Improved media for normal human muscle satellite cells: Serum-free clonal growth and enhanced growth with low serum

Summary 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. This research was supported by a grant from the Muscular, Dystrophy Association. Editor's statement This article describes the optimization of both the basal nutrient medium and growth factor requirements for human muscle cells in vitro. This system is critical for studies of normal muscle cell and molecular biology, as well as for understanding diseases of muscle such as Duchenne, Muscular Dystrophy.     

Mapping of FMR1, the gene implicated in fragile X-linked mental retardation, on the mouse X chromosome

A genetic map of the Cf-9 to Dmd region of the mouse X chromosome has been established by typing 100 offspring from a Mus musculus x Mus spretus interspecific backcross for the four loci Cf-9, Cdr, Gabra3, and Dmd. The following order and genetic distances in centimorgans were determined: (Cf-9)-2.4 +/- 1.7-(Cdr)-2.0 +/- 1.4-(Gabra3)-4.1 +/- 2.0-(Dmd). Six backcross offspring carrying X chromosomes with recombination events in the Cdr-Dmd region were identified. These recombination events were used to define the position of Fmr-1, the murine homologue of FMR1, which is the gene implicated in the fragile X syndrome in man, and that of DXS296h, the murine homologue of DXS296. Both Fmr-1 and DXS296h were mapped into the same recombination interval as Gabra3 on the mouse X chromosome. These findings provide strong support for the concept that the order of loci lying in the Cf-9 to Gabra3 segment of the X chromosome is highly conserved between human and mouse.     

Fingerprinting human chromosomes by polymerase chain reaction-mediated DNA amplification.

We describe here a method for DNA fingerprinting of human chromosomes by Alu-polymerase chain reaction (PCR) amplification of DNA from monochromosomal hybrids, following digestion with restriction endonucleases. DNA digestion with restriction enzymes prior to PCR amplification reduces the total number of amplified fragments. The number and pattern of bands of PCR products observed in an electrophoretic medium are chromosome specific and provide a "fingerprint signature" for individual human chromosomes. Using this approach, we have produced fingerprints for human chromosomes 2, 5, 7, 9, and 12. The applicability of this approach to chromosome identification was assessed by comparing the fingerprints obtained for two different hybrids containing chromosome 7. DNA fragments specific for the long and the short arms of human chromosome 12 have also been identified. In addition, Alu-PCR-generated DNA fragments, specific for different chromosomes, were used to probe Southern blots of a hybrid cell panel to identify human chromosomes present in hybrid cell lines. The chromosomal specificity of these probes permits the identification of intact as well as rearranged chromosomes composed of segments arising from more than one chromosome.     

Genotoxic evaluation of Ara-C by multiple parameters

The cytogenetic effects of the antimetabolite, cytosine arabinoside (Ara-C) are evaluated using in vivo and in vitro test systems and applying multiple parameters. The in vivo assay was carried out on 8-10-week-old inbred Swiss albino male mice using bone marrow as the somatic test system and the cells of testis as the meiotic test system. In vitro human leukocyte cultures were also employed. In vivo experimental doses were computed on surface area basis within the therapeutic dose range and injected intraperitoneally and for in vitro they were calculated on blood volume basis. Evaluation of somatic chromosome mutations included conventional screening for chromosome aberrations, variations in mitotic index and sister-chromatid exchanges (SCEs) by in vivo and in vitro methods besides studies on meiotic test systems using conventional screening for chromosome and sperm-head abnormalities. The quantitative data were subjected to statistical analysis by applying appropriate tests to evaluate their significance. The results of in vivo and in vitro experiments reveal the chromosome mutational activity of the compound. This is further supported by data on SCEs from both systems. However, a comparison of both demonstrated a differential mutagenic response of the drug, more in vivo than in vitro. This is also true for SCEs. Even though the mechanisms involved in causing chromosome aberrations and SCEs are different, the data on both corroborate each other on induction of chromosome mutations.     

The structural mimicry of membrane sterols by tamoxifen: evidence from cholesterol coefficients and molecular-modelling for its action as a membrane anti-oxidant and an anti-cancer agent

The anti-cancer drug tamoxifen is a potent inhibitor of lipid peroxidation induced by Fe(III)-ascorbate in ox-brain phospholipid liposomes. Similar anti-oxidant effects, but with varying potencies, are also shown by 4-hydroxytamoxifen, cholesterol, ergosterol and 17-β-oestradiol. We now describe a computer-graphic fitting technique that demonstrates a structural similarity between the five compounds. In addition, we have quantified the differences (relative to cholesterol) between the anti-oxidant activities of the compounds in terms of a novel expression reffered to here as the cholesterol coefficient (C_c) Finally, we discuss how the inhibitory effect of tamoxifen on lipid peroxidation may result from a membrane stabilization that is associated with a decrease in membrane fluidity. This action may be related to the anti-proliferative effect exerted by tamoxifen on cancer and fungal cells.     

Whole-embryo culture of Drosophila : development of embryonic tissues in vitro

Summary We have devised techniques to culture whole, dissected embryos of Drosophila melanogaster. We examine multiple aspects of the morphological and physiological development of the epidermis, musculature, nervous system, and internal organs in this cultured preparation, and show that in vitro development closely parallels normal embryogenesis. These techniques permit a wide range of experimental manipulations during embryogenesis and allow us to extend observations through late embryonic stages, after cuticle deposition. Applications of this technique are presented.     

A homologue of the Escherichia coli DsbA protein involved in disulphide bond formation is required for enterotoxin biogenesis in Vibrio cholerae

A strain of Vibrio cholerae, which had been engineered to express high levels of the non-toxic B subunit (EtxB) of Escherichia coli heat-labile enterotoxin, was subjected to transposon (TnphoA) mutagenesis. Two chromosomal TnphoA insertion mutations of the strain were isolated that showed a severe defect in the amount of EtxB produced. The loci disrupted by TnphoA in the two mutant derivatives were cloned and sequenced, and this revealed that the transposon had inserted at different sites in the same gene. The open reading frame of the gene predicts a 200-amino-acid exported protein, with a Cys-X-X-Cys motif characteristic of thioredoxin, protein disulphide isomerase, and DsbA (a periplasmic protein required for disulphide bond formation in E. coli). The V. cholerae protein exhibited 40% identity with the DsbA protein of E. coli, including 90% identity in the region of the active-site motif. Introduction of a plasmid encoding E. coli DsbA into the V. cholerae TnphoA derivatives was found to restore enterotoxin formation, whilst expression of Etx or EtxB in a dsbA mutant of E. coli confirmed that DsbA is required for enterotoxin formation in E. coli. These results suggest that, since each EtxB subunit contains a single intramolecular disulphide bond, a transient intermolecular interaction with DsbA occurs during toxin subunit folding which catalyses formation of the disulphide in vivo.     

Changes in soil microflora associated with control of Sclerotium Rolfsii by Furfuraldehyde

The efficacy of 2‐furfuraldehyde for control of Sclerotium rolfsii was studied in laboratory and greenhouse experiments. Mycelial growth of the fungus was reduced proportionally with concentrations of 0.1–0.5 ml furfuraldehyde l^‐1 agar medium, and viability of sclerotia diminished on exposure to 2‐furfuraldehyde vapours. Detectable populations of bacteria and fungi, including Trichoderma spp., were reduced significantly (9=0.05) when furfuraldehyde was added to the agar used for soil dilution plates of untreated soil. Repeated treatments of natural soil with the fumigant significantly increased populations of Trichoderma spp. and bacteria, but diminished numbers of actinomycetes. Increasing dosages applied to soil artificially infested with S. rolfsii caused a reduction of disease on lentil, Lens culinaris. Results indicate that the compound, when applied to field soil, changes the composition of soil microflora and has potential for integrated control of S. rolfsii.     

A Comparison of the Efficacy of Nuclear Polyhedrosis and Granulosis Viruses in Spray and Bait Formulations for the Control of Agrotis segetum (Lepidoptera: Noctuidae) in Maize

Agrotis segetum nuclear polyhedrosis virus (AsNPV) and granulosis virus (AsGV), propagated in laboratory cultures of A. segetum in England and A. ipsilon in Spain, respectively, were applied to plots of maize plants at the one‐ to four‐leaf stage of growth. Plots were arranged in a 6 x 6 Latin square design and infested with second‐instar A. segetum larvae (the common cutworm). Each virus was applied in separate treatments by two application methods; as an aqueous spray containing 0.1% Agral as a wetting agent, and as a bran bait. The NPV was applied at a rate of 4 X 10¹² polyhedra/ha, and the GV at 4 X 10¹³ granules/ha. Soil and plants were sampled for larvae on three occasions following virus treatment: 24 h, 4 days and 11 days. The larvae were reared on diet in the laboratory, until death or pupation, to examine the rate and level of viral infection. Infection data showed 87.5% and 91% NPV infection and 12.5% and 55% GV infection in spray and bait treatments, respectively, in larvae sampled 24 h after treatment. In larvae sampled 4 days after treatment, the results were 78% and 100% NPV infection, and 13% and 6% GV infection. A total of only six larvae were retrieved on day 11. In both treatments larvae infected with AsNPV died significantly more rapidly and at an earlier instar than those infected with AsGV, indicating that AsNPV appears to have better potential as a control agent for A. segetum.     

The root-knot nematode resistance gene (Mi) in tomato: construction of a molecular linkage map and identification of dominant cDNA markers in resistant genotypes

A dominant allele at the Mi locus on chromosome 6 of tomato (Lycopersicon esculentum Mill) confers resistance to three species of root-knot nematodes (Meloidogyne). The resistance, which is associated with a localized necrotic response, was originally introduced into tomato from the wild species Lycopersicon peruvianum. As a step towards the molecular cloning of Mi, we have identified closely linked DNA markers from both cDNA and genomic DNA libraries as restriction fragment length polymorphisms (RFLPs). DNA from tomato populations segregating for nematode resistance was analyzed to generate a high-resolution genetic map of this region. Additional information on gene order was obtained by comparing the size of the introgressed L. peruvianum chromosomal segment within a collection of nematode-resistant tomato lines. Among the four cDNA markers that are tightly linked to Mi, three are dominant, i.e. L. peruvianum-specific. One cDNA marker corresponds to a gene family comprising 20-30 members, one of which is diagnostic for all nematode-resistant genotypes tested. The presence of non-homologous sequences around the Mi gene may contribute to the suppression of recombination in this region of the genome in crosses heterozygous for Mi. The potential of 'walking' from closely linked markers to Mi is discussed.