Purification and proliferation of human myoblasts isolated with fluorescence activated cell sorting.
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Cell Division↗
Biomedical subjects
C Webster
Publications and source records attributed to C Webster.
Accelerated age-related decline in replicative life-span of Duchenne muscular dystrophy myoblasts: implications for cell and gene therapy.
An assessment of the replicative life-span of myoblasts is of fundamental importance in designing treatment strategies for Duchenne muscular dystrophy (DMD) based on cell or gene therapy. To ascertain myoblast life-span, or the total number of cell divisions of which a myoblast was capable, we serially passaged and counted the progeny of individual myoblasts until they senesced. We compared the life-span of myoblasts from eight DMD patients with controls: three individuals with no known neuromuscular disease, three DMD carriers, and three patients with other muscle degenerative diseases. A decline in replicative capacity was observed with increasing donor age, which was markedly accelerated for DMD relative to control myoblasts. The average myoblast from a 5-year-old control was capable of 56 doublings, or a potential yield of approximately 10(17) cells per cell. By contrast, at 2 years of age, the typical age at clinical onset, only 6% of DMD myoblasts had a life-span of 50 doublings in tissue culture, and by age 7 DMD myoblasts capable of 10 doublings were rare. Our results suggest that the myoblasts (satellite cells) of even the youngest DMD patients have undergone extensive division in an attempt to regenerate degenerating myofibers. These findings have implications for therapeutic intervention in DMD involving genetic engineering and myoblast implantation.
Age Factors↗
Conflict and consensus: explaining the British Health Service.
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History, Modern 1601-↗
Doctors, public service and profit: general practitioners and the National Health Service.
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Family Practice↗
The Escherichia coli melR gene encodes a DNA-binding protein with affinity for specific sequences located in the melibiose-operon regulatory region.
Crude extracts, made from Escherichia coli cells carrying a plasmid in which the melR gene was expressed from the galP2 promoter, were used as a source of MelR protein. Using DNase I footprinting and gel retardation assays, we show that MelR binds to two sites located from nucleotides (nt) -49 to -75 and -85 to -113, upstream from the melAB transcription start point. The two sites contain identical 18-bp sequences. Specific binding is unaltered by deletions that remove 1 or 6 amino acids (aa) from the C terminus of MelR, but is abolished by deletion of 16, 24 or more aa residues. Sequence homologies between MelR and other DNA-binding proteins are discussed.
Amino Acid Sequence↗
Protein kinase C--a family affair.
The structural analysis of protein kinase C has led to the identification of a family of related gene products. This family of kinases consists of six unique genes that give rise to at least seven polypeptides. The high degree of conservation and the differential distribution of these mRNAs/proteins suggest that they perform distinct functions in vivo. Characterization of the activities of some of these proteins in vitro shows that there are functional differences with respect to both their regulation and substrate specificity. This indicates that each member of this family may play a unique role in signal transduction.
Amino Acid Sequence↗
Dependence of Ethanolic Fermentation, Cytoplasmic pH Regulation, and Viability on the Activity of Alcohol Dehydrogenase in Hypoxic Maize Root Tips.
We examined the role of alcohol dehydrogenase (ADH) in the metabolism and survival of hypoxic maize (Zea mays L.) root tips. The dependence of the rate of ethanolic fermentation, cytoplasmic pH, and viability on the activity of ADH in maize root tips during extreme hypoxia was determined. Maize lines with ADH activities differing over about a 200-fold range were studied. Effects of genetic background were controlled by comparing pairs of F4 progeny of crosses between mutant (low ADH activity) and reference inbred lines. The capacity of hypoxic root tips to perform ethanolic fermentation exhibited a dependence on ADH activity only at activities found in Adh 1 nulls. The ability of maize root tips to withstand prolonged and extreme hypoxia was like-wise independent of ADH activity, except at the lowest activities. Root tips that exhibited lower tolerance of hypoxia had more acidic cytoplasm during extreme hypoxia. We conclude that the activity of ADH in normal maize root tips does not limit the capacity for energy production via fermentation, and does not determine viability under extreme hypoxia. The significance of the induction of ADH activity in plants by hypoxia is discussed.
Journal Article↗
Transcription from the Escherichia coli melR promoter is dependent on the cyclic AMP receptor protein.
Expression of the melR gene is required for melibiose-dependent stimulation of transcription initiation at the promoter of the melAB operon. Using the S1 nuclease method we have located the melR transcription start point. Transcription from the melR promoter is dependent on cAMP-CRP: specific nucleotide sequences downstream of bp -59 with respect to the melR transcription start are sufficient for full promoter activity. Nucleotide sequence homologies suggest that the cAMP-CRP binding site is located from bp -52 to -31, in exactly the same position as at the galP1 promoter. Using DNase I footprinting we show that cAMP-CRP and RNA polymerase together bind tightly to the melR promoter sequence, creating a strong footprint from bp -70 to +20. Alone, cAMP-CRP binding is hardly detectable, whereas RNA polymerase alone creates a weak footprint centred around the -10 hexamer sequence. When the melR gene is expressed from a cAMP-CRP-independent promoter, melibiose-dependent transcription from the melAB promoter becomes independent of cAMP-CRP, showing that the melR promoter is the primary site of control by cAMP-CRP in the mel regulon.
Base Sequence↗
Fast muscle fibers are preferentially affected in Duchenne muscular dystrophy.
We show that Duchenne muscular dystrophy (DMD) selectively affects a subset of skeletal muscle fibers specialized for fast contraction. Muscle fiber types were characterized immunohistochemically with monoclonal antibodies that distinguish isoforms of fetal and adult-fast or adult-slow myosin heavy chain present in the same fiber. Fetal myosin expression increased with patient age and was not due to arrested development but rather to de novo synthesis, which served as a sensitive indicator of muscle regeneration. A subset of fast fibers were the first to degenerate (type IIb). Extensive fast fiber regeneration occurred before slow fibers were affected. These results suggest that the DMD gene product has a specific function in a subpopulation of muscle fibers specialized to respond to the highest frequency of neuronal stimulation with maximal rates of contraction.
Age Factors↗
Improved media for normal human muscle satellite cells: serum-free clonal growth and enhanced growth with low serum.
We have developed a serum-free medium for clonal growth of normal human muscle satellite cells (HMSC). It consists of an optimized nutrient medium, MCDB 120, plus a serum-free supplement, designated SF, that contains epidermal growth factor (EGF), insulin, dexamethasone, bovine serum albumin, and fetuin. Fibroblast growth factor was needed with dialyzed fetal bovine serum (dFBS) as the only other supplement, but in media containing SF, it was only slightly beneficial, and was omitted from the final medium without significant loss. Clonal growth of HMSC in MCDB 120 plus SF is as good as with 15% serum and 0.5% chicken embryo or bovine pituitary extract. However, growth is further improved by use of a doubly-supplemented (DS) medium containing both SF and 5% dFBS. Clonal growth of HMSC in the DS medium far exceeds that in previous media with any amount of serum, and monolayer growth is at least equal to that in conventional media with higher levels of serum. Cells grown in these media exhibit little differentiation, even when grown to high densities. However, they retain the capacity for extensive fusion and synthesis of increased creatine kinase when transferred to a serum-free differentiation-promoting medium, such as Dulbecco's modified Eagle's medium plus insulin. All experiments were done with clonal cultures of HMSC to insure that observed growth responses were always those of muscle cells.
Cell Differentiation↗
Isolation of human myoblasts with the fluorescence-activated cell sorter.
We have established procedures for the rapid and efficient purification of human myoblasts using the fluorescence-activated cell sorter. Our approach capitalizes on the specific reaction of monoclonal antibody 5.1H11 with a human muscle cell surface antigen. For each of the five samples analyzed, an enrichment of myoblasts to greater than 99% of the cell population was immediately achieved. Following 3 to 4 weeks of additional growth in vitro, sorted myoblast cultures remained 97% pure. Differentiation of the sorted myoblast cultures, assessed by creatine kinase activity and isozyme content, was comparable to that of pure myoblast cultures obtained by cloning, and was significantly greater than that of mixed fibroblast and myoblast cultures. An average of 10(4) viable myoblasts can be obtained per 0.1 g tissue, each with the potential to undergo approximately 40 cell divisions. Accordingly, if only two-thirds of this proliferative capacity is utilized, the potential yield approximates 10(12) myoblasts, equivalent to 1 kg of cells. Human myogenesis in vitro is no longer limited by cell number and is now amenable to molecular and biochemical analysis on a large scale.+
Abortion, Therapeutic↗
Sub-inhibitory and post-antibiotic effects of spiramycin and erythromycin on Staphylococcus aureus.
The antibacterial responses of clinical isolates of Staphylococcus aureus to spiramycin and erythromycin were compared. Conventional MICs showed erythromycin-sensitive strains to be 16-32 times less sensitive to spiramycin. MBCs were only four to eight times higher for spiramycin. Erythromycin resistant S. aureus were more frequently encountered. Concentrations of both macrolides at 1/4 MIC produced antibacterial effects. Post-antibiotic effects were more marked with spiramycin. After 3 h exposure to 4 x MIC of antibiotic the delay in regrowth of S. aureus was 5 h for erythromycin and 9 h for spiramycin. In a continuous cultivation model, spiramycin produced an inhibitory effect on S. aureus for 12 h whereas the effect of erythromycin was only apparent for 6 h. In conclusion, spiramycin is more active against staphylococci in vitro than would be expected by its modest MICs.
Drug Resistance, Microbial↗
Effects of fenfluramine on autistic individuals residing in a state developmental center.
The effects of fenfluramine on 21 maladaptive behaviors in 20 autistic individuals were examined over a 9-month period utilizing a double-blind, cross-over, placebo-controlled design. Raters carried out time-sampled observations in the school and residence. In addition, videotaped data were collected in controlled settings and assessed by the raters at the conclusion of the study. Some individuals displayed negative side effects such as tension, agitation, insomnia, and sweating during the 16-week period they received fenfluramine. The results demonstrated that fenfluramine caused no significant reductions in maladaptive behaviors. The lack of any significant positive results from this medication and the side effects observed strongly indicate the need for caution in the use of fenfluramine with autistic persons.
Adolescent↗
Organisation of the regulatory region of the Escherichia coli melibiose operon.
The regulatory region of the Escherichia coli melibiose operon contains two divergent promoters. One promoter is responsible for the expression of the melR gene, that is essential for melibiose-dependent stimulation of the second promoter. Melibiose-induced transcription from this second promoter initiates at a start point 25 bp upstream from the start codon of the melA gene, encoding an alpha-galactosidase. The nucleotide sequence covering the divergent promoters and the melR gene is reported.
Base Sequence↗
Transcription initiation at the Escherichia coli galactose operon promoters in the absence of the normal -35 region sequences.
The gal operon regulatory region contains two overlapping promoters, P1 and P2, regulated by cyclic AMP and the cyclic AMP receptor protein (cAMP X CRP). Starting with a mutation that eliminated P1, the promoter that is usually dependent on cAMP X CRP, we constructed a series of deletions that substituted increasing amounts of DNA sequence from upstream of P2, the promoter that usually functions in the absence of cAMP X CRP. Expression from P2 in vivo was halved by deletions that replace the -35 region with unrelated sequences, showing that the -35 sequence participates in promoter function, but is not essential. In vitro studies show that replacement of the -35 sequence increases the time for open complex formation at P2, but does not alter the transcription start point. We examined the effects of the same deletions at the wild type gal promoter region: again, the deletion that replaces the -35 region halves expression in vivo. However, in this case, in the absence of cAMP X CRP, the deletion switches expression from the P2 promoter to P1, the promoter that is usually dependent on cAMP X CRP. Moreover, although the deletion also removes the specific cAMP X CRP binding site, this P1 activity is sharply inhibited in a crp+ background. We argue that this is due to a direct contact between CRP and RNA polymerase bound at the P1 Pribnow box, and we discuss the role of the -35 sequence at these and other promoters.
Base Sequence↗
The myoblast defect identified in Duchenne muscular dystrophy is not a primary expression of the DMD mutation. Clonal analysis of myoblasts from five double heterozygotes for two X-linked loci: DMD and G6PD.
We previously proposed the hypothesis that the primary expression of the defect in X-linked Duchenne muscular dystrophy (DMD) occurred in the myoblast, or muscle precursor cell. This was based on the observation that the number of viable myoblasts obtained per gram DMD muscle tissue was greatly reduced and those that grew in culture had decreased proliferative capacity and an aberrant distended flat morphology. Here we test that hypothesis by determining whether the expression of the myoblast defect is X-linked. Muscle cells were obtained from five doubly heterozygous carriers of two X-linked loci, DMD and glucose-6-phosphate dehydrogenase (G6PD), and compared with those from five sex- and age-matched controls heterozygous for G6PD only. A total of 1,355 individual clones were determined to be muscle and evaluated at the single cell level for proliferative capacity, morphology, and G6PD isozyme expression. The results demonstrate that the proportion of defective myoblast clones is significantly increased in DMD carriers. However, since this cellular defect does not consistently segregate with a single G6PD phenotype in the myoblast clones derived from any of the carriers, it is unlikely to be the primary expression of the DMD mutant allele.
Adult↗
Syntheses and in vitro evaluation of 4-(2-aminoethyl)-2(3H)-indolones and related compounds as peripheral prejunctional dopamine receptor agonists.
A series of (beta-aminoethyl)indolones and related compounds was synthesized and evaluated in vitro as peripheral prejunctional dopaminergic agonists in the field-stimulated isolated perfused rabbit ear artery. 4-[2-(Di-n-propylamino)ethyl]-7-hydroxy-2(3H)-indolone was the most potent compound (ED50 = 2 +/- 0.3 nM) tested, while the related secondary amine 24 and the des-OH derivatives 28 and 34 were only slightly less potent. 4-Methoxybenzeneethanamine and 2-methyl-3-nitrophenylacetic acid were employed as starting materials for for the synthesis of the 4-(beta-aminoethyl)indolones. The ring-opened 3-acylamino analogues 46 and 47 were prepared via nitration of the phenethylamine 43 derived from 4-methoxyphenylacetic acid. The inactive isomeric indolones 38, 39, and 41 were derived from 4-nitrobenzeneethanamine and from indolone-6-acetic acid.
Animals↗
Insulin and insulinlike growth factor receptors and responses in cultured human muscle cells.
Specific receptors for insulinlike growth factors I and II (IGF-I and IGF-II) were found on cultured human myoblasts and myotubes. In contrast, myotubes but not myoblasts specifically bound insulin and were stimulated by nanomolar concentrations of insulin to take up deoxyglucose. In addition, in myoblasts, physiological concentrations of IGF-I and -II and, to a lesser extent, insulin stimulated two- to threefold the uptake of the nonmetabolizable amino acid analogue methylaminoisobutyric acid (MAIB). In myotubes, uptake of MAIB was stimulated preferentially by IGF-I. Monoclonal antibodies that preferentially recognize either the insulin receptor or the IGF-I receptor were utilized to examine which receptors mediated the biological effects of these hormones. The effects of insulin on both myoblasts and myotubes appeared to be mediated in part by the insulin receptor and in part by the IGF-I receptor. In myotubes, the effects of IGF-I and -II both appeared to be mediated through the IGF-I receptor. In myoblasts, the effects of the two IGFs appeared to be in part mediated by the IGF-I receptor and in part mediated by either the IGF-II receptor or another type of IGF-I receptor. The present results suggest that cultured human muscle cells provide a useful model system in which to study the biological actions of insulin and the IGFs.
Aminoisobutyric Acids↗