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.     

Climatic adaptation in recently shorn merino sheep

Heart rates, respiratory rates, body temperatures, ad libitum ‘day’ and ‘night’ food consumption and body weight changes have been examined in 15 mature Merino wethers shorn in moderate environmental conditions. All sheep showed a depression in food consumption for two days after shearing. Sheep that gained weight during the next three weeks then increased their food consumption at night by approximately 30% although the average daily consumption was only increased by 5%. Sheep that lost weight showed a depressed food consumption throughout the three week period after shearing. Marked increases in the temperature difference between ear skin and air as well as thermal tachypnoea during the warmest period of the day were recorded in all sheep 14–16 days after shearing. This indicated that the critical temperature for all sheep had decreased by about 10°C. These signs of acclimatisation appeared at similar times in all sheep, suggesting that increased resistance to body cooling developed at similar rates in weight gain and weight loss sheep and independent of the origin of body heat production. The results of the investigations are discussed in relation to the concept that the initial response to cold stress includes a depression in food intake and that the duration of this depression is a function of the cold stimulus and the strain it induces in the sheep.     

Fate of corticotrophins in an isolated adrenal-cell bioassay and decrease of peptide breakdown by cell purification

1. The fate of corticotrophins in a trypsin-dispersed rat adrenal-cell assay system was investigated with a view to establishing whether differences in the rate of inactivation might contribute to potency differences observed between analogues. 2. Corticotrophin-(1-24)-tetracosapeptide and to a lesser extent synthetic 1-39 corticotrophins were found to be inactivated during incubation with cell suspension. 3. Peptide fragments were isolated by using [[(3)H(2)]Tyr(23)]corticotrophin-(1-24)- tetracosapeptide as a marker. The fragments indicate a peptidase with a predominantly tryptic specificity. 4. The peptidase is present in the extracellular fluid and is released from cells when they are damaged. 5. Cells were fractionated on an albumin gradient. Cells from the zona fasciculata and the zona intermedia or reticularis were present in fractions which produced fluorogenic steroids in response to corticotrophin. 6. Purification of the cells by centrifugation through albumin decreased degradation by peptidases, so that if the assay is carried out with a dilute suspension of purified cells peptide breakdown should not affect the observed potencies of adrenocorticotrophin analogues. 7. No binding of [[(3)H(2)]Tyr(23)]corticotrophin-(1-24)- tetracosapeptide to cells could be detected at low concentrations of the peptide. This indicated that less than 120 receptors/cell are occupied during stimulation by a dose that would elicit approx. 80% of the maximal response.     

Some properties of influenza virus nucleocapsids

Nucleocapsids released from influenza virions by sodium deoxycholate sedimented heterogeneously in sucrose gradients. Highly infectious virus (complete) preparations yielded nucleocapsids with peak distributions at 64 and 56S; von Magnus type virus (incomplete) lacked 64S nucleocapsids. Treatment of influenza virus nucleocapsids with pancreatic ribonuclease rendered the associated viral ribonucleic acid (RNA) molecules acid-soluble, indicating that capsid proteins do not completely surround the viral RNA's. However, the capsid proteins remained associated after enzymatic hydrolysis of the RNA, as judged by persistently high sedimentation rates. Sedimentation rates of viral nucleocapsids reflected the sedimentation rates of the associated RNA's: 64S nucleocapsids contained 18S RNA, whereas 56S nucleocapsids contained 15S RNA, although in both cases RNA's sedimenting at 4 to 13S were also recovered. Furthermore, just as incomplete virions lacked 64S nucleocapsids, they also lacked 18S RNA. These findings support the hypothesis that the influenza virus genome is divided among several distinct pieces of RNA.     

Procedure for Evaluating the Effects of 2,450- Megahertz Microwaves upon Streptococcus faecalis and Saccharomyces cerevisiae

Modifications of a commercial 2,450-megahertz microwave oven were made so that 6 ml of microbial suspension could be exposed to the microwave field for various periods of time. The microorganisms were contained in the central tube of a modified Liebig condenser positioned in the approximate geometric center of the oven cavity. Kerosene at -25 C was circulated through the jacket of the condenser during microwave exposure permitting microwaves to reach the microbial suspension. Flow rates of the kerosene were varied to permit the temperature of the suspension to range from 25 to 55 C during microwave exposure. Conductive heating experiments using similar temperatures were also conducted. A thermocouple-relay system was employed to measure the suspension temperature immediately after the magnetron shutoff. Continuous application of microwaves to suspensions of 10(8) to 10(9)Streptococcus faecalis or Saccharomyces cerevisiae per ml appeared to produce no lethal effects other than those produced by heat. Respiration rates of microwave-exposed Scerevisiae were directly related to decreases in viable count produced by increased microwave exposure times.     

On the site of action of phosphatide acyl-hydrolase activity of rat brain homogenates on lecithin

The hydrolysis of 2- [14C]acyl-labelled and [Me-3H]choline-labelled lecithins by rat brain homogenates has been studied. The acyl-labelled substrate was hydrolysed with the production of both radioactive lysolecithin and radioactive free fatty acid in the proportions of 60 per cent and 40 per cent; traces of labelled mono- and diglyceride were also formed. In addition to radioactive lysolecithin the [3H]choline-labelled lecithin also gave rise to much smaller amounts of radioactive water soluble derivatives, consisting almost entirely of free choline and phosphorylcholine with only traces of glycerylphosphorylcholine. The results provide evidence for the production of a mixture of 1- and 2-acyl isomers of lysolecithin by phospholipase action in brain homogenates at pH 7.4, the latter predominating slightly.     

GROWTH STUDIES OF THE ROOT OF INCENSE CEDAR,LIBOCEDRUS DECURRENS. I. THE ORIGIN AND DEVELOPMENT OF PRIMARY TISSUES

Wilcox , Hugh . (State U. Coll. of Forestry, Syracuse, New York.) Growth studies of the root of incense cedar, Libocedrus decurrens. I. The origin and development of primary tissues. Amer. Jour. Bot. 49(3): 221–236. Illus. 1962.—The anatomical features of active and dormant roots of incense-cedar seedlings are described and discussed in relation to various problems of differentiation and morphogenesis. Autoradiographs confirm the presence of a group of relatively inactive cells at the site of the apical initials. During periods of maximum growth activity, the presence of a quiescent center is accentuated by a peak in number of divisions in adjacent tissues. With diminution in growth activity, the peak occurs closer to the quiescent center and the size of the meristem appears to diminish. During dormancy, the configuration of the initial region seems to indicate the existence of apical initial cells which coincide with a minimal constructional center, as determined by studies of cell lineage. Roots whose apical cells retain their meristematic appearance are able to resume growth after a period of dormancy, whereas roots whose apical cells undergo vacuolation are likely to perish. Graphs are presented to show the functional relationships between growth rate and the varying distances from the apical meristem at which the tissues of the root differentiate and mature. Although early differentiation of precursory phloem could be discerned almost as soon as early vacuolation of metaxylem, its recognition was more dependent upon subjective judgment. The functional relationship between differentiation and growth rate was most pronounced in the maturation of protoxylem elements, the development of Casparian strips in the endodermis, the development of suberin lamellae in the endodermis, and by the development of phi layers in the inner cortex.