Two-dimensional tension tests in plant biomechanics--sweet cherry fruit skin as a model system

Splitting of fruits is a function of two-dimensional tension caused by different growth rates of tissues and turgor, especially water uptake shortly before harvest. In order to analyse the mechanical properties of spheroid plant material close to stress-strain conditions in vivo, a new hydraulic two-dimensional testing device was set up. Sweet cherry (Prunus avium L.) fruit skin was chosen as a model system. The recorded pressure-deflection curves were non-linear, with a considerable initial "lag phase" and a distinct increasing end part. Taking into account the special geometry, these curves could be modelled with a newly developed analytical approach based on linear elastic material behaviour. The results demonstrated good correlation if a modulus of elasticity ranging from 160 to 250 MPa for the cherry fruit skin was chosen. In addition, a mean strength value of 47 MPa was calculated based on the theory of thin shells and spheres. The results are compared with mechanical data found for fruits and other plant material. In order to test the theoretical approach, two- and one-dimensional tension tests were performed on packaging PE foil, revealing a mean modulus of 171 MPa in bi-axial tension, and 193 and 242 MPa in uni-axial tension, depending on the test speed. The results demonstrate that it seems to be feasible to use this method to analyse the two-dimensional stress-strain conditions of spheroid plant materials such as cherry fruit skins. It may be applied as a tool for crop testing to elucidate the mechanical basis of cracking susceptibility of fruits.     

Growth and architecture of small honey mesquites under jackrabbit browsing: overcoming the disadvantage of being eaten

Browsing is an important mortality factor in seedlings and small plants. However, the induced changes in the architecture of plant survivors may influence subsequent browsing, opening the possibility of compensating for the damage done. How jackrabbit (Lepus californicus) browsing affects the growth and architecture of small individuals of honey mesquite, Prosopis glandulosa var. torreyana, a tree/shrub that produces spines at every node, was explored. Naturally established mesquites of unknown age were selected in one site, and 2-year-old mesquites were transplanted in another site. In both cases, half of them were exposed to jackrabbits and the other half were excluded as controls. After 4 years, shoot production (height, length and number of derived shoots) and plant growth (height and cover) increased 1.4-2.5-fold in naturally established controls relative to exposed plants, depending on the measured variable. In the transplant experiment, the increases were 2.8-7.1-fold in controls relative to exposed plants 2 years after initiation of the experiment. The net loss of biomass in treatment vs. control plants in these experiments suggests a negative response to browsing which has been defined as under-compensation. Alternative architectures in honey mesquites were evident at the end of the exclusion experiments: controls had long branches and an extended crown cover, while exposed plants had short branches and a compact crown cover. Results indicated that mesquites were able to grow under browser pressure by packing many stems in a compact matrix armed with spines and producing one or more shoots tall and wide enough to escape from jackrabbits.     

Stored elastic energy powers the 60-microm extension of the Limulus polyphemus sperm actin bundle

During the 5 s of the acrosome reaction of Limulus polyphemus sperm, a 60-microm-long bundle of scruin-decorated actin filaments straightens from a coiled conformation and extends from the cell. To identify the motive force for this movement, we examined the possible sources of chemical and mechanical energy and show that the coil releases approximately 10-13 J of stored mechanical strain energy, whereas chemical energy derived from calcium binding is approximately 10-15 J. These measurements indicate that the coiled actin bundle extends by a spring-based mechanism, which is distinctly different from the better known polymerization or myosin-driven processes, and that calcium initiates but does not power the reaction.     

Vascular endothelial cells express a functional fas-receptor due to lack of hemodynamic forces

The fas system is present in atherosclerotic lesions. However, its role in the initiation and progression is still unclear. Here we show that in endothelial cells (EC) the expression of the fas receptor is regulated by flow conditions. The EC of the vascular system are regularly exposed to a range of hemodynamic forces with great impact on cellular structures and functions. Recently it was reported that in endothelial cells the lack of hemodynamic forces as well as irregular flow conditions trigger apoptosis by induction of a mechanosensitive autocrine loop of thrombospondin-1 and the _V3 integrin/integrin-associated protein complex. Here we show that EC cultivated under regular laminar flow conditions are devoid of the fas-receptor whereas cultivation under static conditions as well as under turbulence leads to its expression. Stimulation of the fas-receptor by its ligand increases the amount of apoptotic cells by twofold; the increase can be prevented by blocking the fas-receptor. The availability of the expressed fas receptor for stimulation by its ligand hints at a role as a tool for progression of atherosclerosis.     

A kinetic model for enzyme interfacial activity and stability: pa-hydroxynitrile lyase at the diisopropyl ether/water interface

A kinetic framework is developed to describe enzyme activity and stability in two-phase liquid-liquid systems. In particular, the model is applied to the enzymatic production of benzaldehyde from mandelonitrile by Prunus amygdalus hydroxynitrile lyase (pa-Hnl) adsorbed at the diisopropyl ether (DIPE)/aqueous buffer interface (pH = 5.5). We quantitatively describe our previously obtained experimental kinetic results (Hickel et al., 1999; 2001), and we successfully account for the aqueous-phase enzyme concentration dependence of product formation rates and the observed reaction rates at early times. Multilayer growth explains the early time reversibility of enzyme adsorption at the DIPE/buffer interface observed by both enzyme-activity and dynamic-interfacial-tension washout experiments that replace the aqueous enzyme solution with a buffer solution. The postulated explanation for the unusual stability of pa-Hnl adsorbed at the DIPE/buffer interface is attributed to a two-layer adsorption mechanism. In the first layer, slow conformational change from the native state leads to irreversible attachment and partial loss of catalytic activity. In the second layer, pa-Hnl is reversibly adsorbed without loss in catalytic activity. The measured catalytic activity is the combined effect of the deactivation kinetics of the first layer and of the adsorption kinetics of each layer. For the specific case of pa-Hnl adsorbed at the DIPE/buffer interface, this combined effect is nearly constant for several hours resulting in no apparent loss of catalytic activity. Our proposed kinetic model can be extended to other interfacially active enzymes and other organic solvents. Finally, we indicate how interfacial-tension lag times provide a powerful tool for rational solvent selection and enzyme engineering.     

Mechanically unfolding proteins: the effect of unfolding history and the supramolecular scaffold

The mechanical resistance of a folded domain in a polyprotein of five mutant I27 domains (C47S, C63S I27)(5)is shown to depend on the unfolding history of the protein. This observation can be understood on the basis of competition between two effects, that of the changing number of domains attempting to unfold, and the progressive increase in the compliance of the polyprotein as domains unfold. We present Monte Carlo simulations that show the effect and experimental data that verify these observations. The results are confirmed using an analytical model based on transition state theory. The model and simulations also predict that the mechanical resistance of a domain depends on the stiffness of the surrounding scaffold that holds the domain in vivo, and on the length of the unfolded domain. Together, these additional factors that influence the mechanical resistance of proteins have important consequences for our understanding of natural proteins that have evolved to withstand force.     

Effects of establishing cell cultures and cell culture conditions on the proliferative life span of human fibroblasts isolated from different tissues and donors of different ages

It is well known that normal human cells placed in a culture environment exhibit a limited proliferative capacity. The extent to which the culture environment influences proliferative life span is not understood. This study evaluated the effects of the standard procedures used to establish and maintain cultures on the proliferative life spans of different types of human fibroblast cells established from fetal and adult skin and lung. The results of this study demonstrate that procedures to establish cell cultures use only one of several subpopulations of cells present in biopsy pieces and that the culture conditions routinely employed by most laboratories can exert significant effects on proliferative life-span determinations. The maximum proliferative life span differed significantly when obtained by growing the cells in two commonly used commercial media. Proliferative life span was inversely related to ambient oxygen tension and directly related to seeding density in all of the lines examined although lines established from adult skin were much more resistant to toxicity. Enzymatic antioxidant defense levels of fetal skin fibroblasts were much lower than those observed in adult skin fibroblasts, but the effects of oxygen on their life spans were similar. Hyperoxia induced larger increases in glutathione concentration in cell lines with low antioxidant enzyme levels.     

Environmental and auxin regulation of wood formation involves members of the Aux/IAA gene family in hybrid aspen

Indole acetic acid (IAA/auxin) profoundly affects wood formation but the molecular mechanism of auxin action in this process remains poorly understood. We have cloned cDNAs for eight members of the Aux/IAA gene family from hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) that encode potential mediators of the auxin signal transduction pathway. These genes designated as PttIAA1-PttIAA8 are auxin inducible but differ in their requirement of de novo protein synthesis for auxin induction. The auxin induction of the PttIAA genes is also developmentally controlled as evidenced by the loss of their auxin inducibility during leaf maturation. The PttIAA genes are differentially expressed in the cell types of a developmental gradient comprising the wood-forming tissues. Interestingly, the expression of the PttIAA genes is downregulated during transition of the active cambium into dormancy, a process in which meristematic cells of the cambium lose their sensitivity to auxin. Auxin-regulated developmental reprogramming of wood formation during the induction of tension wood is accompanied by changes in the expression of PttIAA genes. The distinct tissue-specific expression patterns of the auxin inducible PttIAA genes in the cambial region together with the change in expression during dormancy transition and tension wood formation suggest a role for these genes in mediating cambial responses to auxin and xylem development.     

Mechanosensitive induction of apoptosis in fibroblasts is regulated by thrombospondin-1 and integrin associated protein (CD47)

Fibroblasts are cultured in three-dimensional collagen matrices to investigate the effect of mechanical tension on the regulation of apoptosis. Under the influence of mechanical loading, the cells show little apoptosis whereas releasing of tension leads to an increase up to tenfold during the first 24 h and remains constant for further 48 h. An autocrine loop of the integrin _V3/CD47 receptor complex and thrombospondin-1 is identified as the molecular coupling device between mechanical loading and apoptosis: The integrin _V3 is expressed under mechanical loading as well as unloading whereas the CD47 could only be identified after the release of tension. The secreted thrombospondin binds to the active receptor and induces apoptosis. The presented mechanosensitive regulation of apoptosis in fibroblast cultures could be an essential mechanism for the regression of the granulation tissue by apoptosis in the process of wound healing.     

A simple non-enzymatic method for the isolation of high yields of functional rat hepatocytes

A method for isolation of rat hepatocytes using liver perfusion by ethylenediamine tetra-acetic acid (EDTA)-containing sucrose solution and mechanical tissue disaggregation by controlled vibration (MVD) is described. The yields of hepatocytes produced by this method were similar to those obtained using collagenase perfusion. The cells had well preserved membrane integrity as judged by the trypan blue staining test (91 +/- 4%), ATP contents, rates of endogenous respiration and enzyme leakage that indicated they were functional cells. There was little evidence of expression of latent damage when the cells were stored either at 37 degrees C (by pre-incubation) or at 4 degrees C. This method can be used to isolate high yields of functional cells from rat liver if the collagenase perfusion technique is not available.