Tailed forisomes of Canavalia gladiata: a new model to study Ca2+-driven protein contractility.

BACKGROUND AND AIMS: Forisomes are Ca(2+)-dependent contractile protein bodies that form reversible plugs in sieve tubes of faboid legumes. Previous work employed Vicia faba forisomes, a not entirely unproblematic experimental system. The aim of this study was to seek to establish a superior model to study these intriguing actuators. METHODS: Existing isolation procedures were modified to study the exceptionally large, tailed forisomes of Canavalia gladiata by differential interference contrast microscopy in vitro. To analyse contraction/expansion kinetics quantitatively, a geometric model was devised which enabled the computation of time-courses of derived parameters such as forisome volume from simple parameters readily determined on micrographs. KEY RESULTS: Advantages of C. gladiata over previously utilized species include the enormous size of its forisomes (up to 55 microm long), the presence of tails which facilitate micromanipulation of individual forisomes, and the possibility of collecting material repeatedly from these fast-growing vines without sacrificing the plants. The main bodies of isolated Canavalia forisomes were box-shaped with square cross-sections and basically retained this shape in all stages of contraction. Ca(2+)-induced a 6-fold volume increase within about 10-15 s; the reverse reaction following Ca(2+)-depletion proceeded in a fraction of that time. CONCLUSIONS: The sword bean C. gladiata provides a superior experimental system which will prove indispensable in physiological, biophysical, ultrastructural and molecular studies on the unique ATP-independent contractility of forisomes.

Biomimetic actuators: where technology and cell biology merge.

The structural and functional analysis of biological macromolecules has reached a level of resolution that allows mechanistic interpretations of molecular action, giving rise to the view of enzymes as molecular machines. This machine analogy is not merely metaphorical, as bio-analogous molecular machines actually are being used as motors in the fields of nanotechnology and robotics. As the borderline between molecular cell biology and technology blurs, developments in the engineering and material sciences become increasingly instructive sources of models and concepts for biologists. In this review, we provide a--necessarily selective--summary of recent progress in the usage of biological and biomimetic materials as actuators in artificial environments, focussing on motors built from DNA, classical cellular motor systems (tubulin/kinesin, actin/myosin), the rotary motor F1F0-ATPase and protein-based 'smart' materials.

Reversible calcium-regulated stopcocks in legume sieve tubes.

Sieve tubes of legumes (Fabaceae) contain characteristic P-protein crystalloids with controversial function. We studied their behavior by conventional light, electron, and confocal laser scanning microscopy. In situ, crystalloids are able to undergo rapid (<1 sec) and reversible conversions from the condensed resting state into a dispersed state, in which they occlude the sieve tubes. Crystalloid dispersal is triggered by plasma membrane leakage induced by mechanical injury or permeabilizing substances. Similarly, abrupt turgor changes imposed by osmotic shock cause crystalloid dispersal. Because chelators generally prevent the response, divalent cations appear to be the decisive factor in crystalloid expansion. Cycling between dispersal and condensation can be induced in opened cells by repetitive exchange of bathing media containing either Ca(2)+ or chelators. Sr(2)+ and Ba(2)+, but not Mg(2)+, are equally active. In conclusion, the fabacean P-protein crystalloids represent a novel class of mechanically active proteinaceous structures, which provide an efficient mechanism with which to control sieve tube conductivity.

Does growth correlate with turgor-induced elastic strain in stems? A re-evaluation of de Vries' classical experiments.

The correlation between growth and turgor-induced elastic expansion was studied in hypocotyls of sunflower (Helianthus annuus) seedlings under various growth conditions. Turgor-induced elastic cell wall strain was greater in hypocotyls of faster growing seedlings, i.e. in etiolated versus light-grown ones. It also was higher in rapidly growing young seedlings as compared with nongrowing mature ones. However, analysis of the spatial distribution of elastic strain and growth demonstrated that their correspondence was only apparent. Profiles of elastic strain declined steadily from the top of the hypocotyls toward the basis, whereas the profiles of relative elemental growth rate along the hypocotyls showed maxima within the growing zones. In contrast to earlier hypotheses, we conclude that turgor-induced elastic cell wall strain and growth do not correlate precisely in growing hypocotyls.

The mechanic state of "inner tissue" in the growing zone of sunflower hypocotyls and the regulation of its growth rate following excision.

Spontaneous growth of isolated inner tissue from the etiolated sunflower (Helianthus annuus L.) hypocotyl growing zone was investigated. A new preparation technique allowed measurements starting 3 s after excision. Elongation with respect to the turgescent and plasmolized state was quantified in terms of relative growth rates, facilitating comparison to growth in situ. Turgor and turgor-induced strain were determined. Overall longitudinal strain in inner tissues in situ was positive, indicating that compressive forces exerted by peripheral tissues are outweighed by turgor-dependent tensile stress. Inner tissue expansion following isolation depended on water uptake. Extreme plastic extension rates occurred immediately after excision, suggesting that mechanical parameters of inner tissue in situ cannot be extrapolated from the mechanics of excised sections. In the long term, excised inner tissue autonomously established values of turgor, turgor-induced strain, and relative growth rates similar to values in the living plant. These results support historic models of tissue cooperation during organ growth, in which inner tissues actively participate in the control of growth rates.

What makes plants different? Principles of extracellular matrix function in 'soft' plant tissues.

An overview of the biomechanic and morphogenetic function of the plant extracellular matrix (ECM) in its primary state is given. ECMs can play a pivotal role in cellular osmo- and volume-regulation, if they enclose the cell hermetically and constrain hydrostatic pressure evoked by osmotic gradients between the cell and its environment. From an engineering viewpoint, such cell walls turn cells into hydraulic machines, which establishes a crucial functional differences between cell walls and other cellular surface structures. Examples of such hydraulic machineries are discussed. The function of cell walls in the control of pressure, volume, and shape establishes constructional evolutionary constraints, which can explain aspects commonly considered typical of plants (sessility, autotrophy). In plants, 'cell division' by insertion of a new cell wall is a process of internal cytoplasmic differentiation. As such it differs fundamentally from cell separation during cytokinesis in animals, by leaving the coherence of the dividing protoplast basically intact. The resulting symplastic coherence appears more important for plant morphogenesis than histological structure; similar morphologies are realized on the basis of distinct tissue architectures in different plant taxa. The shape of a plant cell is determined by the shape its cell wall attains under multiaxial tensile stress. Consequently, the development of form in plants is achieved by a differential plastic deformation of the complex ECM in response to this multiaxial force (hydrostatic pressure). Current concepts of the regulation of these deformation processes are briefly evaluated.

Monitoring considerations for port-access cardiac surgery.

BACKGROUND: A method for monitoring patients was evaluated in a clinical trial of minimally invasive port-access cardiac surgery with closed chest endovascular cardiopulmonary bypass. METHODS AND RESULTS: Cardiopulmonary bypass was conducted in 25 patients through femoral cannulas. An endovascular pulmonary artery vent was placed in the main pulmonary artery through a jugular vein. For mitral valve surgery, a catheter was placed in the coronary sinus for delivery of cardioplegia. A balloon catheter ("endoaortic clamp," EAC) used for occlusion of the ascending aorta, delivery of cardioplegia, aortic root venting, and pressure measurement was inserted through a femoral artery and initially positioned by use of fluoroscopy and transesophageal echocardiography (TEE). Potential migration of the EAC was monitored by (1) TEE of the ascending aorta, (2) pulsed-wave Doppler of the right carotid artery, (3) balloon pressure, (4) comparison of aortic root pressure and right radial artery pressure, and (5) fluoroscopy. TEE, fluoroscopy, and pressure measurement were effective in monitoring catheter insertion and position. With inadequate balloon inflation, migration of the EAC toward the aortic valve could be detected with TEE. During administration of cardioplegia, TEE showed movement of the balloon away from the aortic valve, and migration into the aortic arch was detectable with loss of carotid Doppler flow. Stability of EAC position was demonstrated with appropriate balloon volume. Cardioplegic solution was visualized in the aortic root, and aortic root pressure changed appropriately during administration of cardioplegia. Venous cannula position was optimized with TEE and endopulmonary vent flow measurement. CONCLUSIONS: An effective method has been developed for monitoring patients and the catheter system during port-access cardiac surgery.

Mitral valve replacement via a right mini-thoracotomy in the dog: use of carbon dioxide to reduce intracardiac air.

OBJECTIVE: To develop a clinically applicable method of minimally invasive mitral valve replacement (MVR) with cardioplegia, and examine the ability of carbon dioxide (CO2) to improve de-airing. METHODS: MVR was performed via a 5 x 3-cm right lateral minithoracotomy in eight greyhounds. Peripheral cardiopulmonary bypass and an ascending aortic balloon catheter (endoaortic clamp) were used for cardioplegia and aortic root venting. The endoaortic clamp was inflated in the ascending aorta under fluoroscopy and cardioplegic solution was infused. In four dogs, CO2 at 2 l/min was used to displace air in the chest. A left atriotomy was made, the valve exposed and a mechanical valve implanted. After left atrial closure, retained intracardiac gas was aspirated from the aortic root and collected in a bubble-trap. The endoclamp was deflated and the animal weaned from bypass. RESULTS: A satisfactory MVR was performed in all cases. The clamp time was 64 +/- 13 min and all dogs were stable post-bypass. In the CO2 group, intrathoracic CO2 was maintained above 86% and 0.1 +/- 0.1 ml of gas was collected, compared to 1.3 +/- 0.8 ml in the non-CO2 group (P < 0.05). CONCLUSIONS: Femoro-femoral bypass and use of the endoaortic clamp allow a safe and efficacious MVR via a right minithoracotomy in the dog. A high intrathoracic CO2 concentration reduces the amount of retained intracardiac gas.

Closed-chest cardiopulmonary bypass and cardioplegia: basis for less invasive cardiac surgery.

BACKGROUND: We developed a method of closed-chest cardiopulmonary bypass to arrest and protect the heart with cardioplegic solution. This method was used in 54 dogs and the results were retrospectively analyzed. METHODS: Bypass cannulas were placed in the right femoral vessels. A balloon occlusion catheter was passed via the left femoral artery and positioned in the ascending aorta. A pulmonary artery vent was placed via the jugular vein. In 17 of the dogs retrograde cardioplegia was provided with a percutaneous coronary sinus catheter. RESULTS: Cardiopulmonary bypass time was 111 +/- 27 minutes (mean +/- standard deviation) and cardiac arrest time was 66 +/- 21 minutes. Preoperative cardiac outputs were 2.9 +/- 0.70 L/min and postoperative outputs were 2.9 +/- 0.65 L/min (p = not significant). Twenty-one-French and 23F femoral arterial cannulas that allowed coaxial placement of the ascending aortic balloon catheter were tested in 3 male calves. Line pressures were higher, but not clinically limiting, with the balloon catheter placed coaxially. CONCLUSIONS: Adequate cardiopulmonary bypass and cardioplegia can be achieved in the dog without opening the chest, facilitating less invasive cardiac operations. A human clinical trial is in progress.

The Determination of Relative Elemental Growth Rate Profiles from Segmental Growth Rates (A Methodological Evaluation).

Relative elemental growth rate (REGR) profiles describe spatial patterns of growth intensity; they are indispensable for causal growth analyses. Published methods of REGR profile determination from marking experiments fall in two classes: the profile is either described by a series of segmental growth rates, or calculated as the slope of a function describing the displacement velocities of points along the organ. The latter technique is usually considered superior for theoretical reasons, but to our knowledge, no comparative methodological study of the two approaches is currently available. We formulated a model REGR profile that resembles those reported from primary roots. We established the displacement velocity profile and derived growth trajectories, which enabled us to perform hypothetical marking experiments on the model with varying spacing of marks and durations of measurement. REGR profiles were determined from these data by alternative methods, and results were compared to the original profile. We find that with our model plotting of segmental relative growth rates versus segment position provides exact REGR profile estimations, if the initial segment length is less than 10% of the length of the whole growing zone, and if less than 20% of the growing zone is displaced past its boundary during the measurement. Based on our analysis, we discuss systematic errors that occur in marking experiments.

Extracorporeal circulation for port-access cardiac surgery.

Minimally invasive techniques for cardiac surgery are a new approach in performing some cardiac operations. Minimally invasive surgery may minimize patient discomfort, length of stay in the hospital and postoperative rehabilitation. These procedures utilize a small thoracotomy for direct visualization of the heart. However, without the use of cardiopulmonary bypass, this approach is limited to some epicardial procedures such as coronary bypass grafting, where the heart rate is pharmacologically reduced. Port-access cardiac surgery is a new approach which provides all the benefits of minimally invasive surgery without sacrificing the advantages of cardiopulmonary bypass and myocardial preservation. Port-access cardiac surgery uses an anterior mediastinotomy and thoracic ports in conjunction with a specially designed set of endovascular catheters. These catheters provide a mode to arrest, preserve and vent the heart through an endoaortic occlusion balloon positioned in the ascending aorta. A pulmonary artery vent and coronary sinus cardioplegia catheter can also be used. These endovascular catheters, integrated with a modified heart-lung machine, provide complete cardiopulmonary support through extrathoracic cannulae inserted in a femoral artery and vein. Maintenance and monitoring of this endovascular cardiopulmonary bypass system requires the use of a kinetic pump in the venous drainage line to augment return to the heart-lung machine. Special guidelines and management parameters exist to optimize bypass with this catheter system. Using this system, port-access, minimally invasive surgery can be applied to a wider range of both epicardial and intracardiac procedures.

Port-access bilateral internal mammary artery grafting for left main coronary artery disease: canine feasibility study.

BACKGROUND: To extend the applications of minimal access cardiac surgery, an endovascular cardiopulmonary bypass (CPB) system that allows cardioplegia delivery and cardiac venting was used to perform bilateral internal mammary artery (IMA) bypass grafting in six dogs. METHODS: The left IMA (LIMA) was taken down thoracoscopically from three left lateral chest ports, followed by the right IMA (RIMA) from the right side. One left-sided port was extended medially 5 cm with or without rib resection, to expose the pericardium. Both IMAs were divided and exteriorized through the left anterior mediastinotomy. Flow and pedicle length were satisfactory in all cases. Femoral-femoral bypass was used and the heart arrested with antegrade delivery of cardioplegic solution via the central lumen of a balloon catheter inflated to occlude the ascending aorta. All anastomoses were made through the mediastinotomy under direct vision. In five studies the RIMA was attached to the left anterior descending artery (LAD) and the LIMA to the circumflex, and in one study the RIMA was tunneled through the transverse sinus to the circumflex and the LIMA was anastomosed to the LAD. All animals were weaned from CPB in sinus rhythm without inotropes. CPB duration was 108 +/- 27 minutes (mean +/- SD) and the clamp duration was 54 +/- 10 minutes. RESULTS: Preoperative and postoperative cardiac outputs were 2.9 +/- 0.71/min and 2.4 +/- 0.31/min, respectively (p = NS), and corresponding pulmonary artery occlusion pressures were 6 +/- 3 mmHg and 7 +/- 2 mmHg, respectively (p = NS). All 12 grafts were demonstrated to be fully patent. Postmortem examination revealed well aligned pedicles and correctly grafted target vessels. CONCLUSION: This canine model demonstrates the potential for a less invasive approach to the surgical management of left main coronary artery disease in humans.

Minimally invasive right heart operations: techniques for bicaval occlusion and cardioplegia.

Peripheral cardiopulmonary bypass with cardioplegia has facilitated minimally invasive coronary artery bypass grafting and mitral valve replacement. The cardiopulmonary bypass system was modified to allow bicaval occlusion for right heart operations. In 4 canine studies, three variants of bicaval cannulation techniques were successfully used for atrial septal defect repair via a right minithoracotomy.

Port-access mitral valve replacement in dogs.

OBJECTIVE: The objective was to assess mitral valve replacement in a minimally invasive fashion by means of port-access technology. METHODS: Fifteen dogs, 28 +/- 3 kg (mean +/- standard deviation), were studied with the port-access mitral valve replacement system (Heartport, Inc., Redwood City, Calif.). Eleven dogs underwent acute studies and were sacrificed immediately after the procedure. Four dogs were allowed to recover and then were sacrificed 4 weeks after operation. Cardiopulmonary bypass was conducted by femoral cannulation with an endovascular balloon catheter for aortic occlusion, root venting, and antegrade delivery of cardioplegic solution. Catheters were inserted in the jugular vein for pulmonary artery venting and retrograde delivery of cardioplegic solution. Through the oval port, a prosthesis (St. Jude Medical, Inc., St. Paul, Minn., or CarboMedics, Inc., Austin, Texas) was inserted through the left atrial appendage and secured to the anulus with sutures. Deairing was performed. RESULTS: Cardiopulmonary bypass duration was 114 +/- 24 minutes and aortic crossclamp time was 68 +/- 14 minutes. All animals were weaned from cardiopulmonary bypass in sinus rhythm. Cardiac output and pulmonary artery occlusion pressure were unchanged (2.8 +/- 0.7 L/min and 7 +/- 3 mm Hg before operation vs 2.6 +/- 0.6 L/min and 9 +/- 4 mm Hg after operation). There was no mitral regurgitation according to left ventriculography in 13 of 15 dogs. In two dogs there was interference with prosthetic valve closure by residual native anterior leaflet tissue. Pathologic examination otherwise showed normal healing without perivalvular discontinuity. Microscopic studies showed no damage to the valve surfaces. Transthoracic echocardiography of the four dogs in the long-term study showed normal ventricular and prosthetic valve function 4 weeks after the operation. CONCLUSION: Mitral valve replacement with a minimally invasive method has been demonstrated in dogs. A clinical trial is in progress.

Port-access coronary artery bypass with cardioplegic arrest: acute and chronic canine studies.

BACKGROUND: Our goal is to perform minimally invasive coronary artery bypass grafting without sacrificing the benefits of myocardial protection with cardioplegia. METHODS: Twenty-three dogs underwent acute studies and 4 dogs underwent survival studies. The left internal mammary artery was taken down using a thoracoscope. Cardiopulmonary bypass was conducted via femoral cannulas and using an endovascular balloon catheter for ascending aortic occlusion, root venting, and delivery of antegrade blood cardioplegia. Pulmonary artery venting was achieved with a jugular vein catheter. An internal mammary artery-to-coronary artery anastomosis was performed using a microscope through a 10 mm port. RESULTS: All animals were weaned from cardiopulmonary bypass in sinus rhythm without inotropes. Cardiopulmonary bypass duration was 104 +/- 28 minutes and aortic clamp duration was 61 +/- 22 minutes. Cardiac output and pulmonary artery occlusion pressure were unchanged. The internal mammary artery was anastomosed to the left anterior descending artery (25) or the first diagonal (2) with patency shown in 25 of 27. One dog in the survival study had a very short internal mammary artery pedicle under tension and was euthanized for excessive postoperative hemorrhage. Three weeks postoperatively the remaining dogs had angiographically patent anastomoses, normal transthoracic echocardiograms, and histologically normal healing and patent grafts. CONCLUSIONS: Endovascular cardiopulmonary bypass using a balloon catheter is effective in arresting and protecting the heart to allow thoracoscopic internal mammary artery-to-coronary artery anastomosis.

The history of tissue tension.

In recent years the phenomenon of tissue tension and its functional connection to elongation growth has regained much interest. In the present study we reconstruct older models of mechanical inhomogenities in growing plant organs, in order to establish an accurate historical background for the current discussion. We focus on the iatromechanic model developed in Stephen Hales' Vegetable Staticks, Wilhelm Hofmeister's mechanical model of negative geotropism, Julius Sachs' explanation of the development of tissue tension, and the differential-auxin-response-hypothesis by Kenneth Thimann and Charles Schneider. Each of these models is considered in the context of its respective historic and theoretical environment. In particular, the dependency of the biomechanical hypotheses on the cell theory and the hormone concept is discussed. We arrive at the conclusion that the historical development until the middle of our century is adequately described as a development towards more detailed explanations of how differential tensions are established during elongation growth in plant organs. Then we compare with the older models the structure of more recent criticism of hormonal theories of tropic curvature, and particularly the epidermal-growth-control hypothesis of Ulrich Kutschera. In contrast to the more elaborate of the older hypotheses, the recent models do not attempt an explanation of differential tensions, but instead focus on mechanical processes in organs, in which tissue tension already exists. Some conceptual implications of this discrepancy, which apparently were overlooked in the recent discussion, are briefly evaluated.

Port-access coronary artery bypass grafting: a proposed surgical method.

Minimally invasive surgical methods have been developed to provide patients the benefits of open operations with decreased pain and suffering. We have developed a system that allows the performance of cardiopulmonary bypass and myocardial protection with cardioplegic arrest without sternotomy or thoracotomy. In a canine model, we successfully used this system to anastomose the internal thoracic artery to the left anterior descending coronary artery in nine of 10 animals. The left internal thoracic artery was dissected from the chest wall, and the pericardium was opened with the use of thoracoscopic techniques and single lung ventilation. The heart was arrested with a cold blood cardioplegic solution delivered through the central lumen of a balloon occlusion catheter (Endoaortic Clamp; Heartport, Inc., Redwood City, Calif.) in the ascending aorta, and cardiopulmonary bypass was maintained with femorofemoral bypass. An operating microscope modified to allow introduction of the 3.5x magnification objective into the chest was positioned through a 10 mm port over the site of the anastomosis. The anastomosis was performed with modified surgical instruments introduced through additional 5 mm ports. In the cadaver model (n = 7) the internal thoracic artery was harvested and the pericardium opened by means of similar techniques. A precise arteriotomy was made with microvascular thoracoscopic instruments under the modified microscope on four cadavers. In three other cadavers we assessed the exposure provided by a small anterior incision (4 to 6 cm) over the fourth intercostal space. This anterior port can assist in dissection of the distal internal thoracic artery and provides direct access to the left anterior descending, circumflex, and posterior descending arteries. We have demonstrated the potential feasibility of grafting the internal thoracic artery to coronary arteries with the heart arrested and protected, without a major thoracotomy or sternotomy.