Regulation of oscillatory contraction in insect flight muscle by troponin.

Insect indirect flight muscle is activated by sinusoidal length change, which enables the muscle to work at high frequencies, and contracts isometrically in response to Ca(2+). Indirect flight muscle has two TnC isoforms: F1 binding a single Ca(2+) in the C-domain, and F2 binding Ca(2+) in the N- and C-domains. Fibres substituted with F1 produce delayed force in response to a single rapid stretch, and those with F2 produce isometric force in response to Ca(2+). We have studied the effect of TnC isoforms on oscillatory work. In native Lethocerus indicus fibres, oscillatory work was superimposed on a level of isometric force that depended on Ca(2+) concentration. Maximum work was produced at pCa 6.1; at higher concentrations, work decreased as isometric force increased. In fibres substituted with F1 alone, work continued to rise as Ca(2+) was increased up to pCa 4.7. Fibres substituted with various F1:F2 ratios produced maximal work at a ratio of 100:1 or 50:1; a higher proportion of F2 increased isometric force at the expense of oscillatory work. The F1:F2 ratio was 9.8:1 in native fibres, as measured by immunofluorescence, using isoform-specific antibodies. The small amount of F2 needed to restore work to levels obtained for the native fibre is likely to be due to the relative affinity of F1 and F2 for TnH, the Lethocerus homologue of TnI. Affinity of TnC isoforms for a TnI fragment of TnH was measured by isothermal titration calorimetry. The K(d) was 1.01 muM for F1 binding and 22.7 nM for F2. The higher affinity of F2 can be attributed to two TnH binding sites on F2 and a single site on F1. Stretch may be sensed by an extended C-terminal domain of TnH, resulting in reversible dissociation of the inhibitory sequence from actin during the oscillatory cycle.

Simulation of guided waves in complex piping geometries using the elastodynamic finite integration technique.

Although many technologies exist for inspecting piping systems, they are most successful on straight pipes and are often unable to accommodate the added complexities of pipe elbows, bends, twists, and branches, particularly if the region of interest is inaccessible. This paper presents a numerical technique based on the elastodynamic finite integration technique for simulating guided elastic wave propagation in piping systems. Comparisons show agreement between experimental and simulated data, and guided wave interaction with flaws, focusing, and propagation in pipe bends are presented. These examples demonstrate the ability of the simulation method to be used to study elastic wave propagation in piping systems which include three-dimensional pipe bends, and suggest its potential as a design tool for designing pipe inspection hardware and ultrasonic signal processing algorithms.

Crystalline yolk spheroids in Drosophila melanogaster oocyte: freeze fracture and two-dimensional reconstruction analysis.

The major sites of energy storage during oogenesis in the Drosophila melanogaster oocyte are the alpha- and beta-yolk spheres. By applying biochemical and transmission electron microscopy (TEM) immunogold techniques we found that the beta-yolk spheres contain mainly polysaccharides, while the three main yolk proteins (YPs) are stored in the alpha-yolk spheres of the developing oocyte. Moreover, by using high-resolution TEM of freeze fractured or cryosectioned follicles, we identified the existence of crystalline structures within the alpha-yolk spheres of the mature oocyte. Our subsequent two-dimensional reconstruction analysis revealed that the unit cell of the crystal is about 113 Angstrom x 113 Angstrom. Assuming that the repeating unit is a cylinder of about 110 Angstrom in length and 25 Angstrom in diameter this cylinder would then have a volume of about 50,000 cubic Angstrom, which corresponds to about 40 kDa of protein. This size fits quite well with the known molecular weight of about 40-45 kDa for each of the three D. melanogaster YPs. Overall, our study identifies for the first time the supramolecular arrangement of the alpha-yolk spheres constituent molecules and provides direct evidence for the "natural" crystallization, and therefore the efficient packaging, of the YPs during oogenesis.

Lamb wave tomography of pipe-like structures.

Lamb waves are guided ultrasonic plate waves that can follow the curvature of pipe-like structures. By transmitting and receiving many helically propagating Lamb waves via longitudinal transducers in contact with the surface of a pipe, crosshole tomographic geometries can be mimicked and tomographic reconstructions performed in order to locate and size flaws. We describe here a meridional-array scheme which mimics a single line of transducers along the exterior surface of the pipe in the axial direction, and show proof of concept results on a pipe sample with an internal wall-thinning. We also demonstrate improved reconstructions for the other helical ultrasound tomography geometry where the transmitters and receivers lie along parallel circumferential rings. We find frequency compounding smoothes out some of the noise and artifacts that appear in the reconstructions.

Multi-mode Lamb wave tomography with arrival time sorting.

Lamb wave tomography has been shown to be an effective nondestructive evaluation technique for platelike structures. A series of pitch-catch measurements between ultrasonic transducers can be taken from different orientations across the sample to create a map of a particular feature of interest such as plate thickness. Most previous work has relied solely on the first arriving mode for the time-of-flight measurements and tomographic reconstructions. The work described here demonstrates the capability of the Lamb wave tomography system to generate accurate reconstructions from multiple modes. Because each mode has different through-thickness displacement values, each is sensitive to different types of flaws, and the information gained from a multi-mode analysis can improve understanding of the structural integrity of the inspected material. However, one of the problems with the extraction of multi-mode arrival times is that destructive interference between two modes may cause one of the modes to seemingly disappear in the signal. The goal of the sorting algorithm presented in this work is to try and counteract this problem by using multiple frequency scans--also known as frequency walking--to sort the arrival times into their correct mode series.

Myofilin, a protein in the thick filaments of insect muscle.

Thick filaments in striated muscle are myosin polymers with a length and diameter that depend on the fibre type. In invertebrates, the length of the thick filaments varies widely in different muscles and additional proteins control filament assembly. Thick filaments in asynchronous insect flight muscle have an extremely regular structure, which is likely to be essential for the oscillatory contraction of these muscles. The factors controlling the assembly of thick filaments in insect flight muscle are not known. We previously identified a thick filament core protein, zeelin 1, in Lethocerus flight and non-flight muscles. This has been sequenced, and the corresponding proteins in Drosophila and Anopheles have been identified. The protein has been re-named myofilin. Zeelin 2, which is on the outside of Lethocerus flight muscle thick filaments, has been sequenced and because of the similarity to Drosophila flightin, is re-named flightin. In Drosophila flight muscle, myofilin has a molecular weight of 20 kDa and is one of five isoforms produced from a single gene. In situ hybridisation of Drosophila embryos showed that myofilin RNA is first expressed late in embryogenesis at stage 15, a little later than myosin. Antibody to myofilin labelled the entire A-band, except for the H-zone, in cryosections of flight and non-flight muscle. The periodicity of myofilin in Drosophila flight muscle thick filaments was found to be 30 nm by measuring the spacing of gold particles in labelled cryosections; this is about twice the 14.5 nm spacing of myosin molecules. The molar ratio of myofilin to myosin in indirect flight muscle is 1:2, which is the same as that of flightin. We propose a model for the association of these proteins in thick filaments, which is consistent with the periodicity and stoichiometry. Myofilin is probably needed for filament assembly in all muscles, and flightin for stability of flight muscle thick filaments in adult flies.

Association of the chaperone alphaB-crystallin with titin in heart muscle.

alphaB-crystallin, a major component of the vertebrate lens, is a chaperone belonging to the family of small heat shock proteins. These proteins form oligomers that bind to partially unfolded substrates and prevent denaturation. alphaB-crystallin in cardiac muscle binds to myofibrils under conditions of ischemia, and previous work has shown that the protein binds to titin in the I-band of cardiac fibers (Golenhofen, N., Arbeiter, A., Koob, R., and Drenckhahn, D. (2002) J. Mol. Cell. Cardiol. 34, 309-319). This part of titin extends as muscles are stretched and is made up of immunoglobulin-like modules and two extensible regions (N2B and PEVK) that have no well defined secondary structure. We have followed the position of alphaB-crystallin in stretched cardiac fibers relative to a known part of the titin sequence. alphaB-crystallin bound to a discrete region of the I-band that moved away from the Z-disc as sarcomeres were extended. In the physiological range of sarcomere lengths, alphaB-crystallin bound in the position of the N2B region of titin, but not to PEVK. In overstretched myofibrils, it was also in the Ig region between N2B and the Z-disc. Binding between alphaB-crystallin and N2B was confirmed using recombinant titin fragments. The Ig domains in an eight-domain fragment were stabilized by alphaB-crystallin; atomic force microscopy showed that higher stretching forces were needed to unfold the domains in the presence of the chaperone. Reversible association with alphaB-crystallin would protect I-band titin from stress liable to cause domain unfolding until conditions are favorable for refolding to the native state.

Guided wave helical ultrasonic tomography of pipes.

Ultrasonic guided waves have been used for a wide variety of ultrasonic inspection techniques. We describe here a new variation called helical ultrasound tomography (HUT) that uses guided ultrasonic waves along with tomographic reconstruction algorithms that have been developed by seismologists for what they call "cross borehole" tomography. In HUT, the Lamb-like guided waves travel the various helical criss-cross paths between two parallel circumferential transducer arrays instead of the planar criss-cross seismic paths between two boreholes. Although the measurement itself is fairly complicated, the output of the tomographic reconstruction is a readily interpretable map of a quantity of interest such as pipe wall thickness. In this paper we demonstrate HUT via laboratory scans on steel pipe segments into which controlled thinnings have been introduced.

Structural and biochemical analysis of the Leptinotarsa decemlineata (Coleoptera; Chrysomeloidea) crystalline chorionic layer.

The developmental aspects of the Leptinotarsa decemlineata crystalline chorionic layer (CCL) morphogenesis, its composition and its supramolecular structure were studied. The mature Leptinotarsa decemlineata eggshell consists of the vitelline membrane and the CCL, while the follicle cell remnants following their degeneration after oogenesis completion constitute the outer chorionic layer. The vitelline membrane and the CCL layers are formed through continuous material deposition from the follicular epithelium, whereas the main morphogenic factor during most insect eggshell formation, namely the follicle cell and oocyte microvilli, are seemingly involved only in vitelline membrane formation. Analysis of the CCL morphogenesis showed that this layer is assembled from a fiber-like pre-crystalline material, which accumulates at the vitelline membrane-follicle cell interface. The mature CCL is about 1 microm thick and exhibits a periodicity of approximately 10 nm, while computer image analysis studies of thin-sectioned CCL revealed the existence of crystalline layers parallel to the CCL surface. Finally, SDS-PAGE-electrophoresis of purified CCLs showed that this crystalline layer is of a proteinaceous nature and is most likely composed of 3-5 polypeptides with a molecular weight ranging in between 28-60 kDa. Overall, these data exemplify for the first time the nature and supramolecular arrangement of a crystalline layer and its constituent molecules in Coleoptera.