The intrinsic stiffness of the in vivo lumbar spine in response to quick releases: Implications for reflexive requirements

Torso muscles contribute both intrinsic and reflexive stiffness to the spine; recent modeling studies indicate that intrinsic stiffness alone is sometimes insufficient to maintain stability in dynamic situations. The purpose of this study was to experimentally test this idea by limiting muscular reflexive responses to sudden trunk perturbations. Nine healthy males lay on a near-frictionless apparatus and were subjected to quick trunk releases from the neutral position into flexion or right-side lateral bend. Different magnitudes of moment release were accomplished by having participants contract their musculature to create a range of moment levels. EMG was recorded from 12 torso muscles and three-dimensional lumbar spine rotations were monitored. A second-order linear model of the trunk was employed to estimate trunk stiffness and damping during each quick release. Participants displayed very limited reflex responses to the quick load release paradigms, and consequently underwent substantial trunk displacements (>50% flexion range of motion and >70% lateral bend range of motion in the maximum moment trials). Trunk stiffness increased significantly with significant increases in muscle activation, but was still unable to prevent the largest trunk displacements in the absence of reflexes. Thus, it was concluded that the intrinsic stiffness of the trunk was insufficient to adequately prevent the spine from undergoing potentially harmful rotational displacements. Voluntary muscular responses were more apparent than reflexive responses, but occurred too late and of too low magnitude to sufficiently make up for the limited reflexes.     

Effect of load position on muscle forces,internal loads and stability of the human spine in upright postures

A novel kinematics-based approach coupled with a non-linear finite element model was used to investigate the effect of changes in the load position and posture on muscle activity, internal loads and stability margin of the human spine in upright standing postures. In addition to 397 N gravity, external loads of 195 and 380 N were considered at different lever arms and heights. Muscle forces, internal loads and stability margin substantially increased as loads displaced anteriorly away from the body. Under same load magnitude and location, adopting a kyphotic posture as compared with a lordotic one increased muscle forces, internal loads and stability margin. An increase in the height of a load held at a fixed lever arm substantially diminished system stability thus requiring additional muscle activations to maintain the same margin of stability. Results suggest the importance of the load position and lumbar posture in spinal biomechanics during various manual material handling operations.     

Size scaling and stiffness of avian primary feathers: implications for the flight of Mesozoic birds

The primary feathers of birds are subject to cyclical forces in flight causing their shafts (rachises) to bend. The amount the feathers deflect during flight is dependent upon the flexural stiffness of the rachises. By quantifying scaling relationships between body mass and feather linear dimensions in a large data set of living birds, we show that both feather length and feather diameter scale much closer to predictions for geometric similarity than they do to elastic similarity. Scaling allometry also indicates that the primary feathers of larger birds are relatively shorter and their rachises relatively narrower, compared to those of smaller birds. Two-point bending tests indicated that larger birds have more flexible feathers than smaller species. Discriminant functional analyses (DFA) showed that body mass, primary feather length and rachis diameter can be used to differentiate between different magnitudes of feather bending stiffness, with primary feather length explaining 63% of variance in rachis stiffness. Adding fossil measurement data to our DFA showed that Archaeopteryx and Confuciusornis do not overlap with extant birds. This strongly suggests that the bending stiffness of their primary feathers was different to extant birds and provides further evidence for distinctive flight styles and likely limited flight ability in Archaeopteryx and Confuciusornis.     

A method for testing the stability of a steady-state system during the calculation of a response to large changes in regulator concentrations

A modification of the ‘finite decomposition’ method (Crabtree and Newsholme (1985) Curr. Top. Cell. Regul. 25, 21–76) for calculating physiological responses from sensitivities is described, to enable the system to be tested for stability at each step of the procedure. Instability is indicated by a change of sign of the determinant of the square matrix (N) in the governing equation for the system. The method cannot be used to predict responses beyond any step at which instability occurs.     

Identification of components of a new stability system of plasmid R1, ParD, that is close to the origin of replication of this plasmid

Summary We provide evidence that a mutation which derepresses an autoregulated system that is located in the vicinity of the basic replicon of R1, stabilizes the ParA^- and ParB^- miniplasmid of R1 pKN1562, without increasing its copy number. The system, which we have called ParD, maps inside the 1.45-kb PstI-EcoRI fragment that is adjacent to the origin of replication of the plasmid. Two protiens whose expression is coordinated are components of the system. The sequence of the PstI-EcoRI fragment was obtained. The wild-type ParD system determines in cis a basal but detectable stability.     

Entamoeba histolytica: impedance measurements and cytotoxicity in the presence of bepridil, verapamil, and cytochalasin D

Entamoeba histolytica, and invasive enteric protozoa, kills mammalian target cells by sequential adherence and cytolytic events. Using platinum plate electrodes with an alternating current source placed in a Wheatstone bridge circuit, the impedance (resistance to ion flow) of a cell suspension of axenic amebae (strain HM1-IMSS) was measured. The impedance of the amebic cell suspension, expressed as resistivity (in ohm-cm), was significantly greater than the test solution and increased with decreasing temperature or greater cell packing (P less than 0.01), indicating that the resistivity measurements reflected the impedance of the amebic surface membrane. Cytochalasin D (10 micrograms/ml), a microfilament inhibitor which inhibited amebic in vitro adherence and cytolysis of target Chinese hamster ovary (CHO) cells (P less than 0.001), also increased resistivity of the amebic suspension (P less than 0.01). Exposure of amebae to bepridil (10(5) M), a slow-channel blocker, inhibited amebic killing of target cells (P less than 0.01) and also increased the resistivity of the amebic suspension (P less than 0.01), but both to a lesser degree than cytochalasin D (P less than 0.001). In contrast, exposure of amebae to verapamil followed by washing had no effect on amebic killing of target cells or resistivity of the amebic suspension. The increased resistivity measured in cytochalasin D or following exposure to bepridil was not due to a change in cell density of the amebic suspension. These studies indicate that changes in impedance of the amebic surface membrane are produced by bepridil and cytochalasin D. The effect of these agents on membrane impedance may contribute directly to the concurrent observed alteration in amebic cytopathogenic capacity or may serve as a parallel marker for the cell membrane alterations induced by such pharmacologic agents which inhibit amebic microfilament function or calcium flux.     

Studies on the degradation pathway of iron-sulfur centers during unfolding of a hyperstable ferredoxin: cluster dissociation,iron release and protein stability

The ferredoxin from the thermoacidophile Acidianus ambivalens is a representative of the archaeal family of di-cluster [3Fe-4S][4Fe-4S] ferredoxins. Previous studies have shown that these ferredoxins are intrinsically very stable and led to the suggestion that upon protein unfolding the iron-sulfur clusters degraded via linear three-iron sulfur center species, with 610 and 520 nm absorption bands, resembling those observed in purple aconitase. In this work, a kinetic and spectroscopic investigation on the alkaline chemical denaturation of the protein was performed in an attempt to elucidate the degradation pathway of the iron-sulfur centers in respect to protein unfolding events. For this purpose we investigated cluster dissociation, iron release and protein unfolding by complementary biophysical techniques. We found that shortly after initial protein unfolding, iron release proceeds monophasically at a rate comparable to that of cluster degradation, and that no typical EPR features of linear three-iron sulfur centers are observed. Further, it was observed that EDTA prevents formation of the transient bands and that sulfide significantly enhances its intensity and lifetime, even after protein unfolding. Altogether, our data suggest that iron sulfides, which are formed from the release of iron and sulfide resulting from cluster degradation during protein unfolding in alkaline conditions, are in fact responsible for the observed intermediate spectral species, thus disproving the hypothesis suggesting the presence of a linear three-iron center intermediate. Kinetic studies monitored by visible, fluorescence and UV second-derivative spectroscopies have elicited that upon initial perturbation of the tertiary structure the iron-sulfur centers start decomposing and that the presence of EDTA accelerates the process. Also, the presence of EDTA lowers the observed melting temperature in thermal ramp experiments and the midpoint denaturant concentration in equilibrium chemical unfolding experiments, further suggesting that the clusters also play a structural role in the maintenance of the conformation of the folded state.     

Differing in vitro biology of equine,ovine, porcine and human articular chondrocytes derived from the knee joint: an immunomorphological study

For lack of sufficient human cartilage donors, chondrocytes isolated from various animal species are used for cartilage tissue engineering. The present study was undertaken to compare key features of cultured large animal and human articular chondrocytes of the knee joint. Primary chondrocytes were isolated from human, porcine, ovine and equine full thickness knee joint cartilage and investigated flow cytometrically for their proliferation rate. Synthesis of extracellular matrix proteins collagen type II, cartilage proteoglycans, collagen type I, fibronectin and cytoskeletal organization were studied in freshly isolated or passaged chondrocytes using immunohistochemistry and western blotting. Chondrocytes morphology, proliferation, extracellular matrix synthesis and cytoskeleton assembly differed substantially between these species. Proliferation was higher in animal derived compared with human chondrocytes. All chondrocytes expressed a cartilage-specific extracellular matrix. However, after monolayer expansion, cartilage proteoglycan expression was barely detectable in equine chondrocytes whereby fibronectin and collagen type I deposition increased compared with porcine and human chondrocytes. Animal-derived chondrocytes developed more F-actin fibers during culturing than human chondrocytes. With respect to proliferation and extracellular matrix synthesis, human chondrocytes shared more similarity with porcine than with ovine or equine chondrocytes. These interspecies differences in chondrocytes in vitro biology should be considered when using animal models.     

Thermodynamic analysis of the contributions of the copper ion and the disulfide bridge to azurin stability: synergism among multiple depletions

The stabilizing potential of the copper ion and the disulfide bridge in azurin has been explored with the aim of inspecting the ways in which these two factors influence one another. Specifically, whether copper and disulfide contributions to protein stability are additive has been examined. To this aim, the thermal unfolding of a copper-depleted mutant lacking the disulfide bridge between Cys3 and Cys26 (apo C3A/C26A azurin) was studied by differential scanning calorimetry. A comparison of the unfolding parameters of holo and apo C3A/C26A azurin with the apo C3A/C26A protein has shown that the effects of simultaneous copper and disulfide depletion are additive only at two temperatures: T=15 degrees C and T=67 degrees C. Within this range the presence of the copper ion and the disulfide bridge has a positive synergistic effect on azurin stability. These findings might have implications for the rational use of the stabilizing potential of copper and disulfides in copper protein engineering.     

Evaluating the stability of disulfide bridges in proteins: a torsional potential energy surface for diethyl disulfide

Disulfide bonds formed by the oxidation of cysteine residues in proteins are the major form of intra- and inter-molecular covalent linkages in the polypeptide chain. To better understand the conformational energetics of this linkage, we have used the MP2(full)/6-31G(d) method to generate a full potential energy surface (PES) for the torsion of the model compound diethyl disulfide (DEDS) around its three critical dihedral angles (χ₂, χ₃, χ₂′). The use of ten degree increments for each of the parameters resulted in a continuous, fine-grained surface. This allowed us to accurately predict the relative stabilities of disulfide bonds in high resolution structures from the Protein Data Bank. The MP2(full) surface showed significant qualitative differences from the PES calculated using the Amber force field. In particular, a different ordering was seen for the relative energies of the local minima. Thus, Amber energies are not reliable for comparison of the relative stabilities of disulfide bonds. Surprisingly, the surface did not show a minimum associated with χ₂～ ? 60°, χ₃～90, χ₂′～ ? 60°. This is due to steric interference between Hα atoms. Despite this, significant populations of disulfides were found to adopt this conformation. In most cases this conformation is associated with an unusual secondary structure motif, the cross-strand disulfide. The relative instability of cross-strand disulfides is of great interest, as they have the potential to act as functional switches in redox processes.     

Effect of methylation on the stability and solvation free energy of amylose and cellulose fragments: a molecular dynamics study

Molecular dynamics (MD) simulations were used to study the stability and solvation of amylose and cellulose fragments. The recently developed gromos carbohydrate force field was further tested by simulating maltose, cellobiose, and maltoheptaose. The MD simulations reproduced fairly well the favorable conformations of disaccharides defined by the torsional angles related with the glycosidic bond and the radius gyration of maltoheptaose. The effects of methylation at different hydroxyl groups on the stability of amylose and cellulose fragments were investigated. The methylations of O-2 and O-3 reduce the stability of a single helix more than methylation at O-6, while the latter reduces the stability of a double helix more. Solvation free-energy differences between the unsubstituted amylose and cellulose fragments and the methylated species were studied using the single-step perturbation method. It was found that methylation at O-2 has the biggest effect, in agreement with experiment.     

Energetic stress and the degree of fluctuating asymmetry: Implications for a long-lasting,honest signal

Summary Duplicate, bilateral structures of individual animals are usually symmetrical. In cases where such structures are asymmetrical, the degree of asymmetry may indicate the propensity of an individual to stray from the genetically programmed outcome during the development of the structure. Asymmetries have recently been assumed to constitute an honest signal of male quality and, as such, a cue for female choice. I tested the assumption that different rates of energy intake would produce differences in the degree of asymmetry by measuring original and induced fourth rectrices of both sides of the body in European nuthatchesSitta europaea. I found no predominance for the right or left side, thus showing the fluctuating nature of the asymmetry at a population level. This was not the case within individuals which consistently had a longer fourth rectrix on one or the other side of the body. Induced rectrices, grown during winter when food availability was relatively low, exhibited a higher degree of asymmetry than did such rectrices grown during winter after hoardable food had been provided earlier during the winter. The original rectrices, grown during relatively benign conditions in late summer, showed the smallest degree of fluctuating asymmetry. This indicates that the degree of asymmetry is affected by the rate of energy intake. Thus, male quality, reflecting the rate with which energy can be secured and shown by differing degrees of asymmetry, can be used as an honest, long-lasting cue by females in their choice of a mate.     

On the NMR structure determination of a 44n RNA pseudoknot: Assignment strategies and derivation of torsion angle restraints

The complete T- and pseudoknotted acceptor arm of the tRNA-like structure of turnip yellow mosaic virus (TYMV) genomic RNA has been studied by NMR spectroscopy. Resonance assignment and the gathering of restraints of the 44-mer are impeded by spectral complexity as well as by line broadening. The latter is caused by local dynamical effects in the pseudoknot domain in the molecule. These specific problems could be solved by using different field strengths and selectively ¹³C/¹⁵ labeled samples. Experiments for assigning the sugar spin systems were adjusted to satisfy the requirements of this system. Furthermore, the quality of the structure could be improved by determining the backbone torsion angles , and , using new approaches that were tailored for use in large RNA molecules.     

The derivation, utility and implications of a divergence index for the fynbos genus Leucadendron (Proteaceae)

The derivation, utility, and implications of a divergence index for the fynbos genus Leucadendron (Proteaceae). The genus Leucadendron is useful for a phylogenetic study because it contains many species (79), is morphologically very diverse and appears to have diverged early on in the history of the family in Africa. Polarity of many characters was determined by outgroup analysis and the ontogenetic method. Exceptionally high levels of convergence made the determination of the phylogeny of the subsections problematic. An index, which is the sum of weight of all derived characters present in a species, was determined.
The genus should be removed from subfamily Aulacinae and placed closer to the Leucospermum 'alliance'. I suggest an arid temperate, shrub-like origin for the African Proteaceae, which is in contrast to some published viewS. Modifications to accepted concepts of fruit homology and evolutionary trends such as pollination and dispersal are presented for the family. The divergence index, if used as a 'morphological clock', appears to be useful for further biogeographic and ecological analyses.     

Trait variability and stress: canalization, developmental stability and the need for a broad approach

Trait variability (particularly fluctuating asymmetry) may provide a general measure of environmental stress applicable across taxa but consistent empirical support is lacking. Historically, stress effects were considered to act independently on trait canalization, developmental noise and trait size. However, in trait comparisons these processes are often assumed to be associated. Here we reconsider this issue and implications for detecting stress effects using trait variability. Published studies that consider multiple environments report little association between the effects of environmental variation on trait canalization and on developmental noise measured as fluctuating asymmetry, sug-gesting that environmental effects often act independently on these processes. To further test the usefulness of trait variability as an indicator of stress, comparisons across environ-ments should take a broad approach and report on several measures of trait variability, rather than focusing on only one index of fluctuating asymmetry as is commonly done.     

Prey-induced changes to a predator's behaviour and morphology: Implications for shell-claw covariance in the northwest Atlantic

Whereas many plasticity studies demonstrate the importance of inducible defences among prey, far fewer investigate the potential role of inducible offences among predators. Here we ask if natural differences in a snail's shell hardness can induce developmental changes to a predatory crab's claw size. To do this, we fed Littorina obtusata snails from either thick- or thin-shelled populations to captive European green crabs Carcinus maenas. The crabs' shell-breaking behaviour dominated among those fed thin-shelled snails, whereas crabs fed thick-shelled snails mostly winkled flesh through the shell opening without damaging the shell itself (a.k.a. aperture-probing behaviour). Significantly, the size of crab crusher claws grew in proportion to the frequency of shell-crushing behaviour and, for a same shell-crushing frequency, crabs fed thick-shelled snails grew larger crusher claws than those fed thin-shelled snails after two experimental moults. Diet and behaviour had no effect on the growth of the smaller cutter claws of same individuals, providing good evidence that allometric changes to crusher claws were indeed a result of differential use while feeding. Findings indicate that both predation habits and claw sizes are affected by green crabs' diet, supporting the hypothesis that prey-induced phenotypic plasticity contributes to earlier accounts of shell-claw covariance between this predator and its Littorina prey in the wild.     

Linkage assignment of a DNA sequence (ERCC2L1) homologous to a human DNA repair gene in Xiphophorus fishes: Implications for the evolutionary derivation of human chromosome 19

Fish gene mapping studies have identified several syntenic groups showing conservation over more than 400 million years of vertebrate evolution. In particular, Xiphophorus linkage group IV has been identified as a homolog of human chromosomes 15 and 19. During mammalian evolution, loci coding for glucosephosphate isomerase, peptidase D, muscle creatine kinase, and several DNA repair genes (ERCC1, ERCC2, and XRCC1) appear as a conserved syntenic group on human chromosome 19. When X. clemenciae and X. milleri PstI endonuclease-digested genomic DNA was used in Southern analysis with a human ERCC2 DNA repair gene probe, a strongly cross-hybridizing restriction fragment length polymorphism was observed. Backcrosses to X. clemenciae from X. milleri × X. clemenciae F₁ hybrids allowed tests for linkage of the ERCC2-like polymorphism to markers covering a large proportion of the genome. Statistically significant evidence for linkage was found only for ERCC2L1 and CKM (muscle creatine kinase), with a total of 41 parents and 2 recombinants (4.7% recombination, χ² = 35.37, P < 0.001); no evidence for linkage to GPI and PEPD in linkage group IV was detected. The human chromosome 19 synteny of ERCC2 and CKM thus appears to be conserved in Xiphophorus, while other genes located nearby on human chromosome 19 are in a separate linkage group in this fish. If Xiphophorus gene arrangements prove to be primitive, human chromosome 19 may have arisen from chromosome fusion or translocation events at some point since divergence of mammals and fishes from a common ancestor.