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We examined the allelopathic activities of Arabidopsis thaliana, ecotype Columbia by two in vitro methods. The effect of dried leaves on the growth of recipient lettuce seedlings was examined by the sandwich method. The allelopathic activity on protoplast growth was examined by co-culture with recipient lettuce leaf protoplasts in 50 µl liquid medium using a 96-well culture plate. Non-spherically enlarged or dividing protoplasts of lettuce were counted under an inverted microscope. Inhibition of yellow accumulation during lettuce protoplast growth was quantitated by image analysis of scanned digital images of 96-well culture plates. The results were described as the percentages of control without A. thaliana. The results were compared with those obtained using several plants which had strong allelopathic activities on recipient lettuce using the same methods. The growth of lettuce protoplasts was inhibited at both 4 and 8 days of culture with protoplasts of A. thaliana. The results suggested the usefulness of the protoplast co-culture method for future studies on allelopathy.

A yellow callus strain was established from hypocotyls of a halophilic mangrove plant, Avicennia alba, by subculture in the dark. Allelopathic activities of yellow A. alba callus were assayed using recipient lettuce protoplasts at three growth stages by the protoplast co-culture method with digital image analysis. The protoplast cultures of yellow A. alba callus were halophilic to NaCl, KCl, and MgCl2 (up to 200 mM) but not to CaCl2. By contrast, NaCl and KCl inhibited the growth of non-salt-tolerant lettuce protoplasts, while CaCl2 and MgCl2 stimulated their growth at low concentrations. Highly salt-tolerant or halophilic mangrove plant cells were expected to have low allelopathic activity, but the protoplasts of yellow A. alba callus had very strong allelopathic activity. The inhibition was strongest at the cell division stage with growth being inhibited to 50% and 9% of the control by 104 mL-1 and 5 × 104 mL-1 of A. alba, respectively. There was less inhibition at the yellow pigment accumulation stage of lettuce. Stimulation was observed at the early cell wall formation stage with up to 105 mL-1 of A. alba. The yellow pigment of yellow A. alba callus was extracted with hexane and its absorption spectrum showed the wavelength peaks of a carotenoid, neoxanthin. Using transmission electron microscopy, specific electron-dense structures were found in yellow A. alba callus, which were similar to the undeveloped ultrastructure of a carotenoid. A carotenoid was strongly suggested to be the putative allelochemical in yellow callus of A. alba.

We examined the allelopathic activities of Arabidopsis thaliana, ecotype Columbia by two in vitro methods. The effect of dried leaves on the growth of recipient lettuce seedlings was examined by the sandwich method. The allelopathic activity on protoplast growth was examined by co-culture with recipient lettuce leaf protoplasts in 50 µl liquid medium using a 96-well culture plate. Non-spherically enlarged or dividing protoplasts of lettuce were counted under an inverted microscope. Inhibition of yellow accumulation during lettuce protoplast growth was quantitated by image analysis of scanned digital images of 96-well culture plates. The results were described as the percentages of control without A. thaliana. The results were compared with those obtained using several plants which had strong allelopathic activities on recipient lettuce using the same methods. The growth of lettuce protoplasts was inhibited at both 4 and 8 days of culture with protoplasts of A. thaliana. The results suggested the usefulness of the protoplast co-culture method for future studies on allelopathy.

The effect of a high concentration of NaCl on the intra- (cytoplasmic matrix and vacuole) and extracellular (cell wall) distribution of Na, Cl, K, Mg, Ca, S, and P was investigated in suspension-cultured cells of the mangrove halophyte Sonneratia alba and compared to cultured cells of glycophytic rice (Oryza sativa). No significant differences were observed in ultrastructural features of cluster cells of both species cultured with and without 50mM NaCl. Quantitative X-ray microanalysis of cryosections of the cells cultured in the presence of 50mM NaCl showed that the Na concentration ([Na]) and Cl concentration ([Cl]) significantly increased in all three cell components measured. In S. alba, the [Na] was highest in the vacuole and lowest in the cytoplasmic matrix, while the [Cl] was highest in the cell wall and lowest in the cytoplasmic matrix. In O. sativa, however, the [Na] and [Cl] were highest in the cell wall, and the [Na] was lowest in the cytoplasmic matrix. Thus, the possible activities for Na and Cl transport from the cytoplasmic matrix into the vacuole were greater in S. alba than in O. sativa, suggesting that halophilic mangrove cells gain salt tolerance by transporting Na and Cl into their vacuoles. In O. sativa, the addition of NaCl to the culture medium caused no significant changes to the intracellular concentrations of various elements, such as K, P, S, Ca, and Mg, which suggests the absence of a direct relationship with the transport Na and Cl. In contrast, a marked decrease in the Ca concentration ([Ca]) in the cytoplasmic matrix and vacuole and an approximately two-fold increase in the P concentration ([P]) in the cytoplasmic matrix were found in S. alba, suggesting that the decrease in the [Ca] is related to the halophilic nature of S. alba (as indicated by the inward movement of Na(+) and Cl(-)). The possible roles of a Na(+)/Ca(2+) exchange mechanism in halophilism and the effect of the [P] on the metabolic activity under saline conditions are discussed.

Various mechanisms are involved in detoxification of heavy metals such as lead (Pb) in plant cells. Most of the Pb taken up by plants accumulates in their roots. However, the detailed properties of Pb complexes in roots remain unclear. We have investigated the properties of Pb deposits in root cell walls of radish (Raphanus sativus L.) seedlings grown on glass beads bed containing Pb pellets, which are the source of Pb-contamination in shooting range soils. Pb deposits were tightly bound to cell walls. Cell wall fragments containing about 50,000 ppm Pb were prepared from the roots. After extracting Pb from the cell wall fragments using HCl, Pb ions were recombined with the Pb-extracted cell wall fragments in a solution containing Pb acetate. When the cell wall fragments were treated with pectinase (E.C. 3.2.1.15) and were chemically modified with 1-ethyl-3-dimethylamino-propylcarboimide, the Pb-rebinding ability of the treated cell wall fragments decreased. When acid-treated cell wall fragments were incubated in a solution containing Pb(2+) and excess amounts of a chelating agent, Pb recombined with the cell wall fragments were measured to estimate the affinity between Pb(2+) and the cell wall fragments. Our data show that Pb(2+) binds to carboxyl groups of cell walls. The source of the carboxyl groups is suggested to be pectic compounds. A stability constant of the Pb-cell wall complex was estimated to be about 10(8). The role of root cell walls in the mechanism underlying heavy metal tolerance was discussed.

SUMMARY Molluscan smooth muscle can maintain tension over extended periods with little energy expenditure, a process termed catch. Catch is thought to be regulated by phosphorylation of a thick filament protein, twitchin, and involves two phosphorylation sites, D1 and D2, close to the N and C termini,respectively. This study was initiated to investigate the role of the D2 site and its phosphorylation in the catch mechanism. A peptide was constructed containing the D2 site and flanking immunoglobulin (Ig) motifs. It was shown that the dephosphorylated peptide, but not the phosphorylated form, bound to both actin and myosin. The binding site on actin was within the sequence L10 to P29. This region also binds to loop 2 of the myosin head. The dephosphorylated peptide linked myosin and F-actin and formed a trimeric complex. Electron microscopy revealed that twitchin is distributed on the surface of the thick filament with an axial periodicity of 36.25 nm and it is suggested that the D2 site aligns with the myosin heads. It is proposed that the complex formed with the dephosphorylated D2 site of twitchin, F-actin and myosin represents a component of the mechanical linkage in catch.

To clarify the contribution of Ca to the gravitropic response, quantitative X-ray microanalyses were performed on cryosections of roots of soybean seedlings reoriented horizontally from their original vertical orientation. After reorientation, the roots bent gradually toward the ground at the elongation zone. The concentrations of Ca in the cell walls, cytoplasmic matrices and central vacuoles of cortical cells were measured in the upper and lower halves of the elongation zone at 0, 30, 60 and 120 min after reorientation. The Ca concentration did not significantly change in the cytoplasmic matrices or vacuoles. Additionally, the Ca concentration did not change significantly in cell walls at 30 min after reorientation; however, beyond 30 min, this concentration significantly increased gradually in the lower half of the elongation zone and decreased in the upper half of the elongation zone, indicating a typical asymmetrical distribution of Ca. These results suggest that Ca moves apoplastically in soybean roots to produce an asymmetrical Ca distribution in the elongation zone, which contributes to root curvature. The possible role of Ca in accelerating or repressing the effect of auxin is also discussed in this study.

SUMMARY To examine intracellular Ca2+ translocation during the contraction–relaxation cycle in vertebrate striated muscle, electron probe X-ray microanalysis was performed on the swimbladder muscle (SBM) fibres of a scorpionfish Sebastiscus marmoratus. The SBM fibres were rapidly frozen at rest, during contraction and at various times after the onset of relaxation. Changes in calcium distribution in the components of the sarcoplasmic reticulum (SR) were examined on the SBM fibre cryosections. In resting fibres, the calcium concentration was highest around the boundary between the A and I bands (A–I boundary), where the terminal cisternae(TC) were located. In contracting fibres, the calcium concentration decreased around the A–I boundary, while it increased in all other regions of the sarcomere, indicating Ca2+ release from the TC into the myoplasm. During relaxation, the calcium concentration first increased around the regions, where the fenestrated collars (FC) and the longitudinal tubules (LT)were located, and then gradually returned to the levels seen in resting fibres. These results support the view that, after the onset of relaxation in the SBM fibres, Ca2+ in the myoplasm is first taken up by the FC and the LT, and then gradually returns to the TC.

The ultrastructural basis for the extremely rapid contraction-relaxation cycle (up to 300 s(-1)) in the swim-bladder muscle (SBM) of a scorpionfish (Sebastiscus marmoratus), producing characteristic sounds for communication, was investigated by electron microscopy. The SBM fibres contained well-developed sarcoplasmic reticulum (SR) showing triadic contacts with well-organized transverse tubules (T tubules). It was newly found that different types of triadic contacts were present within the single SBM fibre. In the middle region of the fibre (approximately 54% of the fibre length), the triadic contacts were located around the level of boundary between the A- and I-bands (AI-type triad). However in the two end regions of the fibre (approximately 21% and approximately 12% of the fibre length), the triadic contacts were seen around the level of the Z-band (Z-type triad). Between the middle and end regions of the fibre, T tubule-SR contacts exhibited the form of pentads composed of a pair of T tubules and three SR elements, and newly found heptads composed of three T tubules and four SR elements. The fractional volume of SR relative to the fibre volume was estimated to be approximately 26% in the middle region of the fibre with the AI-type triads and approximately 15% in the fibre ends with the Z-type triads. These results are discussed in connection with the mechanism, by which the mechanical activity of the SBM muscle is neurally controlled.

Two cultured cell lines were developed from cotyledons of a halophyte mangrove, Avicennia alba.In the high-Ca callus line, which was sub-cultured in amodified amino acid medium containing 3 mM CaCl2, growth of calluses and their protoplasts were both inhibited by low concentrations of CaCl2 in the culture medium. Removal of Ca2+ from the culture medium stimulated callus growth and the calluses could be sub-cultured without CaCl2 (low-Ca callus line). The intra- (cytoplasmic matrix and vacuole) and extra- (cell wall) cellular concentrations of elements, i.e., [Ca], [K], [Cl], [Na], [Mg], [P] and [S] were investigated using quantitative X-ray microanalysis of cryosections of calluses from bothcell lines. [Ca] was high in the cytoplasmic matrix and cell wall of the high-Ca line. [Ca] was lowered in the low-Calineinall cell compartments, though still detected. Ca-containing electron-dense precipitates were accumulated in the middle lamella of cell walls in resin-embedded sections of the high-Ca line. CaCl2 in the medium stimulated protoplast growth only in the low-Caline. These results suggested that a low cellular [Ca] is needed for protoplasts growth of A. alba. The importance of cellular [Ca] for the growth of halophilic mangrove plant cells was discussed.

A 41-year-old man with aplastic anemia and paroxysmal nocturnal hemoglobinuria complex presented with complaints of frequent episodes of acute renal failure associated with hemolytic attacks. In the last admission he suffered from severe renal dysfunction that required hemodialysis for 3 weeks. A renal biopsy sample showed normal appearing of glomeruli and a moderate degree of tubular atrophy, probably due to massive ferrous deposition which was confirmed by x-ray analysis. He was found to have Fanconi's syndrome, with phosphaturia, uricosuria, acidic aminoaciduria, and excessive urinaryβ2-microglobulin excretion. This is a case in which paroxysmal nocturnal hemoglobinuria caused frequent episodes of acute renal failure and a coincident Fanconi's syndrome.

Although muscle contraction is known to result from movement of the myosin heads on the thick filaments while attached to the thin filaments, the myosin head movement coupled with ATP hydrolysis still remains to be investigated. Using a gas environmental (hydration) chamber, in which biological specimens can be kept in wet state, we succeeded in recording images of living muscle thick filaments with gold position markers attached to the myosin heads. The position of individual myosin heads did not change appreciably with time in the absence of ATP, indicating stability of the myosin head mean position. On application of ATP, the position of individual myosin heads was found to move by ≈20 nm along the filament axis, whereas no appreciable movement of the filaments was detected. The ATP-induced myosin head movement was not observed in filaments in which ATPase activity of the myosin heads was eliminated. Application of ADP produced no appreciable myosin head movement. These results show that the ATP-induced myosin head movement takes place in the absence of the thin filaments. Because ATP reacts rapidly with the myosin head (M) to form the complex (M⋅ADP⋅P i ) with an average lifetime of >10 s, the observed myosin head movement may be mostly associated with reaction, M + ATP → M⋅ADP⋅P i . This work will open a new research field to study dynamic structural changes of individual biomolecules, which are kept in a living state in an electron microscope.

The columella cells of soybean roots grown under gravity and simulated microgravity induced by a clinostat were examined using potassium pyroantimonate (PA) and quantitative X-ray microanalysis of cryosections to determine the role of Ca in the regulation of the gravitropic response. Amyloplasts in the columella cells were localized exclusively at the bottom under gravity, but diffusely distributed in the cytoplasmic matrix under simulated microgravity, thus supporting the statolith theory. In the columella cells, PA precipitates containing Ca were diffusely distributed in the cytoplasmic matrix under gravity. Under simulated microgravity, however, they decreased in number and size in the cytoplasmic matrix, whereas increased only in number in the vacuole, indicating that Ca moved from the cytoplasmic matrix into the vacuole. The vacuole of columella cells contained mostly electron-dense granular structures localized along the inner surface of tonoplasts, which closely resembled the tannin vacuole reported in Mimosa pulvinar motor cells. Under simulated microgravity, their configuration changed dramatically from a granular shape to a flat plate. The quantitative X-ray microanalysis of cryosections showed that the vacuolar electron-dense structures contained a large amount of Ca. Under simulated microgravity, the concentration of Ca increased conspicuously in these vacuolar electron-dense structures, concomitantly with a marked decrease of K in the vacuoles and an increase of K in the cell walls. These results suggest that the release of Ca(2+) from, and uptake by, the vacuolar electron-dense structures is closely related to the signal transmission in the gravitropic response and that Ca movement occurs opposite to that of K.

Isometric skinned muscle fibers were activated by the photogeneration of a substoichiometric amount of ATP and their cross-bridge configurations examined during the development of the rigor force by x-ray diffraction and electron microscopy. By the photogeneration of approximately 100 microM ATP, approximately 2/3 of the concentration of the myosin heads in a muscle fiber, muscle fibers originally in the rigor state showed a transient drop of the force and then produced a long-lasting rigor force (approximately 50% of the maximal active force), which gradually recovered to the original force level with a time constant of approximately 4 s. Associated with the photoactivation, muscle fibers revealed small but distinct changes in the equatorial x-ray diffraction that run ahead of the development of force. After reaching a plateau of force, long-lasting intensity changes in the x-ray diffraction pattern developed in parallel with the force decline. Two-dimensional x-ray diffraction patterns and electron micrographs of the sectioned muscle fibers taken during the period of 1-1.9 s after the photoactivation were basically similar to those from rigor preparations but also contained features characteristic of fully activated fibers. In photoactivated muscle fibers, some cross-bridges bound photogenerated ATP and underwent an ATP hydrolysis cycle whereas a significant population of the cross-bridges remained attached to the thin actin filaments with no available ATP to bind. Analysis of the results obtained indicates that, during the ATP hydrolysis reaction, the cross-bridges detached from actin filaments and reattached either to the same original actin monomers or to neighboring actin monomers. The latter cross-bridges contribute to produce the rigor force by interacting with the actin filaments, first producing the active force and then being locked in a noncycling state(s), transforming their configuration on the actin filaments to stably sustain the produced force as a passive rigor force.

In horseshoe crab skeletal muscle fibers, the extension of SEC at the maximum isometric force P 0 is about 6% of the slack fiber length L 0 (sarcomere length, 7 μm), i.e., about 210 nm per half-sarcomere, being too large to be explained by the cross-bridge and the thin filament elasticities. Cinematographic studies of isometrically contracting myofibril bundes indicate that the highly compliant SEC mostly originates from the “elastic” thick filament misalignment in each A-band during isometric force generation. Possible contribution of the titin filaments to the “elastic” thick filament misalignment is discussed.

Differential scanning calorimetry (DSC) was performed to investigate thermodynamic properties of three carp fast skeletal light meromyosin (LMM) isoforms expressed in Escherichia coli by recombinant DNAs. Three isoforms were the 10 degreesC-, intermediate-, and 30 degreesC-type LMM predominantly expressed in carp acclimated to 10, 20, and 30 degreesC. The isoforms expressed in E. coli by recombinant DNAs exhibited a typical pattern of alpha-helix in CD spectroscopy with two minima at 222 and 208 nm. Moreover, the three isoforms formed paracrystals typical of LMM, suggesting that expressed proteins retained intact structural properties. When the LMM isoforms were subjected to DSC analysis, the 10 degreesC and 30 degreesC types showed endotherms having transition temperatures (Tm) at 35.1 and 39.5 degreesC, respectively, which are responsible for thermal unfolding of alpha-helix. The intermediate type exhibited two comparable endotherms with Tm values at 34.9 and 40.6 degreesC, implying that it has intermediate thermodynamic properties between those of 10 degreesC and 30 degreesC types. However, a chimeric LMM having the 10 degreesC and 30 degreesC type as N- and C-terminal halves, respectively, showed the DSC pattern typical of the whole 30 degreesC-type molecule. On the other hand, another chimeric LMM composed of the N-terminal 30 degreesC type and C-terminal 10 degreesC type gave the pattern of the full 10 degreesC type. These results suggest that thermodynamic properties of the C-terminal half largely account for thermal unfolding of the whole molecule.

Using a gas environmental (hydration) chamber, in which biological specimens can be kept in wet state, we succeeded in recording images of ‘living’ muscle thick filaments with gold position markers attached to the myosin heads. The position of individual myosin heads did not change appreciably with time in the absence of ATP, indicating stability of the myosin head mean position. On application of ATP, the position of individual myosin heads was found to move by ∼20 nm along the filament axis, while no appreciable movement of the filaments was detected. The ATP-induced myosin head movement was not observed in filaments in which ATPase activity of the myosin heads was eliminated. Application of ADP produced no appreciable myosin head movement. These results show that the ATP-induced myosin head movement takes place in the absence of the thin filaments. Since ATP reacts rapidly with the myosin head (M) to form the complex (M ADP Pi) having average lifetime of >10 s, the observed myosin head movement may be mostly associated with reaction, M + ATP → M ADP Pi. This work will open a new research field to study dynamic structural changes of individual biomolecules which are kept in ‘living’ state in an electron microscope.

Transverse ultrathin sections and the freeze-fracture deep-etch method were used to study the structure of vertebrate thick filaments. We examined intact frog semitendinosus and glycerinated rabbit psoas muscle fibers. In transverse thin sections, using standard staining methods, cores were moderately well revealed. The cores were particularly distinct when sections were stained with potassium permanganate followed by differentiation with bleach. For freeze-etching, the specimens were generally fractured transversely, and occasionally obliquely, to filament axis, and were then deep-etched and rotary-shadowed. In both transverse and oblique images, thick filaments were made up of a central core, surrounded by an annular region. Since cores are characteristic of invertebrate thick filaments, it is tempting to suggest a similarity. On the other hand, the cores observed here seem simplest to interpret as being hollow, as in microtubules.

To give information about the changes of cross-bridge (myosin head) orientation during muscle contraction, mechanically skinned frog muscle fibers were rapidly frozen at various states, and the cross-bridges were observed on freeze-etch replicas. The number of cross-bridges per unit length of thick filament in relaxed state was less than one-third of that in contracting and rigor states. The interval between adjacent cross-bridges was maximum around 35 nm, a value close to the crossover repeat of actin helix. The cross-bridge angle distribution, as measured with a digital image processor, showed a peak around 90 degrees in all the states examined. The proportion of cross-bridges with angles around 90 degrees decreased either after stretch or after release of fibers in rigor state. The axial spacing of actin monomers in the thin filament was found to increase with increasing rigor force, to give the thin filament stiffness (times unit length) of about 1.8 x 10(4) pN. These results are discussed in connection with the mechanical properties of cross-bridges.

To give information about changes in orientation of myosin heads (cross-bridges) during contraction, mechanically skinned frog muscle fibers were rapidly frozen in various states, and cross-bridge angles were measured on the freeze-etch replicas. Histograms of cross-bridge angle distribution showed a peak around 90° in relaxed, contracting and rigor states. The proportion of cross-bridges taking angles around 90° decreased when rigor fibers were stretched or released before freezing. These results are explained by assuming the stretch-induced tilting of cross-bridges due to elastic recoil of the thin filaments in the I-band.

When the anterior byssal retractor muscle of Mytilus edulis is stretched by 5-20% during submaximal activation with 100 mM K+ or 10(-5) M acetylcholine, it exhibited a delayed force development after the completion of stretch. The effect of ions and drugs on the stretch-induced activation suggests the stretch-induced release of Ca2+ from the inner surface of the plasma membrane.

The swimbladder muscle of a teleost fish,Sebasticus marmoratus, contracts rapidly to produce sounds for communication and contains extremely well developed sarcoplasmic reticulum (SR), providing a material suitable for studying the intracellular Ca translocation during the contraction-relaxation cycle. We examined the change of Ca distribution along the sarcomere at various states of the fibers during the contractionrelaxation cycle in the swimbladder muscle by means of quantitative electron probe X-ray microanalysis of cryosections.The muscle fibers were rapidly frozen at rest, during sustained contraction and at 0.1 and 1.0 sec after the onset of relaxation with a dual-jet liquid propane freezing device. Cryosections (200 nm thick) were cut from the middle portion of the frozen fibers at -110°C on a LKB NOVA cryoultramicrotome, and placed on thin carbon supporting films on Nigrids. Then the cryosections were freeze-dried at 10-6torr and below -80°C. Electron probe X-ray microanalysis was performed on a liquid N2-cooled Be-stage at -130°C in a JEOL 2000FX electron microscope operated at 80 kV and equipped with a Tracor Northern TN5500 energy dispersive X-ray microanalyzer.

The physiological properties of contraction and the ultrastructure of the longitudinal retractor muscle (LRM) of a sea cucumber Stichopus japonicus were studied to give information about the sources of activator Ca in echinoderm somatic smooth muscles. The magnitude of ACh- and K-induced contractures was dependent on [Ca]0, and both contractures were eliminated in Ca-free solution, while they were not markedly influenced by Mn ions (10 mM) and low pH (4·0). Procaine (5 mM) decreased ACh-contracture tension with a long lasting after-effect, which was removed by the application of high [K]o. ACh-contractures were markedly potentiated shortly after the termination of mechanical response to the removal of external Na. The LRM could also be made to contract by caffeine (10 mM), the removal of external divalent cations and hypertonic solutions, indicating the presence of intracellularly stored Ca available for the activation of the contractile mechanism. The LRM fibres contain poorly developed intracellular membranous structures. The inner surface of the plasma membrane and small vesicles located underneath the plasma membrane seem to be two of the sources of activator Ca.

The ultrastructure of frog semitendinosus muscle was explored using the freeze-fracture, deep-etch, rotary-shadowing technique. Mechanically skinned fibers were stretched to decrease or eliminate the overlap of thick and thin filaments before rapid freezing with liquid propane. In relaxed, contracting, and rigor fibers, a significant number of bridgelike interconnections, distinct from those observed in the M-region, were observed between adjacent thick filaments in the non-overlap region. Their half-length and diameter corresponded approximately to the known dimensions of the cross-bridge (or myosin S-1). The interconnection may thus be formed by the binding of two apposed cross-bridges projecting from adjacent thick filaments. Fixation with 0.5% glutaraldehyde for 5-10 min before freezing effectively preserved these structures. The results indicate that the interconnections are genuine structures that appear commonly in stretched muscle fibers. They may play a role in stabilizing the thick filament lattice, and possibly in the contractile process.

The fine structure of plastids in the fruit of cherry peppers was studied during the various stages of ripening. The color change of fruit during ripening is due to the quantitative change of such pigment components as chlorophyll, carotenoid and anthocyanin. Plastid metamorphosis takes place in relationship to the disappearance of chlorophyll and the new formation of carotenoids. The membrane system of plastids degenerates through ripening, although a little differentiation is observed in young plastids of creamy fruits. In parallel wity the color change of fruit from cream to orange, the osmiophilic globules increase in both number and size. As ripening proceeds further, the large osmiophilic globules seem to be gradually transformed into the needle shaped crystalloids of carotenoid pigments which are the remarkable feature of the chromoplasts in red-ripe fruit. The relationship between the development of chromoplasts and the increase and decrease of some pigments is also discussed.

Quick stretches applied to the helical strips of rabbit cerebral artery produced marked delayed tension development. The stretch-induced contraction was more resistant to the removal of [Ca2+]o and Ca antagonists than the mechanical response to high [K+]o and electrical stimulation, and was absent in chemically skinned preparations, indicating that it is not an inherent property of the contractile mechanism per se. The effect of the removal of [Ca2+]o and drugs such as dantrolene and ryanodine indicated that the intracellular origin of Ca2+ was involved in the stretch-induced contraction. To explore the above possibility, the intracellular Ca localization in rabbit cerebral artery smooth muscle was examined by the pyroantimonate method. In muscle cells fixed at rest, the pyroantimonate precipitate containing Ca was localized along the inner surface of the plasma membrane, while in muscle cells fixed during the stretch-induced contraction the precipitate was diffusely distributed in the myoplasm. These results strongly suggest that the stretch-induced mechanical response is associated with the Ca2+ release from the inner surface of the plasma membrane.

Correlated physiological and electron-microscopic studies were made on the source of calcium activating the contractile system (activator calcium) in dog coronary artery smooth muscle fibers. The magnitude of contracture tension induced by 100 mM K+ was dependent on external Ca2+ concentration and reduced or eliminated by factors known to reduce the Ca2+ spike or ca2+ influx. Little or no mechanical response was elicited by treatments known to cause release of intracellularly stored calcium. These results indicated that the contractile system is mainly activated by the inward movement of extracellular calcium. In accordance with the physiological experiments, electron-opaque pyroantimonate precipitate containing calcium was found in the lumina of caveolae, but not in any intracellular structures close to the plasma membrane, when the relaxed fibers were fixed in a 1% osmium tetroxide solution containing 2% potassium pyroantimonate. If the contracted fibers were fixed in the same solution, the pyroantimonate precipitate was diffusely distributed in the myoplasm in the form of numerous particles, while the precipitate in the caveolar lumina was scarcely seen. These findings are discussed in connection with the regulation of intracellular Ca2+ concentration in dog coronary artery smooth muscle.

1. In the anterior byssal retractor muscle of Mytilus edulis , the relation between extent of recovery and [K] 0 was a mirror image of that between tension and [K] 0 . 2. High [Ca] 0 shifted the tension vs [K] 0 curve to the left, and the tension vs ACh concentration curve to the right. 3. Caffeine in subthreshold concentrations had no effect on K- and ACh-contractures. 4. Low temperature reduced the maximal K- and ACh-contractures without changing mechanical threshold. 5. These results are consistent with the view that potassium- and ACh-contractures are mainly due to inward movement of extracellular Ca and release of intracellularly stored Ca respectively.

The extensibility of the myofilaments in vertebrate skeletal muscle was studied by stretching glycerinated rabbit psoas muscle fibers in rigor state and examining the resulting extension of sarcomere structures under an electron microscope. Although stretches applied to rigor fibers produced a successive yielding of the weakest sarcomeres, the length of the remaining intact sarcomeres in many myofibrils was fairly uniform, being definitely longer than the sarcomeres in the control, nonstretched part of rigor fibers. The stretch-induced increase in sarcomere length was found to be taken up by the extension of the H zone and the I band, whereas the amount of overlap between the thick and thin filaments did not change appreciably with stretches of 10-20%. The thick filament extension in the H zone was localized in the bare regions, whereas the thin filament extension in the I band appeared to take place uniformly along the filament length. No marked increase in the Z-line width was observed even with stretches of 20-30%. These results clearly demonstrate the extensibility of the thick and thin filaments. The possible contribution of the myofilament compliance to the series elastic component (SEC) in vertebrate skeletal muscle fibers is discussed on the basis of the electron microscopic data and the force-extension curve of the SEC in rigor fibers.

1. The effect of ions and drugs on the initiation and the conduction of peristaltic wave was studied on the heart of a tunicate, Ciona intestinalis . 2. The conduction of peristaltic wave was blocked by the replacement of [Na] o by choline, but not by the addition of Mn ions (20 mM) or the removal of [Ca] o . 3. Procaine (1 mM) inhibited the pacemaker activity, but did not block the conduction of peristaltic wave. The heart-beat was not influenced by tetrodotoxin (10 −6 g/ml) or picrotoxin (10 −3 g/ml). 4. These results indicate that the action potential in the muscle cells is mainly associated with the regenerative increase in Na-permeability.

Regulatory mechanism of contraction in the proboscis retractor muscle of Phascolosoma scolops was studied by physiological measurements and cytochemical electron microscopy. The magnitude of K+-contracture was dependent on external Ca2+ concentration and the contracture disappeared in Ca2+-free solution. The K+-contracture was suppressed by application of procaine and Mn2+. Caffeine induced contracture even when external Ca2+ was absent. Ultrastructural observations of the retractor muscle cells showed the presence of a large number of vesicles (subsarcolemmal vesicles), corresponding to the sarcoplasmic reticulum in vertebrate skeletal muscle, underneath the plasma membrane. For the cytochemical electron microscopy, the muscle fibers were fixed with 1% OsO4 solution containing 2% K-pyroantimonate. In the relaxed fibers, pyroantimonate precipitates were localized along the inner surface of plasma membrane and in the subsarcolemmal vesicles. In the contracting fibers, the precipitates were uniformly distributed in the myoplasm. The X-ray microanalysis revealed that the precipitates contained Ca. These results suggest that the contractile system is activated by the influx of extracellular Ca2+ as well as by the release of Ca2+ from the intracellular structures such as the inner surface of the plasma membrane and subsarcolemmal vesicles.

The intracellular localization of activator Ca and its translocation during the mechanical activity were studied on vertebrate and invertebrate smooth muscles by fixing muscle fibers with a 1% OsO4 solution containing 2% potassium pyroantimonate for electron microscopic examination. When guinea-pig tacnia coli, Mytilus anterior byssal retractor muscle, and Dorabella longitudinal body wall muscle were fixed during the relaxed state, electron-opaque pyroantimonate precipitate containing Ca was localized along the inner surface of the plasma membrane and at other membranous structures in close apposition to the plasma membrane, in accordance with physiological evidence that these muscles contain intracellularly stored activator Ca. When they were fixed during the contracted state, the precipitate was distributed diffusely in the myoplasm in the form of small particles, indicating the release of activator Ca from the peripheral structures. The contraction in dog coronary artery smooth muscle appears to be associated with the inward movement of extracellular Ca. In accordance with this, the resting coronary artery muscle fibers exhibited the precipitate in the lumen of the caveolae, i.e., the bottle-shaped plasma membrane invaginations, but not at the peripheral intracellular structures, though the contracted fibers showed the diffuse distribution of the precipitate in the myoplasm. These results indicate that the pyroantimonate method is very effective in studying the translocation of activator Ca in various types of smooth muscle.

The physiological properties of mechanical responses and the intracellular localization and translocation of calcium as a pyroantimonate precipitate were studied in the longitudinal retractor muscle (LRM) of a Bermuda sea cucumber. Acetylcholine (ACh)-induced contraction was reduced by lowering the external Ca concentration, and suppressed completely by prolonged soaking in Ca-free solution. The magnitude of ACh-induced contraction was decreased by Mn and La ions. Furthermore, procaine reduced the ACh-induced contraction. The complete removal of Ca and Mg ions from the external medium induced a so called Ca . Mg-removal contraction. Electron microscopically, numerous subsarcolemmal vesicles were observed in the LRM fibers. In the resting fibers, pyroantimonate precipitates were localized in the subsarcolemmal vesicles and along the inner surface of plasma membrane. While, in the fiber fixed during mechanical activity, the pyroantimonate precipitates were decreased remarkably in the subsarcolemmal vesicles and at the plasma membrane, and diffusely distributed in the myoplasm. Electron-probe X-ray microanalysis showed that the precipitate contains Ca in a significant amount. These results indicate that the contraction of the LRM fibers is caused not only by Ca-influx but also by Ca-release from the intracellular storage sites, such as the subsarcolemmal vesicles and the inner surface of plasma membrane.