Physical Perturbation Research Articles

Bax-induced cytochrome c release from mitochondria depends on alpha-helices-5 and -6

The pro-apoptotic protein Bax plays a key role in the mitochondrial signalling pathway. Upon induction of apoptosis, Bax undergoes a conformational change and translocates to mitochondrial membranes, where it inserts and mediates the release of cytochrome c from the intermembrane space into the cytosol. However, the domains of Bax that are essential for the induction of cytochrome c release are still elusive. Therefore various Bax deletion mutants were generated and expressed in Escherichia coli. The proteins were then purified in order to delineate the function of the transmembrane domain, the BH3 (Bcl-2 homology 3) domain and the putative pore-forming alpha-helices-5 and -6. These proteins were used to analyse the mechanism of Bax-induced cytochrome c release from mitochondria. None of the Bax proteins caused cytochrome c release merely through physical perturbation of the mitochondrial outer membrane. The alpha-helices-5 and -6 of Bax were shown to mediate the insertion of the protein into mitochondrial membranes and to be essential for the cytochrome c -releasing activity of Bax. In contrast, neither the transmembrane domain nor a functional BH3 domain is required for the Bax-mediated release of cytochrome c from mitochondria.

Semianalytical models of volt-ampere characteristics of diffuse low-current low-pressure discharges

We perform calculations of volt-ampere (V-A) characteristics of low-current low-pressure diffuse (Townsend) discharges in hydrogen and compare them to the experiment. The basis for the calculation is the numerical procedure for electric field calculation by solving integral equations for the surface charges. We have used analytic solutions for the homogeneous field as the initial approximation and made a cycle of calculations until the charge profile and the field were self-consistent. This procedure allows us to remove the field expansion from the physical perturbation theory of such discharges and to study the causes for negative differential resistances, oscillations, and nonlinear effects. Furthermore, we have developed a nonlinear version of the physical model of Phelps and coworkers which helps us identify the features of the ionization coefficient and secondary electron yield that give rise to nonlinear development of the V-A characteristics of Townsend regime discharges.

Optical nanosensors--an enabling technology for intracellular measurements.

Optical nanosensors have been designed to utilise the sensitivity of fluorescence for making quantitative measurements in the intracellular environment, using devices that are small enough to be inserted into living cells with a minimum of physical perturbation. Advantages over widely used fluorescence dye based methods are observed because the nanosensor matrix imparts two key benefits; (1) protection of the sensing component from interfering species within the intracellular environment and (2) protection of the intracellular environment from any toxic effects of the sensing component. This Highlight article discusses the recent developments in nanosensor technology and investigates the use of more complex sensing schemes that will expand the range of analytes that can be detected and quantified.

4] Fluorescence correlation spectroscopy

Publisher Summary This chapter describes fluorescence correlation spectroscopy (FCS). FCS is an attractive technique for intracellular applications. First, FCS determines kinetic processes from equilibrium fluctuations. Thus, no external perturbation is required to obtain kinetic information. Second, FCS provides excellent spatial resolution. FCS characterizes any kinetic process that leads to changes in the fluorescence, because the spontaneous fluctuations at thermodynamic equilibrium are governed by the same laws that describe the kinetic relaxation of a system to equilibrium. Thus, FCS offers a very convenient method for determining kinetic properties at equilibrium without requiring a physical perturbation of the sample. This is especially important for systems in which the use of perturbation techniques is extremely difficult and challenging, such as measurements in living cells. The concept of measuring signal fluctuations has direct consequences for the experimental realization of FCS. When performing FCS measurements inside the nucleus, it can be found that the fluorescence intensity is very stable. In the cytoplasm, however, the fluorescence intensity depends on the spatial location, and sometimes strong fluctuations, which persist for a few seconds, are observed, whereas at other times the intensity is as stable as inside the nucleus.

Coupling of the Dolichol-P-P-Oligosaccharide Pathway to Translation by Perturbation-sensitive Regulation of the Initiating Enzyme, GlcNAc-1-P Transferase

In mammalian cells, inhibition of translation interferes with synthesis of the lipid-linked oligosaccharide (LLO) Glc3Man9GlcNAc2-P-P-dolichol as measured with radioactive sugar precursors. Conflicting hypotheses have been proposed, and the fundamental basis for this regulation has remained elusive. Here, fluorophore-assisted carbohydrate electrophoresis (FACE) was used to measure LLO concentrations directly in cells treated with translation blockers. Further, LLO biosynthetic enzymes were assayed in vitro with endogenous acceptor substrates using either cells gently permeabilized with streptolysin-O (SLO) or microsomes from homogenized cells. In Chinese hamster ovary (CHO)-K1 cells treated with translation blockers, FACE did not detect changes in concentrations of Glc3Man9GlcNAc2-P-P-dolichol or early LLO intermediates. These results do not support earlier proposals for feedback repression of LLO initiation by accumulated Glc3Man9GlcNAc2-P-P-dolichol, or inhibition of a GDP-mannose dependent transferase. With microsomes from cells treated with translation blockers, there was no interference with LLO initiation by GlcNAc-1-P transferase (GPT), mannose-P-dolichol synthase, glucose-P-dolichol synthase, or LLO synthesis in vitro, as reported previously. Surprisingly, inhibition of all of these was detected with the SLO in vitro system. Additional experiments with the SLO system showed that the three transferases shared a limited pool of dolichol-P that was trapped as Glc3Man9GlcNAc2-P-P-dolichol by translation arrest. Overexpression of GPT was unable to reverse the effects of translation arrest on LLO initiation, and experiments with FACE and the SLO system showed that overexpressed GPT was not functional in vivo, although it was highly active in microsomal assays. Thus, the combined use of the SLO in vitro system and FACE showed that LLO biosynthesis depends upon a limited primary pool of dolichol-P. Physical perturbation associated with microsome preparation appears to make available a secondary pool of dolichol-P, masking inhibition by translation arrest, as well as activating a nonfunctional fraction of GPT. The implications of these results for the organization of the LLO pathway are discussed.

Seasonal fluctuations in the photosynthetic capacity of photosystem II in symbiotic dinoflagellates in the Caribbean reef-building coral Montastraea

The photosynthetic capacity of photosystem II (PS II) in symbiotic dinoflagellates (Symbiodinium sp.), as measured by analysis of chlorophyll fluorescence, was investigated in the primary Caribbean reef-building corals, Montastraea annularis and Montastraea faveolata, for 5 years and Montastraea franksi over 2 years in the Bahamas. Significant seasonal fluctuations in the quantum yield of charge separation (Fv/Fm) of PS II were found in all species at all depths, with the highest photosynthetic capacity consistently recorded between mid-winter and early spring and the lowest photosynthetic capacity occurring in the mid to late summer. Corals residing in shallow depths of 1–2 m showed the greatest fluctuations in Fv/Fm, whereas deeper corals (3–4 and 14 m depths) had consistently higher values of Fv/Fm. Densities of symbiotic dinoflagellates and photosynthetic pigments followed a similar pattern. Fluctuations of photosynthetic capacity showed a strong correlation with seasonal patterns of water temperature and light. Such seasonal shifts in photosynthetic capacity are most likely due to several biochemical processes in the algae that lead to alterations of both photoprotection and photodamage. While symbiont density changed significantly on a seasonal basis, visual signs of coral bleaching were noted only in the fall of 1995 and the spring and summer of 1998. Comparisons of photosynthetic capacity and the decrease in the number of symbionts and their subsequent recovery indicated that symbiont populations in this study had the ability to recover quickly following bleaching events, as long as continued physical perturbation (e.g. thermal stress) did not shorten the recovery phase. Large-scale bleaching events are best viewed as the end points of seasonal physiological variation in which photosynthetic capacity and density of symbiotic dinoflagellates are reduced to a lower level than during "non-bleaching" years.

Physical and Enzymatic Perturbation of the Architecture of the Tunica media Destroys its Inherent Thromboresistance

The media layer of the arterial cryo-cross sections, is defective for vWf-dependent platelet adhesion. Exposure of the same layer by stripping off the most inner portions of the vessel wall results in a highly thrombogenic surface. Stripping or balloon dilation was applied to porcine arteries prior to functional assays. Cryosections of treated or untreated arteries were perfused with porcine blood at 3,350 s(-1) and platelet deposition was detected by indirect immunofluorescence. Following balloon dilation, vWf-dependent platelet deposition increased; covering 9.08 +/- 1.36% of the total media surface area, this value for untreated vessels was 0.88 +/- 0.14%. A 10-fold increase was also found in the binding of vWf-coated fluorescent beads to the media. In addition to mechanical procedures, treatment by serine-proteases like trypsin, chymotrypsin and proteinase 3, or by chondroitinase ABC, but not by heparitinase also resulted in a 7-10-fold increase in platelet coverage over the media. Collagen in the media may be complexed with another vessel wall component shielding the vWf-binding sites. Mechanical or biochemical processes unmask these sites, and increase the thrombogenicity of the vessel wall.

Bilirubin directly disrupts membrane lipid polarity and fluidity, protein order, and redox status in rat mitochondria

Background/Aims : Unconjugated bilirubin (UCB) impairs crucial aspects of cell function and induces apoptosis in primary cultured neurones. While mechanisms of cytotoxicity begin to unfold, mitochondria appear as potential primary targets. Methods : We used electron paramagnetic resonance spectroscopy analysis of isolated rat mitochondria to test the hypothesis that UCB physically interacts with mitochondria to induce structural membrane perturbation, leading to increased permeability, and subsequent release of apoptotic factors. Results : Our data demonstrate profound changes on mitochondrial membrane properties during incubation with UCB, including modified membrane lipid polarity and fluidity ( P<0.01 ), as well as disrupted protein mobility ( P<0.001 ). Consistent with increased permeability, cytochrome c was released from the intermembrane space ( P<0.01 ), perhaps uncoupling the respiratory chain and further increasing oxidative stress ( P<0.01 ). Both ursodeoxycholate, a mitochondrial-membrane stabilising agent, and cyclosporine A, an inhibitor of the permeability transition, almost completely abrogated UCB-induced perturbation. Conclusions : UCB directly interacts with mitochondria influencing membrane lipid and protein properties, redox status, and cytochrome c content. Thus, apoptosis induced by UCB may be mediated, at least in part, by physical perturbation of the mitochondrial membrane. These novel findings should ultimately prove useful to our evolving understanding of UCB cytotoxicity.

The contribution of epigenetic changes to abnormal centrosomes and genomic instability in breast cancer.

The centrosome is the major microtubule organizing center of the cell and as such it plays an important role in cytoskeletal organization and in the formation of the bipolar mitotic spindle. Centrosome defects, characterized by abnormal size, number, and microtubule nucleation capacity, are distinguishing features of most high grade breast tumors and have been implicated as a possible cause for the loss of tissue architecture and the origin of mitotic abnormalities seen in solid tumors in general. Centrosome defects arise through uncoupling of centriole duplication and the cell cycle as a result of either genetic alterations or through physical or chemical perturbation of centrosome function. Centrosomes manifest unique epigenetic properties whereby positional or structural information can be propagated through somatic cell lineages by way of nongenetic pathways. Because aberrant centrosome function can result in chromosomal instability, these properties may have important implications for the origin of malignant breast tumors.

Lysophosphatidylcholine-induced cellular injury in cultured fibroblasts involves oxidative events

Lysophosphatidylcholine (lysoPC), formed during LDL oxidation and located within atherosclerotic plaques, induces numerous cellular responses, but via unknown mechanisms. Cellular events involved in sublethal lysoPC-induced injury were examined because these are relevant to mechanisms by which lysoPC alters cell behavior. LysoPC evoked transient membrane permeabilization in fibroblasts within 10 min. Cells underwent reversible rounding within 2 h, returning 3 h later to grossly normal appearance and a normal response to growth stimulation. We asked whether this sublethal permeabilization resulted from physical perturbation of the plasma membrane or if it required cellular events. LysoPC induced leakage of fluorescent dye from unilamellar phospholipid vesicles, suggesting physical membrane perturbation was a significant contributor. To characterize this further we increased the cholesterol content of cells and vesicles to stabilize membranes, and found decreased lysoPC-induced permeabilization in both cell and cell-free systems as cholesterol levels increased. Interestingly, vitamin E, a known antioxidant, blunted lysoPC-induced permeabilization and morphological changes in cells. Thus, lysoPC appeared to cause an unexpected oxidant stress-dependent enhancement of cell injury. To confirm this, several structurally distinct antioxidants, including N, N'-diphenyl-1,4-phenylenediamine, Desferal, Tiron, and 4-hydroxy TEMPO, were applied and these also were inhibitory. Oxidant stress was observed by a lysoPC-induced increase in fluorescence of 5- and 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, an intracellular marker of reactive oxygen species. Lysophosphatidylethanolamine (lysoPE) caused qualitatively similar morphological changes to cells and induced permeabilization, but injury by lysoPE was not inhibited by antioxidants. These data suggest that generation of intracellular reactive oxygen species follows lysoPC-induced plasma membrane destabilization and that this lysoPC-specific oxidant stress enhances cell injury. This intracellular oxidant stress in response to lysoPC may be an integral part of the multiple influences lysoPC has on gene expression and cell function.

An algorithm for robust noninteracting control of ship propulsion system

In this paper, a new algorithm for noninteracting control system design is proposed and applied to ship propulsion system control. For example, if a ship diesel engine is operated by consolidated control with controllable pitch propeller (CPP), the minimum fuel consumption is achieved satisfying the demanded ship speed. For this, it is necessary that the ship is operated on the ideal operating line which satisfies the minimum fuel consumption, and the both pitch angle of CPP and throttle valve angle are controlled simultaneously. In this context of view, this paper gives a controller design method for a ship propulsion system with CPP based on noninteracting control theory. Where, linear matrix inequality (LMI) approach is introduced for the control system design to satisfy the given H~ constraint in the presence of physical parameter perturbation and disturbance input. To the end, the validity and applicability of this approach are illustrated by the simulation in the all operating ranges.

Influence of a Pulmonary Surfactant on the In Vitro Activity of Tobramycin against Pseudomonas aeruginosa

Delivery of therapeutic concentrations of drugs to the lung periphery is crucial in treatment of many infectious and inflammatory diseases of the lung. Conventional drug treatment for serious pulmonary infections consists of systemic administration of drug in order to achieve concentrations in the bloodstream that will reach therapeutic results. Advantages to topical drug administration to the lungs via aerosol are well known (3). However, the amount of drug that can be inhaled as aerosol is frequently small, thereby limiting the amount of drug delivered to the distal portion of the lung (1). Intratracheal instillation of surfactant, in the animal model, increased the fractional area of lung exposed and improved the uniformity of spread of radioactive label in the lung compared to the saline control (2). We examined the effect of physical perturbation with the mixture, antibiotic, and surfactant, via heat and sonication and stability over time as a function of the MIC for Pseudomonas. Tobramycin and surfactant (Exosurf suspension) were combined in cationic adjusted Mueller-Hinton broth (CAMHB) to create mixtures containing 8, 16, 32, or 64 μg of surfactant per ml, all with 4 μg of tobramycin per ml (4). One aliquot of each mixture was heated at 80°C for 30 min, one was sonicated at room temperature for 30 min, and a third was held at room temperature for 30 min without additional treatment. Serial twofold dilutions of each aliquot were prepared in CAMHB in microdilution trays (final volume, 100 μl/well) and inoculated with approximately 5 × 105 CFU of Pseudomonas aeruginosa ATCC 27853. Serial dilutions of tobramycin in CAMHB and separate serial dilutions of surfactant in CAMHB were inoculated with the same organism to serve as controls. All trays were incubated for 18 h at 35°C, and the MIC was determined visually. When tobramycin and surfactant were mixed, sonicated, and heated to 80°C for 30 min, there was no in vitro reduction in MIC for P. aeruginosa. To assess the stability of mixtures of tobramycin and surfactant, we prepared a stock mixture containing 27 mg each of tobramycin and surfactant per ml. Dilutions of this mixture were prepared immediately to give final concentrations of tobramycin ranging from 0.06 to 8 μg/ml in CAMHB. Controls, inoculation, incubation, and examination were identical to those described above. The remaining stock mixture was stored at 4°C for 3 weeks, and the MIC was determined at 1 week and again at 3 weeks. No decline in the MIC of the mixture was found when it was stored in a refrigerated environment for up to 3 weeks. van’t Veen and coworkers (5) found a reduction in bactericidal activity of tobramycin when tobramycin was mixed with surfactant which could be compensated for by increasing the tobramycin concentration. A possible explanation of the difference from our study may be the difference in the surfactants. Exosurf, a synthetic compound, does not contain protein, whereas van’t Veen et al. used a surfactant obtained from the bovine lung containing a protein component.

Use of ‘functional habitats’ to link ecology with morphology and hydrology in river rehabilitation

1. This paper examines the influence of channel morphology and hydraulics on the occurrence and diversity of ‘functional habitats’ (also called ‘meso-habitats’) in semi-natural and physically degraded rivers. 2. The depths and velocities at which each functional habitat is most likely to be found are described using ‘occurrence matrices’. In most cases, each habitat was associated with distinct depth-velocity conditions. 3. The relationship between habitat occurrence, diversity and river degradation was examined. The deviation of a site from its predicted natural summer wetted width was used as an index of physical degradation. Habitat diversity (measured as the Shannon index) was low for sites that were wider than expected for natural conditions. This was independent of the type of physical perturbation causing the width deviation. 4. Two main types of physical perturbation were identified in the degraded river: over-widening and ponding. Over-widened sites were shallow, slow and dominated by silt and emergent macrophyte habitats. Ponded sites were deep, slow and dominated by floating-leaved macrophyte and macroalgal habitats. 5. River reaches of uniform depth had low habitat diversity compared with reaches containing both shallow and deep areas. 6. It is recommended that objective assessment of the type and extent of physical degradation, prior to restoration, should be made by measurement of depth, velocity, width and functional habitat distribution. Changes in channel morphology should then be designed to create the necessary range of depths and widths, and therefore velocities and habitat types, deemed desirable for that river site. Habitats that are over-dominant or rare can be identified and the ‘occurrence matrices’ used to inform decisions about the change in physical conditions needed to enhance or suppress that habitat. 7. The approach is a cost-effective way to link ecology with morphology and hydrology in river channel rehabilitation. Copyright © 1999 John Wiley & Sons, Ltd.

Macrobenthic communities impacted by anthropogenic activities in an intertidal sand flat on the west coast (Yellow Sea) of Korea

Impacts on benthic macrofaunal communities by pollution and physical perturbation following seawall construction were assessed for an intertidal sand flat in Kyeonggi Bay, Korea near an industrialized harbour city. Despite the apparent input of a considerable amount of industrial effluents, no traces of heavy metal contamination were detected in either sediments or resident animals, probably owing to overall low percentages of mud. A seasonal cycle of silty sediment deposition and erosion also appears to play a role in alleviating metal accumulation in the sand flat. Recent seawall constructions, however, seem to have influenced the sedimentary environment. Sediment grain size became significantly coarser after seawall constructions and, associated with the grain size change, some shifts in dominance of abundant species were recorded. In addition, both total abundance and biomass significantly increased. In particular, abundance of the filter-feeding bivalve Mactra veneriformis increased ten times, seemingly benefiting from the coarser grain size. Changes in community structure were also reflected in k-dominance abundance curves.

Prediction of Phase Equilibria in Binary Aqueous Systems Containing Alkanes, Cycloalkanes, and Alkenes with the Cubic-plus-Association Equation of State

The cubic-plus-association (CPA) equation of state (EoS) is applied in this study to binary aqueous mixtures containing hydrocarbons. The CPA EoS combines the Soave−Redlich−Kwong (SRK) cubic equation of state for the physical part and perturbation theory for the chemical (association) part. Rigorous expressions for the contribution of the association term to the pressure and to the chemical potential, which do not include any derivatives of the mole fraction of molecules i not bonded at site A (XAi), are presented. Three different association models for water have been considered depending on the number of hydrogen bonding sites per water molecule: the two-, three-, and four-site models. Successful correlation of both vapor pressures and saturated liquid volumes is obtained with all three models. However, satisfactory correlation results of the mutual solubilities of water/aliphatic hydrocarbon systems are obtained only with the four-site model using a single interaction parameter (kij) in the attractive...

Effect of a non-linear boundary layer on the radiation from earthquakes and underground nuclear explosions

A constitutive equation in the immediate source region is postulated. It is proposed that between the permanently deformed source volume and the linear zone there exists a microfractured boundary layer having the constitution of a five-parameter Murnaghan solid that exhibits both geometrical finite strain and physical cubic terms in the energy density function. Waves excited by the source carry the signature of the boundary layer into the linear zone, all the way to the far field. The mathematical realization of this programme consists of the application of the longwave Born–Rayleigh approximation to the Landau–Goldberg equation. The perturbed field, governed by an additional force system, is represented as a multipole expansion of eigenvector spherical harmonics of the unperturbed background field. Our solution aims at the spatial characteristics of the 3-D perturbed field (e.g. radiation patterns, amplitude amplification, etc.) together with its frequency-content features. The results show, in general, that for every spatial mode with indices (ℓ1, m1) fed into the non-linear boundary layer system, there emerges in the elastic zone the output modes (ℓ1±2s, 2m1), s=0,2,4,…. Compressional and shear modes generate each other (‘mode crossing’), and spatial field harmonics of lower and higher orders than originally existed in the primary field appear. In addition, one encounters the expected frequency doubling in the spectral displacements of the first-order perturbation. Detailed calculations are made, especially for dipolar sources, which include explosion (ℓ1=0,m1=0) and shear dislocations (ℓ1=2, m1=0,1,2). It is revealed that the boundary layer tends to amplify the source field by a factor that may reach 4 for explosions in pre-existing cavities and 2 for earthquakes. Hence, the amplification will effectively limit the true seismic decoupling of underground nuclear explosions to a limiting value of under 70 and tend to boost the true seismic moments of earthquakes. In the latter case the non-linear theory suggests the introduction of two new source parameters: a virtual radius, r0, associated with the yield limit of rocks, and a larger radius associated with the elastic limit of rocks. It is shown that for shear dislocations, the fault's area is interpreted geometrically as a virtual spherical surface of radius r0. As long as small finite strains are assumed inside a thin boundary layer, scrambling of the wavefield (that is displacement amplification, generation of higher-degree multipoles and mode crossing) is the most conspicious physical perturbation, independent of any non-classical modification of the constitutive law. In this sense, the salient gross features of our final results are model-independent. Our results serve as a bridge between the microscopic and macroscopic theories of fracture and hopefully pave the road towards a dynamical theory of earthquake-source mechanisms.

Moderately High Temperatures Inhibit Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) Activase-Mediated Activation of Rubisco

We tested the hypothesis that light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is inhibited by moderately elevated temperature through an effect on Rubisco activase. When cotton (Gossypium hirsutum L.) or wheat (Triticum aestivum L.) leaf tissue was exposed to increasing temperatures in the light, activation of Rubisco was inhibited above 35 and 30 degreesC, respectively, and the relative inhibition was greater for wheat than for cotton. The temperature-induced inhibition of Rubisco activation was fully reversible at temperatures below 40 degreesC. In contrast to activation state, total Rubisco activity was not affected by temperatures as high as 45 degreesC. Nonphotochemical fluorescence quenching increased at temperatures that inhibited Rubisco activation, consistent with inhibition of Calvin cycle activity. Initial and maximal chlorophyll fluorescence were not significantly altered until temperatures exceeded 40 degreesC. Thus, electron transport, as measured by Chl fluorescence, appeared to be more stable to moderately elevated temperatures than Rubisco activation. Western-blot analysis revealed the formation of high-molecular-weight aggregates of activase at temperatures above 40 degreesC for both wheat and cotton when inhibition of Rubisco activation was irreversible. Physical perturbation of other soluble stromal enzymes, including Rubisco, phosphoribulokinase, and glutamine synthetase, was not detected at the elevated temperatures. Our evidence indicates that moderately elevated temperatures inhibit light activation of Rubisco via a direct effect on Rubisco activase.

EFFECTS OF PERIODIC DISTURBANCES FROM TRAMPLING ONROCKY INTERTIDAL ALGAL BEDS

We investigated the ability of an assemblage of animals and plants on rocky shores in southeastern Australia to resist and/or recover from repeated pulse disturbances in the form of trampling. Disturbances of four different intensities were applied experimentally over six summers, with no human access at other times of the year. The dominant intertidal plant, the brown alga Hormosira banksii, was affected by trampling, but the effects were heterogeneous between sites. At two sites, a series of pulse disturbances produced a series of pulse responses, although the effect of a given pulse varied among years, possibly related to the severity of summer desiccating conditions each year. At the third site, pulse disturbances produced a press response; at high levels of trampling, Hormosira was almost eliminated within 2 yr, and at two intermediate levels of trampling, cover was reduced from >90 to 60–70%, where it remained for 4 yr. Effects of trampling showed little small-scale spatial variation. Untrampled areas did fluctuate through time, often as a result of summer burnoff of algae. Natural disturbances occurred irregularly through the study, and their effects varied on very small spatial scales (among plots <30 m apart). Trampling enhanced the densities of a range of herbivorous mollusks, especially limpets, and reduced the abundance of articulated coralline algae, which were abundant in the understory of Hormosira mats. These effects varied among sites but showed much less variation on smaller spatial scales. The reductions in coralline algae may be a direct effect of trampling, but increases in mollusk abundance occurred some time after changes to Hormosira cover, and those changes may be an indirect effect of trampling. We compared the effects of trampling on areas of the shore that had been trampled for two and four summers, to test whether a past history of disturbance influenced the effect of a new disturbance. No significant effects were found on algae or mobile animals, although a mild summer may have made our test of history relatively weak. Hormosira banksii fits the definition of a keystone species or engineer and, as such, is an appropriate focus for management and as an indicator. Spatially heterogeneous effects of a constant physical perturbation, however, mean that management of these rocky shores requires more complex models and indicate that caution should be used in adopting this species as a uniform indicator of environmental change.

Design of protein function by physical perturbation method.

Based on three-dimensional structure of proteins, a rational strategy to design the protein function by physical perturbation method was proposed and tested on one of the well-examined enzymes, thermolysin for higher catalytic activity. An attempt was made to change the electrostatic potential and the dynamic property of three-dimensional structure around the active sites by single-amino-acid mutations, and the physical property of the mutants was then evaluated. Several mutants were found to have remarkably higher enzymatic activity than wild type. The multiple mutation was introduced and the logarithm of the activity was found to be almost additive. A ten times higher active mutant was realized by simultaneously introducing three single-mutations. This strategy can be easily extended to not only other enzymes but also other kinds of proteins than enzymes to modify or control the protein function based on their three-dimensional structures.

Design of protein function by physical perturbation method

Based on three-dimensional structure of proteins, a rational strategy to design the protein function by physical perturbation method was proposed and tested on one of the well-examined enzymes, thermolysin for higher catalytic activity. An attempt was made to change the electrostatic potential and the dynamic property of three-dimensional structure around the active sites by single-amino-acid mutations, and the physical property of the mutants was then evaluated. Several mutants were found to have remarkably higher enzymatic activity than wild type. The multiple mutation was introduced and the logarithm of the activity was found to be almost additive. A ten times higher active mutant was realized by simultaneously introducing three single-mutations. This strategy can be easily extended to not only other enzymes but also other kinds of proteins than enzymes to modify or control the protein function based on their three-dimensional structures.