Bioengineered human skeletal muscle capable of functional regeneration.

BACKGROUND: Skeletal muscle (SkM) regenerates following injury, replacing damaged tissue with high fidelity. However, in serious injuries, non-regenerative defects leave patients with loss of function, increased re-injury risk and often chronic pain. Progress in treating these non-regenerative defects has been slow, with advances only occurring where a comprehensive understanding of regeneration has been gained. Tissue engineering has allowed the development of bioengineered models of SkM which regenerate following injury to support research in regenerative physiology. To date, however, no studies have utilised human myogenic precursor cells (hMPCs) to closely mimic functional human regenerative physiology. RESULTS: Here we address some of the difficulties associated with cell number and hMPC mitogenicity using magnetic association cell sorting (MACS), for the marker CD56, and media supplementation with fibroblast growth factor 2 (FGF-2) and B-27 supplement. Cell sorting allowed extended expansion of myogenic cells and supplementation was shown to improve myogenesis within engineered tissues and force generation at maturity. In addition, these engineered human SkM regenerated following barium chloride (BaCl2) injury. Following injury, reductions in function (87.5%) and myotube number (33.3%) were observed, followed by a proliferative phase with increased MyoD+ cells and a subsequent recovery of function and myotube number. An expansion of the Pax7+ cell population was observed across recovery suggesting an ability to generate Pax7+ cells within the tissue, similar to the self-renewal of satellite cells seen in vivo. CONCLUSIONS: This work outlines an engineered human SkM capable of functional regeneration following injury, built upon an open source system adding to the pre-clinical testing toolbox to improve the understanding of basic regenerative physiology.

Thermomechanical modification of diffraction gratings.

The most accurate approaches to fabrication of diffraction gratings are known to be the lithographic and holographic methods. The lithographic methods allow fabrication of arbitrarily chirped gratings whose performance, however, is degraded by stitching errors. The holographic methods are free from stitching errors; however, they are limited in the achievable spatial variations of their grating periods. We suggest a method of diffraction grating modification by nonuniform heating and stretching that is much more flexible than the holographic approach and does not suffer from the problem of stitching error. We demonstrate our approach for quartz phase masks that have a characteristic grating period of 1 microm and a length of several centimeters. Our approach allows the grating periods of the phase masks to vary in a range from a few picometers to a few nanometers and a spatial resolution of a few millimeters. It is shown that the grating period can be modified with a negligible effect on the profile of the gratings.

The use of projective techniques in pediatric nursing research from 1984 to 1993.

The purpose of this study was to review and analyze the use of projective techniques in published nursing studies in which children were the subjects. A search of the pediatric nursing research literature revealed 27 studies that were published between 1984 and 1993 in which projective techniques were used to investigate children's responses. The research purposes, characteristics of subjects, projective techniques employed and their validity and reliability, data analysis strategies, and major findings of the studies reviewed are analyzed and summarized. Recommendations for the use of projective techniques in research and clinical practice with children are offered.

Differences in color preferences of well school-age children and those in varying stages of illness.

The purpose of this descriptive comparative study was to determine the preferences in color of 89 children ages 7-12 who were well and those in varying states of ill health. The authors collected information about diagnoses, days hospitalized, siblings, and projective information. Data were analyzed using chi-square statistics. Independent variables of sex, age, and health were examined in a multivariate manner by fitting logistic regression models having indicator regressor variables to represent the various levels of these factors. The only regressor for these models that was significant was health state. Significant differences were found (p < .05 level) in color preferences of (a) children with adjustment problems and those who were well; (b) children with adjustment problems and those who were ill; (c) acutely ill and chronically ill children; and (d) physically disabled and acutely ill children. Significant differences were also found in color preferences of children ages 7-9 and 10-12.

"High-tech" home care for children with chronic health conditions: a pilot study.

The purposes of this study were to pilot test the instrument and methods for a major study of caregivers of children who are technology dependent and to test constructs proposed for the major study in a discrete sample. The major study will be implemented in 13 cities in the United States. The pilot was conducted in three small- to moderate-size metropolitan areas in the Southeast and Midwest. Seventy-three primary caregivers provided data during a structured telephone interviews. The children represented four groups of children dependent on technology as defined by the Office of Technology Assessment (OTA) (1987). The findings supported the methodology and the viability of the instrument. Analysis suggests that caring for a child who is technology dependent affects family functioning and increases family stress levels in some families. The financial burden is also significant. It is suggested that nurses can be a critical source of support and information to these families.

Signal transduction by G proteins in cardiac tissues.

The role of G proteins in mediating the responses of the heart to circulating catecholamines and to the influences of the autonomic nervous system is of special interest to cardiologists. It is evident that G proteins are essential links in the cascade of biochemical events that ensure when neurotransmitters and hormones interact with receptors on myocardial cells. It is likely [corrected] that dysfunction of G proteins plays a role in cardiovascular pathophysiology. With current methodologies, especially molecular biological and recombinant DNA techniques, and with transgenic animal models that can relate physiological function and specific gene dosage, some cardiovascular diseases may be traced to G protein-related defects.

Selective parasympathectomy increases the quantity of inhibitory guanine nucleotide-binding proteins in canine cardiac ventricle.

In mammalian heart, vagal stimulation or the direct application of acetylcholine produces profound direct effects on the electro-physiologic characteristics of atrial myocytes. At the tissue level, these effects are observed as shortening of atrial action potential duration. Despite anatomic, biochemical, and physiologic evidence for significant vagal input to the mammalian ventricle, similar direct effects of acetylcholine on the ventricular action potential have been difficult to demonstrate. Chronic denervation via cervical vagotomy is one method that has been shown to render previously unresponsive ventricular myocytes sensitive to acetylcholine, but the molecular mechanism has not been defined. In the experiments described, selective cardiac para-sympathectomy was performed on mongrel dogs. Five to seven days after parasympathectomy, the dogs were sacrificed, electrophysiologic responses to acetylcholine were measured, and sarcolemmal vesicles were prepared. After parasympathectomy, ventricular myocytes were responsive to the effects of acetylcholine, manifested as shortening of the action potential duration. A quantitative and functional assessment of the transmembrane signalling mechanisms of the muscarinic receptor was carried out. After parasympathectomy, the density of muscarinic receptors in the sarcolemma was increased, compared with control ventricles. After parasympathectomy, ventricular sarcolemma displayed significant increases in both basal and oxotremorine-stimulated GTPase activity. ADP-ribosylation revealed significantly increased quantities of the pertussis toxin substrates Gi and Go. The quantity of ADP ribose incorporated was correlated with the increased level of GTPase activity in control and oxotremorine-stimulated membranes. Quantitation of the alpha and beta gamma subunits of the guanine nucleotide-binding proteins by immunoblot confirmed the increase in density of inhibitory guanine nucleotide-binding proteins following parasympathectomy. The results offer new insights into possible mechanisms of altered electrophysiologic responsiveness to acetylcholine following cardiac parasympathectomy.

Muscarinic cholinergic-receptor stimulation of specific GTP hydrolysis related to adenylate cyclase activity in canine cardiac sarcolemma.

One component of muscarinic receptor inhibition of the function of cardiac ventricles is mediated by the inhibition of activated adenylate cyclase activity in sarcolemma. We have shown previously that muscarinic agonists inhibit GTP- but not Gpp(NH)p-activated adenylate cyclase activity, and various studies in other tissues indicate that nonhydrolyzable GTP analogues prevent inactivation of the enzyme. These data have suggested a role for GTP hydrolysis in the mechanism of inhibition of adenylate cyclase. The present study demonstrates that purified canine cardiac sarcolemma displays high-affinity GTPase activity that is reciprocally related to adenylate cyclase activity. The high-affinity GTPase activity was stimulated by muscarinic agonists and blocked by atropine. Furthermore, the one-half maximal effects of oxotremorine for binding to muscarinic receptors, stimulation of high-affinity GTPase activity, and inhibition of adenylate cyclase activity were similar. Muscarinic stimulation of GTPase activity and inhibition of adenylate cyclase activity required functional activity of the pertussis toxin (IAP) substrate(s). Treatment of sarcolemmal membranes with IAP attenuated the ability of oxotremorine to both stimulate high-affinity GTPase activity and inhibit adenylate cyclase activity. These studies indicate that muscarinic receptor stimulation of high-affinity GTPase activity dependent on functional IAP substrate(s) is closely linked to the mechanism of muscarinic inhibition of adenylate cyclase activity.

Pertussis toxin-treated dog: a whole animal model of impaired inhibitory regulation of adenylate cyclase.

We have shown previously that stimulation of high-affinity GTP hydrolysis and inhibition of adenylate cyclase activity by muscarinic agonists are mediated by pertussis toxin (IAP) substrates (Gi and Go) in canine cardiac sarcolemma. We have now used the pertussis toxin-treated dog as a whole animal model in which Gi- and Go-mediated biochemical mechanisms can be studied. Mongrel dogs were injected intravenously with IAP 48 hours prior to death and isolation of left ventricular sarcolemma. Treatment of the animal in vivo with the toxin prevented subsequent in vitro IAP-catalyzed [32P]ADP-ribosylation of substrates in cardiac, erythrocytic, and renal cortical plasma membranes, suggesting that ADP-ribosylation occurred in vivo from endogenous substrate. Consistent with our previous results obtained by treating sarcolemma in vitro with IAP, muscarinic receptor-mediated stimulation of high-affinity GTP hydrolysis and inhibition of GTP-activated adenylate cyclase activity were attenuated in sarcolemma purified from the toxin-treated animals. Proximal to adenylate cyclase, guanine nucleotide regulation of muscarinic receptor affinity for agonists was also abolished in membranes from the toxin-treated animals. In addition, the ability of oxotremorine to attenuate GTP regulation of stimulation of adenylate cyclase activity by magnesium ions was abolished in sarcolemma from the IAP-treated dogs. Thus, cardiac sarcolemma isolated from the IAP-treated animals displayed biochemical characteristics of an adenylate cyclase system in which inhibitory regulatory pathways had been attenuated. The cardiac biochemical studies and the in vivo ADP-ribosylation of noncardiac IAP substrates also suggests considerable potential use of this model in the physiological and biochemical study of regulatory mechanisms mediated by GTP-binding proteins in other systems.

Restoration of adenylate cyclase responsiveness in murine myeloid leukemia permits inhibition of proliferation by hormone. Butyrate augments catalytic activity of adenylate cyclase.

Mechanisms of leukemic cell clonal dominance may include aberrations of transmembrane signaling. In particular, neoplastic transformation has been associated with reduced capacity for hormone-stimulated adenylate cyclase activity. In the present study, prostaglandin E, a hormonal activator of adenylate cyclase that has antiproliferative activity in myeloid cells, and cholera toxin, an adenylate cyclase agonist that functions at a postreceptor site by activating the adenylate cyclase stimulatory GTP-binding protein (Gs), were studied for antiproliferative activity in two murine myeloid cell lines. FDC-P1, an interleukin 3 (IL 3)-dependent myeloid cell line and a tumorigenic IL 3-independent subline, FI, were resistant to these antiproliferative agents. The in vitro ability of the "differentiation" agent, sodium butyrate, to reverse their resistance to adenylate cyclase agonists was studied. The antiproliferative action of butyrate involved augmentation of transmembrane adenylate cyclase activity. Increased adenylate cyclase catalyst activity was the primary alteration of this transmembrane signaling group leading to the functional inhibitory effects on leukemia cells, although alterations in regulatory G-proteins appear to play a secondary role.

Color drawings: a means of identifying problems in children.

Between the ages of seven and nine years, the child develops a schema or way to draw his version of the world. Drawings are revealing in that they can help assess a child's behavior. Figure drawings are useful in determining general cognitive development, self-image, and level of maturity (Harris, 1963; DiLeo, 1973; DiLeo, 1983; and Koppitz, 1968). As the child progresses through developmental stages in perceiving and using color, she similarly progresses in her drawing abilities. Distinction, identification and aesthetic reaction to color are thus functions of the cortex; they are developmental and educational results rather than instinctual and reactive responses (Scott, 1969).

Differences in color preferences of school-age children in varying stages of health: a preliminary study.

The purpose of the study was to determine the preferences in color of children ages 7-12 who were physically well and those in varying states of ill health. Data were collected on a convenience sample of 72 subjects. Children were asked to name their favorite color, and then select the color they preferred from a set of color squares and the picture they preferred from two sets of colored pictures. General information about the child's diagnosis, number of days hospitalized, and number of siblings was also obtained along with other projective information. Data were analyzed using chi-square to test the hypotheses. Logistic regression models were used to examine the independent variables sex, age group, and health state in a multivariate manner by fitting the models with the choices of red, blue, or green as the first or second color on the Luscher Color Test as the dependent variable. This analysis was repeated with the Picture Test as the dependent variable. The only regressor for these models that was significant was health state. Significant differences were found (p less than 0.05 level) in color preferences of physically well children and those who were ill; in acutely ill and chronically ill children; in physically disabled and acutely ill children; and in children ages 7-9 and 10-12.

Muscarinic receptor regulation of cardiac adenylate cyclase activity.

Stimulation and inhibition of adenylate cyclase activity are mediated by the guanine nucleotide regulatory proteins Gs and Gi, respectively. Two general mechanisms have been proposed for the inhibition of activated adenylate cyclase: direct inhibition of the catalyst by Gi, and indirect inhibition of the activated catalyst mediated by Gi inhibition of Gs. We have assessed direct inhibition of adenylate cyclase by evaluating the ability of Gpp(NH)p to inhibit the forskolin-stimulated enzyme in the presence of various concentrations of magnesium ions and the guanine nucleotide. Gpp(NH)p inhibition of adenylate cyclase activity was only observed in the presence of forskolin and low concentrations of MgCl2. Muscarinic agonists did not increase Gpp(NH)p inhibition of the forskolin-stimulated enzyme, even in the presence of low concentrations of MgCl2 and guanine nucleotide (near the respective Kact or Ki). Whether in the absence or presence of muscarinic agonists, no concentration of Gpp(NH)p was found to inhibit basal adenylate cyclase activity in the absence of forskolin. In addition, muscarinic agonists had no effect on the rate constant (kon) for Gpp(NH)p activation of the enzyme. In contrast to these data, the muscarinic agonist methacholine stimulated the inactivation rate constant (koff) for isoproterenol plus GTP-activated adenylate cyclase activity 15-fold, and the increase in koff was blocked by atropine. Moreover, the sarcolemma displayed specific, high affinity GTP hydrolytic activity which was stimulated by methacholine activation of muscarinic receptors. These data further support our original hypothesis, indicating that although direct inhibition of the catalyst by Gi may occur in cardiac sarcolemma, physiologically relevant attenuation of adenylate cyclase activity by muscarinic agonists occurs by a mechanism linked to GTP hydrolysis.