Interaction of force transmission and sarcomere assembly at the muscle-tendon junctions of carp (Cyprinus carpio): ultrastructure and distribution of titin (connectin) and α-actinin

At muscle-tendon junctions of red and of white axial muscle fibres of carp, new sarcomeres are found adjacent to existing sarcomeres along the bundles of actin filaments that connect the myofibrils with the junctional sarcolemma. As the filament bundles that transmit force to the junction originate proximal to new sarcomeres, they probably relieve these new sarcomeres from premature loading. In red fibres, these filament bundles are long (up to 20 m) and dense, permitting light-microscopical immunohistochemistry (double reactions: anti-titin or anti--actinin and phalloidin). New sarcomeres have clear I bands; their A band lengths are similar to those of older sarcomeres and the thick filaments lie in register. T tubules are found at the distal side of new sarcomeres but terminal Z lines are absent. The late addition of -actinin suggests that -actinin mainly has a stabilizing role in sarcomere formation. The presence of titin in the terminal fibre protrusions is in agreement with its supposed role in sarcomere formation, viz. the integration of thin and thick filaments. The absence of a terminal Z line from sarcomeres with well-registered A bands suggests that this structure is not essential for the anchorage of connective (titin) filaments.     

Polarized distribution of γ interferon-stimulated MHC antigens and transferrin receptors in a clonal cell line isolated from Fisher rat thyroid (FRT cells)

Summary The fine structure of single identified muscle fibers and their nerve terminals in the limb closer muscle of the shore crab Eriphia spinifrons was examined, using a previous classification based on histochemical evidence which recognizes a slow (Type-I) fiber and three fast (Type-II, Type-III, Type-IV) fibers. All four fiber types have a fine structure characteristic of crustacean slow muscle, with 10–12 thin filaments surrounding each thick filament and sarcomere lengths of 6–13 m. Type-IV fibers have sarcomere lengths of 6 m while the other three types have substantially longer sarcomeres (10–13 m). Structural features of nerve terminals revealed excitatory innervation in all four fiber types but inhibitory innervation in Type-I, Type-II, and Type-III fibers only. Thus fibers with longer sarcomeres receive the inhibitor axon but those with shorter sarcomeres do not. Amongst the former, synaptic contact from an inhibitory nerve terminal onto an excitatory one, denoting presynaptic inhibition, was seen in Type-I and Type-II fibers but not in Type-III and Type-IV fibers. Inhibitory innervation of the walking leg closer muscle is therefore highly differentiated: some fibers lack inhibitory nerve terminals, some possess postsynaptic inhibition, and some possess both postsynaptic and presynaptic inhibition.     

Quantitativ-morphologische Untersuchungen an Herzmuskelzellen von normalen und hypoxischen Ratten

Zusammenfassung Normale und hypoxische Herzmuskelzellen aus der Wand des linken Ventrikels der Ratte wurden quantitativ-morphologisch anhand von elektronenmikroskopischen Längsschnitten nach Perfusionsfixierung untersucht. In normalen Zellen waren alle Myofibrillen relaxiert, die mittlere Sarcomerlänge betrug 2,2 m. Die Schnittfläche wurde zu 55% von Myofibrillen, zu 27% von Mitochondrien und zu 18% von Grundplasma und Reticulum eingenommen. Die zwischen den Myofibrillen liegenden Mitochondrien waren längsoval und im Mittel 2,3mal so lang wie breit. Es bestand kein Unterschied zwischen subendokardial und subepikardial gelegenen Zellen.10 min nach Erstickung der Tiere waren in den sonst unauffälligen Muskelzellen die Glycogengranula vermindert. Nach 20 min führte die Hypoxie zu einer Zunahme der relativen Schnittfläche der Mitochondrien um etwa 16% und zu einer beginnenden Kontraktur der Myofibrillen (Sarcomerlänge 2,0 m). 20 min Hypoxie in Hypothermie (25–30°C intrathorakal) veränderte die normale Zellstruktur dagegen kaum. Wenn die Herzen während der 20 min dauernden Hypoxie in Normothermie mit einer procainhaltigen sauerstoff- und glucosefreien Blutersatzlösung durchspült wurden, waren die Myofibrillen relaxiert, die Schwellung der Mitochondrien dagegen wurde nicht reduziert. 30 min nach Erstickung wurde die Kontraktur stärker (Sarcomerlänge 1,7 m). Nach 60 min bildeten sich Superkontraktionsknoten, einzelne Myofibrillen waren in Höhe der I-Bänder unterbrochen. Die Cristae der Mitochondrien wichen auseinander, die Schnittfläche der Mitochondrien hatte um 27% zugenommen.Während in Normotherapie eine Asphyxie des Tieres bereits nach 10 min die Herzmuskelzellen funktionell schwer schädigt, ist die Schädigung morphologisch erst nach 20 min eindeutig. Das bedeutet, daß für die elektronenmikroskopische Präparation eine Hypoxie von unter 10 min bedeutungslos ist. Hinsichtlich der morphologischen Manifestationszeit für die Unterbrechung der Sauerstoffversorgung stimmen unsere Befunde an Herzmuskelzellen gut mit vergleichbaren Angaben an Leberzellen überein.

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Quantitative-morphological investigations of heart muscle cells of normal and asphyctic rats

Summary In heart muscle cells of the left ventricle of rats the distribution of cell organelles and their reaction to hypoxia were investigated by electron microscopy.In normal hearts fixed by perfusion with aldehydes, the mean sarcomere length was 2.2 m. 27% of the longitudinal sectional area was occupied by mitochondria, 55% by myofibrils and 18% by sarcoplasmic reticulum and ground plasm. The mitochondria situated in rows between the fibrils were oval and measured 2.3 times more in length than in width. There was no difference between cells from subendocardial and subepicardial regions.10 min hypoxia (complete occlusion of the trachea) did not affect the appearance of muscle cells but diminished the number of glycogen granules. After 20 minutes the area occupied by mitochondria was increased by 16%, the mitochondria between the myofibrils were more spherical and only 1.5 times longer than wide. The sarcomeres shortened to 2.0 m. With hypothermia (25–30°C) hypoxia of 20 minutes duration did not affect the cell structure. Perfusion of the heart by a saline solution, which contained procaine but neither oxygen nor glucose, for 20 minutes prevented shortening of the sarcomeres but not swelling of the mitochondria. 30 minutes after occlusion of the trachea the myofibrils shortened to a sarcomere length of 1.7 m. After 60 minutes irregularly and excessively contracted myofibrils appeared and some sarcomeres were interrupted at the level of the I-bands. In some of the swollen mitochondria the cristae were widely separated. The increase of the area occupied by mitochondria was 27%.Asphyxia affects heart muscle cells severely with respect to function within 10 min, but morphologically it takes 20 min before a definite effect can be noticed. As to the time after which lack of oxygen is manifested morphologically, our results are consistent with findings in liver cells.

     

Ultrastructure of the neurogenic heart of Limulus polyphemus

Summary The ultrastructure of Limulus cardiac muscle was examined. The hearts were fixed in situ by perfusion with isotonic glutaraldehyde solution while in relaxed, contracted, or stretched states. The sarcomeres are relatively long, varying in length from about 2.5 to 6.6 . The average A-band length is 2.46 . M lines are absent, and H zones are poorly distinguished. Thick and thin filament diameters average about 200 Å and 50 Å, respectively; each thick filament is surrounded by 8–12 thin ones. Superficial invaginations of the sarcolemma occur, making contact with the Z lines of the outermost myofibrils. There is an extensive sarcoplasmic reticulum and transverse (T) tubules. Some T tubules run longitudinally and some open into deep sarcolemmal invaginations which extend into the fiber interior. The T tubules swell markedly in hypertonic solution. Single neurons and small bundles of neurons are observed in close apposition with myocardial cells. Intercalated disks are found in Limulus heart at regions of contact between contiguous myocardial cells lying end to end; semitight or gap junctions are essentially absent. Prominent differences in sarcomere lengths sometimes occur across the disk, thus indicating that the disks demarcate cells functionally. Hence, in addition to direct motoneuron activation, there may be some transfer of excitation across the intercalated disks in accord with our previous finding that propagating, overshooting action potentials can be induced in this heart.Supported by grants from the American Heart Association and from the Public Health Service (HE-11155 and HE-05815). I thank Mrs. Jan Redick for expert technical assistance.     

Immunofluorescence staining of thin-filament sections not participating in actomyosin crossbridges: Studies by use of a monoclonal antibody specific to actin

Summary Monoclonal antibodies (mcab) were produced in vitro by fusing mouse X63-Ag8.653 plasmacytoma cells with spleen cells from a Balb/c mouse immunized with primary cultures of chick skeletal muscle (pmcc). After cloning on agar, stable clones were obtained, the antibodies of which stain specifically the I-band of myofibrils in the immunofluorescence (IF) procedure. For further characterization of these mcab their affinities to muscle proteins were tested by immunoblotting and by enzyme-linked immunosorbent assay (ELISA). Mcab specific for actin were revealed by these criteria. One of the anti-actin antibodies, mcab 647, reveals a variety of IF-staining patterns on myofibrils. On rest-length myofibrils the I-band is labeled only. However, at sarcomere lengths below 2 m, where the thin filaments meet in the middle of the A-band and form a region of double overlap, an additional fluorescent band appears in this position. The fluorescence intensity of this band is increased significantly in shorter sarcomeres. Finally, when the I-band has disappeared at a sarcomere length of 1.5 m, fluorescence is located exclusively in the middle of the A-band. These IF-staining patterns suggest that only those sections of the thin filament are stained that do not participate in actomyosin crossbridges.     

The membrane systems of the cardiac muscle cell of Euchaeta norvegica Boeck (Crustacea,Copepoda)

Summary The membrane systems of the cardiac muscle cell of the copepod Euchaeta norvegica Boeck are described. The heart wall, which is between 0.12 and 1.36 m thick, consists of an epicardium and a single layer of muscle cells. Invaginations of the sarcolemma forming transverse tubules have been found at all levels of the sarcomere with the exception of the H-band level. The longitudinal tubules of the same system are closely associated with the sarcoplasmic reticulum to form interior couplings at the A-I level of the sarcomere. Triadic couplings at the Z band level were not seen in E. norvegica, but peripheral couplings were demonstrated. Nexuses were found in the intercalated discs.     

The fine structure of red and white myotomal muscle fibres of the coalfish (Gadus virens)

Summary The fine structure of the red and white myotomal muscles of a marine teleost, the coalfish Gadus virens, has been examined and ultrastructural measurements and analyses carried out. The sarcomere lengths of the red and white fibres were found to be 1.60 minimum, 1.82 maximum and 1.70 minimum, 1.85 maximum, respectively. No significant difference was found between the red and white fibres in their percentage of sarcoplasmic reticulum and T system. Both were found to have regularly occurring triads at the Z disk level, to have distinctive M lines and to be multiply innervated. Ultrastructurally the two fibres can be distinguished by the thicker Z line and more abundant mitochondria of the red fibre, and by the ribbon-shaped peripheral myofibrils of the white fibres. The structure of the fibres in these two types of muscle is discussed in relation to their possible role in swimming.This work was supported by a research grant from the National Environmental Research Council.     

Effects of glyburide (glibenclamide) on myocardial function in Langendorff perfused rabbit heart and on myocardial contractility and slow calcium current in guinea-pig single myocytes

Glyburide, also known as glibenclamide, was shown to have positive inotropic effect in human and animal hearts. The objectives of the present study was to investigate the effects of glyburide on developed left ventricular pressure (DLVP), coronary flow (CF), and heart rate (HR), in isolated rabbit heart as well as its effects on myocardial contractility and L-type calcium current, i_Ca, in guinea pig myocytes. Rabbit hearts were mounted on Langendorff apparatus and perfused with an oxygenated Krebs for 30 min until reaching steady state to be followed by 20 min of experimental perfusion divided into 5 min of control perfusion and 15 min of perfusion with Glyburide (10 M). Ventricular myocytes were isolated by enzymatic dispersion technique and superfused in an oxygenated Tyrode solution. Cells were voltage-clamped at holding potential –40 mV to inactivate Na⁺ current and a step depolarizations, 200 msec duration, to 0 mV was applied to elicit i_Ca. The contractions of the myocytes were measured by optical methods. Glyburide significantly increased DLVP by 30% and CF by 36% but had no effect on HR. Glyburide increased cell contractility by 7 ± 6, 18 ± 7, 28 ± 9 and 54 ± 15% for 0.1, 1, 10 and 100 M respectively, p < 0.001. Meanwhile it depressed i_Ca by 9 ± 6 and 19 ± 8% for 1 and 10 M respectively. In conclusion, glyburide increased contractility of guinea pig single myocytes and of isolated rabbit heart, as indicated by increased developed left ventricular pressure while it depressed i_Ca. It is hypothesized that an elevation in intracellular calcium, which caused increased myocardial contractility, could be attributed to an increase in intracellular Na⁺ that could increase intracellular calcium via Na⁺/Ca²⁺ exchange.     

Organization of connectin/titin filaments in sarcomeres of differentiating chicken skeletal muscle cells

Very long, elastic connectin/titin molecules position the myosin filaments at the center of a sarcomere by linking them to the Z line. The behavior of the connectin filaments during sarcomere formation in differentiating chicken skeletal muscle cells was observed under a fluorescent microscope using the antibodies to the N terminal (located in the Z line), C terminal (M line), and C zone (myosin filament) regions of connectin and was compared to the incorporation of -actinin and myosin into forming sarcomeres. In early stages of differentiating muscle cells, the N terminal region of connectin was incorporated into a stress fiber-like structure (SFLS) together with -actinin to form dots, whereas the C terminal region was diffusely distributed in the cytoplasm. When both the C and N terminal regions formed striations in young myofibrils, the epitope to the C zone of A-band region, that is the center between the A-I junction and the M-line, initially was diffuse in appearance and later formed definite striations. It appears that it took some time for the N and C terminal regions of connectin to form a regular organization in a sarcomere. Thus the two ends of the connectin filaments were first fixed followed by the specific binding of the middle portion onto the myosin filament during sarcomere formation.     

Oxygen requirements,morphology, cell coat and membrane permeability of calcium-tolerant myocytes from hearts of adult rats

Summary The morphological, functional, and biochemical properties of freshly isolated heart muscle cells were examined. A reproducible method for the separation and purification of such cells isolated from adult rat heart was developed. It yields an average of 5×10⁶ striated rectangular cells which retain normal morphology (range) 2.5 to 11×10⁶ and 4×10⁶ calcium-tolerant cells (range) 2.5 to 5.5×10⁶ per heart. After purification, 85 to 95% of the cells retain normal morphology in solutions of calcium ion activity equal to 10M, and 65 to 79% of the cells are rectangular in solutions of calcium ion activity equal to 1 mM.Under the light microscope we were able to identify functionally intact individual cells that are calcium-tolerant and contract only in response to electrical stimulation, as well as dying myocytes that beat spontaneously. The examination of such cells under the electron microscope permitted us to address the question: What is the sequence of structural changes in a dying cell? The sarcomere lengths measured both in the living state and after preparation for electron microscopy are in the physiological range. In steady states of oxygen tension, respiration of the intact cells is undiminished from 50 torr to 2 torr. The oxygen tension for half maximal respiration is 0.15 torr. Therefore, the limitation of oxygen diffusion to the mitochondria of isolated heart muscle cells must be remarkably small.This work was supported in part by NIH HL 19299 (BAW), and NIH HL 24336 and N.Y. Heart Grant in Aid (TFR.)Dr. Thomas F. Robinson is the recipient of NIH, Research Career Development Award HL 00568     

Morphological changes in cultured myotubes treated with agents that interfere with lysosomal function

Summary Treatment of cultured muscle cells with the inhibitors of lysosomal function, leupeptin, and chloroquine, decrease the degradation of acetylcholine receptors (AChR) and causes accumulation of undegraded receptors intracellularly. Under these conditions the number of cytoplasmic coated vesicles, i.e. structures that appear to transport this receptor within the cultured muscle cell, increases in parallel. This study investigates the effects of leupeptin and chloroquine on the morphology of cultured myotubes in order to learn more about the turnover of acetylcholine (ACh) receptors and the origin of the coated vesicles. Chloroquine causes involution of the plasma membrane, disorganization in the arrangement of sarcomeres, vacuolization, and enlargement of dense lysosome-like bodies in myotubes. The diameter of dense bodies in untreated myotubes is 0.36 ± 0.01 m (mean ± SEM) compared with 2 ± 0.12 m after 48 h of incubation with chloroquine. Leupeptin does not disrupt the normal architecture of sarcomeres and does not cause vacuolization of the myotubes. However, leupeptin does enlarge the dense bodies, although to a lesser extent than chloroquine (average diameter after 48 h treatment, 1.0 ± 0.06 m, p < 0.01). Untreated myotubes appear to contain equal numbers of large and small coated vesicles. After chloroquine treatment 95% of coated vesicles are large (80–120 nm in diameter), whereas after leupeptin treatment the majority of coated vesicles are small (40–70 nm in diameter). After incubation with horseradish peroxidase (HRP) 62% ± 9 of coated vesicles in chloroquinetreated cells contain the tracer, whereas in control cells only 11% ± 4 of coated vesicles contain HRP reaction product. These observations indicate that chloroquine causes accumulation of coated vesicles and interferes with degradation of AChR by preventing fusion of lysosomes with coated vesicles originating by endocytosis.     

Adrenocorticotropin production by the intermediate lobe of the rat pituitary

Summary Tissue from the four chambers of the heart of the plaice (Pleuronectes platessa, L.) has been examined in the electron microscope in order to describe the morphology of the heart at a fine structural level.The sinus venosus is a thin walled chamber between 60–90 thick consisting of a connective tissue matrix in which are situated the plexus of the parasympathetic cardiac ganglion and localised bundles of myocardial cells. The myocardial cells do not form a continuous layer but are associated in particular with the region of the cardiac ganglion and are innervated by it.The sino-auricular junction has hitherto been described as a pacemaker region but the myocardial cells in this region are identical in morphology to myocardial cells in other parts of the heart. There is a large complex of nerves, derived from the cardiac plexus, that runs around the junction before branching to innervate the auricle.The myocardial tissues consist of an outer layer of myocardium forming the wall of the heart and a profusion of trabeculae. The endocardium invaginates into the endocardium to divide up the cells into populations of approximately 25 cells in profile. There is no well-defined coronary blood supply although capillaries are occasionally seen. The myocardial cells themselves are small in diameter (3.5–5.5 ) and show some primitive features which are: a short sarcomere (1.4–2.0 ), the absence of any sarcoplasmic reticulum, and very scarce fasciae occludentes. In the atrium in particular, there are many groups of 1500 Å membrane-bound, dense-cored vesicles in the myocardial cells. Ventricular cells contain more myofilaments and mitochondria than do atrial cells and have many vesicles of 0.1–0.3 diameter whose function and contents are unknown.Connective tissue is very evident in the plaice heart, being an integral part of the sinus venosus and the auriculo-ventricular junction and being the sole constituent of the auriculoventricular valve and the bulbus arteriosus.This investigation was carried out during the tenure of an S. R. C. studentshyp awarded to R. M. S.     

Formation and alignment of Z lines in living chick myotubes microinjected with rhodamine-labeled alpha-actinin

We have used fluorescence analogue cytochemistry in conjunction with time lapse recording to study the dynamics of alpha-actinin, a major component of the Z line, during myofibrillogenesis. Rhodamine-labeled alpha-actinin microinjected into living cultured chick skeletal myotubes became localized in discrete cellular structures within 1 h and remained specifically associated with structures for up to 4 d, allowing individual identified structures to be followed during development. In the most immature cells used, alpha-actinin was found in diffuse aggregates, some of which displayed sarcomeric periodicity. Aggregates were observed to coalesce into better defined structures (Z bands) that were approximately 1.0-micron wide. Z bands condensed into narrow, more intensely fluorescent Z lines in 4-48 h. During this period, Z lines grew laterally, primarily by the addition of small beads of alpha-actinin to existing Z lines or by the merging of small Z lines. In more mature cells, alpha-actinin added to Z lines without going through a visible intermediary structure. Mean sarcomere length did not change significantly during the stages examined, although the variability of sarcomere length did decrease markedly over time for identified sets of sarcomeres. At early stages, myofibrils frequently shifted position in both the longitudinal and lateral directions. Neighboring myofibrils were frequently associated for one or more sarcomeres sporadically along their length, such that the intervening sarcomeres were often misaligned. Associations between myofibrils were often transitory. Shifts in myofibril location in conjunction with the formation, breaking, and reformation of lateral associations between myofibrils facilitated the alignment of Z lines through a trial and error process.     

Role of EDTA in a simple method for determining toxicity using a bacterial indicator organism

A method to determine toxicity using a bacterium as the indicator organism previously developed (Botsford 1998) perceives most divalent cations as being toxic. Mercury is perceived as the most toxic, followed by cadmium, zinc and copper. It was found that adding 2.5 m EDTA to the reaction would relieve the toxicity of the 15 divalent cations tested. This effect does not appear to be simple chelation. One micromolar EDTA eliminated the toxicity of 1.6 m calcium or 0.006 m mercury. Thirty-six chemicals were tested for their toxicity in the presence and absence of 2.5 m EDTA and 25 ppm calcium. Twenty-one were less toxic and two of these, p-aminobenzoic acid and tetrachloroethylene would no longer appear to be toxic according to the assay when these additions were present. Six chemicals had the same toxicity with and without the additions. Nine chemicals were more toxic when the EDTA and calcium were present. This experiment was repeated with six chemicals and ten times the EDTA concentration and ten times the calcium concentration. The toxicity with 10× was compared with the toxicity with 1× the additions. The toxicity of 4 of the six chemicals changed with the higher concentration of EDTA and calcium when the absorbancy values observed in samples with the lower levels were compared with samples with the higher levels. Obviously before EDTA can be added to mitigate the toxicity of divalent cations, it must be determined how much EDTA is required to eliminate the toxicity by the ions present in the sample. Alternatively, if the nature of the contaminating organic chemical is known, it can be determined what the effect of EDTA and the divalent cation present is on the apparent toxicity of the compound.     

Cellular organization of the embryonic lobster heart

Summary The cellular organization of the embryonic heart of the lobster Homarus americanus was examined in 6-week and 6-month-old animals. The heart wall consists of an outer adventitial layer of fibroblast cells and an inner layer of transversely striated myocardial cells. Present in close association with the myocardium are cardiac neurons, hemocytes and so-called storage cells.Adjacent fibroblasts form fasciae adhaerentes and gap junctions. Adherent junctions also occur between fibroblasts and myocardial cells. Intercalated discs and differentiated membrane regions of close apposition (4 nm) occur between adjacent myocardial cells.The cardiac neurons form a ganglion that contains four small and five large somata. Regions of neuropil are present. Motor axons arising from the cardiac ganglion form neuromuscular synapses with the myocardial cells.The storage cells contain large inclusions and form gap junctions with the myocardial cells. They may supply nutritive material to the developing myocardium.The heart at 6 weeks is about 200 m long and 160 m wide. At 6 months, it is about 300 m long and 250 m wide. The myocardium at 6 weeks is one cell layer thick, and the cells are from 2–6 m in maximum width. At 6 months the myocardium is 2–4 cells thick, and the cells are from 6–12 m in width. Therefore, the myocardium grows by an increase in the number and size of the myocardial cells.     

Activatory effect of regucalcin on GTPase activity in rat liver plasma membranes

The effect of regucalcin, a regulatory protein of Ca²⁺ signaling, on guanosine-5-triphosphatase (GTPase) activity in isolated rat liver plasma membranes was investigated. GTPase activity was significantly increased by the addition of Ca²⁺ (25–100 M) in the enzyme reaction mixture. Such an increase was not seen by other metals (Mg, Co, Zn, Cu, Ni, and Mn) with 50 M. The activatory effect of calcium (50 M) was significantly decreased by calmodulin (2.5 and 5 g/ml), indicating that it does not depend on calmodulin. The presence of regucalcin (0.1–0.5 M) in the enzyme reaction mixture caused a significant increase in GTPase activity. This increase was not significantly enhanced by calcium (50 M). GTPase activity was significantly increased by dithiothreitol (DTT; 5 mM), a protecting reagent of thiol (SH)-groups, while it was decreased by N-ethylmaleimide (NEM; 5 mM), a modifying reagent of SH-groups. The effect of calcium or regucalcin in increasing GTPase activity was not seen in the presence of NEM. Also, the activatory effect of calcium or regucalcin on GTPase was not seen in the presence of vanadate, an inhibitor of protein phosphorylation, which could inhibit GTPase activity. Moreover, the effect of regucalcin was not seen in the presence of digitonin (0.01%), a solubilizing reagent of membranous lipids, while the effect of calcium was not inhibited by digitonin. The present study demonstrates that regucalcin has an activatory effect on GTPase activity independently of Ca²⁺ in rat liver plasma membranes.     

(Z)-9-tetradecenal: a potent inhibitor of pheromone-mediated communication in the oriental tobacco budworm moth,Helicoverpa assulta

The behavioural significance of (Z)-9-tetradecenal to male H. assulta was tested by comparing the number of moths attracted to lures containing a standard synthetic female sex pheromone with lures in which (Z)-9-tetradecenal was also added. The standard pheromone mixture used contained 1000 g (Z)-9-hexadecenal, 50 g (Z)-11-hexadecenal, 300 g (Z)-9-hexadecenyl acetate and 15 g (Z)-11-hexadecenyl acetate impregnated on rubber septa. Addition of (Z)-9-tetradecenal to the standard pheromone was shown to significantly reduce the caught of male H. assulta when added in amounts greater than 10 g or 1% of the major pheromone component in both field and net-house experiments. The reduction in catch was found to be dependent on the quantity of (Z)-9-tetradecenal added to the standard pheromone. The implications of these results on conspecific and inter-specific pheromone-mediated communication in H. assulta and related sympatric heliothine species is discussed.Abbreviations Z9-16:AL (Z)-9-hexadecenal - Z11-16:AL (Z)-11-hexadecenal - Z9-16:AC (Z)-9-hexadecenyl acetate - Z11-16:AC (Z)-11-hexadecenyl acetate - Z9-14:AL (Z)-9-tetradecenal - Z9-16:OH (Z)-9-hexadecen-1-ol - Z11-16:OH (Z)-11-hexadecen-1-ol - RH relative humidity     

The response of the atrium to direct mechanical wounding in the adult heart of the newt,Notophthalmus viridescens

Summary Wound repair and proliferation were examined in the injured newt atrium with light- and electron-microscopic techniques including autoradiography. Hearts were injured by removing a piece approximately 0.5 mm² of the atrial wall. The five-day wound was an endothelial and mesothelial-lined blood clot bordered by a 150-m necrotic zone. Repair progressed from the periphery inward with areas of macrophage activity replaced by fibroblasts and connective tissue. The wound at 25 days consisted of a scar with few myocytes. There was no difference in the proliferative behavior between the right and left atria. Proliferative cells were localized to a 500-m reactive zone surrounding the wound. The maximum mesothelial cell thymidine-labeling index of 20.5% and mitotic index of 1.4% were seen 5 days after injury. The peak connective tissue cell thymidine-labeling index of 10.2% and mitotic index of 0.4% were seen 10 days after wounding. The peak thymidine-labeling index of 9.8% for myocardial cells was recorded 10 days after injury with a mitotic index of 0.2%. Proliferation returned to control levels by 25 days post-injury. Electron microscopy demonstrated that myocytes engaged in DNA synthesis were indistinguishable from control myocytes. Z-band material was not observed in mitotic myocytes, but myofilaments and junctions were present.     

De novo myofibrillogenesis in C2C12 cells: evidence for the independent assembly of M bands and Z disks

We studied the distribution of the giant sarcomeric protein obscurin during de novo myofibrillogenesis in C₂C₁₂ myotubes to learn when it is integrated into developing sarcomeres. Obscurin becomes organized first at the developing M band and later at the mature Z disk. Primordial M bands consisting of obscurin, myomesin, and M band epitopes of titin assemble before adult fast-twitch sarcomeric myosin is organized periodically and nearly concurrently with primitive Z disks, which are composed of -actinin and Z disk epitopes of titin. Z disks and M bands can assemble independently at spatially distant sites. As sarcomerogenesis proceeds, these structures interdigitate to produce a more mature organization. Fast-twitch muscle myosin accumulates in the myoplasm and assembles into A bands only after Z disks and M bands assume their typical interdigitated striations. The periodicities of M bands remain constant at 1.8 µm throughout sarcomerogenesis, whereas distances between Z disks increase from 1.1 µm in early sarcomeres to 1.8 µm in more mature structures. Our findings indicate for the first time that primitive M bands self-assemble independently of Z disks, that obscurin is a component of these primitive M bands in skeletal muscle cells, and that A bands assemble only after M bands and Z disks integrate into maturing sarcomeres. obscurin; titin; myosin; myomesin; -actinin; A band     

Biochemical mechanism of irreversible cell injury caused by free radical-initiated reactions

Effects of oxidative stress on isolated rat ventricular myocytes were studied. Myocyte viability was determined by the ability of these cells to retain rod-shaped morphology and to exclude trypan blue. The mean life time of myocytes was quantitated using the Weibull distribution function. Superfusion with 200 M tert-butyl hydroperoxide (t-BHP) led to a time-dependent loss of cell viability, generation of the products of lipid peroxidation, oxidation of protein and non-protein thiols, a decrease in [ATP]_i and in the cellular energy charge. Dithiothreitol (DTT, 5 mM) prolonged survival of myocytes exposed to t-BHP, attenuated oxidation of protein and non-protein thiols, and preserved the energy charge. Exposure to DTT did not affect the concentration of t-BHP-generated lipid peroxidation products. Promethazine (1 M) prevented t-BHP-induced increase in the concentration of lipid peroxidation products, but did not prevent either loss of thiols or loss of cell viability. Superfusion with N-ethylmaleimide (NEM, 5 M) also led to loss of cell viability, with accompanying decreases in protein and non-protein thiols, ATP and energy charge without the accumulation of the products of lipid peroxidation. Superfusion with FeSO₄ (400 M) and ascorbate (1 mM), (Fe-Asc) did not result in loss of cell viability or a decrease protein thiols or the energy charge. Superfusion with Fe-Asc, did, however, lead to a slight decrease in the concentration of non-protein thiols and ATP and a large increase in the concentration of lipid peroxidation products. Accumulation of lipid peroxidation products induced by Fe-Asc was prevented by promethazine. These results indicate that free radical-induced irreversible cell injury results from a loss of protein thiols. Changes in the cellular energy charge and lipid peroxidation do not bear a simple relationship to the survival of cardiac myocytes under oxidative stress.