Sustained Tension Research Articles

Biomechanical response of bovine temporomandibular joint disc to prolonged tensile stress

This study was designed to evaluate the influence of prolonged tensile stress on the viscoelasticity of the temporomandibular joint (TMJ) disc. Twenty discs from 10, 3-year-old cattles were used. Tensile stress of 1.5 MPa was applied to specimens from the discs for 10, 20, 40 and 60 min. Following the prescribed period of tension for creep, the specimens were removed from the tension device and any recovery observed for 20 min. In all specimens, strain increased at the onset of stress application and reached almost steady conditions after 5 min. Although, the strain became slightly larger when the creep time was longer, no significant differences were found in the strains between any two tests with different periods of creep. The residual strain increased significantly with creep duration, and similarly the degree of recovery decreased significantly. In 10- and 20-min creep tests, the residual strains were 0.1 and 1.0%, the specimens in 40- and 60-min tests revealed irreversible changes in length. It was concluded that continuous loading for >40 min causes creep damage in bovine TMJ disc, and that prolonged sustained tension affects the recovery of joint homeostasis.

Dimethyl sulphoxide enhances the effects of P(i) in myofibrils and inhibits the activity of rabbit skeletal muscle contractile proteins.

In the catalytic cycle of skeletal muscle, myosin alternates between strongly and weakly bound cross-bridges, with the latter contributing little to sustained tension. Here we describe the action of DMSO, an organic solvent that appears to increase the population of weakly bound cross-bridges that accumulate after the binding of ATP, but before P(i) release. DMSO (5-30%, v/v) reversibly inhibits tension and ATP hydrolysis in vertebrate skeletal muscle myofibrils, and decreases the speed of unregulated F-actin in an in vitro motility assay with heavy meromyosin. In solution, controls for enzyme activity and intrinsic tryptophan fluorescence of myosin subfragment 1 (S1) in the presence of different cations indicate that structural changes attributable to DMSO are small and reversible, and do not involve unfolding. Since DMSO depresses S1 and acto-S1 MgATPase activities in the same proportions, without altering acto-S1 affinity, the principal DMSO target apparently lies within the catalytic cycle rather than with actin-myosin binding. Inhibition by DMSO in myofibrils is the same in the presence or the absence of Ca(2+) and regulatory proteins, in contrast with the effects of ethylene glycol, and the Ca(2+) sensitivity of isometric tension is slightly decreased by DMSO. The apparent affinity for P(i) is enhanced markedly by DMSO (and to a lesser extent by ethylene glycol) in skinned fibres, suggesting that DMSO stabilizes cross-bridges that have ADP.P(i) or ATP bound to them.

Dimethyl sulphoxide enhances the effects of Pi in myofibrils and inhibits the activity of rabbit skeletal muscle contractile proteins

In the catalytic cycle of skeletal muscle, myosin alternates between strongly and weakly bound cross-bridges, with the latter contributing little to sustained tension. Here we describe the action of DMSO, an organic solvent that appears to increase the population of weakly bound cross-bridges that accumulate after the binding of ATP, but before Pi release. DMSO (5–30%, v/v) reversibly inhibits tension and ATP hydrolysis in vertebrate skeletal muscle myofibrils, and decreases the speed of unregulated F-actin in an in vitro motility assay with heavy meromyosin. In solution, controls for enzyme activity and intrinsic tryptophan fluorescence of myosin subfragment 1 (S1) in the presence of different cations indicate that structural changes attributable to DMSO are small and reversible, and do not involve unfolding. Since DMSO depresses S1 and acto–S1MgATPase activities in the same proportions, without altering acto–S1 affinity, the principal DMSO target apparently lies within the catalytic cycle rather than with actin–myosin binding. Inhibition by DMSO in myofibrils is the same in the presence or the absence of Ca2+ and regulatory proteins, in contrast with the effects of ethylene glycol, and the Ca2+ sensitivity of isometric tension is slightly decreased by DMSO. The apparent affinity for Pi is enhanced markedly by DMSO (and to a lesser extent by ethylene glycol) in skinned fibres, suggesting that DMSO stabilizes cross-bridges that have ADP·Pi or ATP bound to them.

Experimental procedures and cavitation mechanisms in tensiometer measurements

In recent years tensiometers for direct measurement of matric suction have been developed at Imperial College and later on at the University of Saskatchewan. The major drawback of these instruments is water cavitation which may occur before pressure equalisation. A better understanding of the mechanisms that control cavitation inside the tensiometer may therefore help optimise their design and define adequate experimental procedures. This paper presents some of the experiences gained over the past four years using three tensiometers manufactured by Imperial College. Some of the anomalous responses recorded by tensiometers are first discussed. These were probably due to inadequate saturation of the porous ceramic rather than malfunctioning of the instruments. The results from a series of tests conducted with two or three tensiometers positioned on the same sample are then presented. These were helpful in defining suitable experimental procedures. Finally, the history of tension breakdown of each tensiometer is examined to identify some of the factors that affect the maximum sustainable tension as well as measurement duration. On the basis of data recorded before and after cavitation, a possible mechanism of cavitation in a tensiometer is described.

Use of the tensile strength of water for the direct measurement of high soil suction: Reply

Discussions The authors express their appreciation for the additional information provided by the Discussers on the subject of the tensile strength of water. This is a new subject area to the geotechnical literature and one that initially appears to go counter to the basic physics related to the behaviour of water. The test results and findings of the ongoing research pro gram on this subject, at Imperial College, U.K., have proven to be extremely valuable in better understanding the behaviour of water that has been conditioned through the use of pressurization process. The Discussers have pointed out that the sustainable tension in the direct measurement, high suction probe appears to be related to the air entry value of the ceramic. All of the tests performed by the authors were performed using a 1500 kPa ceramic. The maximum sustainable tension appeared to be limited by the ceramic; however, a repeated cavitation of the sensor appeared to reduce the maximum sustainable tension. Also, different ceramics with the same air entry value (i.e., 1500 kPa) often produced a different response both in terms of magnitude and response pattern. For example, in one case, it was not possible to sustain a maximum tension in excess of 300 kPa, no matter what attempts were made to pressurize and saturate the system. Even with the use of 10 000 kPa, the maximum sustainable tension could not be increased, no matter what attempts were made to saturate the system. The experience of the authors was that the 24 h pressurization period did not provide much advantage over the 12 h or even the 6 h pressurization period. The Discussers have emphasized the benefit of having a small water compartment. The authors found it difficult to determine the exact amount of water in the compartment, but a space ranging from 0.1 to 0.3 mm would appear to be favorable. Further studies, over a long period of time — under both field and in situ conditions — will prove extremely valuable in encouraging the direct measurement of high tensions in the water phase (i.e., high soil suctions). The Imperial College Suction Probe, as well as other probes, are indeed a welcome device in the application of unsaturated soil mechanics to geotechnical engineering.

Direct observations of the mechanical behaviors of the cytoskeleton in living fibroblasts.

Cytoskeletal proteins tagged with green fluorescent protein were used to directly visualize the mechanical role of the cytoskeleton in determining cell shape. Rat embryo (REF 52) fibroblasts were deformed using glass needles either uncoated for purely physical manipulations, or coated with laminin to induce attachment to the cell surface. Cells responded to uncoated probes in accordance with a three-layer model in which a highly elastic nucleus is surrounded by cytoplasmic microtubules that behave as a jelly-like viscoelastic fluid. The third, outermost cortical layer is an elastic shell under sustained tension. Adhesive, laminin-coated needles caused focal recruitment of actin filaments to the contacted surface region and increased the cortical layer stiffness. This direct visualization of actin recruitment confirms a widely postulated model for mechanical connections between extracellular matrix proteins and the actin cytoskeleton. Cells tethered to laminin-treated needles strongly resisted elongation by actively contracting. Whether using uncoated probes to apply simple deformations or laminin-coated probes to induce surface-to-cytoskeleton interaction we observed that experimentally applied forces produced exclusively local responses by both the actin and microtubule cytoskeleton. This local accomodation and dissipation of force is inconsistent with the proposal that cellular tensegrity determines cell shape.

Fire-Resistant Booms: From Testing to Operations1

ABSTRACT A great deal of concern and effort has gone into testing various fire resistant booms since the 1993 Newfoundland Offshore Burn Experiment (NOBE), when it became apparent that there were potential limitations in the performance of commercially manufactured fire booms. One of the major questions that arose after this experiment was the capability of fire booms to adequately support real in situ burn operations. Towing experiments on selected booms both at sea and in test tanks, coupled with data from burn tests based on proposed ASTM-F20 Standards, have begun to new reveal facts about the performance of these booms. Results of the at-sea towing tests indicate that, in general, booms with higher buoyancy-to-weight ratios attained higher critical tow speeds, sustained higher towing tensions, and maintained better wave conformance. Results of towing tests of booms containing oil at the OHMSETT test tank facility suggest that fire booms should perform successfully when tow speeds of less than 1.0 knot is maintained. Burn tests at the U.S. Coast Guard Fire and Test Detachment revealed that fire booms could be expected to maintain some structural stability and freeboard for at least three 1-hour burns during a deployment.

Contractile and Relaxant Effects of Tetrapentylammonium Ions in Rat Isolated Mesenteric Artery

Both contractile and relaxant responses to tetrapentylammonium ions (TPA⁺) were studied in rat isolated mesenteric artery. TPA⁺ (5–10 µmol/l) caused a sustained increase of muscle tension. The contractile effect of TPA⁺ (10 µmol/l) was dependent upon the presence of extracellular Ca²⁺ but independent of the presence of endothelium. TPA⁺ (10–50 µmol/l) induced biphasic contraction, and the amplitude of peak and sustained tension decreased with increasing TPA⁺ concentration. TPA⁺ (100–300 µmol/l) only produced monophasic contraction. TPA⁺ (50 µmol/l) abolished the transient contraction induced by caffeine (10 mmol/l) or phenylephrine (1 µmol/l) in the absence of extracellular Ca²⁺. Nifedipine and verapamil concentration-dependently reduced the TPA⁺-induced contraction with respective IC₅₀ values of 1.34 ± 0.24 and 9.46 ± 1.36 nmol/l, these values were similar to 1.35 ± 0.21 and 16.07 ± 1.71 nmol/l, respectively, for the inhibitory effects of nifedipine and verapamil on the high K⁺ (60 mmol/l)-induced contraction. TPA⁺ (>10 µmol/l) concentration-dependently reduced the phenylephrine (1 µmol/l)-, U46619 (30 nmol/l)-, endothelin I (10 nmol/l)- and high K⁺ (60 mmol/l)-induced sustained tension with respective IC₅₀ values of 53.7 ± 9.5, 31.9 ± 5.3, 30.9 ± 3.4 and 20.9 ± 2.8 µmol/l. The present results indicate that TPA⁺ at low concentrations could contract the arterial smooth muscle probably through promoting Ca²⁺ influx. At higher concentrations (>20 µmol/l), TPA⁺ relaxes arterial smooth muscle probably through inhibition of both nifedipine-sensitive Ca⁺ channels and internal Ca²⁺ release. TPA⁺, unlike other quaternary ammonium ions, could therefore act at multiple sites in arterial smooth muscle.

Mechanical Behavior and High‐Temperature Performance of a Woven Nicalon™/Si‐N‐C Ceramic‐Matrix Composite

A modern ceramic‐matrix composite (CMC) has been extensively characterized for a high‐temperature aerospace turbine‐engine application. The CMC system has a silicon‐nitrogen‐carbon (Si‐N‐C) matrix reinforced with Nicalon fibers woven in a balanced eight‐harness satin weave fabric. Tensile tests have demonstrated that this CMC exhibits excellent strength retention up to 1100°C. The room‐temperature fatigue limit was 160 MPa, ∼80% of the room‐temperature tensile strength. The composite reached run‐out conditions under cyclic (105 cycles at 1 Hz) and sustained tension (100 h) conditions at a stress of 110 MPa, which was ∼35 MPa above the proportional limits at temperatures up to 1100°C in air. At stress levels >110 MPa, cyclic loading at 1000°C caused a more severe reduction in life, based on time, compared with sustained tension. Further life degradation was observed in the 1000°C fatigue specimens that were exposed to a salt‐fog environment. This degradation decreased the fatigue life ∼85% at the stress levels that were tested.

Role of endothelium and K+ channels in dobutamine-induced relaxation in rat mesenteric artery.

1. In order to examine the possible involvement of the endothelium and K+ channel activation in the relaxation induced by dobutamine, a beta 1-adrenoceptor agonist, in rat isolated mesenteric arteries, the effects of inhibitors of nitric oxide (NO) activity, blockers of K+ channels and high extracellular K+ were studied by measuring isometric tension in both endothelium-intact and -denuded arteries. 2. Dobutamine inhibited the phenylephrine (PE)-induced sustained tension with a pEC50 of 7.40 +/- 0.08 in endothelium-intact arteries. Removal of functional endothelium attenuated the effect of dobutamine. The relaxation induced by dobutamine was inhibited by the beta 1-adrenoceptor antagonist CGP 20712A (3 mumol/L) but not by the beta 2-adrenoceptor antagonist ICI 118,551 (3 mumol/L) in endothelium-denuded arteries. 3. Pretreatment with NG-nitro-L-arginine (L-NNA; 100 mumol/L) or methylene blue (3 mumol/L) induced a similar degree of inhibition of the dobutamine-induced relaxation in endothelium-intact arteries, while NG-nitro-D-arginine (100 mumol/L) and indomethacin (10 mumol/L) had no effect. In contrast, pretreatment with L-NNA (100 mumol/L) did not affect the relaxation induced by sodium nitroprusside (SNP) or forskolin. Methylene blue (3 mumol/L) inhibited the relaxant response to SNP. 4. Charybdotoxin (CTX; 100 nmol/L), iberiotoxin (IBX; 100 nmol/L) and tetraethylammonium ions (TEA+; 3 mmol/L) significantly reduced the dobutamine-induced relaxation. Tetrapentylammonium ions (TPA+; 5 mumol/L) markedly inhibited the relaxant effect of dobutamine. The pEC50 values for control and in the presence of TPA+ in endothelium-intact arteries were 7.35 +/- 0.11 and 6.14 +/- 0.17, respectively, and 6.35 +/- 0.09 and 5.87 +/- 0.17 for control and in the presence of TPA+ in endothelium-denuded arteries, respectively. In contrast, glibenclamide (3 mumol/L) was ineffective. At 5 mumol/L, TPA+ also inhibited the relaxation induced by forskolin. 5. The maximal relaxation of PE-contracted arteries induced by 3 mumol/L dobutamine was completely abolished in the 60 mmol/L K(+)-contracted arteries with and without endothelium, while dobutamine at a concentration greater than 3 mumol/L induced inhibition of the high-K+ response. 6. The present results indicate that endothelium, probably NO but not prostacyclin, was involved in the dobutamine-induced relaxation in rat mesenteric arteries. Activation of CTX-, IBX- and TPA(+)-sensitive K+ channels contributed towards the observed relaxation. Loss of the ability to relax the 60 mmol/L K(+)-contracted arteries suggests that endothelium-derived vasoactive factors affected by concentrations of dobutamine less than 3 mumol/L may also act through K+ channels in our preparations. Higher concentrations of dobutamine may have a direct, endothelium-independent relaxant effect.

Application of the Orthotropic Damage Growth Rule to Variable Principal Directions

The classical orthotropic brittle damage law [Kachanov (1958)] is applicable for principal directions of the stress tensor. When shear stresses are accounted, the principal directions of the stress tensor rotate with time and, hence, a tensorial formulation of the damage is required [Chow and Lu (1992)]. In general, current principal directions of the stress tensora, and of the damage tensorp3B do not coincide, however, when the principal axes of stress rotate due to the shear effect, the principal axes of damage follow them. The symmetric second rank damage tensor 2 [Murakami and Ohno (1981)] is applied, and the objective derivative Q of the damage tensor is adopted, to account for the effect of rotation of principal directions on the damage accumulation process. Then, a current transformation to the global coordinate system (sampling coordinate space) is performed. The similarity of deviators based on the flow theory and on the time hardening hypothesis, associated with the orthotropic damage growth rule, both written for current principal directions of stress, are taken as constitutive relationships for creep [Boyle and Spence (1983), Kachanov (1986)]. Three examples are considered: rotationally-symmetric disk subjected to stationary radial and tangential forces, disk subjected to sustained radial tension and multiple reverse torsion. and disk subjected to alternating body forces due to acceleration/breaking cycles.

The Conversational Circle: Rereading the English Novel, 1740-1775

The Conversational Circle offers a model for exploring a range of novels that experiment with narrative patterns. It makes a compelling case that teleological approaches to novel history that privilege the conflict between the individual and society are, quite simply, ahistorical. Twentieth-century historians of the early novel, most prominently Ian Watt, Mikhail Bakhtin, and Terry Castle, have canonized fictions that portray the individual in sustained tension with the social environment. Such fictions privilege a strongly linear structure. Recent reexaminations of the canon, however, have revealed a number of early novels that do not fit this mold. Betty Schellenberg identifies another kind of plot, one that focuses on the social group-the conversational circle-as a model that can affirm traditional values but just as often promotes an alternative sense of community. Schellenberg selects a group of mid-eighteenth-century novels that experiment with this alternative plot structure, embodied by the social circle. Both satirical and sentimental, canonical and non-canonical, these novels demonstrate a concern that individualistic desire threatened to destabilize society. Writing that reflects a circular structure emphasizes conversation and consensus over individualism and conquest. As a discourse that highlights negotiation and harmony, conversation privileges the social group over the individual. These fictions of the conversation circle include lesser-known works by canonical authors (Henry Fielding's Amelia and Richards's Sir Charles Grandison as well as his sequel to Pamela ), long-neglected novels by women (Sarah Fielding's David Simple and its sequel Volume the Last, and Sarah Scott's Millenium Hall ), and Tobias Smollet's last novel, Humphrey Clinker . Because they do not fit the linear model, such works have long been dismissed as idealogicall flawed and ideologically irrelevant.

Tomographic three-dimensional reconstruction of insect flight muscle partially relaxed by AMPPNP and ethylene glycol.

Rigor insect flight muscle (IFM) can be relaxed without ATP by increasing ethylene glycol concentration in the presence of adenosine 5'-[beta'gamma- imido]triphosphate (AMPPNP). Fibers poised at a critical glycol concentration retain rigor stiffness but support no sustained tension ("glycol-stiff state"). This suggests that many crossbridges are weakly attached to actin, possibly at the beginning of the power stroke. Unaveraged three-dimensional tomograms of "glycol-stiff" sarcomeres show crossbridges large enough to contain only a single myosin head, originating from dense collars every 14.5 nm. Crossbridges with an average 90 degrees axial angle contact actin midway between troponin subunits, which identifies the actin azimuth in each 38.7-nm period, in the same region as the actin target zone of the 45 degrees angled rigor lead bridges. These 90 degrees "target zone" bridges originate from the thick filament and approach actin at azimuthal angles similar to rigor lead bridges. Another class of glycol-PNP crossbridge binds outside the rigor actin target zone. These "nontarget zone" bridges display irregular forms and vary widely in axial and azimuthal attachment angles. Fitting the acto-myosin subfragment 1 atomic structure into the tomogram reveals that 90 degrees target zone bridges share with rigor a similar contact interface with actin, while nontarget crossbridges have variable contact interfaces. This suggests that target zone bridges interact specifically with actin, while nontarget zone bridges may not. Target zone bridges constitute only approximately 25% of the myosin heads, implying that both specific and nonspecific attachments contribute to the high stiffness. The 90 degrees target zone bridges may represent a preforce attachment that produces force by rotation of the motor domain over actin, possibly independent of the regulatory domain movements.

STUDIES ON VASORELAXATION BY TETRAPENTYLAMMONIUM IONS IN RAT AORTIC RINGS

Tetrapentylammonium ions (TPA +) relaxed the isolated rat aortic rings precontracted with phenylephrine and high extracellular K + in a concentration-dependent manner with respective IC 50 values of 38.9 ± 3.9 μM and 40.2 ± 2.9 μM. Other quaternary ammonium ions with a carbon side chain of varying length did not induce relaxation. The relaxant effect of TPA + was independent of the presence of the endothelium, and was unaffected by various putative blockers of K + channels such as iberiotoxin (100 nM), glibenclamide (3 μM) and 4-aminopyridine (1 mM). In addition, tetrodotoxin (3 μM), indomethacin (10 μM) and methylene blue (10 μM) had no effect on the TPA +-induced relaxation. TPA + (50 μM) and procaine (10 mM) completely abolished the phasic contractile response to caffeine in Ca 2+-free solution. In the absence of extracellular Ca 2+, phorbol 12,13-diacetate (PDA) evoked a sustained tension and TPA + concentration-dependently reduced the contraction with IC 50 of 30.7 ± 3.1 μM. TPA + reduced the sustained tension of the similar magnitude induced by phenylephrine, 60 mM K + and active phorbol ester with similar potencies. These results indicate that TPA + could act as a non-selective relaxant in arterial smooth muscle. This vasorelaxant effect is unique for TPA + since other quaternary ammonium ions did not show the similar action in the rat aorta.

Use of the tensile strength of water for the direct measurement of high soil suction

Discussions The authors express their appreciation for the additional information provided by the Discussers on the subject of the tensile strength of water. This is a new subject area to the geotechnical literature and one that initially appears to go counter to the basic physics related to the behaviour of water. The test results and findings of the ongoing research pro gram on this subject, at Imperial College, U.K., have proven to be extremely valuable in better understanding the behaviour of water that has been conditioned through the use of pressurization process. The Discussers have pointed out that the sustainable tension in the direct measurement, high suction probe appears to be related to the air entry value of the ceramic. All of the tests performed by the authors were performed using a 1500 kPa ceramic. The maximum sustainable tension appeared to be limited by the ceramic; however, a repeated cavitation of the sensor appeared to reduce the maximum sustainable tension. Also, different ceramics with the same air entry value (i.e., 1500 kPa) often produced a different response both in terms of magnitude and response pattern. For example, in one case, it was not possible to sustain a maximum tension in excess of 300 kPa, no matter what attempts were made to pressurize and saturate the system. Even with the use of 10 000 kPa, the maximum sustainable tension could not be increased, no matter what attempts were made to saturate the system. The experience of the authors was that the 24 h pressurization period did not provide much advantage over the 12 h or even the 6 h pressurization period. The Discussers have emphasized the benefit of having a small water compartment. The authors found it difficult to determine the exact amount of water in the compartment, but a space ranging from 0.1 to 0.3 mm would appear to be favorable. Further studies, over a long period of time — under both field and in situ conditions — will prove extremely valuable in encouraging the direct measurement of high tensions in the water phase (i.e., high soil suctions). The Imperial College Suction Probe, as well as other probes, are indeed a welcome device in the application of unsaturated soil mechanics to geotechnical engineering.

Endothelium-dependent rhythmic contractions induced by cyclopiazonic acid in rat mesenteric artery

The action of cyclopiazonic acid, the putative inhibitor of the Ca 2+-ATPase of endoplasmic reticulum, on phenylephrine-evoked isometric contractions in rat isolated mesenteric arteries were investigated. Cyclopiazonic acid (3 μM) induced an initial relaxation followed by rhythmic contractions of the phenylephrine-precontracted arteries with intact endothelium. Removal of endothelium abolished the effect of cyclopiazonic acid. Pretreatment of tissues with N G-nitro- l-arginine (100 μM) abolished the initial relaxation but not the rhythmic contractions. Indomethacin and glibenclamide did not affect the cyclopiazonic acid-induced response. Charybdotoxin (100 nM) converted the cyclopiazonic acid-induced rhythmic contractions to the sustained tension in the absence or presence of N G-nitro- l-arginine (100 μM). Pretreatment of charybdotoxin (100 nM) abolished cyclopiazonic acid-induced rhythmic activity but not the initial relaxation. Nifedipine (10 nM) abolished the effect of cyclopiazonic acid. Moderate increase of extracellular K + (20 mM) reduced the initial relaxation but completely abolished rhythmic contractions induced by cyclopiazonic acid. The remaining relaxation was reversed or prevented by N G-nitro- l-arginine (100 μM). The results of the present investigation indicate that cyclopiazonic acid caused endothelium-dependent response in rat isolated mesenteric arteries probably by releasing nitric oxide responsible for the initial relaxation, and probably by releasing endothelium-derived hyperpolarizing factors primarily responsible for activation of charybdotoxin-sensitive K + channels and induction of rhythmic contractile activity.

Influence of endothelium in contraction induced by phorbol ester in isolated rat aortic rings

The present study aimed to investigate the possible role of endothelium in contractile response to phorbol 12,13-diacetate (PDA) by measuring the contractile force in rat isolated aortic rings. PDA at low concentrations induced small and sustained tension in arteries with intact endothelium. N G-Nitro-L-arginine (100 μM), a nitric oxide synthase inhibitor and methylene blue (10 μM), an O 2 − generator induced a large increase in tension in the presence of PDA. The magnitude of contractions in response to N G-Nitro-L-arginine was related to concentrations of PDA. Staurosporine (10 nM), an inhibitor of protein kinase C completely inhibited contractile response to PDA as well as potentiating effects of N G-Nitro-L-arginine and methylene blue. Removal of the endothelium abolished contractile responses to both N G-Nitro-L-arginine and methylene blue in the presence of PDA. Removal of extracellular Ca 2+ suppressed contractile responses to both PDA and N G-Nitro-L-arginine. On the other hand, N G-Nitro-L-arginine (100 μM) did not induce contractions of the rat aorta pre-treated with 4-α-phorbol 12-myristate 13-acetate, an inactive form of phorbol ester. Acetylcholine concentration-dependently induced reduction of tension induced by PDA (0.3 μM) in rat aorta. N G-Nitro-L-arginine or removal of endothelium prevented the effect of the acetylcholine-induced relaxation. Indomethacin (1 μM), glibenclamide (3 μM) and charybdotoxin (100 nM) did not affect the PDA response in rat aorta. These results indicate that in rat aorta, the basal release of nitric oxide could modulate the PDA-induced contraction, which is likely to accounts for the smaller contractile response induced by PDA in arteries with intact endothelium compared to much larger contractions seen in arteries without endothelium.

Modeling and measuring the elastic properties of an archaeal surface, the sheath of Methanospirillum hungatei, and the implication of methane production.

We describe a technique for probing the elastic properties of biological membranes by using an atomic force microscope (AFM) tip to press the biological material into a groove in a solid surface. A simple model is developed to relate the applied force and observed depression distance to the elastic modulus of the material. A measurement on the proteinaceous sheath of the archaebacterium Methanospirillum hungatei GP1 gave a Young's modulus of 2 x 10(10) to 4 x 10(10) N/m2. The measurements suggested that the maximum sustainable tension in the sheath was 3.5 to 5 N/m. This finding implied a maximum possible internal pressure for the bacterium of between 300 and 400 atm. Since the cell membrane and S-layer (wall) which surround each cell should be freely permeable to methane and since we demonstrate that the sheath undergoes creep (expansion) with pressure increase, it is possible that the sheath acts as a pressure regulator by stretching, allowing the gas to escape only after a certain pressure is reached. This creep would increase the permeability of the sheath to diffusible substances.

Effects of U46619 on intracellular Ca ++ concentration and tension in human umbilical artery

OBJECTIVE: The aim was to examine the effects of U46619, a thromboxane A 2 analog, on intracellular Ca ++ concentration and tension in human umbilical artery. STUDY DESIGN: By means of front-surface fluorometry and human umbilical arterial strips loaded with fura-2 we simultaneously measured intracellular Ca ++ concentration and tension. RESULTS: In the presence of extracellular Ca ++ (1.25 mmol/L), U46619 elevated intracellular Ca ++ concentrations and tension in a concentration-dependent manner. Oscillations of both intracellular Ca ++ concentration and tension were evident in many cases. In the absence of extracellular Ca ++ U46619 induced a transient elevation of intracellular Ca ++ concentration that was associated with a transient increase and a sustained tension. At any given intracellular Ca ++ concentration level the tension induced by the cumulative application of extracellular Ca ++ during depolarization with high external K + in the presence of U46619 was greater than that in the absence of this drug; thus the intracellular Ca ++ concentration-tension relationship in the presence of U46619 shifted to the left. CONCLUSION: The tension developed by U46619 depends both on an influx of Ca ++ from the extracellular space and on release of intracellular Ca ++, and U46619 increased Ca ++ sensitivity of the contractile apparatus.

Recombinant human chromogranin A: expression, purification and characterization of the N-terminal derived peptides

Chromogranin A (CGA) is an ubiquitous 48 kDa secretory protein stored and released from most endocrine cells and is present in nanomolar concentration in the human vascular system. Recent data suggest that CGA may be the precursor of several peptides with a defined biological activity. The present report describes the expression of human CGA in Escherichia coli using the pET3a vector system, the purification and characterization of the recombinant protein and the production of antibody against the expressed protein. The expressed CGA was purified by a multi-step protocol including heat treatment, gel filtration and high performance-anion exchange chromatography and two-dimensional gel electrophoresis. Two major forms of recombinant human CGA (rhCGA) were purified from the bacterial cytosol: a 70 kDa form which corresponded to the native full-length CGA and a major proteolytic 63 kDa product recognized by antibodies raised against the 70 kDa rhCGA or to synthetic peptides localized in the N-terminal part of the bovine CGA sequence. This E. coli expression system provides a method for producing a suitable protein which will permit the identification of CGA-derived peptides with defined biological function in human. Fragments containing the N-terminal domain were generated by acidic cleavage of the two forms of rhCGA. A two-step purification using high-performance reverse-phase chromatography yielded 6 peptide bands ranging in apparent molecular mass from 7 to 18 kDa. Four components (molecular mass range 12–18 kDa) were immunostained with antibodies directed against synthetic sequences of bovine vasostatin II (bCGA 1–113) while the two others (molecular mass range 7–8 kDa) were immunostained only with antibodies directed against vasostatin I (bCGA 1–76). From protein staining the ratio vasostatins II/I was 10:1. The vasoinhibitory activity of this preparation was examined on isolated human saphenous vein segments. An inhibitory effect was obtained in paired vessel segments from 7 patients undergoing surgery for coronary artery bypass, however with low potency for supression of the endothelin-1 evoked sustained tension in these vessels.