The force exerted by a muscle cross-bridge depends directly on the strength of the actomyosin bond

Myosin produces force in a cyclic interaction, which involves alternate tight binding to actin and to ATP. We have investigated the energetics associated with force production by measuring the force generated by skinned muscle fibers as the strength of the actomyosin bond is changed. We varied the strength of the actomyosin bond by addition of a polymer that promotes protein-protein association or by changing temperature or ionic strength. We estimated the free energy available to generate force by measuring isometric tension, as the free energy of the states that precede the working stroke are lowered with increasing phosphate. We found that the free energy available to generate force and the force per attached cross-bridge at low [Pi] were both proportional to the free energy available from the formation of the actomyosin bond. We conclude that the formation of the actomyosin bond is involved in providing the free energy driving the production of isometric tension and mechanical work. Because the binding of myosin to actin is an endothermic, entropically driven reaction, work must be performed by a "thermal ratchet" in which a thermal fluctuation in Brownian motion is captured by formation of the actomyosin bond.     

A nonpeptide provides insight into mechanisms that regulate Drosophila melanogaster heart contractions

Here we report the effect of a nonpeptide, benzethonium chloride (bztc), on Drosophila melanogaster larval, pupal, and adult heart rates in vivo. Benzethonium chloride reduced the frequency of spontaneous contractions in the D. melanogaster pupal heart, but not in the larval heart or the adult heart as measured in noninvasive whole animal preparations. When applied directly to the D. melanogaster heart, in the absence of hemolymph, bztc reduced the frequency of spontaneous contractions in larval, pupal, and adult hearts. These findings are consistent with the conclusion that bztc acts through or is regulated by different mechanisms in these three developmental stages. An alternative explanation is that larval hemolymph and adult hemolymph contain a material that interferes with the effect of the nonpeptide on heart contractions. Bztc mimicked the effect of the peptide dromyosuppressin (DMS) on the heart at an equivalent concentration; in contrast, 103-fold more nonpeptide is required to mimic the effect of DMS on fly gut. These findings are consistent with the presence of tissue-specific myosuppressin receptors or mechanisms.     

Hawai‘i’s Mountain-to-Sea Ecosystems: Social–Ecological Microcosms for Sustainability Science and Practice

There is an urgent need to develop the underlying theory and principles of “sustainability science,” based on an understanding of the fundamental interactions between nature and humans. This requires a new research and education paradigm that embraces biocomplexity, integrates the physical, biological, and social sciences, and uses a coupled, human–natural systems approach. An initiative aligned with this paradigm and approach, and centered on the Hawaiian Island’s unique mountain-to-sea ecosystems, is developing at the University of Hawai‘i. These ecosystems, extending from upland tropical forests to the fringing coral reefs, correspond to the roughly wedge-shaped catchments, traditionally called ahupua‘a in the Hawaiian language. Despite the collapse of the ahupua‘a system and, tragically, the Native Hawaiian population, its legacy of ecological and cultural stewardship remains. This legacy, and the potential of these ecosystems as microcosms for addressing the core questions of sustainability science, has provided the impetus for a growing number of projects employing a social–ecological systems perspective. An overview of three projects that employ a “learning community” approach and cultural stewardship perspective inspired by the ahupua‘a system is provided. These include the Ecosystems Thrust Area of Hawai‘i EPSCoR, a U.S. National Science Foundation research infrastructure program, focused on ecosystem research and monitoring activities; a sustainability curriculum program, Mālama I Ka ‘Āina, of the College of Education; and a project that builds on programs of the Division of Ecology and Health and its affiliated Asia-Pacific Center for Infectious Disease Ecology, linking ecosystem resilience and infectious diseases.     

CaATP as a substrate to investigate the myosin lever arm hypothesis of force generation

In an effort to test the lever arm model of force generation, the effects of replacing magnesium with calcium as the ATP-chelated divalent cation were determined for several myosin and actomyosin reactions. The isometric force produced by glycerinated muscle fibers when CaATP is the substrate is 20% of the value obtained with MgATP. For myosin subfragment 1 (S1), the degree of lever arm rotation, determined using transient electric birefringence to measure rates of rotational Brownian motion in solution, is not significantly changed when calcium replaces magnesium in an S1-ADP-vanadate complex. Actin activates S1 CaATPase activity, although less than it does MgATPase activity. The increase in actin affinity when S1. CaADP. P(i) is converted to S1. CaADP is somewhat greater than it is for the magnesium case. The ionic strength dependence of actin binding indicates that the change in apparent electrostatic charge at the acto-S1 interface for the S1. CaADP. P(i) to S1. CaADP step is similar to the change when magnesium is bound. In general, CaATP is an inferior substrate compared to MgATP, but all the data are consistent with force production by a lever arm mechanism for both substrates. Possible reasons for the reduced magnitude of force when CaATP is the substrate are discussed.     

Evidence for conformers of rabbit muscle adenylate kinase

Changes of the apparent Mr values and the circular dichroism patterns suggest the existence of three relatively stable conformers of rabbit muscle adenylate kinase (RMAK). The effects of dithiothreitol (DTT) which stimulates activity, pH, the substrates, and ATP on the Mr value and the Stokes radius of RMAK were determined from gel filtration data, and apparent Mr values near 22,000, 26,000, and 29,000 resulted. Substrates generated multiple Mr values, suggesting the presence of multiple conformers of RMAK. The higher apparent Mr values were obtained in the presence of DTT and at the higher substrate concentrations, indicating more open conformations. The effect of the substrates on the conformation of RMAK is discussed in relation to the kinetic mechanism of this random bireactant system. Circular dichroism studies were undertaken in order to observe any changes in the secondary structures of RMAK in relation to changes of the Mr values. The secondary structure composition of RMAK, determined under our conditions, does not agree with results determined from crystallographic studies. The gel filtration and the CD studies suggest that above pH 7 a more open conformation of RMAK obtains in the presence of DTT. The results of these studies are discussed with reference to the location of the active sites.     

Interactions between fungal growth,substrate utilization,and enzyme production during solid substrate cultivation of Phanerochaete chrysosporium on cotton stalks

Fungal pretreatment, using lignin-degrading microorganisms to improve lignocellulosic feedstocks with minimal energy input, is a potential alternative to physiochemical pretreatment methods. Identifying the kinetics for fungal pretreatment during solid substrate cultivation is needed to help establish the processing conditions for effective scale up of this technology. In this study, a set of mathematical models were proposed for describing the interactions between holocellulose consumption, lignin degradation, cellulase, ligninolytic enzyme, and the growth of Phanerochaete chrysosporium during a 14 day fungal pretreatment process. Model parameters were estimated and validated by the System Biology Toolbox in MatLab. Developed models provided sufficiently accurate predictions for fungal growth (R ² = 0.97), holocellulose consumption (R ² = 0.97), lignin degradation (R ² = 0.93) and ligninolytic enzyme production (R ² = 0.92), and fair prediction for cellulase production (R ² = 0.61). The models provide valuable information for understanding the interactive mechanisms in biological systems as well as for fungal pretreatment process scale up and improvement.     

Adult stem cells and bioengineering strategies for the treatment of cerebral ischemic stroke

The adult central nervous system (CNS) contains a population of neural stem cells, yet unlike many other tissues, has a very limited capacity for self-repair. Promoting tissue repair and functional recovery following CNS injury or disease is a high priority as there are currently no effective treatments towards this end for the treatment of disorders such as stroke, traumatic brain injury and spinal cord injury. Recent advances in stem cell biology have offered a number of enticing potential avenues and we will discuss these possibilities along with the associated challenges as they pertain to stroke. We will consider exogenous therapies involving the transplantation of adult stem cells, and the mobilization of endogenous stem cells, as well as drug delivery and tissue engineering strategies that enhance and complement the cell based strategies.     

Effect of surgery to implant motion and force sensors on vertical ground reaction forces in the ovine model

Activities of daily living (ADLs) generate complex, multidirectional forces in the anterior cruciate ligament (ACL). While calibration problems preclude direct measurement in patients, ACL forces can conceivably be measured in animals after technical challenges are overcome. For example, motion and force sensors can be implanted in the animal but investigators must determine the extent to which these sensors and surgery affect normal gait. Our objectives in this study were to determine (1) if surgically implanting knee motion sensors and an ACL force sensor significantly alter normal ovine gait and (2) how increasing gait speed and grade on a treadmill affect ovine gait before and after surgery. Ten skeletally mature, female sheep were used to test four hypotheses: (1) surgical implantation of sensors would significantly decrease average and peak vertical ground reaction forces (VGRFs) in the operated limb, (2) surgical implantation would significantly decrease single limb stance duration for the operated limb, (3) increasing treadmill speed would increase VGRFs pre- and post operatively, and (4) increasing treadmill grade would increase the hind limb VGRFs pre- and post operatively. An instrumented treadmill with two force plates was used to record fore and hind limb VGRFs during four combinations of two speeds (1.0 m/s and 1.3 m/s) and two grades (0 deg and 6 deg). Sensor implantation decreased average and peak VGRFs less than 10% and 20%, respectively, across all combinations of speed and grade. Sensor implantation significantly decreased the single limb stance duration in the operated hind limb during inclined walking at 1.3 m/s but had no effect on single limb stance duration in the operated limb during other activities. Increasing treadmill speed increased hind limb peak (but not average) VGRFs before surgery and peak VGRF only in the unoperated hind limb during level walking after surgery. Increasing treadmill grade (at 1 m/s) significantly increased hind limb average and peak VGRFs before surgery but increasing treadmill grade post op did not significantly affect any response measure. Since VGRF values exceeded 80% of presurgery levels, we conclude that animal gait post op is near normal. Thus, we can assume normal gait when conducting experiments following sensor implantation. Ultimately, we seek to measure ACL forces for ADLs to provide design criteria and evaluation benchmarks for traditional and tissue engineered ACL repairs and reconstructions.