Crab Leg Muscle Research Articles

Mechanistic studies to evaluate the size dependent toxicity of titanium dioxide nanoparticles (TiO2 NPs) on the soil nematode, Caenorhabditis elegans

In recent years, polybrominated diphenyl ethers (PBDEs) are ubiquitous environmental contaminants, but its bioaccumulation and debromination in biota have remained largely unclear. In this study, we analyzed six PBDEs (BDE47, 99, 100, 153, 154, and 209) in various tissues (i.e., viscera, muscle, and gill) of 11 types of marine organisms including zooplankton, invertebrate and fish. The concentrations of six PBDE including BDE209 in marine organisms ranged from 0.75 to 7.29 ng/g dry weight, BDE209 from 0.46 to 6.78 ng/g dry weight, respectively. BDE209 was the dominant congener in all samples, followed by BDE47. The concentration ratio of BDE47, 99, 154 to ΣPBDEs in various tissues of organisms (i.e., Rapana venosa, shrimp, crab, cuttlefish, octopus, Synechogobius hasta, tonguefish and wolfish) increased, while the concentration ratio of BDE209 to ΣPBDEs decreased. Large differences of the concentration ratios between BDE99 and BDE100 in tissues of crab was found, ranging from 32:68 in crab viscera to 83:17 in crab leg muscle. Biomagnification factors for individual PBDE congeners ranged from 0.16 to 78.6. In general, the BMFs for BDE209 in muscle were higher than those in viscera within feeding relationships. The study results suggesting BDE209 can be biodegraded to BDE47 through BDE154 and BDE99 in marine organism, its metabolite importantly influenced by organism type not trophic level; higher percentage of BDE154 was found in viscera than that in other tissues in the analyzed marine organisms of Bohai Bay.

Bioaccumulation characteristics of polybrominated diphenyl ethers in the marine food web of Bohai Bay

In recent years, polybrominated diphenyl ethers (PBDEs) are ubiquitous environmental contaminants, but its bioaccumulation and debromination in biota have remained largely unclear. In this study, we analyzed six PBDEs (BDE47, 99, 100, 153, 154, and 209) in various tissues (i.e., viscera, muscle, and gill) of 11 types of marine organisms including zooplankton, invertebrate and fish. The concentrations of six PBDE including BDE209 in marine organisms ranged from 0.75 to 7.29 ng/g dry weight, BDE209 from 0.46 to 6.78 ng/g dry weight, respectively. BDE209 was the dominant congener in all samples, followed by BDE47. The concentration ratio of BDE47, 99, 154 to ΣPBDEs in various tissues of organisms (i.e., Rapana venosa, shrimp, crab, cuttlefish, octopus, Synechogobius hasta, tonguefish and wolfish) increased, while the concentration ratio of BDE209 to ΣPBDEs decreased. Large differences of the concentration ratios between BDE99 and BDE100 in tissues of crab was found, ranging from 32:68 in crab viscera to 83:17 in crab leg muscle. Biomagnification factors for individual PBDE congeners ranged from 0.16 to 78.6. In general, the BMFs for BDE209 in muscle were higher than those in viscera within feeding relationships. The study results suggesting BDE209 can be biodegraded to BDE47 through BDE154 and BDE99 in marine organism, its metabolite importantly influenced by organism type not trophic level; higher percentage of BDE154 was found in viscera than that in other tissues in the analyzed marine organisms of Bohai Bay.

The isolation of a plasma membrane-rich fraction from the skeletal muscle of two species of marine crab, Carcinus maenas and Cancer pagurus

1. 1. A rapid method for the isolation of a plasma membrane-rich fraction from crab leg muscle, with high purity and yield recovery was developed. 2. 2. The method is based on sodium iodide extraction of the crude homogenate, followed by centrifugation on Percoll self-creating gradient. 3. 3. ( Na + K +)ATPase and alkaline phosphodiesterase I were used as marker enzymes for the plasma membrane and revealed levels of purification of approximately 13-fold and yields recovery of the total activity in the crude muscle homogenate of approximately 18%, for both species of crab studied.

31P-NMR saturation transfer study of the in vivo kinetics of arginine kinase in Carcinus crab leg muscle

The kinetics of the reaction catalyzed by arginine kinase have been determined at 9.5 and 23°C for in vivo leg muscle of Carcinus maenas (the common shore crab) using the noninvasive technique of 31P-NMR spectroscopy. Concentrations of mobile phosphorus metabolites were the same at both temperatures: 78.7 mM for arginine phosphate, 9.0 mM for adenosine triphosphate (ATP), and 2.6 mM for inorganic phosphate (P i), as estimated from NMR resonance intensities and literature values for ATP concentration as assayed by traditional biochemical methods. Apparent unidirectional rate constants for formation of ATP from arginine phosphate and ADP were 0.09 s −1 at 9.5°C and 0.27 s −1 at 23°C. Pseudo-first-order rate constants for arginine phosphate generation from Arg and ATP were 0.38 and 1.10 s −1 at 9.5 and 23°C, respectively. In vivo Q 10 for the arginine kinase reaction between 9.5 and 23°C was thus 2.2 for both directions. When the kinetic data are analyzed using the Arrhenius equation, activation energies of 126 kJ/mol for ATP formation and 105 kJ/mol for arginine phosphate formation are found. The measured chemical fluxes through arginine kinase in the forward reaction (arginine phosphate hydrolysis) were twice those in the reverse reaction, consistent with either compartmentation of substrates or participation of substrates in alternative metabolic pathways.

On the intensity reversal of the "tropomyosin reflexions" in X-ray diffraction patterns from crab striated muscle.

X-ray diffraction patterns have been recorded from skinned single fibres obtained from crab leg muscle, and the outer parts of layer-lines indexed as orders of 38.5 nm, which have been assigned to tropomyosin and actin, were examined. Fibres at normal length in the presence of ATP (gamma-S), and over-stretched fibres in rigor solutions, show no intensity reversal of the 2nd and the 3rd layer-lines when the Ca2+ concentration is raised. The results are discussed with reference to the mechanism of Ca2+ regulation. Fibres in the presence of Mg-ADP and vanadate ion (Vi) and fibres pretreated at low pH, though generating no substantial tension at high Ca2+ concentration, give rise to rigor-like patterns which are dependent on Ca2+ concentration.

The excitatory actions of a series of piperidine dicarboxylates on central neurons of the rat, snail, leech and horseshoe crab and on crab neuromuscular junction

1. 1. Intracellular recordings were made from Helix, Hirudo and Limulus central neurons and from Eupagurus leg muscle and extracellular recordings from rat lateral geniculate neurons. The actions of 2,3-, ,2,4-, 2,5- and 2,6-piperidine dicarboxylates were tested on those preparations for both direct effects and interactions with the response to L-glutamate. 2. 2. All four piperidine analogues were devoid of either reproducible direct or indirect effects on Helix and Limulus neurons save that the 2,5-analogue did mimic the action of L-glutamate on Limulus neurons which were inhibited by glutamate. 3. 3. On Hirudo and rat neurons and on Eupagurus muscle, all four piperidines mimicked the action of L-glutamate, all were weak agonists on rat neurones while on Hirudo neurons the 2,5-analogue was the most potent but on Eupagurus muscle, the 2,3- and 2,6-analogues were the most potent. 4. 4. All four piperidines potentiated the action of L-glutamate on rat and Hirudo neurons and on Eupagurus leg muscle and of muscle ejps. 5. 5. The piperidine analogues indicate that there are differences between the glutamate receptors used in the present study and provide some supportive evidence that the preferred conformation for glutamate on crab leg muscle is partially folded while for Hirudo neurons it is more extended.

Structural changes in thin filaments of crab striated muscle

Low angle X-ray diffraction patterns were recorded from crab leg muscle in living resting state and in rigor (glycerol-extracted). Both resting and rigor patterns showed a series of layer-lines arising from a helical arrangement of actin subunits in the thin filaments. In the resting state, the crossover repeat of the long-pitch actin helices was 36.6 nm, and the symmetry of the genetic actin helix was an intermediate between 26 12 and 28 13 . When the muscle went into rigor, the crossover repeat changed to 38.3 nm and the helical symmetry to 28 13 . In the living resting pattern, six other reflections were observed on the meridian and in the near-meridional region. These were indexed as orders of 2 × 38.2 nm and could be assigned to troponin molecules; the spacings and the intensity distributions of these reflections could be explained by the model proposed by Ohtsuki (1974) for the arrangement of troponin molecules in the thin filaments. The muscle in rigor gave meridional and near-meridional reflections at orders of 2 × 38.3 nm. These were identified as the same series of reflections as was assigned to troponin in the living resting pattern, but were more intense and could be seen up to higher orders. We consider that the myosin heads attached to the thin filament at regular intervals along its axis also contribute to these reflections in the rigor pattern.

Fast-axon synapses of a crab leg muscle.

Neuromuscular synapses of the "fast" excitatory axon supplying the main extensor muscle in the leg of the shore crab Pachygrapsus crassipes were studied with electrophysiological and electron-microscopic techniques. Electrical recording showed that many muscle fibers of the central region of the extensor muscle responded only to stimulation of the fast axon, and electron microscopy revealed many unitary subterminal axon branches. Maintained stimulation, even at a low frequency, resulted in depression of the excitatory junctional potentials (EJPs) set up by the fast axon but EJPs of different muscle fibers depressed at different rates, indicating some physiological heterogeneity among the fast-axon synapses. Focal recording at individual synaptic sites on the surfaces of the muscle fibers showed quantal contents ranging from 1.4 to 5.5 at different synapses; these values are relatively high in comparison with similar determinations made in the crayfish opener muscle. Synapse-bearing nerve terminals were generally relatively small in diameter and filiform, with many individual synaptic contact areas of uniform size averaging 0.6 micron2. All of the individual synapses had a presynaptic "dense body" at which synaptic vesicles clustered. If these structures represent release points for transmitter quanta, the initial high quantal content would have an ultrastructural basis. The mitochondial content of the nerve terminals, the synaptic vesicle population, and the specialized subsynaptic sarcoplasm were all much reduced in comparison with tonic axon synaptic regions in this and other crustaceans. The latter features may be correlated with the relatively infrequent use of this axon by the animal, and with rapid fatigue.

Conformational changes of F-actin in the thin filaments of muscle induced in Vivo and in Vitro by calcium ions

Ultraviolet linear dichroism measurements were undertaken to investigate conformational changes of F-actin in the thin filaments of muscle induced by calcium ions. The absorption of two polarized light beams was measured in a glycerinated single fiber of crab leg muscle, which has a very long sarcomere, using an optical microscope. The dichroism spectrum of I-band was very similar to that of an oriented gel of pure F-actin from the crab muscle; that is, it had two negative peaks (larger absorption of light polarized perpendicular to the fiber axis). One appeared at about 260 nm, which is due to ADP bound to actin, and the other at about 295 nm, which is probably due to a specific tryptophan residue in actin. These negative peaks of dichroism of the I-band became smaller in the presence of Ca 2+ than in its absence, both by about 30%. The dichroism measurement was also undertaken on the reconstituted F-actin-tropomyosin-troponin complex from rabbit muscle oriented by flow. The negative dichroism of the complex at about 260 nm and 295 nm became smaller in the presence of Ca 2+ than in its absence, both by about 20%. The magnitude of dichroism of F-actin in the complex in the presence of Ca 2+ was the same as that of F-actin alone. The decrease in dichroism is evidence for a change in the state of actin in the muscle fiber induced by Ca 2+. It is caused either by a change of intramonomer conformation or intermonomer arrangement in F-actin or by increased fluctuation of monomer orientation. H-band (the non-overlapping region of A-band) gave positive dichroism between 260 nm and 300 nm with a peak at about 280 nm, which is due to tyrosine and tryptophan residues in the thick filament. This dichroism did not depend on the Ca 2+ concentration (in the absence of ATP). In addition, a remarkable difference was found in the dichroism around 280 nm between F-actins from rabbit and from crab, suggesting a difference in the structure of actin monomers.

'Glycerol effect' in various kinds of muscle cell.

We reported in the previous paper (FUJINO et al., 1961) the phenomenon that, after the completion of immersion in a hypertonic glycerol-Ringer, fibers of frog skeletal muscle give a selective inhibition of contraction in the presence of action potential. In the present paper, the original results on this description are presented, and several observations on the mechanism of the phenomenon are also given:(1) A quite similar phenomenon occurs when urea is substituted for glycerol, indicating that the phenomenon can be produced regardless of the kind of non-electrolyte used;(2) an inhibition of contraction due to the addition of glycerol followed by its removal is observable in crab leg muscle fiber and in frog stomach, where it occurs relatively obscurely, but it is not found in heart ventricle of frog. From the present results and other experimental observations by us, it is deduced that the phenomenon can occur widely in various kinds of muscle cell regardless of whether the transverse tubule system exists or not, two factors are considered to be essential for the appearance of the phenomenon:(1) penetrability of the substance or non-electrolyte used through ‘membrane’ system, which might be in contact with extracellular fluid and (2) osmotic pressure.

Electron Microscope Studies of Insect Muscle II. Flight and Leg Muscles of Belostoma and Periplaneta

The greater part of our information concerning the fine structure of striated muscle has been derived from a study of the vertebrates. Insect, or invertebrate other than mollusc, muscle has been little studied with the electron microscope. Richards, Anderson and Hance (1942) illustrated a microtome sectioning technique for electron microscopy with sections of cockroach striated muscle. Farrant and Mercer (1952) compared grasshopper wing muscle with crab leg muscle, obtaining excellent photographs of myofilaments but failing to observe the classical lines and bands described for vertebrate myofibrils. In recent studies (Edwards, Souza Santos, Souza Santos, and Sawaya, 1953; 1954) of the muscles of the water beetle, Hydrophilus piceus, it was found that the flight muscle fibril differs from that of the coxa in color, form, size and number of cross lines; that the presence of cross lines depends upon the state of contraction; and that the insect muscle fibril differs considerably from that of the vertebrate.

Electron Microscope Studies of Insect Muscle. I. Flight and Coxal Muscle of Hydrophilus Piceus

The ultrastructure of vertebrate muscle has been, of recent years, under intensive study (Hall, Jakus and Schmitt, 1946; Jakus and Hall, 1947; Szent-Gyorgyi, 1947; 1953; Jones and Barer, 1948; Draper and Hodge, 1949; Rozsa, Szent-Gyorgyi and Wyckoff, 1950; Bluhm and Sitaramayya, 1951; Perry and Home, 1952; Weber and Portzehl, 1952). As a result we now have a good idea of the structural organization of vertebrate muscle fibers and a knowledge of the relation between certain morphological and chemical events in the fiber during its action. To date there are few published studies of the ultrastructure of invertebrate striated muscle. The first was that of Richards, Anderson and Hance (1942) who used insect muscle to illustrate a fine sectioning technique. Later, Farrant and Mercer (1952) studied the filamentous structure of grasshopper wing muscle and crab leg muscle, but failed to show the classical cross striations in the fibrils. More recently in a comparative study of invertebrate and vertebrate fibrils (Edwards, Souza Santos, Souza Santos and Sawaya, 1953a; 1953b) it was found that muscles from different regions of the insect present differences in fibrillar structure, and that the insect fibril differs considerably from that of the vertebrate. Differences between insect and vertebrate muscle fibers are not too surprising in view of the fact that ryanodine, a drug acting specifically on the contractile processes in striated muscle, produces different responses in the frog and cockroach muscle (Edwards, Weiant, Slocombe and Roeder, 1948; Edwards and Theodorides, 1949), some of these differences being summarized in Table I. Further differences between insect and vertebrate muscle may be encountered in the innervation, intermediary metabolism, oxygen supply, etc. ( cf . Roeder, 1953). Thus a study of the ultrastructure of various types of insect muscles seems warranted.

Studies on the structure of muscle: II. Arthropod muscles

An electron microscopical examination of mechanically disintegrated insect wing muscle and crab leg muscle shows that these arthropod muscles are broken down into relatively dense myofibrils in which A and Z bands may be distinguished. After tryptic digestion crab muscle fibrils can be further resolved into sheets of filaments with a strong lateral association; insect muscle similarly treated resolves into bundles of filaments held together by the remnants of the Z-hands. Further digestion releases free filaments in a form suitable for high resolution study. The density and coherence of the myofibrils seems to be due to the presence of an “accessory protein” which may be extracted by salt solutions from insect material leaving behind a bundle of filaments. It is possible that the proteins soluble in salt solution represent the myosin fraction of the muscle and therefore that the fibrillar remnant is the actin. This picture of the muscle fiber as consisting of a basic fibrillar frame work to which the myosin is attached is in agreement with the suggestions made by Rozsa, Szent-Györgyi and Wyckoff. Stained and shadowed specimens of single isolated filaments of crab muscle seem to consist of a core of one protein which is enveloped by a second protein. It is suggested that the quasi-periodic nodulation of the filaments of these arthropod muscles and also the more regular nodulation of toad, rat, etc. muscles is due to the distribution of the accessory protein (myosin?) along the basic fibrils (actin?). The basic fibrils themselves, unlike those of certain kinds of artificially formed actin fibrils are smooth and structureless.