Expression of ACh-activated channels and sodium channels by messenger RNAs from innervated and denervated muscle

Xenopus oocytes were used to express polyadenylated messenger RNAs (mRNAs) encoding acetylcholine receptors and voltage-activated sodium channels from innervated and denervated skeletal muscles of cat and rat. Oocytes injected with mRNA from denervated muscle acquired high sensitivity to acetylcholine, whereas those injected with mRNA from innervated muscle showed virtually no response. Hence the amount of translationally active mRNA encoding acetylcholine receptors appears to be very low in normally innervated muscle, but increases greatly after denervation. Conversely, voltage-activated sodium currents induced by mRNA from innervated muscle were about three times larger than those from denervated muscle; this result suggests that innervated muscle contains more mRNA coding for sodium channels. The sodium current induced by mRNA from denervated muscle was relatively more resistant to block by tetrodotoxin. Thus a proportion of the sodium channels in denervated muscle may be encoded by mRNAs different from those encoding the normal channels.     

Lack of differentiation following mouse serum treatment of cultured chick limb myoblasts

This investigation was conducted to assess the effects of mouse serum on chick skeletal muscle cell differentiation. In light of earlier findings of altered membrane phospholipid metabolism following mouse serum treatment of Friend erythroleukemic and chick chondrogenic cells, it was of interest to determine whether similar changes would modulate the fusion of mononucleated myoblasts, which is necessary for the formation of multinucleated skeletal muscle fibers. When mouse serum is added to low density cultures of enriched chick myoblasts shortly following cell attachment to the substratum, fusion is inhibited and neutral lipid accumulation ensues. There is an early inhibitory effect on DNA synthesis but not on protein synthesis. There is no increase in the uptake of 2-deoxyglucose following insulin stimulation of the cells, which suggests that while the cells are accumulating large amounts of lipid, they are not being converted into typical adipocytes. Finally, even in cultures of mouse serum-treated cells that undergo significant fusion, one observes thinner myotubes that do not spontaneously contract as do those of control cultures, as well as a disorganization of fluorescently stained actin and myosin myofilaments. These findings demonstrate that mouse serum acts in a dose-dependent manner, is not cytotoxic to the cells, but is capable of modulating normal developmental events of myoblasts as reported for other cell and tissue types.     

Taking care of patients--does it matter whether the physician is a woman?

Researchers have recently begun to compare male and female physicians'' attitudes toward patients, medical knowledge, and practice styles. Although women start medical school with more "humanistic views," the conservative effect of medical socialization on both male and female students attenuates these differences. While some studies suggested that men are more scientifically knowledgeable, recent studies showed no significant differences in physicians'' medical knowledge. Male and female physicians also had comparable diagnostic and therapeutic behavior. In the intimate world of physicians and patients, however, there were notable differences. Women physicians seemed better able to communicate sensitivity and caring to patients, which may account for the common perception that women are more caring and empathic physicians. Medical educators may wish to study more closely female physicians'' communication styles to identify these behaviors and inculcate them into all physicians.     

Loss of low-affinity serotonin receptors upon storage of human platelets

Platelets actively accumulate virtually all plasma serotonin within their dense granules. As a readily isolated, homogeneous cell type, platelets have served as a model for serotonin uptake into neurological tissue, in addition to defining the role of serotonin in hemostasis. The number of serotonin receptor types on the platelet membrane and the function of these receptors has not been conclusively demonstrated. The presence of different receptor types that may be altered or lost in disease or upon aging (in vitro storage or in vivo) could have significant physiological effects on platelet function. This report demonstrates that at least two receptor types are present on freshly prepared human platelets. However, after 3 to 4 days of storage in autologous plasma, the low-affinity, high-capacity serotonin receptor appears to be lost. This phenomenon probably accounts for some of the discrepancies reported in the literature. The high-affinity receptor present in both freshly isolated and stored platelets binds about 9 x 10(3) serotonin molecules per platelet. Binding can be completely blocked by imipramine; however, some passive diffusion appears to occur even at the low level of extracellular serotonin concentrations employed in these studies (nanomolar range). The influx of serotonin into platelets appears to be poorly reversible, even in reserpine-treated cells, where the extravesicular cytoplasmic concentration would be high. The loss of the low-affinity serotonin receptor type reported in these studies may be directly or indirectly associated with the reduced responsiveness observed in stored platelets.     

Short-term entrainment of ventilation to the walking cycle in humans

We describe a breath-by-breath method to test for entrainment of breathing and walking cycles. Thirty-eight normal subjects walked comfortably on a treadmill while breathing through a pneumotachograph. We analyzed the time intervals between heel strikes and the onset of inspiration (or expiration) for evidence of phase locking between steps and breaths, using Monte Carlo simulation to model the probability that n consecutive inspirations (or expirations) would begin at a constant time interval +/- 0.10 s from heel strikes by chance. We developed empirical criteria for rhythm synchronization during series of four or more breaths, while maintaining an estimated specificity of 95%. The majority of subjects showed some evidence of entrainment (29 +/- 23% of breaths on average), which occurred intermittently, usually lasting less than 10 breaths at a time. The precision of phase locking during spontaneous entrainment was similar to that in 10 subjects who attempted to maintain deliberate entrainment. The results suggest that the walking cadence provides a persuasive, but not dominant, input to the central breathing pattern generator. The present method can detect entrainment even when it occurs sporadically or with varying coupling pattern.     

Human recombinant insulin-like growth factor I. II. Binding characterization and radioreceptor assay development using BALB/c 3T3 mouse embryo fibroblasts

Summary The binding of human recombinant insulin-like growth factor I (IGF-I) to BALB/c 3T3 mouse embryo fibroblasts has been characterized, resulting in the development of a radioreceptor assay. Binding of radioiodinated IGF-I (¹²⁵I-IGF-I) to washed monolayers of BALB/c 3T3 cells was specific, time dependent, and stable, being maximal after a 10-h incubation at 15°C with no loss of bound ligand or cells through 25 h. Scatchard analysis identified a class of high affinity binding sites with K _d =59.6 pM and an estimated 1.57×10⁵ receptors/cell. Half-maximal displacement of bound¹²⁵I-IGF-I occurred with 15 to 20 ng/ml unlabeled IGF-I competitor. Insulin-like growth factor II and insulin were far less effective competitors, providing halfmaximal displacement at concentrations of 130 to 170 ng/ml and 2 to 3 μg/ml, respectively. Epidermal growth factor, transforming growth factor type α, and acidic and basic fibroblast growth factors did not compete for¹²⁵I-IGF-I binding at 1 μg/ml. Cells fixed with glutaraldehyde before ligand binding did remain attached to culture dishes more tightly; however such pretreatment destroyed approximately 70% of ligand binding. Crosslinking data indicated that¹²⁵I-IGF-I binds specifically to a 330-kDalton receptor as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. This receptor dissociated into 130-kDalton subunits when analyzed in the presence of dithiothreitol. This work was supported by a contract from IMCERA Bioproducts, Inc.     

Modulation of differentiation markers in human choriocarcinoma cells by extracellular matrix: on the role of a three-dimensional matrix structure

Abstract. During spontaneous or chemically induced differentiation human choriocarcinoma cells express typical characteristics of the normal differentiating trophoblast: 1) increased production of peptide and steroid hormones (chorionic gonadotropin, placental lactogen, estrogens, progesterone); 2) increased activity of cellular alkaline phosphatase; 3) morphological transition from cytotrophoblast to syncytiotrophoblast-like cells; and 4) arrested cell proliferation. Since the extracellular matrix is known to control gene expression we have examined the effects of different substrates composed of matrix macromolecules on the differentiation of BeWo choriocarcinoma cells. Matrices tested were; fibronectin, laminin, collagens type I and type IV, the basement membrane-like complex matrix Matrigel, and a complex matrix extracted from human term placenta. Irrespective of the type of molecule(s), it was consistently found that, whenever the matrix molecules were presented as threedimensional structures (as opposed to protein coatings on tissue culture plastic) the response of affected differentiation markers monitored was highly pronounced. Morphology was changed from monolayers to rounded colonies, cell proliferation was reduced, and the secretion of chorionic gonadotropin was increased up to tenfold. Heterogeneous effects were observed on progesterone secretion and on the activity of cellular alkaline phosphatase. Cell adhesion to matrix molecules, however, did not depend on the structure of the matrix. This study demonstrates that gene expression in these tumor cells can be modified by extracellular matrix and highlights that not only the presence of effector molecules in the matrix but also the three-dimensional structure of the matrix is important for the induction of differentiation.