beta-Catenin and actin reorganization in HGF/SF response of ST14A cells.

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic factor that activates proliferation, differentiation, and migration of various cell types. Its action is mediated by c-Met, a receptor endowed with tyrosine kinase activity that activates complex signaling cascades and mediates diverse cell responses. Although HGF action was first demonstrated in epithelial cells, expression of HGF and c-Met receptor has also been described in developing and adult mammalian brain. In the developing central nervous system, areas of HGF and c-Met expression are coincident with the migratory pathway of precursor cells. In the present article we report that the interaction between c-Met and HGF/SF in striatal progenitor ST14A cells triggers a signaling cascade that induces modification of cell morphology, with decreased cell-cell interactions and increased cell motility; in particular, we analyzed the reorganization of the actin cytoskeleton and the delocalization of beta-catenin and N-cadherin. The testing of other neurotrophic factors (NGF, BDNF, NT3, and CNTF) showed that the observed modifications were peculiar to HGF. We show that phosphoinositide 3-kinase inhibitor treatment, which blocks cell scattering induced by HGF/SF, does not abolish actin and beta-catenin redistribution. The effects of HGF/SF on primary spinal cord cell cultures were also investigated, and HGF/SF was found to have a possible motogenic effect on these cells. The data reported suggest that HGF could play a role in the early steps of neurogenesis as a motogenic factor.

Effects of blood lipid lowering pharmaceuticals (bezafibrate and gemfibrozil) on immune and digestive gland functions of the bivalve mollusc, Mytilus galloprovincialis.

Fibrates are hypolipidemic pharmaceuticals that have been detected as contaminants in wastewaters and surface waters. In this work, the possible effects of two fibrates, Bezafibrate (BEZA) and Gemfibrozil (GEM) in the bivalve mollusc Mytilus spp were investigated. In the immune cells, the hemocytes, addition of both compounds in vitro induced rapid lysosomal membrane destabilization, extracellular lysozyme release, NO production and decreased phagocytic activity. The effect of fibrates were partly mediated by activation of ERK and p38 MAPKs (Mitogen Activated Protein Kinases), as demonstrated by the use of specific inhibitors of different kinases. The effects of fibrates on hemocyte function were confirmed in vivo, in the hemocytes of mussels injected with 0.01, 0.1 and 1 nmol/animal (corresponding to nominal concentrations of 3.61, 36.18 and 361.8ng/g dry weight for BEZA and of 2.50, 25.03 and 250.35 ng/g dry weight for GEM, respectively) and sampled at 24h post-injection. Both compounds induced a concentration-dependent lysosomal destabilization and extracellular lysozyme release; an increase in phagocytosis was observed at the highest concentration. In vivo exposure to fibrates also induced significant effects on mussel digestive gland, the key metabolic organ in bivalves. Both BEZA and GEM increased the activity of the glycolytic enzymes phosphofructokinase (PFK) and pyruvate kinase (PK), and of Glutathione transferase (GST) glutathione reductase (GSR), and total glutathione content. A significant increase in the peroxisomal enzyme catalase was observed; however, BEZA exposure decreased Palmytoyl CoA oxidase activity, whereas GEM was ineffective. The results indicate that in mussels environmental concentrations of hypolipidemic drugs can affect the immune function, as well as glycolysis, redox balance and peroxisomal function.

Acetylcholinesterase modulates neurite outgrowth on fibronectin.

Acetylcholinesterase (AChE) has been reported to be involved in the modulation of neurite outgrowth. To understand the role played by different domains, we transfected neuroblastoma cells with three constructs containing the invariant region of AChE, differing in the exon encoding the C-terminus and therefore in AChE cellular fate and localization. All isoforms increased neurite extension, suggesting the involvement of the invariant domain [A. De Jaco, G. Augusti-Tocco, S. Biagioni, Alternative AChE molecular forms exhibit similar ability to induce neurite outgrowth, J. Neurosci. Res. 70 (2002) 756-765]. The peripheral anionic site (PAS) is encoded by invariant exons and represents the domain involved in non-cholinergic functions of AChE. Masking of PAS with fasciculin results in a significant decrease of neurite outgrowth in all clones overexpressing AChE. A strong reduction was also observed when clones were cultured on fibronectin. Treatment of clones with fasciculin, therefore masking PAS, abolished the fibronectin-induced reduction. The inhibition of the catalytic site cannot revert the fibronectin effect. Finally, when clones were cultured on fibronectin in the presence of heparin, a ligand of fibronectin, the inhibitory effect was completely reversed. Our results indicate that PAS could directly or indirectly mediate AChE/fibronectin interactions.

Hepatocyte growth factor stimulates cell motility in cultures of the striatal progenitor cells ST14A.

Hepatocyte growth factor/scatter factor (HGF/SF) is a growth factor with pleiotropic effects on different cell types. It acts as a mitogen and motility factor for many epithelial cells. HGF/SF and its receptor Met are present in the developing and adult mammalian brain and control neuritogenesis of sympathetic and sensory neurons. We report that the striatal progenitor ST14A cells express the Met receptor, which is activated after binding with HGF/SF. The interaction between Met and HGF/SF triggers a signaling cascade that leads to increased levels of c-Jun, c-Fos, and Egr-1 proteins, in agreement with data reported on the signaling events evoked by HGF in other cellular types. We also studied the effects of the exposure of ST14A cells to HGF/SF. By time-lapse photography, we observed that a 24-hr treatment with 50 ng/ml HGF/SF induced modification in cell morphology, with a decrease in cell-cell interactions and increase of cell motility. In contrast, no effect on cell proliferation was observed. To investigate which intracellular pathway is primarily involved we used PD98059 and LY294002, two specific inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAP-kinase/ERK-kinase) and phosphoinositide 3-OH kinase (PI3-K), respectively. Cell motility in HGF/SF treated cultures was inhibited by LY294002 but not by PD98059, suggesting that PI3-K plays a key role in mediating the HGF/SF-induced dissociation of ST14A cells. Previous evidence of HGF stimulation of motility in nervous system has been obtained on postmitotic neurons, which have already acquired their specificity. Data reported here of a motogenic response of ST14A cell line, which displays properties of neuronal progenitors, seem of interest because they suggest that HGF could play a role in very early steps of neurogenesis.

Dystrophin localization and gene expression in the developing nervous system of the chick.

The presence and distribution of dystrophin was studied in selected areas of the chick embryo nervous system and in primary cultures. Dystrophin was examined at the protein level by immunocytochemistry and at the transcriptional level by a semiquantitative reverse transcriptase-polymerase chain reaction analysis. Immunofluorescence staining shows that dystrophin is present early during embryogenesis in dorsal root ganglia, spinal cord, and ciliary ganglia and colocalizes with neurofilament subunits. Cultured dorsal root ganglion, spinal cord, and ciliary ganglion neurons show immunoreactivity for dystrophin, both in cell bodies and along fibers. Dystrophin mRNA level in ciliary and dorsal root ganglia is higher than in spinal cord throughout development and shows a tissue-specific pattern of expression. In primary cultures of dorsal root ganglia and ciliary ganglia, dystrophin mRNA level increases with time in vitro. However, in spinal cord cultures, dystrophin mRNA drastically decreases with time in vitro, but it is significantly increased when embryonic muscle extract is added to the cultures. Our results show that dystrophin is present in neurons from different areas of embryonic chick nervous system and that its mRNA level is developmentally regulated both in vivo and in vitro.

CNTF variants with increased biological potency and receptor selectivity define a functional site of receptor interaction.

Human CNTF is a neurocytokine that elicits potent neurotrophic effects by activating a receptor complex composed of the ligand-specific alpha-receptor subunit (CNTFR alpha) and two signal transducing proteins, which together constitute a receptor for leukemia inhibitory factor (LIFR). At high concentrations, CNTF can also activate the LIFR and possibly other cross-reactive cytokine receptors in the absence of CNTFR alpha. To gain a better understanding of its structure-function relationships and to develop analogs with increased receptor specificity, the cytokine was submitted to affinity maturation using phage display technology. Variants with greatly increased CNTFR alpha affinity were selected from a phage-displayed library of CNTF variants carrying random amino acid substitutions in the putative D helix. Selected variants contained substitutions of the wild-type Gln167 residue, either alone or in combination with neighboring mutations. These results provide evidence for an important functional role of the mutagenized region in CNTFR alpha binding. Affinity enhancing mutations conferred to CNTF increased potency to trigger biological effects mediated by CNTFR alpha and enhanced neurotrophic activity on chicken ciliary neurons. In contrast, the same mutations did not potentiate the CNTFR alpha-independent receptor actions of CNTF. These CNTF analogs thus represent receptor-specific superagonists, which should help to elucidate the mechanisms underlying the pleiotropic actions of the neurocytokine.

Extracellular glycoproteins at acetylcholine receptor clusters of rat myotubes are organized into domains.

Cultured neonatal rat myotubes develop acetylcholine receptor (AChR) clusters where they adhere to the substrate. These clusters are often linear, with AChR-rich domains alternating with AChR-poor "contact domains" closer to the tissue culture substrate. We have used sequential detergent extraction and immunofluorescence microscopy to localize extracellular matrix components within these two domains. Laminin, heparan sulfate proteoglycan, type IV collagen, and fibronectin are present at AChR-rich domains; nerve cell adhesion molecule is present at both AChR and contact domains. Extracts of contact domains are enriched in a 36-kDa concanavalin A binding protein and in a 90-kDa polypeptide recognized by antibodies against rat muscle adherons. These results suggest that extracellular components at substrate-apposed AChR clusters are organized into distinct domains that parallel the organization of the cluster bilayer.

Acetylcholinesterase in the development of chick dorsal root ganglia.

Acetylcholinesterase is expressed in chick dorsal root ganglia neurons very early in development. Since the physiological role of the enzyme in these cells is still obscure, it appeared of interest to investigate its modifications in the course of development. The specific activity of acetylcholinesterase in chick dorsal root ganglia increases, during in ovo development, from day E5 to day E13; after day E13 there is a decrease. Conversely, when acetylcholinesterase activity was expressed on a per ganglion basis, a continuous increase in the level of the enzyme until day E20 was observed. Acetylcholinesterase is a polymorphic enzyme and its molecular forms have different cellular localizations. Two globular forms, a tetramer (G4) and a dimer (G2), are present in the ganglia, as in chick brain. G4 is the major form at day E5, where it represents about 85% of the activity. This form shows a progressive decrease since day E8, and at day E20 exhibits activity levels similar to those of G2. It is known that acetylcholinesterase-producing cells are also able to release the enzyme in the extracellular space. We determined the release of acetylcholinesterase by cultured dorsal root ganglia neurons at various developmental stages: acetylcholinesterase release is significantly increased at day E20, as compared to younger stages, and 90% of the enzyme released is G4.

Muscle acetylcholinesterase in a familial myopathic disease.

Three sisters with myopathy characterized by different degrees of weakness, hypotonia, cramps and a significant hypertrophy of the calves underwent clinical tests. Laboratory examinations (nerve conduction velocity, electromyography and serum enzymes), serial histochemical analyses of muscle specimens and tests for muscular acetylcholinesterase (AChE) activity and its molecular forms were performed. AChE activities did not differ significantly from those of controls, while sedimentation patterns evidenced the disappearance of 16 S, 13 S and 10 S molecular forms in the elder sisters. The genealogical tree of the patients is described and their cases compared to those of others with calf hypertrophy reported in the literature.

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.

Differential and stage-related expression in embryonic tissues of a new human homoeobox gene.

The homoeobox is a 183 base-pair (bp) DNA sequence conserved in several Drosophila genes controlling segmentation and segment identity. Homoeobox sequences have been detected in the genome of species ranging from insects and anellids to vertebrates and homoeobox containing genes have been cloned from Xenopus, mouse and man. We recently isolated human homoeobox containing complementary DNA clones, that represent transcripts from four different human genes. One clone (HHO.c10) is selectively expressed in a 2.1 kilobase (kb) polyadenylated transcript in the spinal cord of human embryos and fetuses 5-10 weeks after fertilization. We report the characterization of a second cDNA clone, termed HHO.c13, that represents a new homoeobox gene. This clone encodes a protein of 255 amino-acid residues, which includes a pentapeptide, upstream of the homoeo domain, conserved in other Drosophila, Xenopus, murine and human homoeobox genes. By Northern analysis HHO.c13 detects multiple embryonic transcripts, which are differentially expressed in spinal cord, brain, backbone rudiments, limb buds and heart in 5-9-week-old human embryos and fetuses, in a striking organ- and stage-specific pattern. These observations suggest that in early mammalian development homoeobox genes may exert a wide spectrum of control functions in a variety of organs and body parts, in addition to the spinal cord.

A muscle cell line from dystrophic mice expressing an altered phenotype in vitro.

The isolation and characterization of a myogenic cell line from C57BL/6J/dydy mice is described. This line (DyA4) maintains the morphological, biochemical and electrophysiological characteristics of the primary cultured cells, at least for 20 passages. The cells actively divide as long as they are subcultured in media supplemented with horse serum and embryo extract. If the cells are not subcultured for a few days, they fuse into multinucleated contracting myotubes, which readily synthesize specific muscle products such as acetylcholinesterase and acetylcholine receptor. This dystrophic cell line expresses in vitro the same altered phenotype that is characteristic of dystrophic muscle cells in primary cultures, namely reduced acetylcholine sensitivity and reduced acetylcholine receptor expression. Because they can be grown in large amounts, and represent a pure muscle cell population which express an altered phenotype in an in vitro aneural avascular environment, DyA4 cells provide a very useful model system for investigating the pathogenesis of murine muscular dystrophy.

Membrane acetylcholinesterase in murine muscular dystrophy In vivo and in cultured myotubes.

Murine muscular dystrophy is characterized by a reduction of the 10S molecular form of acetylcholinesterase (AChE); this reduction occurs in both strains of dystrophic mice and at the time of the phenotypic appearance of the disease. In the present study we have analyzed the biochemical features, the cellular distribution and the developmental appearance of the AChE alteration. Sequential extractions with low salt, detergent and high salt revealed that this alteration affects only membrane-bound forms (those requiring Triton X-100 for solubilization), while both the low salt soluble and the high salt soluble forms appeared almost identical in normal and dystrophic muscles. Specific activity, sensitivity to different ions, pH dependence and Km were found to be identical in the enzymes from normal and dystrophic muscles, suggesting that the catalytic site of the 10S form is probably not altered. Further analysis, by non-denaturing gel electrophoresis, of the detergent soluble forms separated by sedimentation, revealed a single band for the 4S, a doublet for the 6S and three bands for the 10S peaks, indicating the existence of charge heterogeneity in AChE molecular forms. The corresponding molecular forms from dystrophic muscles behaved identically upon electrophoresis: the residual activity in the detergent soluble 10S form could still be separated into three bands, comigrating with their normal counterparts. Neuraminidase treatment resulted in a reduction of migration of both the 6S and 10S derived bands, but not of the 4S species, showing that sialic acid is added only to polymeric forms. Interestingly, the reduction of the 10S form appears to be linked to a developmental stage not reached in cell cultures, as cultured myotubes from muscles of dystrophic mice contained normal amounts of membrane-bound AChE forms. The molecular mechanism underlying the reduction of the tetrameric membrane bound AChE form in dystrophic muscle and the possible functional consequences are discussed.