Differentiation properties of pure populations of human dystrophic muscle cells

The interpretation of the majority of studies of Duchenne muscular dystrophy (DMD) has been complicated by the heterogeneous composition of the cultures used. In addition to muscle cells, muscle tissue contains adipocytes and fibroblasts and the proportion of these cell types varies, especially in disease states. To overcome this problem we developed culture conditions which permitted isolation and characterization of pure populations of clonally derived human muscle cells [1, 2]. Here we report the successful application of these methods to muscle cells from biopsies of individuals with diagnosed DMD. The normal and mutant human muscle cells were used in experiments of muscle differentiation in the same manner as cell lines. Frozen-stored cells were thawed, plated in a series of replicate plates, and allowed to differentiate under similar culture conditions. Yet, in contrast with cell lines, the cells were karyotypically normal, not altered by adaptation to long-term culture, and had a finite lifespan. We have systematically analysed specific properties of the normal and DMD muscle cells which differentiated in culture. The kinetics and extent of myoblast fusion, myotube morphology, and the accumulation and distribution of membrane acetylcholine receptors were monitored. In addition, the isozyme composition of creatine kinase and its intracellular and extracellular distribution were determined. Our results indicate that DMD muscle cells are fully capable of initiating myogenesis in culture and do not differ from normal muscle in several important parameters of differentiation.     

The effect of concanavalin A on egestion of food vacuoles in Tetrahymena

The ability of concanavalin A (conA) to disrupt food vacuole elimination at the cytoproct of Tetrahymena pyriformis, strain GL-C, was investigated using fluorescence microscopy and thin section electron microscopy. ConA was found to induce "tails" in Tetrahymena. These tails were specifically stained by fluorescent conA. Thin section observations of conA-treated cells revealed that these tails were the result of abnormal egestion of food vacuole contents at the cytoproct. Tail formation appears to result from an inhibition of endocytosis of food vacuole membrane during egestion. Instead, the food vacuole membrane appears to be cast out of the cell, along with the contents of the vacuole. The mechanism of this inhibition may be related to an apparent absence of microtubules or microfilamentous mat in the cytoproct region of conA-treated cells. Although conA is ingested into food vacuoles in large amounts, conA appears to affect endocytosis only from outside the cell; ingested conA does not appear to be effective. ConA may exert its influence by binding to the cytoproct region. The ability of conA to induce tail formation is inhibited by sugars specific to it. Numerous membranous vesicles are found in association with the oral cilia and cytoproct region of conA-treated cells. These vesicles may be the conA-binding material reported to be secreted by Tetrahymena.     

Evoked Release of Proteins from Central Neurons In Vivo

Push-pull cannulae were implanted in both substantiae nigrae and caudate nuclei of the halothane-anesthetized cat. The release of total protein, acetylcho-linesterase, and nonspecific cholinesterases was examined. Following direct application of potassium to one substantia nigra, changes occurred in the local release of total protein and acetylcholinesterase, but not nonspecific cholinesterases; changes also were observed in both caudate nuclei and the contralatera/ substantia nigra. The local evoked release of acetylcholinesterase and of total protein differed in the extent to which they were calcium-dependent. Control studies suggest that release of these compounds, both spontaneous and evoked, is related, at least in part, to neuronal activity. The significance of the neuronal release of proteins is discussed.     

Light Enhances the Turnover of Phosphatidylinositol in Rat Retinas

Light stimulation of isolated rat retinas is shown to enhance the turnover of phosphatidylinositol (PI) as demonstrated by a light-dependent increase in [³H]inositol incorporation and concurrent hydrolysis of existing PI. Studies with rat retinas incubated with [³H]inositol and then microdissected at the level of the outer plexiform layer into photoreceptor cell and inner retina layers indicated that the light-enhanced incorporation of [³H]inositol was associated with the photoreceptor cell layer. The rate of PI hydrolysis in retinas prelabeled in vivo with [³H]inositol was higher in light than in dark incubations and was higher in the photoreceptor cell layer than within the inner retina. Within the photoreceptor cell layer, PI turnover involved 2%/min of the total PI contentin dark and 6–8%/min in light. In contrast to what has been reported for stimulus-enhanced turnover of PI in some tissues, this light-enhanced turnover of PI in the retina was not associated with detectable reductions in PI content. Parallel studies of sodium (²²Na) uptake demonstrated that the photoreceptor cells remained functional during these incubations as they retained the capacity to restrict the entry of ²²Na in light but not in dark.     

Effects of decapitation and ovariectomy on the regulation of juvenile hormone synthesis in the cockroach, Diploptera punctata

Corpora allata from Diploptera punctata females at adult ecdysis or at the end of the last-larval stadium, when implanted into decapitated females, underwent a cycle of juvenile hormone synthesis similar in timing and magnitude to that of glands implanted into control animals which had been starved and allatectomized. Starvation did not alter the cycle in rates of juvenile hormone synthesis of sham-operated animals.Decapitation of ovariectomized animals resulted in no cycle in rates of juvenile hormone synthesis by implanted adult corpora allata; however, implantation of an ovary along with the corpora allata into decapitated, ovariectomized hosts resulted in a cycle of juvenile hormone synthesis. In control animals, which retained their heads but were starved and allatectomized as well as ovariectomized, the implanted corpora allata showed a cycle of juvenile hormone synthesis only when implanted with an ovary. The maximal rates of juvenile hormone synthesis by the corpora allata in both experimental and control conditions were lower than normal, likely due to the repeated trauma of surgery. However, at no time from eclosion to the end of the first gonotrophic period was the brain necessary for the cyclic response of the corpora allata to the presence of the ovary.     

Conjugal Transfer of Plasmid-Borne Multiple Antibiotic Resistance in Streptococcus faecalis var. zymogenes

A strain of Streptococcus faecalis var. zymogenes, designated JH1, had high-level resistance to the antibiotics streptomycin, kanamycin, neomycin, erythromycin, and tetracycline. These resistances were lost en bloc from approximately 0.1% of cells grown in nutrient broth at 45 C. The frequency of resistance loss was not increased by growth in the presence of the "curing" agents acriflavine or acridine orange, but after prolonged storage in nutrient agar 17% of cells became antibiotic sensitive. Covalently closed circular deoxyribonucleic acid (DNA) molecules were isolated from the parental strain and from antibiotic-sensitive segregants by using cesium chloride-ethidium bromide gradients. DNA molecular species were identified by using neutral sucrose gradients. Strain JH1 contained two covalently closed circular DNA species of molecular weights 50 x 10(6) and 38 x 10(6). An antibiotic-sensitive segregant, strain JH1-9, had lost the larger molecular species. A second sensitive segregant, strain JH1-5, had also lost the larger molecular species but a new molecular species of approximate molecular weight 6 x 10(6) was present. The antibiotic resistances that were curable from the parental strain were transferred to antibiotic-sensitive strains of S. faecalis and to strain JH1-9, during mixed incubation in nutrient broth at 37 C. Data to be described are interpreted to suggest that the transfer is by a conjugal mechanism. Analysis of the plasmid species in recipient clones showed that all had received the plasmid of molecular weight 50 x 10(6). Strain JH1-5 was not a good recipient. Analysis of one successful recipient clone of JH1-5 revealed that it had gained the 50 x 10(6) molecular weight plasmid but lost the 6 x 10(6) molecular weight species. These data are interpreted to mean that the multiple antibiotic resistance is borne by a transferable plasmid of 50 x 10(6) molecular weight, and that in clone JH1-5 this plasmid suffered a large deletion leaving only a 6 x 10(6) remnant which was incompatible with the complete replicon.     

Oestradiol uptake and retention, and high-affinity binding sites in cultured rabbit uterus

Oestradiol uptake and turnover was examined in rabbit uterus maintained in organ culture for up to 3 days. Serum decreased the uptake of [(3)H]oestradiol, whereas insulin had no significant effect. During the first 24h of culture unoccupied high-affinity receptors for oestradiol were markedly depleted in the cytosol. Nuclear binding sites remained high during the first day of culture, and were still present after 3 days. The stability of nuclear-bound oestradiol was confirmed by examining the turnover of radioactivity during culture of uteri of rabbits injected with [(3)H]oestradiol 6h before death. Over half of the radioactivity was retained for as long as 3 days in tissue cultured in the absence of oestrogen. In tissue cultured for 24h with unlabelled oestrogen, there was a progressive increase in the displacement of [(3)H]oestradiol as the concentration of unlabelled hormone in the medium was increased from 0.1 to 5nm. Higher concentrations of oestradiol had little additional effect. The oestradiol involved in this displacement reaction was associated with macromolecules, characterized by Sephadex G-25 chromatography and sucrose-density-gradient ultracentrifugation of the 0.4m-KCl extract of the nuclear pellet.     

Studies on the lipid composition of the rat liver endoplasmic reticulum after induction with phenobarbitone and 20-methylcholanthrene

1. The cholesterol content, proportions of different phospholipids and fatty acid components of phosphatidylcholine and phosphatidylethanolamine were studied in rat liver endoplasmic-reticulum membrane, after a single injection of 20-methylcholanthrene or injections of phenobarbitone for 5 days. 2. A marked decrease in the proportion of cholesterol occurred 5 days after injection of 20-methylcholanthrene or phenobarbitone. 3. The proportion of phosphatidylcholine was increased by injection of phenobarbitone and minor changes occurred in other phospholipids. 4. Phenobarbitone caused the proportion of linoleic acid in phosphatidylcholine and phosphatidylethanolamine to increase to 120-125% of the control and the proportion of oleic acid, arachidonic acid and docosahexaenoic acid to decrease. 5. 20-Methylcholanthrene caused an increase in the proportion of oleic acid in phosphatidylcholine and ethanolamine to 125-140% of the control, 1 day after injection. 6. The increased proportion of linoleic acid in phosphatidylcholine after phenobarbitone injection occurs simultaneously with the increase of cytochrome P-450 concentration, the rate of oxidative demethylation of aminopyrine and the rate of hydroxylation of biphenyl. It is therefore considered that distinct species of phosphatidylcholine or phosphatidylethanolamine containing linoleic acid in the beta position are essential in the endoplasmic-reticulum membrane for optimal activity of oxidative demethylation.     

Adenylate cyclase in sea anemone implication for chemoreception

Adenylate cyclase of the sea anemoneAnthopleura elegantissima was found to be associated with the heavy particulate fraction of the cell and to be activated by NaF and 2-mercaptoethanol. Reduced glutathione, which elicits the ciliary swallowing response during feeding, also activated adenylate cyclase in particles from the oral disc and pharynx. The GSH effect was dependent on homogenization procedure, whereas the NaF and 2-mercaptoethanol activation was not. The activation of adenylate cyclase from the oral disc and pharynx by GSH was correlated with increased Ca²⁺ binding to the particulate fraction. When activation by GSH was abolished by mechanical homogenization, no increasea in Ca²⁺ binding was observed in the presence of GSH. It is suggested that chemoreception for the swallowing response of this organism is mediated by cyclic AMP control of Ca²⁺ distribution in the cell.