagrel, an inhibitor of thromboxane A2 synthase. 4 Biochemical assay indicated that the production of thromboxane B2, a stable metabolite of thromboxane A2, was increased 17 times in response to stretch only when the endothelium was intact. The production of thromboxane B2 was also inhibited by aspirin or ozagrel. 5 The production of 6-keto prostaglandin F1α, a stable metabolite of prostacyclin, was also increased in response to stretch in the preparation with intact endothelium. However, ozagrel showed no apparent effect on the production of 6-keto prostaglandin F1α. 6 These results suggest that a mechanical stimulus like stretch can act on endothelial cells of rabbit pulmonary artery to cause contraction by activation of arachidonic acid metabolism via the cyclo-oxygenase pathway and subsequent release of thromboxane A2 and/or an increase in the ratio of thromboxane A2/prostacyclin. British Journal of Pharmacology (1997) 122, 199–208; doi:10.1038/sj.bjp.0701362

Adipogenesis is a fundamental process to develop adipose tissues via commitment of mesenchymal stem cells (MSCs) to direction of preadipocytes and production of terminally differentiated lipid-laden adipocytes. While adipose tissues play important roles for energy and metabolic homeostasis in our body, dysregulated adiposities become considerable risk factors for various metabolic and cardiovascular diseases. In view of both preventive and therapeutic aspects, clinical interventions have been mostly directed at control of adiposity such as weight control by improved balance of calorie intake and energy expenditure. Physical exercise has been considered to be an effective approach to improve the systemic energy balance; however, the effects of locally generated mechanical stress on adipose tissues that are directly or indirectly accessible by the exercise and/or massage had remained obscure. It has now become apparent that stretching and other mechanical stimuli activate various cellular signals, including matrix elasticity/stiffness and cytoskeletal control, extracellular matrix–integrin interaction, the extracellular signal-regulated protein kinase/mitogen activated protein kinase (ERK/MAPK), Rho–Rho-kinase pathway, tension-induced/inhibited proteins (TIPs), the cyclooxygenase pathway, and Wnt signaling; all of them are involved in the mechanotransduction pathways and have a significant influence on adipogenesis. The stretching shows bidirectional effects of either inhibition or stimulation on adipogenesis that is presumably depending on the strength, duration, and timing of mechanical inputs, as well as cellular statuses of differentiation. The ERK/MAPK plays a crucial role in the bidirectional outcomes, and other coexisting signals adjust and determine the commitment and adipogenic statuses of MSCs and preadipocytes. These results imply that the mechanical stimulation would modulate the adipose tissue functions through the bidirectional control of adipocyte renewal, adiposity, endocrine function of adipocytes, and fine-tuning of drug actions.

Mechanotransduction is the molecular and cellular processes that transduce mechanical/physical forces into biochemical signals followed by diverse intracellular signalings and cell responses. The cardiovascular system is known to be particularly sensitive to mechanical stimuli such as blood pressure and blood flow. Interestingly, pancreatic β-cells, adipocytes, and skeletal muscle cells, all of which are related to the core concerns in metabolic syndrome causing cardiovascular diseases, are also responsive to mechanical stimuli, such as osmotic change and stretching, and show a variety of functions, including insulin release from β-cells, depression of adipocyte differentiation, and translocation of glucose transporter 4 (GLUT4) in skeletal muscle cells. Furthermore, the fish-oil-derived ω-3 polyunsaturated fatty acid such as eicosapentaenoic acid (EPA) combined with cyclic stretching significantly reduces the adipocyte differentiation. The present article is enable to unitarily discuss three peripheral organs from the viewpoint of mechanosensitivity, which aids in recognizing the importance of biomechanical factors in physiological and pathological conditions, and may potentially have therapeutic consequences.

The fatigue crack growth behavior of a high-strength aluminum alloy, 7075-T651, was examined in deaerated and aerated 3.5% NaCl solution, in addition to in air. The relation between stress intensity range and crack growth rate in the same environment depended on stress ratio. For long cracks, the crack growth rate was always higher at higher stress ratio, while it was sometimes lower at higher stress ratio for short surface cracks. The effect of oxygen concentration was not observed for either long cracks or short surface cracks. The growth rate of long cracks in corrosion fatigue was almost the same as that in air when the stress intensity range was smaller than 7 MPam1/2. In this region, however, the growth rate of short surface cracks in the solution was higher than that in air, and the growth rate of short surface cracks was higher than that of long cracks in the same environment. Although the difference in crack closure was considered to be responsible for the acceleration of short surface crack growth in air, the acceleration of short surface crack growth rate in corrosion fatigue could not be explained solely by the difference in crack closure. The difference in chemical environment within the crack should be considered.

Involvement of secretory phospholipase A2 (sPLA2) in the stretch-induced production of untransformed prostaglandin H2 (PGH2) in the endothelium of rabbit pulmonary arteries was investigated. The stretch-induced contraction was significantly inhibited by indoxam, a selective inhibitor for sPLA2, and NS-398, a selective inhibitor for cyclooxygenase-2 (COX-2). Indoxam inhibited the RGD-sensitive-integrin–independent production of untransformed PGH2, but did not affect the RGD-sensitive-integrin–dependent production of thromboxane A2 (TXA2). These results suggest that the stretch-induced contraction and untransformed PGH2 production was mediated by sPLA2–COX-2 pathway, making it a new possible target for pharmacological intervention of pulmonary artery contractility. Keywords:: stretch-induced pulmonary artery contraction, secretory phospholipase A2 (sPLA2), indoxam

The development of obesity involves multiple mechanisms. Here, we identify adipocyte signaling through the guanosine triphosphatase Rho and its effector Rho-kinase as one such mechanism. Mice fed a high-fat diet (HFD) showed increased Rho-kinase activity in adipose tissue compared to mice fed a low-fat diet. Treatment with the Rho-kinase inhibitor fasudil attenuated weight gain and insulin resistance in mice on a HFD. Transgenic mice overexpressing an adipocyte-specific, dominant-negative form of RhoA (DN-RhoA TG mice) showed decreased Rho-kinase activity in adipocytes, decreased HFD-induced weight gain, and improved glucose metabolism compared to wild-type littermates. Furthermore, compared to HFD-fed wild-type littermates, DN-RhoA TG mice on a HFD showed decreased adipocyte hypertrophy, reduced macrophage recruitment to adipose tissue, and lower expression of mRNAs encoding various adipocytokines. Lipid accumulation in cultured adipocytes was associated with increased Rho-kinase activity and increased abundance of adipocytokine transcripts, which was reversed by a Rho-kinase inhibitor. Direct application of mechanical stretch to mature adipocytes increased Rho-kinase activity and stress fiber formation. Stress fiber formation, which was also observed in adipocytes from HFD-fed mice, was prevented by Rho-kinase inhibition and in DN-RhoA TG mice. Our findings indicate that lipid accumulation in adipocytes activates Rho to Rho-kinase (Rho-Rho-kinase) signaling at least in part through mechanical stretch and implicate Rho-Rho-kinase signaling in inflammatory changes in adipose tissue in obesity. Thus, inhibition of Rho-Rho-kinase signaling may provide a therapeutic strategy for disrupting a vicious cycle of adipocyte stretch, Rho-Rho-kinase signaling, and inflammation of adipose tissue that contributes to and aggravates obesity.

To elucidate the mechanism of tumor necrosis factor (TNF) production, we analyzed proteins produced in macrophages sharing the epitope of TNF according to the priming and triggering of TNF production. Rabbit alveolar macrophages primed with Bacillus Calmette-Guerin (BCG) were isolated and cultured in vitro with 35S-methionine, and the proteins produced were analyzed using anti-rabbit TNF monoclonal antibody. Primed with BCG, alveolar macrophages synthesized two proteins with molecular sizes of 50 and 17 kilodaltons (kDa) (p50 and p17) sharing the same epitope with mature TNF within the cells. These two proteins were released into the medium where other proteins were detected without TNF-activity. Cultured with lipopolysaccharide (LPS triggering), the primed alveolar macrophages released TNF-activity into the medium where p17 together with many larger proteins was detected by immunoprecipitation. In vitro translation of messenger ribonucleic acid (mRNA) from BCG-primed macrophages showed that primary TNF has a molecular size of 28 kDa (p28). These results suggest that active TNF of p17 is secreted when triggered via post-translational processing of the precursor molecules synthesized through priming with BCG.

To simulate multi-scale free surfaces, we developed a hybrid particle/grid method by which the free surfaces within sub-grid regions are simulated by the particle method, and other regions are simulated with the grid method. The particle method uses two types of particles to model gas and liquid fluids in order to simulate the interaction between them. We tested the new method on fragmentation of a water column, and the predicted configurations of the water column are consistent with measurements of Koshizuka and Oka. We also simulated the fuel spray near the outlet of an automobile-engine fuel injector and found that this method qualitatively simulated the breakup of the liquid film.Copyright © 2007 by ASME

This study investigated the effects of cyclic stretching on adipocyte differentiation of mouse preadipocyte 3T3-L1 cells. Confluent 3T3-L1 cells were treated with dexamethasone, 3-isobutyl-1-methylxanthine and insulin for 45 hours (induction period), followed by incubation with insulin for 9 additional days (maturation period). A transient burst of CCAAT/enhancer-binding protein (C/EBP) beta and C/EBPdelta at an early stage (approximately 3 hours) and a delayed induction (approximately 45 hours) of C/EBPalpha and PPARgamma(2) were sequentially provoked during the induction period. Application of cyclic stretching during the entire induction period or only during the final 15 hours of the induction period significantly retarded the induction of glycerol-3-phosphate dehydrogenase (GPDH) activity and the accumulation of intracellular triglycerides by the end of the maturation period. Cyclic stretching for the entire induction period, as well as that applied during the final 15 hours of the induction period, significantly reduced the expression of PPARgamma(2) mRNA, whereas reduction in the expression of C/EBPdelta mRNA was only observed in response to stretching that had been applied during the entire induction period. The expression of C/EBPalpha and C/EBPbeta mRNA did not change in response to stretching. Stretching induced the phosphorylation of extracellular-signal-regulated protein kinases 1 and 2 (ERK1/2), which are members of the mitogen-activated-protein kinase (MAPK) family, during the induction period. PD98,059, a MAPK/ERK kinase inhibitor, reversed the stretch-induced reduction of PPARgamma(2) at both mRNA and protein levels achieved during the induction period. PD98,059 also restored GPDH activity and lipid droplet accumulation. Furthermore, the differentiation inhibited by the stretching was also restored by synthetic PPARgamma ligand. Collectively, these results suggest that the inhibition of adipocyte differentiation in response to stretching is mainly attributable to the reduced expression of PPARgamma(2), which is mediated by activation of the ERK/MAPK system.

We propose a spray pattern control method for swirl-type injectors in direct injection (Dl) gasoline engines. An L-cut orifice nozzle (L-Step nozzle) that produces a horseshoe spray pattern is used to create a rich and lean concentration region. To further control the distribution of fuel, the relationship between the nozzle geometry and the spray pattern is investigated. The mechanism for a horseshoe spray formation is hypothesized and verified through experiment. The spray shape and fuel distribution is found to be controllable by configuring the L-cut step-walls. Furthermore, it is discovered that independent control of rich and lean region distribution is possible by arranging the step-wall position and height.

The autoregulatory mechanism, including myogenic response, so-called "Bayliss effect", is well developed in the brain circulatory area, where also, cerebral vasospasm is often encountered after subarachnoid hemorrhage. In the cerebral artery smooth muscle, protein kinases, such as Rho-associated kinase, tyrosine kinase, and protein kinase C, are activated in response to mechanical stresses, including stretch, pressure and flow. All of these kinases are also activated in due course of time after development of the vasospasm. Myogenic response is a kind of reception and subsequent reaction to mechanical stress, whereas cerebral vasospasm is primarily caused by oxyhemoglobin, i.e., oxidative stress. Thus both myogenic and vasospastic episodes imply a common stress-responding mechanism. It seems possible that various kinases activated by mechanical stress act as not only a physiological signaling but also a proatherogenic/remodeling one. The stiffness of vasospastic artery was enormously increased in particular in the late phase of vasospasm, indicating the augmented process of pathologic remodeling. Therefore, "Bayliss effect" in modern sense and cerebral vasospasm can be argued in terms of a stress-reaction of cerebral artery.

To predict motions of micro and macro free surfaces simultaneously within gas-liquid flows, we have developed a particle/CIP (Cubic Interpolated Propagation) hybrid method. The micro free surfaces (smaller than grid sizes) were simulated by the particle method, and the macro free surfaces (larger than grid sizes) were simulated by the CIP method. And then the particles used in the particle method were assigned near the macro free surfaces by using volume fraction of liquid that was calculated by the CIP method. The developed method was used to predict the collapse of a liquid column. Namely, it predicted both the large deformation of the liquid column and the fragmentation of it simultaneously, and the predicted configurations of the liquid column agreed well with the experimentally measured ones. It was also used to predict breakup of liquid films in a fuel injector used for engines of automobiles, and the predicted profile of the liquid film was sharp in an air region where the thickness of the liquid film became thinner than the grid sizes.

Blood vessels are always subjected to hemodynamic stresses including blood pressure and blood flow. The cerebral artery is particularly sensitive to hemodynamic stresses such as pressure and stretch, and shows contractions that are myogenic in nature; i.e., the mechanical response is generated by the vascular smooth muscle itself. The artery constricts in response to an increase in intraluminal pressure, and dilates in response to a decrease in the intraluminal pressure. We provide herein some insights into the mechanotransduction of vascular tissue; i.e., we discuss how the tissue is receptive to mechanical force and how the latter induces the specific signals leading to myogenic contraction in terms of mechanosensor action and subsequent intracellular signaling. The interactive role of tyrosine kinase, protein kinase C, and Rho/Rho-kinase systems in the mechanotransduction process is discussed, which systems also seem to play an important role in the development of experimental cerebral vasospasm. The study of the mechanotransduction in vascular tissue may aid in clarifying the mechanisms underlying vasospastic episodes and pathologic remodeling in cardiovascular diseases, and may potentially have therapeutic consequences.

To clarify the mechanism of contractile strengthening by endothelin-1 (ET-1), we measured translocation of protein kinase C (PKC) from the cytosol to the membrane fraction in the porcine coronary artery. ET-1 potentiated the serotonin- (5-hydroxytryptamine, 5-HT) induced contraction without any additional increase in myosin light chain phosphorylation. Four PKC isoforms (alpha, beta1, delta, and zeta) were identified but not PKC epsilon. Only PKC delta was translocated from the cytosolic to the membrane fraction during the contractile potentiation by ET-1. Our results suggest that the activity of PKC delta but not PKC epsilon is involved in the contractile strengthening by ET-1 in the porcine coronary artery.

OBJECTIVE We examined the hypothesis that a plasma protease-antiprotease imbalance contributes to the formation of saccular cerebral aneurysms and the suggestion that the assay of these enzymes might be a screening tool for people at higher risk for aneurysm formation. METHODS From June 1997 through May 1998, the plasma leukocyte elastase, which is an important proteolytic enzyme, and alpha-antitrypsin and alpha2-macroglobulin, which are important antiproteolytic enzyme plasma proteins, were examined in 18 patients with ruptured aneurysms, 9 patients with unruptured aneurysms, and 22 controls. RESULTS The elastase:alpha1-antitrypsin ratio and the elastase:alpha2-macroglobulin ratios were significantly higher in patients with ruptured aneurysms within 24 hours after subarachnoid hemorrhage (SAH) than in the controls. The protease-antiprotease imbalance depended on the elevation of the elastase level, which might be correlated with leukocytosis after SAH. The elastase level decreased to the control level 3 months after the onset of SAH. No significant difference in the elastase:alpha1-antitrypsin and elastase:alpha2-macroglobulin ratios was observed between the patients with unruptured aneurysms and the controls. CONCLUSION These results do not support the hypothesis that a plasma protease-antiprotease imbalance is a potential marker to predict the formation of saccular cerebral aneurysms. The increase in plasma elastase levels in patients with ruptured aneurysms might be attributable to leukocytosis after SAH.

The authors have previously shown that 26-kDa membrane-bound tumour necrosis factor precursor (proTNF) on the cell-surface of primed human monocytic cell line THP-1 is involved in positive feedback regulation of lipopolysaccharide (LPS)-dependent TNF-production. Here, we provide direct evidence for modulation of responsiveness of the THP-1 cells against LPS by membrane-bound proTNF. When THP-1 cells were cocultivated with a heterogeneous cell line (proTNF/3T3 cells) which constitutively expressed membrane-bound proTNF, LPS-dependent TNF-production by THP-1 cells was significantly suppressed and the normal level was restored by the presence of anti-TNF antibody during cocultivation. The proTNF-3T3-induced decline of TNF-production of THP-1 was observed primarily at the mRNA level, although no difference was observed in the mRNA level of interleukin 1β, another LPS-inducible cytokine. These results suggest that proTNF could also be involved in the negative feedback regulation of LPS-dependent TNF-production through cell-to-cell contact. The augmentation of LPS-dependent TNF-production accompanied by the production of endogenous proTNF induced by exogenous agent was inhibited by protein kinase C inhibitor, whereas proTNF/3T3-induced suppression of TNF production could not be restored to the normal level. It thus seems possible that proTNF might act on macrophages as a bidirectional regulator of its production by THP-1 cells depending on co-induced signals.

In order to determine whether protein tyrosine kinase mechanisms are involved in pressure-induced contraction, we compared effects of three structurally unrelated tyrosine kinase inhibitors and orthovanadate, a tyrosine phosphatase inhibitor, on the pressure-induced contraction of the posterior cerebral artery isolated from rats. The change in vessel diameter was continuously measured with a width analyzer. Herbimycin A inhibited the pressure-induced contraction, while it only slightly inhibited contractions produced by potassium chloride or 9,11-dideoxy-11α,9α-epoxymethano prostaglandin F 2α (U46619). Genistein inhibited not only the pressure-induced contraction but also the U46619-induced one. Tyrphostin 23 significantly attenuated contractions in response to three different stimuli, i.e., pressure, potassium chloride and U46619. Orthovanadate potentiated the pressure-induced contraction. These results suggest that herbimycin A is a specific and potent inhibitor of the pressure-induced contraction and that a protein tyrosine kinase mechanism may play an important role in the genesis of the pressure-induced contraction of the rat cerebral artery.

To clarify the biological significance of tumour necrosis factor alpha (TNF-alpha) precursor, we analysed its expression at the primed and triggered stages using human monocyte-like cell line THP-1. To prime them, THP-1 cells were treated with either recombinant human interferon gamma (rIFN-gamma) or recombinant human tumour necrosis factor alpha (rTNF-alpha). At the primed stage, transient accumulation of TNF-alpha, mRNA and a small amount of 26-KDa TNF-alpha precursor was observed, and the precursor molecule was located on the cell surface. Following treatment of the primed cells with bacterial lipopolysaccharide (LPS), augmentation of transcription of TNF-alpha mRNA and production of a larger amount of TNF-alpha precursor were observed followed by secretion of a larger amount of mature TNF-alpha (17-KDa) than secreted by the unprimed cells (triggered stage). This suggests that with priming THP-1 cells might be changed to a stage where they are ready for production of a larger amount of TNF-alpha at the triggered stage. When either primed or unprimed THP-1 cells were pretreated with anti-TNF-alpha antibody, augmentation of TNF-alpha production by primed THP-1 cells was specifically suppressed, suggesting that TNF-alpha precursor itself may play an important role in the enhancement of TNF-alpha production by the primed macrophages after treatment with LPS.

To study its biological functions, tumor necrosis factor precursor (proTNF) with a molecular size of 26-KDa was obtained as a recombinant protein from Escherichia coli. The recombinant proTNF was successfully accumulated in the insoluble form, corresponding to about 10–15% of total E. coli proteins. Solubilization, gel filtration and anion exchange chromatography were performed under denatured conditions followed by dialysis in phosphate-buffered saline. These processes removed most of the contaminating bacterial proteins, yielding proTNF with a purity of about 70–80%. This recombinant proTNF is expected to be useful for functional studies on activated macrophages with membrane integrated proTNF.