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Changes in sarcoplasmic metabolite concentrations and pH associated with the catch contraction and relaxation of the anterior byssus retractor muscle of Mytilus edulis measured by phosphorus-31 nuclear magnetic resonance.

The sarcoplasmic concentrations of phosphorus metabolites and pH (pHin) were measured in the anterior byssus retractor muscle (ABRM) of Mytilus edulis by 31P nuclear magnetic resonance spectroscopy. During an active contraction induced by 10(-3) acetylcholine, the concentration of arginine phosphate ([Arg-P]in) decreased from the resting value of 7.47 +/- 0.26 (mean +/- SE, n = 8) to 6.67 +/- 0.29 (n = 6) mumol g-1, and that of inorganic phosphate (Pi) consistently increased from 0.84 +/- 0.06 (n = 7) to 1.61 +/- 0.12 (n = 5) mumol g-1. In the 'catch' state following the active contraction, these concentrations were close to their resting levels, indicating that the catch is an inactive state. 5-hydroxytryptamine caused a rapid relaxation of the catch, which was associated with a slight decrease in [Arg-P]in and an increase in pHin by ca 0.2 units. The sarcoplasmic concentration of ATP (mean, 1.6 mumol g-1) did not change throughout the contraction-relaxation cycle.

Adenosine Triphosphate↗

The 'catch' mechanism in molluscan muscle: an electron microscopy study of freeze-substituted anterior byssus retractor muscle of Mytilus edulis.

A method for quick-freezing muscles while observing their mechanical properties until the moment of freezing is described. This method was used to freeze the anterior byssus retractor muscle (ABRM) of Mytilus edulis. Intact muscle in the presence of sucrose as a cryoprotectant was freeze-substituted in acetone, fixed and embedded for electron microscopy. ABRM was frozen in a number of mechanical states including 'catch', the state of high passive tension particularly associated with some molluscan muscles. Transverse sections were examined to determine the distribution of filaments in the muscle cells. In the relaxed muscle thick and thin filaments are fairly randomly distributed. Groups of thin filaments and of thick filaments are often seen, and there is no obvious association between the two types of filaments. In contrast, in rigor muscles, both glycerol-extracted and intact, most of the thin filaments were found to lie in rings or rosettes around the thick filaments. In some places bridges between thick and thin filaments could be distinguished. In actively contracting muscle (phasic contraction) the appearance is intermediate between that of the relaxed and rigor muscles. Many thick filaments are surrounded by rosettes of thin filaments but many of the thin filaments are grouped and have no connections with thick filaments. The 'catch' state, left after a period of tonic contraction, is similar in its distribution of thick and thin filaments to the active state, many of the thin filaments lying between the thick. Frequently thick and thin filaments seem to be closer together than in other states of the muscle where a pronounced exclusion zone is present around the thick filaments. There is no evidence for association between the thick filaments. The different distribution of thin filaments in the different states is consistent with the previously described X-ray diffraction data if it is assumed that most of the contribution to the equatorial reflection at 12 nm comes from the groups of thin filaments. Our data support a model for catch in which there is a change in the association between thick and thin filaments, rather than one in which thick filaments are clumped.

Actin Cytoskeleton↗

Further identification of bioactive peptides in the anterior byssus retractor muscle of Mytilus: two contractile and three inhibitory peptides.

1. Two contractile and three inhibitory peptides were newly isolated from the anterior byssus retractor muscles (ABRMs) of the bivalve mollusc Mytilus edulis by using the muscle as the bioassay system. 2. The structures of the two contractile peptides were GPFGTHIKamide (GPFG-8) and GPFGLNKHGamide (GPFG-9). The contractile response of the ABRM to the first-time application of GPFG-8 or GPFG-9 was of considerable size. The threshold concentrations of the peptides were around 10(-9) M. However, the contractile response to the second-time application was far smaller than that to the first-time application in both cases. Namely, the muscle showed tachyphylaxis to the peptides. 3. Two of the three inhibitory peptides were members of the Mytilus-inhibitory-peptide (MIP) family. Their structures were RAPLFIamide (MIP6) and RSPMFVamide (MIP7). The peptides, as well as the other MIPs previously identified, showed a potent inhibitory effect on phasic contraction of the ABRM in response to repetitive electrical stimulation. The remaining one was an MIP-related peptide (MIP-RP) having the sequence of MRYFVamide. The MIP-RP was less potent than the two MIPs in inhibiting the contraction of the ABRM.

Amino Acid Sequence↗

A comparative assessment of heavy metal accumulation in soft parts and byssus of mussels from subarctic, temperate, subtropical and tropical marine environments.

Existing data on metal concentrations in mussels from subarctic, temperate, subtropical and tropical waters were analyzed using multivariate statistics in order to assess regional variations in metal contamination. Potential errors were reduced by only analyzing data from surveys that employed the same protocols, analytical methodologies and analysts. Factor analysis demonstrated that mussels inhabiting extremely contaminated areas (e.g. from Japanese and Swedish metallurgy sources) could be separated from mussels from other contaminated areas, and that metals such as Cd, Pb, Cu and Zn could be used to identify heavily contaminated samples while Co, Fe, Cr and Ni concentrations were good markers for exposure to inputs from different industrial sources. Furthermore byssus, like soft tissue, selectively and sensitively reflects variations of certain metal concentrations in ambient waters and thus serves as a reliable biomonitor for these contaminants in a variety of coastal and estuarine areas.

Animals↗

Carbachol and KCl-induced changes in intracellular free calcium concentration in isolated, fura-2 loaded smooth-muscle cells from the anterior byssus retractor muscle of Mytilus edulis.

Intracellular free calcium concentration [( Ca2+]1) was measured in suspensions of fura-2 loaded smooth-muscle cells isolated from the anterior byssus retractor muscle of Mytilus edulis. Successive application of 5mM carbachol (CCh) and 100mM KCl to the cells transiently elevated [Ca2+]1 from the resting value of 124 +/- 4.5nM (mean +/- S.E., n = 14) to 295 +/- 15.3 and 383 +/- 20.5 nM, respectively. The response to CCh was concentration-dependent with an ED50 of 10(-5) M. Under the microscope, 67 +/- 3.0 and 83 +/- 1.3 % of fura-2 loaded cells contracted on the addition of 5mM CCh and 100mM KCl, respectively. In Ca2+ -free sea water, the CCh induced change in [Ca2+]1 was partially suppressed whereas that induced by KCl was completely abolished, suggesting an agonist-evoked release of stored Ca2+.

Animals↗

Mercury and other trace metals (Ag, Cr, Co, and Ni) in soft tissue and byssus of Mytilus edulis from the east coast of Kyushu Island, Japan.

Concentrations of Hg and other metals such as Ag, Cr, Co and Ni in soft tissues and byssi of the blue mussel (Mytilus edulis) from three sites along the east coast of Kyushu Island, Japan, were determined by a cold-vapor technique. Large inter-regional differences in metal concentrations in both soft tissues and byssi were observed. The tissue concentrations of Hg were significantly greater in M. edulis from the most contaminated locations (Saganoseki) compared to those from a non-industrialized area (Urashiro). It is evident that, compared to the soft tissue, byssus is a more sensitive biomonitor for Hg, Cr, Co and Ni. From the data obtained in the present study evidently results that the soft tissue and especially byssi of M. edulis appear to be good bioindicators for identification of coastal areas exposed to Hg contaminants.

Animals↗

Can the byssus of green-lipped mussel Perna viridis (Linnaeus) from the west coast of Peninsular Malaysia be a biomonitoring organ for Cd, Pb and Zn? Field and laboratory studies.

Concentrations of cadmium (Cd), lead (Pb) and zinc (Zn) in total soft tissues (ST) and byssus (BYS) of the green-lipped mussel Perna viridis from 11 different geographical locations off the west coast of Peninsular Malaysia were determined. The metal concentrations distributed between the BYS and ST were compared. The results of this study indicated that higher levels of Cd (1.31 microg/g), Pb (38.49 microg/g) and Zn (206.52 microg/g) were accumulated in the BYS than in the total ST (Cd: 0.29 microg/g; Pb: 8.27 microg/g; Zn: 102.6 microg/g). Semi-static and short period controlled laboratory experiments were also conducted for the accumulation and depuration of Cd, Pb and Zn in the total ST and BYS of P. viridis. The ratios (BYS/ST) for Pb and Cd from the laboratory experiments showed that the total ST accumulated more metals than the BYS. Therefore, these laboratory results disagreed with those found for the field samples. However, the laboratory results for the Zn ratio (BYS/ST) agreed with those of the field samples. It was evident that when compared to the ST, the BYS was a more sensitive biomonitoring organ for Zn while it could be a complementary organ for Cd and Pb in the total ST. Since total ST of P. viridis had been reported to have regulative mechanism for Zn, its BYS can be used as a biomonitoring organ for the identification of coastal areas exposed to Zn pollution.

Animals↗

The peculiar collagens of mussel byssus.

The byssal collagens of marine mussels are extracorporeal collagens that function in byssal threads under tension. Each byssal thread resembles a shock absorber in its mechanical design: it is strong and stiff at one end and pliably elastic at the other. Primary structures of three of these collagens (preCols), deduced from cDNAs, reveal signal peptide sequences, but no N-glycosylation sites or propeptides typical of procollagens. The collagen domain (40-50 kDa) represents roughly half the mass of the mature molecules and is distinguished by its central location, abundant Gly-Gly-X repeats, and "flaws" (usually Gly deletions). Flanking the collagen domains on both sides are structural domains that resemble elastin in preCol-P, spider drag-line silk in preCol-D, and Gly-rich cell wall proteins in preCol-NG. Not surprisingly, studies of preCol distribution in byssal threads suggest preCol-P enhancement in the elastic proximal portion, while preCol-D predominates in the stiffer distal portion. PreCol-NG, in contrast, is evenly distributed. Although no data are yet available on the fibrillogenesis and cross-linking of the preCols, the quarter-stagger assembly of fibrillar interstitial collagens does not pertain since preCols lack the terminal peptides of tropocollagen. Metal-binding by histidines may mediate the initial inter- and intramolecular stabilization of preCols in the byssus.

Animals↗

Proteins in load-bearing junctions: the histidine-rich metal-binding protein of mussel byssus.

Building complex load-bearing scaffolds depends on effective ways of joining functionally different biomacromolecules. The junction between collagen fibers and foamlike adhesive plaques in mussel byssus is robust despite the strikingly dissimilar connected structures. mcfp-4, the matrix protein from this junction, and its presecreted form from the foot tissue of Mytilus californianus were isolated and characterized. mcfp-4 has a mass of approximately 93 kDa as determined by MALDI-TOF mass spectrometry. Its composition is dominated by histidine (22 mol %), but levels of lysine, arginine, and aspartate are also significant. A small amount of 3,4-dihydroxyphenyl-l-alanine (2 mol %) can be detected by amino acid analysis and redox cycling assays. The cDNA-deduced sequence of mcfp-4 reveals multiple variants with highly repetitive internal structures, including approximately 36 tandemly repeated His-rich decapeptides (e.g., HVHTHRVLHK) in the N-terminal half and 16 somewhat more degenerate aspartate-rich undecapeptides (e.g., DDHVNDIAQTA) in the C-terminal half. Incubation of a synthetic peptide based on the His-rich decapeptide with Fe3+, Co2+, Ni2+, Zn2+, and Cu2+ indicates that only Cu is strongly bound. MALDI-TOF mass spectrometry of the peptide modified with diethyl pyrocarbonate before and after Cu binding suggests that histidine residues dominate Cu binding. In contrast, the aspartate-rich undecapeptides preferentially bind Ca2+. mcfp-4 is strategically positioned to function as a macromolecular bifunctional linker by using metal ions to couple its own His-rich domains to the His-rich termini of the preCOLs. Ca2+ may mediate coupling of the C-terminus to other calcium-binding plaque proteins.

Amino Acid Sequence↗

Rotational echo double resonance detection of cross-links formed in mussel byssus under high-flow stress.

13C2H rotational echo double resonance NMR has been used to provide the first evidence for the formation of quinone-derived cross-links in mussel byssal plaques. Labeling of byssus was achieved by allowing mussels to filter feed from seawater containing L-[phenol-4-13C]tyrosine and L-[ring-d4]tyrosine for 2 days. Plaques and threads were harvested from two groups of mussels over a period of 28 days. One group was maintained in stationary water while the other was exposed to turbulent flow at 20 cm/s. The flow-stressed byssal plaques exhibited significantly enhanced levels of 5, 5'-di-dihydroxyphenylalanine cross-links. The average concentration of di-dihydroxyphenylalanine cross-links in byssal plaques is 1 per 1800 total protein amino acid residues.

Animals↗

Probing the adhesive footprints of Mytilus californianus byssus.

California mussels Mytilus californianus owe their tenacity to a holdfast known as the byssus, a fibrous extracellular structure that ends distally in flattened adhesive plaques. The "footprints" of freshly secreted plaques deposited onto glass coverslips were shown by matrix-assisted laser desorption ionization time of flight mass spectrometry to consist chiefly of proteins ranging in mass from 5200 to 6700 Da. These proteins, variants of a family known as mcfp3 (M. californianus foot protein 3), were purified from acetic acid/urea extracts of plaques and foot tissue. Mcfp3 appears to sort into fast and slow electrophoretic variants. Both are rich in Gly and Asn and exhibit post-translational hydroxylation of Tyr and Arg to Dopa and 4-hydroxyarginine, respectively, with the fast variant containing more than twice as much Lys + Arg. Both the slow and fast variants were partially sequenced from the N terminus, and the complete sequences of 12 variants were deduced from cDNA using degenerate oligonucleotides, PCR, and rapid amplification of cDNA ends. Mcfp3s are highly polar molecules and contain up to 28 mol % Dopa, which remains intact and may be crucial for adhesion to metal and mineral surfaces.

Acetic Acid↗

Direct observation of a central bare zone in a native thick filament isolated from the anterior byssus retractor muscle of Mytilus edulis using fluorescent ATP analogue.

To investigate the existence of a central bare zone in native thick filaments isolated from the anterior byssus retractor muscle (ABRM) of blue mussels (Mytilus edulis), the filaments were observed by fluorescence and dark-field microscopy after being incubated in the presence of Ca2+ with the fluorescent ATP analogue, Cy3-EDA-ATP. Filaments appeared under dark-field illumination as thin rods with tapered ends of length 5-30 microm. Fluorescence microscopy revealed that Cy3-EDA-ATP was bound to these filaments, except near their center. Although the boundary between this central non-fluorescent zone and fluorescent regions was not clearly defined, there was a trend for the width of the central non-fluorescent zone to increase with thick filament length (correlation coefficient = 0.45; n = 142). When Cy3-EDA-nucleotides bound to thick filaments were displaced by excess ATP, fluorescent images disappeared with a rate constant of 0. 024 s-1, close to the turnover rate of Cy3-EDA-ATP by myosin on the native thick filaments. These results indicate that each native thick filament isolated from the ABRM has a central bare zone, but its boundary was not sharply resolved.

Adenosine Triphosphate↗

Depressant effect of active shortening in the anterior byssus retractor muscle of Mytilus edulis.

The effect of shortening during activity, previously characterized in vertebrate striated muscle, was investigated in the anterior byssus retractor muscle (ABRM) of the mollusc Mytilus edulis. This muscle is considered to have an essentially myosin-linked Ca2+-regulatory system. Release steps of different amplitude were performed during isometric phasic contraction, and force redevelopment was recorded at a muscle length L1, defined as 90% of the muscle length at which a slight resting tension, approximately 1 mN, appeared in the presence of 2.5 X 10(-5) M 5-HT. Active shortening caused a graded depression of the contractile force without affecting the total duration of the mechanical response. Peak redeveloped force after muscle shortening of 0.06 L1 and 0.18 L1 was reduced by approximately 1.5% and 7.0%, respectively, of the isometric tension value at L1. The shortening effect was fully reversible, and had a lifetime of approximately 8 to 9 s. The depressant effect of active shortening was augmented at a reduced degree of activation of the muscle. The presence of caffeine and dantrolene and altered tonicity of the extracellular medium (0.9 T-1.2 T) did not significantly affect the shortening induced depression obtained at maximum phasic activation of the preparation. The nature of the shortening effect is compared to that obtained in vertebrate striated muscle and is discussed on the basis of differences in Ca2+-regulation of the contractile system in these two muscles.

Animals↗

The respiration of the anterior byssus retractor muscle of Mytilus edulis (ABRM) after a phasic contraction.

1. The oxygen consumption of isolated anterior byssus retractor muscle of Mytilus edulis (ABRM) has been measured at rest and after phasic contractions induced by a.c. stimulation.2. The respiration was measured with a Clark oxygen electrode in successive periods of 5 or 15 min, at 20 degrees C.3. The resting respiration is 71.8 +/- 2.4 n-moles O(2)/g wet weight.min (mean +/- S.E., n = 70). It is increased by a release and decreased by a passive stretch.4. After phasic stimulation of up to 30 sec the respiration is increased and returns to a slightly higher level than the resting level in an exponential fashion with a time constant of about 10 min.5. The duration of stimulation does not change the time course of the excess respiration but it affects its magnitude. The amount of extra oxygen consumed, in n-moles O(2)/g, is made up of a constant amount, 449 +/- 102, and an amount that depends on the duration of stimulation (t, sec), which is given by t x 13.2 +/- 4.3. When due account is taken for the tension developed, these parameters become 83.1 +/- 20.7 and t x 1.24 +/- 0.66 n-moles O(2)/g muscle and kg/cm(2) of tension. This regression analysis is based on forty-eight data, with a residual error based on 5 degrees of freedom.6. Release of the tension after the last stimulus of a 30 sec tetanus reduces by half the extra oxygen consumed during the recovery whereas the same release applied 5 min later has a much smaller effect. This suggests that relaxation is an active process.7. From these measurements of the recovery metabolism the energy cost of the contraction was estimated and compared with this cost in vertebrate striated muscle. The constant item has about the same magnitude, but the item related to the duration of stimulation is about 250 times smaller.

Animals↗

Tension and heat production during isometric contractions and shortening in the anterior byssus retractor muscle of Mytilus edulis.

1. Tension and heat production were measured during phasic isometric contractions and isovelocity shortening in the anterior byssus retractor muscle (ABRM) of Mytilus edulis at 20 degrees C. 2. Isometric tension at lo was 550 +/- 40 mN/mm2 (S.D. for 173 observations in nine muscles), while the isometric maintenance heat rate was 1.0 +/- 0.2 mW/g wet wt. (S.D. for seventy-eight observations in eight muscles). 3. Isometric tension and heat production were measured as functions of muscle length over a range of 0.79--1.14 lo and were found to bear a linear relation to each other. 4. The force-velocity relation was determined in isovelocity releases imposed during tetanic stimulation and was found to fit the Hill equation with parameters alpha/Po = 0.07 +/- 0.01 and b/lo = 0.016 +/- 0.0007 sec-1 (S.E. from non-linear least-squares regression of the pooled data from seven experiments). 5. Heat production measured in the same experiments showed that shortening heat is produced with a shortening heat coefficient alpha/Po of 0.15. Shortening heat does not appear to be force-dependent, and separate experiments confirmed that it is a linear function of the amount of shortening.

Animals↗

Extensible collagen in mussel byssus: a natural block copolymer.

To adhere to solid surfaces, marine mussels produce byssal threads, each of which is a stiff tether at one end and a shock absorber with 160 percent extensibility at the other end. The elastic extensibility of proximal byssus is extraordinary given its construction of collagen and the limited extension (less than 10 percent) of most collagenous materials. From the complementary DNA, we deduced that the primary structure of a collagenous protein (preCol-P) predominating in the extensible proximal portion of the threads encodes an unprecedented natural block copolymer with three major domain types: a central collagen domain, flanking elastic domains, and histidine-rich terminal domains. The elastic domains have sequence motifs that strongly resemble those of elastin and the amorphous glycine-rich regions of spider silk fibroins. Byssal thread extensibility may be imparted by the elastic domains of preCol-P.

Alanine↗

45Ca efflux from anterior byssus retractor muscle in phasic and catch contraction.

Phasic or catch contractions in Mytilus anterior byssus retractor muscle (ABRM) were activated by acetylcholine (ACh) and catch relaxation was initiated by 5-hydroxytryptamine (5HT). During phasic contraction and early in catch there is a brief increase in 45Ca efflux. When catch occurs, there is a subsequent drop in 45Ca efflux which then slowly recovers as catch tension declines. With catch relaxation by 5HT there is a biphasic increase in 45Ca efflux, identical to that seen when 5HT is applied to resting muscle. Compartment analyses based on the magnitude of pairs of these responses at varying times of the washout indicated that the increase in 45Ca efflux with activation originates from a compartment with the same time constant as the intermediate (80--100 min) compartment already described by previous resting efflux experiments. The decrease in 45Ca efflux during catch also involves this compartment. The increase in 45Ca efflux with 5HT originates from a more slowly exchanging Ca store with a time constant of approximately200 min.

Acetylcholine↗

Net calcium fluxes in anterior byssus retractor muscle with phasic and catch contraction.

Calcium in the artificial seawater bathing whole Mytilus anterior byssus retractor muscles (ABRM) was measured by a specific Ca electrode under various conditions of activation, catch, and catch relaxation. Activation in response to ACh was associated with uptake of Ca by the muscles. Phasic contractions produced a small Ca uptake; catch contractions produced a larger and sustained Ca uptake. After tension relaxation, the muscle lost an amount of Ca roughly equal to that gained. Catch relaxation by 5-hydroxytryptamine (5HT) was associated with Ca release. ACh at identical concentrations, applied to the muscle for increasingly longer times, produced increasing amounts of Ca uptake. Regardless of the previous gain of Ca by the muscle, 5HT applied for a constant interval caused release of the same amount of Ca. A model for the Ca control system in ABRM based on this and previously obtained 45Ca efflux data is proposed.

Acetylcholine↗