Ralph E. Stephens

Myoblast Transfer in the Treatment of Duchenne's Muscular Dystrophy

BACKGROUND Myoblast transfer has been proposed as a technique to replace dystrophin, the skeletal-muscle protein that is deficient in Duchennes muscular dystrophy. Donor myoblasts injected into muscles of affected patients can fuse with host muscle fibers, thus contributing their nuclei, which are potentially capable of replacing deficient gene products. Previous controlled trials involving a single transfer of myoblasts have been unsuccessful. METHODS We injected donor muscle cells once a month for six months to the biceps brachii muscles of one arm of each of 12 boys with Duchennes muscular dystrophy. The opposite arms served as sham-injected controls. In each procedure 110 million cells donated by fathers or brothers were transferred. The patients were randomly assigned to receive either cyclosporine or placebo. Strength was measured by quantitative isometric muscle testing. Six months after the final myoblast transfer, the presence of dystrophin was assessed with the use of peptide antibodies specific to the deleted exons of the dystrophin gene. RESULTS There was no significant difference in muscle strength between arms injected with myoblasts and sham-injected arms. In one patient, 10.3 percent of muscle fibers expressed donor-derived dystrophin after myoblast transfer. Three other patients also had a low level of donor dystrophin (< 1 percent); eight had none. CONCLUSIONS Myoblasts transferred once a month for six months failed to improve strength in patients with Duchennes muscular dystrophy. The value of exon-specific peptide antibodies in the interpretation of myoblast-transfer results was demonstrated in a patient with Duchennes muscular dystrophy who had a high percentage of donor-derived dystrophin. Specific variables affecting the efficiency of myoblast transfer need to be identified in order to improve upon this technique.

A microgel electrophoresis technique for the direct quantitation of DNA damage and repair in individual fibroblasts cultured on microscope slides

We demonstrate by single-cell microgel electrophoresis that the 2 main techniques, trypsinization and scraping, used to collect normal diploid mammalian cells cultured in monolayer induce DNA damage. To minimize this potential interference with studies on DNA damage and repair, we have standardized the single-cell gel electrophoretic (SCG) technique for the in situ quantitation of DNA single-strand breaks and alkali-labile sites in cultured human-fibroblasts. To demonstrate the utility of this technique, human neonatal foreskin-derived fibroblasts were allowed to attach to frosted microscope slides and then either irradiated with X-rays (25-200 rad) or treated for 1 h with hydrogen peroxide (2.2-140.8 mumoles). Treatment with either agent induced a dose-dependent increase in DNA migration. At equal levels of DNA damage, cell-to-cell variability in DNA migration was more heterogeneous for hydrogen peroxide-treated cells than for X-irradiated cells. A time course study to evaluate the kinetics of DNA repair for X-ray (200 rad)-induced damage indicated that the damage was completely repaired within 2 h. Applications of this technique for in vitro toxicology are discussed.

Modifications of alkaline microgel electrophoresis for sensitive detection of DNA damage

The alkaline microgel electrophoresis technique was modified to achieve a substantial increase in sensitivity for the detection of radiation-induced DNA damage in human lymphocytes. This increased sensitivity was achieved through: (1) the addition of free radical scavengers to the electrophoresis solution to reduce DNA damage generated during alkaline unwinding and electrophoresis; (2) the modification of the electrophoresis unit to achieve a more uniform electric field; (3) the use of YOYO-1, a DNA dye, producing fluorescence 500-fold more intense than ethidium bromide; and (4) the introduction of an image analysis system for the quantitation of DNA migration. In addition to increasing sensitivity, these modifications have increased the speed with which observations can be quantified, and improved reproducibility from experiment to experiment. In human lymphocytes, these modifications have resulted in an increased sensitivity of several fold, allowing the detection of DNA damage in the range of 50 mGy. This increased sensitivity for the detection of DNA damage should extend the utility of this technique.

Distress and DNA Repair in Human Lymphocytes

This research assessed differences in DNA repair in lymphocytes from high-and low-distressed individuals. A median split on Minnesota Multiphasic Personality Inventory (MMPI) Scale 2 divided 28 newly admitted nonpsychotic psychiatric inpatients into high- and low-distress subgroups. The high-distress subgroup had significantly poorer DNA repair in lymphocytes exposed to X-irradiation than low-distress subjects. We also found that lymphocytes obtained from this psychiatric sample had significantly poorer DNA repair than lymphocytes from nonpsychiatric control subjects when compared 5 hr after X-irradiation. A high level of distress therefore appears to be associated with significant dysfunctional differences at the molecular level which may have important implications for health. These data provide evidence for a direct pathway through which distress could influence the incidence of cancer.

Acidic hydrolysis of plasmalogens followed by high-performance liquid chromatography

A simple, quantitative method for determining the plasmalogen content of small samples is reported here. The method uses the different susceptibility to acid-catalyzed hydrolysis of the alkyl, alkenyl and acyl linkages to separate the plasmalogen subclass from the other two non-labile subclasses. Hydrolysis of plasmenylethanolamine and plasmenylcholine was complete after 4 and 1 min of acid treatment, respectively. The acid-catalyzed hydrolysis did not alter the phospholipid fatty acid composition, making this method useful for fatty acid compositional analysis of the plasmalogen subclass. High-performance liquid chromatography was used for separations, and phospholipids were quantitated by assay of lipid phosphorus or by direct quantitation of peak area. Using this method, small amounts (10 nmol) of ethanolamine glycerophospholipid and choline glycerophospholipid are subjected to acid-catalyzed hydrolysis and subsequent separation of the resulting lysocompounds obtained from plasmalogens from the more acid-stable alkylacyl and diacyl glycerophospholipid fractions. Our values for plasmalogens from commercial preparations of choline and ethanolamine glycerophospholipids agree with literature values. The usefulness of the method is demonstrated for small glycerophospholipid samples that are equivalent to samples from cultured neural cells.

Visual quantification of DNA double-strand breaks in bacteria.

In this paper, we describe a method for the visualization of double-strand breaks in a single electrostretched Escherichia coli DNA molecule. We also provide evidence that electrostretched or migrated DNA under neutral microgel electrophoresis conditions is made up of individual chromosomes. Using the neutral microgel electrophoresis technique, DNA migration (stretching) was measured and the number of DNA double-strand breaks were counted following exposure of E. coli cells to 0, 12.5, 25, 50, or 100 rad of X-rays. The use of an intense fluorescent dye, YOYO and custom-made slides have helped us in visualizing individual bacterial DNA molecules. Bacterial DNA appears similar in structure compared to electrostretched DNA from human lymphocytes. We were able to detect changes in DNA migration (stretching) induced by an X-ray dose as low as 12.5 rad and an increase in the number of DNA breaks induced by a dose as low as 25 rad. The extent of DNA migration and number of breaks were directly correlated to X-ray dosage.

Cell cycle phases of a novel human neural cell line and the effect of exogenous gangliosides

Cell cycle phases of a new cell line (10-1) derived from a gemistocytic astrocytoma were analysed with computer-generated curves fit to the percentage of labeled metaphases at various times after a [3H]-thymidine pulse. These large slowly growing tumor cells in vitro had a mitotic index of 2.8%, a confluent density of 34400 cells/cm2 and an average DNA content of 7.91 pg/cell. Ganglioside treatment (50 uM in the culture medium) prolonged the generation time (26 hrs to 33 hrs) as well as all the phases of the cell cycle. The most noticeable effect of ganglioside exposure was to increase the G1 phase by 40%. Therefore we conclude that exogenously added membrane components, such as gangliosides, can lengthen the gemistocytic astrocytoma cell cycle in vitro and modulate the proliferation of these neural cells.

Phospholipid composition of cultured human endothelial cells

Detailed analyses of the phospholipid compositions of cultured human endothelial cells are reported here. No significant differences were found between the phospholipid compositions of cells from human artery, saphenous and umbilical vein. However, due to the small sample sizes, relatively large standard deviations for some of the phospholipid classes were observed. A representative composition of endothelial cells is: phosphatidylcholine 36.6%, choline plasmalogen 3.7%, phosphatidylethanolamine 10.2%, ethanolamine plasmalogen 7.6%, sphingomyelin 10.8%, phosphatidylserine 7.1%, lysophosphatidylcholine 7.5%, phosphatidylinositol 3.1%, lysophosphatidylethanolamine 3.6%, phosphatidylinositol 4,5-bisphosphate 1.8%, phosphatidic acid 1.9%, phosphatidylinositol 4-phosphate 1.5%, and cardiolipin 1.9%. The cells possess high choline plasmalogen and lysophosphatidylethanolamine contents. The other phospholipids are within the normal biological ranges expected. Phospholipids were separated by high-performance liquid chromatography and quantified by lipid phosphorus assay.

SUBSTANCE P AND NEUROKININ A METABOLISM BY CULTURED HUMAN SKELETAL MUSCLE MYOCYTES AND FIBROBLASTS

A recent study determined that cultured human skeletal muscle adult myoblasts, myotubes, and fibroblasts degraded angiotensins and kinins via neutral endopeptidase-24.11 (NEP-24.11: EC 3.4.24.11) and aminopeptidase N (APN: EC 3.4.11.2). Due to the possible importance of other peptides to skeletal muscle blood flow and function, the present study looked specifically at the metabolism of the neurokinins substance P (SP) and neurokinin A (NKA) by skeletal muscle peptidases. The results show that SP is degraded not only by NEP-24.11, but also sequentially by dipeptidyl(amino)peptidase IV (DAP IV: EC 3.4.14.5)/APN. NKA is unaffected by DAP IV but is metabolized by NEP-24.11 and APN. NEP-24.11 was inhibited by phosphoramidon (IC50 = 80 nM), thiorphan and ZINCOV, DAP IV by diprotin A (IC50 = 8 microM), and APN by amastatin (IC50 = 50 nM) and bestatin (IC50 = 100 microM). Skeletal muscle myocyte and fibroblast metabolism of SP and NKA may regulate local skeletal muscle vascular and extravascular functions including SP- and NKA-mediated nerve-induced vasodilation. Inhibition of both NEP-24.11 and DAP IV/APN may increase skeletal muscle blood flow and decrease peripheral vascular resistance via potentiation of local neurokinin levels.

Angiotensin and bradykinin metabolism by peptidases identified in cultured human skeletal muscle myocytes and fibroblasts

Angiotensin (ANG) and kinin metabolizing enzymes, angiotensin-converting enzyme (ACE; EC 3.4.15.1), neutral endopeptidase-24.11 (NEP-24.11; EC 3.4.24.11), and aminopeptidase M (AmM; EC 3.4.11.2), have recently been identified in a purified skeletal muscle glycoprotein fraction. We have analyzed the cellular localization of these enzymes. In cultured human skeletal muscle adult myoblasts, myotubes, and fibroblasts, kinins and angiotensins were metabolized by NEP-24.11 and AmM but not by ACE. NEP-24.11 degraded ANG II, ANG III. and bradykinin (BK) and converted ANG I to the active metabolite ANG(1-7). ANG III was converted to the novel ANG IV metabolite [des-Arg1]ANG III by AmM. These data suggest that, due to their abundance in the body, skeletal muscle myocytes and fibroblasts may play a major role in modulation of the systemic and local effects of angiotensins and kinins. This role could be particularly important in individuals receiving treatment with ACE inhibitors.