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Notes: The structure of the caudal muscle in the tadpole larva of the compound ascidian Distaplia occidentalis has been investigated with light and electron microscopy. The two muscle bands are composed of about 1500 flattened cells arranged in longitudinal rows between the epidermis and the notochord. The muscle cells are mononucleate and contain numerous mitochondria, a small Golgi apparatus, lysosomes, proteid-yolk inclusions, and large amounts of glycogen. The myofibrils and sarcoplasmic reticulum are confined to the peripheral sarcoplasm.Myofibrils are discrete along most of their length but branch near the tapered ends of the muscle cell, producing a Felderstruktur. The myofibrils originate and terminate at specialized intercellular junctional complexes. These myomuscular junctions are normal to the primary axes of the myofibrils and resemble the intercalated disks of vertebrate cardiac muscle. The myofibrils insert at the myomuscular junction near the level of a Z-line. Thin filaments (presumably actin) extend from the terminal Z-line and make contact with the sarcolemma. These thin filaments frequently appear to be continuous with filaments in the extracellular junctional space, but other evidence suggests that the extracellular filaments are not myofilaments.A T-system is absent, but numerous peripheral couplings between the sarcolemma and cisternae of the sarcoplasmic reticulum (SR) are present on all cell surfaces. Cisternae coupled to the sarcolemma are continuous with transverse components of SR which encircle the myofibrils at each I-band and H-band. The transverse component over the I-band consists of anastomosing tubules applied as a single layer to the surface of the myofibril. The transverse component over the H-band is also composed of anastomosing tubules, but the myofibrils are invested by a double or triple layer. Two or three tubules of sarcoplasmic reticulum interconnect consecutive transverse components.Each muscle band is surrounded by a thin external lamina. The external lamina does not parallel the irregular cell contours nor does it penetrate the extracellular space between cells. In contracted muscle, the sarcolemmata at the epidermal and notochordal boundaries indent to the level of each Z-line, and peripheral couplings are located at the base of the indentations. The external lamina and basal lamina of the epidermis are displaced toward the indentations.The location, function, and neuromuscular junctions of larval ascidian caudal muscle are similar to vertebrate somatic striated muscle. Other attributes, including the mononucleate condition, transverse myomuscular junctions, prolific gap junctions, active Golgi apparatus, and incomplete nervous innervation are characteristic of vertebrate cardiac muscle cells.

Notes: The locomotor function of the caudal muscle cells of ascidian larvae is identical with that of lower vertebrate somatic striated (skeletal) muscle fibers, but other features, including the presence of transverse myomuscular junctions, an active Golgi apparatus, a single nucleus, and partial innervation, are characteristic of vertebrate myocardial cells.Seven stages in the development of the compound ascidian Distaplia occidentalis were selected for an ultrastructural study of caudal myogenesis. A timetable of development and differentiation was obtained from cultures of isolated embryos in vitro.The myoblasts of the neurulating embryo are yolky, undifferentiated cells. They are arranged in two bands between the epidermis and the notochord in the caudal rudiment and are actively engaged in mitosis.Myoblasts of the caudate embryo continue to divide and rearrange themselves into longitudinal rows so that each cell simultaneously adjoins the epidermis and the notochord. The formation of secretory granules by the Golgi apparatus coincides with the onset of proteid-yolk degradation and the accumulation of glycogen in the ground cytoplasm.Randomly oriented networks of thick and thin myofilaments appear in the peripheral sarcoplasm of the muscle cells of the comma embryo. Bridges interconnect the thick and thin myofilaments (actomyosin bridges) and the thick myofilaments (H-bridges), but no banding patterns are evident. The sarcoplasmic reticulum (SR), derived from evaginations of the nuclear envelope, forms intimate associations (peripheral couplings) with the sarcolemma.Precursory Z-lines are interposed between the networks of myofilaments in the vesiculate embryo, and the nascent myofibrils become predominantly oriented parallel to the long axis of the muscle cell.Muscle cells of the papillate embryo contain a single row of cortical myofibrils. Myofibrils, already spanning the length of the cell, grow only in diameter by the apposition of myofilaments. The formation of transverse myomuscular junctions begins at this stage, but the differentiating junctions are frequently oriented obliquely rather than orthogonally to the primary axes of the myofibrils.With the appearance of H-bands and M-lines, a single perforated sheet of sarcoplasmic reticulum is found centered on the Z-line and embracing the I-band. The sheet of SR establishes peripheral couplings with the sarcolemma.In the prehatching tadpole, a second collar of SR, centered on the M-line and extending laterally to the boundaries with the A-bands, is formed. A single perforated sheet surrounds the myofibril but is discontinuous at the side of the myofibril most distant from the sarcolemma. To produce the intricate architecture of the fully differentiated collar in the swimming tadpole (J. Morph., 138: 349, 1972). the free ends of the sheet must elevate from the surface of the myofibril, recurve, and extend peripherally toward the sarcolemma to establish peripheral couplings.Morphological changes in the nucleus, nucleolus, mitochondria, and Golgi bodies are described, as well as changes in the ground cytoplasmic content of yolk, glycogen, and ribosomes.The volume of the differentiating cells, calculated from the mean cellular dimensions, and analyses of cellular shape are presented, along with schematic diagrams of cells in each stage of caudal myogenesis. In an attempt to quantify the differences observed ultrastructurally, calculations of the cytoplasmic volume occupied by the mqjor classes of organelles are included.Comparison is made with published accounts on differentiating vertebrate somatic striated and cardiac muscles.

Notes: Larval compound (jointed) setae of the polychaete Nereis vexillosa Grube were examined by scanning and transmission electron microscopy and by polarization microscopy. Long-bladed spinigers and short-bladed falcigers are described. The proximal shaft of each of these types of setae flares distally into a serrated collar and encloses the proximal end of a toothed blade. The collar projects on one side as a boss. The blade and the cortex of the shaft have longitudinal channels. A large excentric cavity in the shaft (the medullary channel) contains a loose meshwork of trabeculae. In the distal part of the shaft these trabeculae are aggregated into diaphragms. The seta is invested with an electron dense layer of enamel. Juvenile setae contain both chitin and protein. With respect to the long axis of the seta, the blade and the cortex of the shaft are positively birefringent and the medullary diaphragms are negatively birefringent. KOH extraction renders the setae negative to a test for protein and reverses the sign of birefringence of the cortical material of the shaft.

Notes: Six types of hemocytes were identified in fifth instars of the Indian meal moth, Plodia interpunctella. The morphology of these cells was characterized by phase contrast and electron microscopy, with Sudan black B, Giemsa, Janus green B, and periodic acid-Schiff staining. Reaction of the hemocytes with seven fluorescing lectin conjugates revealed distinctive binding patterns by their plasma and nuclear membranes and cytoplasmic inclusions. A direct line of descent from prohemocytes to plasmatocytes to granulocytes is suggested from these morphological observations.

Notes: Hydrocortisone modulates the binding capacity of HeLa cells for 125I-labeled epidermal growth factor (EGF). A twofold increase in 125I-labeled EGF binding is observed within 24 hours after the addition of pharmacological concentration of hydrocortisone (5 × 10-8-1 × 10-6 M). This enhancement of binding is reversible, and occurs when the cells are cultured in either serum-supplemented or completely defined, serum-free, hormone-supplemented medium. Scatchard analysis of the binding data indicates that the number of 125I-EGF binding sites is increased, and that no appreciable change in the affinity of the EGF receptor for labeled EGF occurs. In the serum-free condition hydrocortisone stimulates the growth of HeLa cells, but we have observed no connection between this growth stimulation and the enhancement of EGF binding. The growth response to hydrocortisone is independent of EGF, and the concentration dependency of the growth response to EGF is unaltered by the addition of hydrocortisone to the medium. Hydrocortisone elicits the growth response at a concentration as low as 5 × 10-9 M, while a concentration higher than 5 × 10-8 M is required to affect the binding capacity for 125I-EGF. These effects are specific for glucocorticoid steroids. Similar concentrations of progesterone, testosterone, or estradiol produce no measurable response. Although the elevation of EGF receptor levels in the serum-supplemented medium is similar to that observed in the serum-free cultures, hydrocortisone is growth-inhibitory under these conditions. This growth inhibition occurs at pharmacological concentrations of hydrocortisone with a concentration dependency that is similar to that of the EGF receptor modulation.

Notes: The polypeptide hormone insulin and the binding unit of cholera toxin (CTB) were coupled via a disulfide bond. This hybrid molecule had 1/30 the ability of native insulin to bind to the insulin receptor and 1/30 the biological activity of native insulin in H35 rat hepatoma cells and rat adipocytes. Thus, in these two cell types that are very sensitive to insulin, the biological activity of the hybrid molecule was as predicted on the basis of the ability of the molecule to interact with the insulin receptor. In contrast, in HTC rat hepatoma cells and rat thymocytes, two poorly responsive cell types, the insulin-CTB conjugate had 1/3 the biological activity of native insulin, a value 10 times greater than its insulin receptor binding potency. This increased activity of the conjugate did not appear to be due to cholera toxin in the preparation, since a control of uncoupled CTB had no biological activity. Furthermore, native cholera toxin increased intracellular levels of cAMP by 20-fold, whereas the conjugate had no effect on cAMP levels. The CTB moiety did, however, contribute to the biological activity of the conjugate, since the activity of the hybrid molecule, like cholera toxin, was inhibited by gangliosides, whereas the activity of native insulin was not. Finally, the binding to thymocytes of insulin-CTB conjugate, but not insulin, was inhibited by gangliosides. Thus, a hybrid hormone molecule has been constructed which has insulin-like biological activity with the receptor specificity of cholera toxin in poorly responsive cells.

Notes: Previous studies have suggested that heparin-like glycosaminoglycans may be endogenous inhibitors of smooth muscle proliferation in the vessel wall. The purpose of this study was to determine the effects of exogenous glycosaminoglycans on rat vascular (aortic) smooth muscle cell migration following wounding in vitro. Our data indicate that heparin and related molecules (iota carrageenan, dextran sulfate), but not other glycosaminoglycans (hyaluronate, chondroitin, and dermatan sulfates), inhibit smooth muscle cell motility in a cell-specific, dose-dependent, and reversible fashion. The effect of heparin was maximal (60% inhibition) at 10 μg/ml; a half-maximal effect was observed at 1 μg/ml; Heparin did not significantly affect the migration of bovine aortic endothelium or Swiss 3T3 cells. These observations support the concept that heparin-like glycosaminoglycans may be important regulators of vascular smooth muscle cell function.

Notes: A study of the uptake of exogenous proteins, peroxidase, ferritin, and myoglobin by rabbit blastomeres of different developmental stages was undertaken to determine some of the means by which these stages ingest protein. Exposure of embryos in preimplantation stages, ranging from fertilized ovum to late blastocyst, was carried out in vitro with selected in vivo controls. Blastomeres of early cleavage stages up to the morula show little uptake of peroxidase. However, the endocytosis of peroxidase greatly increases with the morula stages and continues at an elevated level through the blastocyst stages. The uptake of the tracer is initially accomplished via micropinocytotic vesicles and tubules and can have several subsequent fates. The tracer can pass into larger vacuoles and be transported into the cavity of the blastocyst, or can pass into multivesicular bodies where it is presumably degraded by the lysosomal system for cellular use. The use of myoglobin at selected blastocyst stages yielded results similar to those obtained with peroxidase. However, the response by the blastomeres to ferritin is different. Endocytosis of ferritin is scant at all preimplantation stages, even though the ferritin has no difficulty reaching the surface of the blastomeres. The experiment with mechanically denuded blastocysts indicated that ferritin did not adsorb to the cell surface.

Notes: Experiments have been carried out to examine the submandibular glands in mice with hereditary muscular dystrophy. Radioimmunoassay data confirm biological studies which show that submandibular glands in mice with muscular dystrophy contain less nerve growth factor (NGF) than glands of normal animals. Male dystrophics have half as much submandibular NGF as unafflicted mice, while females have only 10% of control levels. Gel filtration and electrophoretic studies detect no differences in the molecular properties of NGF in gland extracts from normal and dystrophic mice. Furthermore, NGF from both sources show equal activity in the sensory ganglion bioassay. Together, these results suggest that NGF deficits in submandibular glands of dystrophic mice are not due to measurement artifacts arising from alterations in the structure of the molecule.Morphological studies have uncovered a cytological basis for chemical deficits within submandibular glands of dystrophic mice. Stereological analysis of light and electron microscopic sections revealed that growth factor containing granular tubule cells (GTC) take up a smaller portion of the total gland volume, are smaller in size, and contain fewer secretory granules than comparable cells in glands from controls. Furthermore, the ultrastructure of GTC in dystrophic animals suggests that the cells are less active in producing secretory protein than GTC in glands from normal animals. These results are consistent with the idea that growth factor deficits arise from cellular abnormalities in the granular tubule segment of the gland.