The effect of colchicine on myogenesis in vivo in Rana pipiens and Rhodnius prolixus (Hemiptera).

The effect of colchicine on myogenesis in vivo has been studied in the regenerating tadpole tail of the frog, Rana pipiens, and in the abdominal molting muscles of a blood-sucking bug, Rhodnius prolixus Stål. Colchicine is shown to disrupt microtubules in the differentiating muscle cells of both these organisms. The disruption of microtubules is correlated with a loss of longitudinal anisometry in the myoblasts and myotubes of the regeneration blastema in the tadpole tail. Before colchicine treatment, the myotubes contain longitudinally oriented myofibrils. After colchicine treatment, rounded, multinucleate myosacs containing randomly oriented myofibrils are present. It is suggested that the primary function of microtubules in myogenesis in the Rana pipiens tadpole is the maintenance of cell shape. The abdominal molting muscles of Rhodnius undergo repeated phases of differentiation and dedifferentiation of the sarcoplasm. However, the longitudinal anisometry of the muscle fibers is maintained in all phases by the attachments of the ends of the fibers to the exoskeleton, and microtubule disruption does not alter cell shape. The orientation of the developing myofibrils is also unaltered, indicating that the microtubules do not directly align or support the myofibrils in this system.

Microtubular organization in elongating myogenic cells.

Microtubule organization has been studied in serially sectioned myogenic cells in the tail muscle regeneration blastema of Rana pipiens tadpoles. In mesenchymal cells and in some premyoblasts, microtubules radiate from centriolar satellites in a cell center, while in more mature myoblasts and myotubes the centrioles no longer appear to serve as organizing centers for microtubules. In all elongate, fusiform myogenic cells, the microtubules are predominately oriented in the longitudinal axis of the cell. Counts of microtubules in transverse sections spaced at regular intervals along the cells show that the absolute number of microtubules is greatest in the thickened midregions of the cells and decreases relatively smoothly toward the tapered ends of the cells. Close paraxial association of microtubules (within 40 nm surface-to-surface) occurs along the entire lengths of cells but appears with greatest frequency in their tapered ends. In two myoblasts, serial sections were used to trace all microtubules in 8-microm long segments of the cells located about midway between the nucleus and one end of the cell. Since tracings show that as many as 50% of the microtubules terminate within the 8-microm long segment, it seems unlikely that any microtubules extend the entire length of the cell. It is proposed that lateral interactions between paraxial microtubules stabilize the overall microtubular apparatus and contribute to maintenance of the bipolar form of the cells. A three-dimensional model of the complete microtubular array in one of the 8-microm long segments of a myoblast has been constructed. The model reveals that a few microtubules within the segment are bent into smooth curves and loops that could be generated by sliding interaction between paraxial microtubules.

Microtubules in cone myoid elongation in the teleost retina.

The myoids of retinal cone cells of the blue-striped grunt (Haemulon sciurus) undergo significant elongation during dark adaptation of the retina. Longitudinally oriented microtubules are present in myoids both before and after elongation. Injection of colchicine into the vitreous of the eye in vivo disrupts the microtubules in the myoids and prevents dark-adaptive myoid elongation. Counts of microtubules in transverse sections along the lengths of elongating myoids show that there is a uniform decrease in the number of microtubules at any one point along the myoid as the myoid elongates. The magnitude of the decrease is proportional to the extent of the elogation. The product of the mean myoid microtubule number (determined from counts at progressive intervals along the myoid) and the myoid length remains essentially constant during myoid elongation, indicating that the total quantity of microtubules in the myoid does not increase with elogation. Serial section tracings of the microtubules along the myoids suggest that individual microtubules do not extend the length of the myoid and that the myoid microtubular apparatus consists of bundles of overlapping shorter microtubules. We propose that elongation of the myoid is accompanied by sliding redistribution of microtubules along the length of the myoid, and that the sliding may be generated by interaction between microtubules in regions where they closely overlap in bundles. We find no evidence for the involvement of discrete, electron-dense microtubular organizing centers in myoid elogation.

Organization of tubulin in normal and transformed rat kidney cells.

We have carried out a quantitative biochemical and ultrastructural study of tubulin and microtubules in a normal rat kidney (NRK) cell line and its viral transformant (442) in culture. Under equivalent culture conditions, both cell lines contain the same amount of tubulin according to a colchicine-binding assay. The normal and transformed cells differ significantly, however, with respect to the state of organization of their tubulin. Counts of microtubules in sectioned cells indicate that NRK cells have almost twice as many microtubules per unit area of cytoplasm as the 442 cells. Centrifugation studies, on the other hand, show that 442 cells have almost twice as much pelletable tubulin as the NRK cells. We propose, therefore, that the transformed cells contain a large amount of tubulin which is in some alternative aggregate form that is not morphologically detectable as microtubles in the cytoplasm

Structural and biochemical aspects of cell motility in amebas of Dictyostelium discoideum.

Amebas of Dictyostelium discoideum contain both microfilaments and microtubules. Microfilaments, found primarily in a cortical filament network, aggregate into bundles when glycerinated cells contract in response to Mg-ATP. These cortical filaments bind heavy meromyosin. Microtubules are sparse in amebas before aggregation. Colchicine, griseofulvin, or cold treatments do not affect cell motility or cell shape. Saltatory movement of cytoplasmic particles is inhibited by these treatments and the particles subsequently accumulate in the posterior of the cell. Cell motility rate changes as Dicytostelium amebas go through different stages of the life cycle. Quantitation of cellular actin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the quantity of cellular actin changes during the life cycle. These changes in actin are directly correlated with changes in motility rate. Addition of cyclic AMP to Dictyostelium cultures at the end of the feeding stage prevents a decline in motility rate during the preaggregation stage. Cyclic AMP also modifies the change in actin content of the cells during preaggregation.

Actin content and organization in normal and transformed cells in culture.

The amount of actin and total protein per cell in normal rat kidney (NRK) cells in culture is initially high in very low density cultures, but rapidly decreases as the cells come into contact in higher density cultures. In a viral transformant of NRK (442), the level of actin and total protein does not change significantly from low to high density cultures. NRK cells, which are flattened against the substrate, have prominent bundles of actinlike microfilaments in the basal cytoplasm adjacent to the substrate. 442 cells, which adhere poorly and are more spherical in shape, lack well-organized basal microfilament bundles, but may display microfilament bundles in cytoplasmic processes extending from the cell body. The percentage of insoluble actin is less than 20% in both cell lines, and 442 cells consistently contain smaller amounts than NRK cells.

Lack of tropomyosin correlates with the absence of stress fibers in transformed rat kidney cells.

We have utilized epithelial rat kidney cells and their Kirsten viral transformant (442) to examine the role of actin-binding proteins in cellular morphogenesis. Normal rat kidney cells are well spread while the transformed cells are more spherical, poorly adherent, and lack actin stress fibers (Rubin, R.W., Warren, R.H., Lukeman, D.S. and Clements, E. (1978) J. Cell Biol. 78, 28-35). By immunofluorescence, antitropomyosin prominently stains normal rat kidney cell stress fibers while only a weak, nonspecific fluorescence is observed in 442 cells. Using two-dimensional gel electrophoresis, tropomyosin can be detected in normal rat kidney cells homogenates. The tropomyosin subunits are enriched in Triton-extracted filamentous normal rat kidney cell models, and in extracts of normal rat kidney cell homogenate produced by using a rapid myosin affinity technique to isolate actin and actin-associated proteins. The identity of the tropomyosin subunits has been confirmed by electrophoretic mobility, lack of proline, and the peptide map generated by limited proteolysis. None of these techniques have detected tropomyosin in the corresponding 442 preparations. Our results suggest that the transformation of normal rat kidney cells has led to an overall reduction in tropomyosin content. This may be related to the inability of 442 cells to organize filamentous actin stress fibers.

Tropomyosin in peripheral ruffles of cultured rat kidney cells.

Tropomyosin distribution has been studied in two normal lines and one transformed line of rat kidney cells during the early phases of substrate attachment and growth. One non-motile normal line, which spreads rapidly after attachment, immediately begins to assemble prominent stress fibers that contain tropomyosin. It displays small peripheral ruffles that are not noticeably stained with anti-tropomyosin. The other normal line is motile and produces large ruffles that are brightly stained with anti-tropomyosin. Large numbers of tropomyosin-positive stress fibers assemble only after the cells stop moving and lose the peripheral ruffles. The transformed line does not assemble stress fibers but does contain large numbers of actin filament bundles in ruffles on the cell surface that are stained with anti-tropomyosin. These observations indicate that cytoskeletal tropomyosin is not restricted in distribution to stress fibers, and may undergo re-organization along with actin during the transition from motile to non-motile behavior.

Adrenergic and cholinergic receptors of cerebral microvessels.

The presence of alpha- and beta-adrenergic and muscarinic cholinergic receptors in cerebral microvessels of the rat and pig was assessed by ligand binding techniques. The results demonstrate the presence of specific binding to alpha 2- and beta-adrenergic receptors but no appreciable specific binding to alpha 1-adrenergic or muscarinic cholinergic receptors. beta-Adrenergic receptors of pig cerebral microvessels are similar to those of the brain and other organs in their binding characteristics to the tritiated ligand and in their stereospecificity of binding to the biologically active isomers of beta-adrenergic agonists. Further evidence derived from the differential potency of binding displacement by the various beta-adrenergic agonists and selective beta 1- and beta 2-adrenergic antagonists indicates that beta-adrenergic receptors of pig cerebral microvessels are mostly of the beta 2-subtype.

Breathing patterns in infants utilizing respiratory inductive plethysmography.

Respiratory inductive plethysmography (RIP) is a method that can be used to assess breathing patterns in infants without an airway connection. Ribcage and abdomen transducers are used which require gain factor calculation for calibration. We employed a single position graphic (SPG) calibration technique for gain factor calculation in RIP to obtain breathing pattern data for 70 infants in the quietly awake state. The SPG technique utilizes selection of two breaths from a 20s run of breaths with different ribcage/pneumotachograph (RC/PNT) and abdomen/pneumotachograph (AB/PNT) ratios for the gain factor calculation. Validation of gain factors was performed by comparing volumes obtained simultaneously by RIP and PNT. In 46 of the infants, maintenance of gain factor accuracy was confirmed following position reversal. Revalidation after position change could not be accomplished in 24 infants who were aroused into an agitated state. Breathing patterns were collected by RIP alone on the 46 infants who remained accurately calibrated in the supine and prone positions. No significant correlations were found between breathing pattern data and anthropometric characteristics. When the infants were repositioned, no consistent pattern of change could be identified. This study suggests that the SPG technique provides time-efficient and accurate calibration of RIP in the newborn infant. Furthermore, accuracy is maintained through position change if the infant remains in the same behavioral state. Breathing pattern data presented is representative of normative values in the quietly awake state for our study population.

Calibration of the respiratory inductive plethysmograph with the single position graphic technique. Accuracy in different behavior states in lambs.

Respiratory inductive plethysmography (RIP) can measure breathing patterns noninvasively. Calibration is required for rib cage and abdomen transducers utilizing breaths with different compartment contribution correlated with tidal volume measured by integrated pneumotachography (PNT). This study was performed to determine if RIP remains accurate during sleep states following calibration in the quietly awake state. We used our single position graphic calibration technique (SPG) to calculate gain factors in seven tracheostomized lambs. Validation of gain factors was accomplished by comparing tidal volume obtained simultaneously by RIP and PNT during quiet wakefulness (QW), quiet sleep (QS) and active sleep (AS). Results of the study showed that RIP was accurately calibrated during QW. Accuracy was decreased during QS and AS.

Somatomedin activity in cystic fibrosis and reserpinized rats: possible explanation for growth retardation.

Somatomedin activity in children who have cystic fibrosis is reduced to approximately 50 percent of the levels found in normal children. In contrast, the growth hormone concentration in these patients, both the resting and the stimulated levels, was found to be no different from normal children (17.2 and 18.4 ng per ml, respectively). Reserpinized rats have been proposed as a model for cystic fibrosis. Serum somatomedin activity in rats treated with reserpine (0.50 mg per kg per d x 7 days) was reduced to 30 percent of the levels measured in control rats. Reserpine also decreased radiosulfate incorporation into cartilage glycosaminoglycan (GAGS) in vivo and in vitro. Fasting decreased serum somatomedin activity as well as the concentration of GAGS in rat cartilage. Refeeding for 24 hours restored these parameters to normal. These data suggest that one mechanism for the growth retardation occurring in patients who have cystic fibrosis may be explained by decreased serum somatomedin activity.

Myoblasts are aligned with collagen fibrils in regenerating frog tadpole tails.

Myoblasts in the regenerating frog tadpole tail differentiate from mesenchymal cells that lie next to the basement membrane of the epidermis of the tail. As these cells elongate and form myotubes, they orientate uniformly in the longitudinal axis of the tail. The collagen fibrils of the basement membrane adjacent to the myogenic cells are also orientated in the tail axis just prior to and during the time when the myogenic cells are elongating. This has been demonstrated by transmission electron microscopy of thin sections, by differential interference contrast microscopy of isolated basement membranes, and by scanning electron microscopy of the inner surface of the basement membrane. Since elongating myoblasts are in contact with the longitudinally orientated fibrils, the latter could provide directional cues to the elongating myoblasts. This proposition is supported by the finding that isolated basement membranes readily orientate cells that are cultured upon their inner surfaces.

Microtubules and actin in giant nerve fibers of the spiny lobster, Panulirus argus.

Giant axons of the spiny lobster, Panulirus argus, are filled with microtubules that are decorated with fine, irregular filaments. Mitochondria and membrane-limited clear vesicles are the only other distinguishable elements in the axoplasm and are located around the periphery of the axon near the axolemma. Neither 100 A neurofilaments nor 70 A microfilaments are evident in fixed, intact axons or in negatively stained axoplasm. Actin-like microfilaments are a prominent constituent of the glial cells that closely ensheathe the axons, and gel electrophoresis studies suggest that most of the actin in the nerve fibers is located in the glia rather than in the axons. Studies of isolated axoplasm indicate that microtubules are the primary elements stabilizing the axoplasm. The microtubules in the isolated axoplasm are disrupted by Ca2+ in the medium in the presence of protease inhibitors.

Chest wall motion in neonates utilizing respiratory inductive plethysmography.

After calibration, respiratory inductive plethysmography can accurately measure breathing patterns noninvasively by transmitting ribcage and abdomen compartment changes caused by ventilation through oscillator circuitry. We measured the breathing pattern of nine quietly awake healthy newborn infants and assessed components reflecting asynchrony, paradoxic motion, and overall phasic relations between ribcage and abdomen compartments. Breathing pattern data (mean +/- SD) on 136 total tidal volume (Vt) breaths revealed: Vt, 14.4 +/- 3.40 ml; frequency, 52.1 +/- 11.5 beats/min; ribcage contribution to Vt, 32.2% +/- 13.4%; maximum compartmental amplitude/Vt, 1.01 +/- 0.01; phase angle, 13.2 +/- 9.50 degrees; inspiratory asynchrony index, 0.26 +/- 0.20 ml2/ml; expiratory asynchrony index, 0.42 +/- 0.3 ml2/ml; and average asynchrony index, 0.34 +/- 0.20 ml2/ml. Results demonstrated a high degree of synchrony between ribcage and abdomen movement during quietly awake breathing. Outward motion of the abdomen preceded that of the ribcage for almost every measured breath.

Pediatric aerosol therapy guidelines. Indications, techniques, and dosages.

Delivery of medication in aerosol form to the pediatric population is an important therapeutic module. Aerosol therapy allows rapid medication effects, reduces systemic side effects, and provides uniform results in comparable clinical presentations if preparation techniques and dosages are appropriate. The effectiveness of aerosol therapy is dependent upon several key factors, and techniques developed to emphasize these factors will maximize aerosol delivery into the tracheobronchial tree. Indications for medical aerosol therapy are specific for children, and individualized treatment can be structured for a wide variety of pulmonary disorders. Proper and successful administration of aerosol therapy to the infant or child requires a comprehensive amount of skill and knowledge on the part of the respiratory therapy practitioner. Guidelines discussed in this paper will assist the respiratory care practitioner in achieving optimal results in treating airway disease in the pediatric patient.

A pulmonary monitoring and treatment plan for children with Duchenne-type muscular dystrophies.

The Pulmonary Medicine Section of the Department of Pediatrics of the University of Arkansas for Medical Sciences has recently developed an association with the Muscular Dystrophy Association Clinic held at Arkansas Children's Hospital. The slowly progressive, insidious onset of pulmonary problems associated with Duchenne-type muscular dystrophies and other degenerative muscle disorders indicated a need for a aggressive monitoring and treatment plan for these children and their caregivers. We have developed a Respiratory Care Handbook for families with information on the pulmonary consequences of these diseases including pathophysiology, pulmonary function tests, respiratory treatments including mechanical ventilatory support, and anticipation and prevention of pulmonary crises. In addition, we have introduced for the physician a formal monitoring and treatment regimen driven by changes in the vital capacity lung volume. The substance of this plan is presented in this manuscript.