Expression of adult fast pattern of acetylcholinesterase molecular forms by mouse satellite cells in culture

The pattern of acetylcholinesterase (AChE) molecular forms, obtained by sucrose gradient sedimentation, was studied at different in vitro developmental stages of myogenic cells isolated from adult mouse skeletal muscle. Only the globular forms were present in rapidly dividing satellite cells during the first days in culture. After myotube formation, a pattern similar to that described in mammalian fast-twitch skeletal muscle was observed. This pattern did not change during the following period in culture (up to 1 month) nor could it be modified by co-culturing with spinal cord motoneurons or by addition of brain-derived extracts. The internal-external localization of AChE molecular forms has been determined by the use of echothiophate iodide, a membrane-impermeant irreversible inhibitor of AChE. Echothiophate-treated cultures showed about 40% of both asymmetric and globular forms localized on the sarcolemma, with their active sites oriented outward. Analysis of culture medium from untreated cultures revealed the presence of both asymmetric and globular forms. When the same analysis was repeated on cultures of myoblasts derived from 16-day-old mouse embryos, the pattern of AChE forms was different. The myotubes derived from these cells exhibit a very small proportion of asymmetric form, which was not released into the medium. This pattern was not further modified during the following days of culture, nor by co-cultures with spinal cord motoneurons or by incubations with brain-derived extracts. Thus, the myotubes derived from myoblasts express in culture a clear phenotypic difference when compared to the corresponding myotubes from satellite cells, supporting the view that these two myogenic cells are endowed with different developmental programs.     

Ethylene Synthesis and Regulated Expression of Recombinant Protein in Synechocystis sp. PCC 6803

The ethylene-forming enzyme (EFE) from Pseudomonas syringae catalyzes the synthesis of ethylene which can be easily detected in the headspace of closed cultures. A synthetic codon-optimized gene encoding N-terminal His-tagged EFE (EFEh) was expressed in Synechocystis sp. PCC 6803 (Synechocystis) and Escherichia coli (E. coli) under the control of diverse promoters in a self-replicating broad host-range plasmid. Ethylene synthesis was stably maintained in both organisms in contrast to earlier work in Synechococcus elongatus PCC 7942. The rate of ethylene accumulation was used as a reporter for protein expression in order to assess promoter strength and inducibility with the different expression systems. Several metal-inducible cyanobacterial promoters did not function in E. coli but were well-regulated in cyanobacteria, albeit at a low level of expression. The E. coli promoter P_trc resulted in constitutive expression in cyanobacteria regardless of whether IPTG was added or not. In contrast, a Lac promoter variant, P_A1lacO-1, induced EFE-expression in Synechocystis at a level of expression as high as the Trc promoter and allowed a fine level of IPTG-dependent regulation of protein-expression. The regulation was tight at low cell density and became more relaxed in more dense cultures. A synthetic quorum-sensing promoter system was also constructed and shown to function well in E. coli, however, only a very low level of EFE-activity was observed in Synechocystis, independent of cell density.     

'Early' mammalian myoblasts are resistant to phorbol ester-induced block of differentiation

Mesenchymal cells were isolated from somites and limbs of mouse embryos at different developmental stages. When grown in tissue culture, some of the cells underwent muscle differentiation as indicated by synthesis of sarcomeric myosin, acetylcholine receptor and, in the case of limb cells, fusion into multinucleated myotubes. When the tumour promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) was added to these cultures, it caused differential effects, depending upon the age of the embryo from which cells were isolated. In cultures of somites or limb bud from embryos up to 12 days post coitum, TPA did not interfere with the appearance of differentiated muscle cells. When TPA was added to cultures from older embryos, it inhibited muscle differentiation with an efficiency which increased with the age of the embryo, reaching about 90% inhibition at 15 days. After this period, a new population of myogenic cells appeared in the limb, which were able to differentiate in the presence of TPA and represented the great majority of myoblasts after day 18 of embryonic development. The simplest interpretation of these data can be based on the existence of three major classes of myogenic cell precursors, which appear sequentially during muscle histogenesis: 'early' myoblasts, which appear resistant to tumour promoters; 'late' myoblasts, whose differentiation is inhibited by tumour promoters and 'satellite' cells which, like early myoblasts, show no sensitivity to TPA.     

An immunohistochemical study of the distribution of ABH antigens in human submandibular glands at the light and electron microscopic levels.

The distribution of blood group antigens ABH in submandibular glands was studied at light and electron microscopy levels by applying ImmunoGold Silver Staining (IGSS) and post-embedding ImmunoGold (IGS) methods, respectively. In IGSS treated samples, a cytoplasmic and a surface form of antigen localization were discernible in the glandular parenchyma. The former was restricted to most mucous cells and to scattered serous cells: A and B antigens were demonstrated in mucous cells of A and B type glands, while H antigen appeared in most mucous and occasional serous elements regardless of the blood type of donors. The latter appeared as a strong H reactivity on cell surfaces of serous acini and ducts regardless of the patient blood type. The IGS method was applied both on non-osmicated samples embedded in LR White resin and on osmicated, Epon embedded samples. In non-osmicated tissues, antigen labelling was revealed in secretory granules and cell surfaces. Positive secretory granules were found in most mucous cells and occasional serous, intercalated, and striated duct cells. A and B antigens weakly reacted in mucous cells of A and B type glands, respectively, while strong H reactivity was seen in mucous, serous, intercalated and striated duct cells of glands of all types. Surfaces labelled with H antigen were found on both lumenal and basolateral membranes of striated ducts in glands of all types. IGS method applied on osmicated, Epon embedded samples, selectively revealed blood group antigens in secretory granules of serous cells but not in the apical vesicles of striated ductal cells. Cell surfaces were completely unreactive.     

Properties of acetylcholine-receptor activation in human Duchenne muscular dystrophy myotubes

In human myotubes cultured from biopsies of normal subjects and dystrophic patients we investigated, with the patch-clamp technique, the activation properties of the nicotinic acetylcholine receptor (AChoR) in the presence of acetylcholine and suberyldicholine. The single-channel conductance and the lifetime of the openings were not found to differ. In contrast, the average frequency of openings was about four times higher in Duchenne muscular dystrophy (DMD) myotubes in the presence of equal amounts of acetylcholine, but not of suberyldicholine. The most reasonable conclusion from this observation is that the behaviour of the AChoR is not altered in DMD cells but that there is a greater average concentration of ACho molecules present around AChoRs. This leads to the tentative conclusion that the activity of the enzyme acetylcholinesterase (AChoE) is impaired by some unknown mechanism in the dystrophic myotube.     

Involvement of the Ca2+-dependent K+ channel activity in the hyperpolarizing response induced by epidermal growth factor in mammary epithelial cells

Epidermal growth factor (EGF) induces a hyperpolarizing response of 5-20 mV amplitude in mouse mammary epithelial cells in culture. The amplitude of the hyperpolarizing response was reduced by more than 60% within several minutes after addition of blockers of voltage and/or Ca2+-dependent K+ channels such as tetraethylammonium (7 mM) or quinine (0.29 mM). Both nifedipine (0.15 mM), a blocker of the Ca2+ channel, and ruthenium red (2 mM), an inhibitor of the Ca2+-binding site, also reduced the amplitude of the hyperpolarizing response by more than 60%. The Ca2+ ionophore, A23187 (3.8 microM), induced a large hyperpolarization, which was 25-40 mV and lasted about 3 min. These data suggest that activity of the Ca2+-dependent K+ channel was involved in the EGF-induced hyperpolarizing response of the mammary epithelial cells.     

Identification of Hb Hamilton or beta 11(A8)Val----Ile gene by the polymerase chain reaction amplification technique.

Amplification of the beta-globin gene by the PCR technique, followed by the enzymatic digestion of the DNA fragment obtained, was used to easily identify the human beta-globin variant Hb Hamilton which is characterized by the valine to isoleucine substitution at position 11. The result revealed the predicted G to A transition at codon 11 which abolishes a MaeIII restriction site. This mutation, which is rather common among Sardinians, is at the level of one of the five CpG dinucleotides of the beta-globin gene.