Stefano Alemà - Academia.edu (original) (raw)

Papers by Stefano Alemà

Research paper thumbnail of Role of cell division in differentiation of myoblasts infected with a temperature-sensitive mutant of Rous sarcoma virus

The EMBO Journal, 1984

The relationship between a potential requirement for cell DNA synthesis and the expression of dif... more The relationship between a potential requirement for cell DNA synthesis and the expression of differentiated muscle cell functions was investigated using primary chicken embryo myoblasts infected with a temperature-sensitive mutant of Rous sarcoma virus (RSV). Under optimized conditions, transformed myoblasts growing at the permissive temperature could differentiate into multinucleated myotubes, express muscle-specific myosin, desmin and acetylcholine receptors in the absence of DNA synthesis and cell division following a shift to the non-permissive temperature. Furthermore, the experiments demonstrate that individual RSV-infected myoblasts have two options: either to divide and express the transformed phenotype or to withdraw from the cell cycle and differentiate into myotubes. The choice between these options appears to depend on the protein-kinase activity of pp6(c, the src gene product. Key words: cell division/mitomycin C/myogenic differentiation/pp6Osrc/Rous sarcoma virus 1975a; Boettiger et at., 1977; Pacifici et at., 1977), it is not clear whether transformed cells constitute lineage compartments distinct from or similar to those of normal cells (Holtzer et al., 1980a, 1980b). Hence, the quantal cell cycle model has also been applied to the transition from transformed myoblasts to multinucleated myotubes of cells transformed by ts-RSV and shifted to the restrictive temperature (Fiszman and Fuchs, 1975; Holtzer et al., 1975a; Moss, et al., 1979). Moss et al. (1979) found that >95% of the nuclei incorporated into myotubes could be labelled with [3H]thymidine after temperature shift up, whereas Holtzer et al. (1975a), in a similar experiment, found 75% of labelled nuclei in myotubes. The presence of unlabelled nuclei could be explained by the finding that some of the RSV-transformed myoblasts can spontaneously differentiate at the permissive temperature (Tato et al., 1983). Both reports suggested that a cell cycle separates the transformed myoblast state from the differentiated myotubes; however, these experiments did not demonstrate whether a cell cycle was necessary for the transition to occur. In this paper we have addressed two intriguing questions that are raised by the above-mentioned observations: (i) does a cell cycle obligatorily separate ts-transformed myoblasts from temperature shift-induced myotubes, and (ii) is RSVinduced block of differentiation the cause or the consequence of transformation-associated altered proliferation?

Research paper thumbnail of Erratum: p120 catenin is required for growth factor-dependent cell motility and scattering in epithelial cells (Molecular Biology of the Cell (2003) 14 (1964-1977))

Molecular Biology of the Cell, 2003

Research paper thumbnail of Mechanisms underlying the block of differentiation in transformed quail myoblasts

Cell Biology International Reports, 1990

The early development of the ventral nervous system in the Drosophila embryo is characterized by ... more The early development of the ventral nervous system in the Drosophila embryo is characterized by a fundamental cell fate decision. Approximately one fourth of the cells in the ventral ~_~ ~~ _ ~~_ ~_ .~~

Research paper thumbnail of Acetylcholine in Intercostal Muscle from Myasthenia Gravis Patients and in Rat Diaphragm after Blockade of Acetylcholine Receptors

Progress in Brain Research, 1979

Publisher Summary This chapter discusses whether the reduction in the number of AChRs in MG is ac... more Publisher Summary This chapter discusses whether the reduction in the number of AChRs in MG is accompanied by changes in the synthesis and release of ACh. It also investigates rat diaphragms, whose AChRs were blocked in vitro by a-bungarotoxin, or by immunization with AChR purified from Torpedo electroplax. ACh was extracted from the intercostal muscles from patients with acquired and congenital myasthenia gravis (MG) and from control patients with no clinical signs of muscle disease. ACh was estimated by mass fragmentography. It was found that the concentration of ACh was about two times higher in muscle from acquired MG than from congenital MG or control patients. Muscle from acquired MG and control patients was also incubated in Ringer with 50 mM KCl in order to stimulate the release of ACh. During the first 15 min of incubation with KCl more ACh was released from myasthenic than from control muscle, but this difference was not sustained on prolonged incubation. It was found that tetrodotoxin depressed the amount of ACh released into the medium in the presence of 50 mM KCI. Diaphragms from normal rats were treated with a-bungarotoxin, or taken from the animals that had been immunized against the ACh receptor protein from Torpedo marmorata. It was found that from these preparations KCl released about twice as much ACh as from control diaphragms.

Research paper thumbnail of Properties of end-plate channels in rats immunized against acetylcholine receptors

The Journal of Physiology, 1981

S. ALEMA AND OTHERS channel complexes is reduced without modification of single channel propertie... more S. ALEMA AND OTHERS channel complexes is reduced without modification of single channel properties. In this respect the immunized rat end-plate is a good model for myasthenia gravis affected human end-plates.

Research paper thumbnail of X-ray absorption spectroscopy (EXAFS and XANES) a structural tool for calcium binding proteins: effect of Mg2+ on Ca2+ binding sites of troponin-C

Progress in clinical and biological research, 1982

Research paper thumbnail of Proceedings: Studies on the interaction of nerve growth factor with tubulin and actin

Hoppe-Seyler's Zeitschrift für physiologische Chemie, 1975

Research paper thumbnail of Effects of retroviral oncogenes on myogenesis

Cell Biology International Reports, 1990

Expression of the proto-oncogene c-fos by ovine conceptus was analyzed by Northern and slot blots... more Expression of the proto-oncogene c-fos by ovine conceptus was analyzed by Northern and slot blots in relation with the expression of an interferon a-II (IFN a-II), named ovine Trophoblastin Protein (oTP). This IFN is produced only by the trophectoderm during the peri-implantation period. The proto-oncogene

Research paper thumbnail of Neuronal differentiation of P19 embryonal cells exhibits cell-specific regulation of neurotrophin receptors

NeuroReport, 1995

During development, the regulation of expression of the Trk family of tyrosine kinase receptors p... more During development, the regulation of expression of the Trk family of tyrosine kinase receptors plays an important role in defining the cellular responses to neurotrophin action. We report here that neurotrophin receptors are differentially expressed in distinct populations of retinoic acid-differentiated P19 cells. TrkB, the tyrosine kinase receptor for brain-derived neurotrophic factor, and LNGFR, the low-affinity receptor for all neutrophins, are preferentially expressed in P19-derived neurones. In contrast, retinoic acid induces the expression of TrkA, the high-affinity receptor for nerve growth factor, and of a non-catalytic form of TrkB, in non-neural subsets of differentiated cells. We propose P19 cells as a model system to study the mechanisms controlling the expression of neurotrophin receptors and the responsiveness of developing neurones to a specific neurotrophin.

Research paper thumbnail of Distinctive effects of the viral oncogenes myc, erb, fps, and src on the differentiation program of quail myogenic cells

Proceedings of the National Academy of Sciences, 1985

The relationship between susceptibility to transformation in vitro by different oncogenes and ter... more The relationship between susceptibility to transformation in vitro by different oncogenes and terminal differentiation was analyzed in embryonic quail myogenic cells. Infection with Rous sarcoma virus (RSV), Fujinami sarcoma virus (FSV), avian erythroblastosis virus (AEV), and the avian myelocytomatosis virus MC29 led to rapid and massive transformation. Transformed cells had distinctive morphological alterations, increased proliferation rates, and the ability to grow in agar suspension. Furthermore, homogeneously transformed cultures failed to fuse into multinucleated myotubes and to express muscle-specific genes. However, cloned populations of RSV-, FSV-, and AEV-transformed myogenic cells could, under appropriate culture conditions, partially differentiate into atypical "revertant" myotubes. In contrast, competence for terminal differentiation was completely and irreversibly suppressed on transformation by MC29. The specificity of action of a given oncogenic sequence on...

Research paper thumbnail of Black widow spider toxin-induced calcium fluxes and transmitter release in a neurosecretory cell line

Research paper thumbnail of Differentiation of PC12 phaeochromocytoma cells induced by v-src oncogene

Nature, 1985

PC12 rat phaeochromocytoma cells are a model system that can be used to study both neuronal diffe... more PC12 rat phaeochromocytoma cells are a model system that can be used to study both neuronal differentiation and the mechanism of action of nerve growth factor (NGF). PC12 cells respond to NGF protein by shifting from a chromaffin-cell-like phenotype to a neurite-bearing sympathetic neurone-like phenotype. Here we present data on the effect of infection of PC12 cells with retroviruses carrying the src oncogene of Rous sarcoma virus. Previous studies have demonstrated that the expression of src severely affects the synthesis and accumulation of differentiated cell products in a variety of cell types. We show that in the PC12 cell system, expression of v-src appears to have an inductive effect on differentiation that resembles the action of a 'physiological' growth factor.

Research paper thumbnail of The v-myc oncogene is sufficient to induce growth transformation of chick neuroretina cells

Nature, 1987

A number of studies have shown that full transformation of non-established rodent fibroblasts can... more A number of studies have shown that full transformation of non-established rodent fibroblasts can be efficiently achieved in vitro by the concerted action of two oncogenes belonging to different complementation groups. Extension of the two-genes carcinogenesis model to other differentiated cell types, presumably endowed with different controls of growth, is desirable for a better understanding of questions such as the host cell selectivity of oncogene action. A recent report claimed that cooperation between two oncogenes, v-myc and v-mil, is required to achieve transformation of chicken embryo neuroretina cells, which are characterized by a limited growth capacity in monolayer culture. Here we present evidence that the v-myc oncogene alone is sufficient to induce growth transformation of glial and neuronal precursor cell types from chick neuroretina. We also report that induction of transformation by v-myc is accompanied by faithful preservation of some of the differentiated functions of the chick cells.

Research paper thumbnail of Distinct Effects of Rac1 on Differentiation of Primary Avian Myoblasts

Molecular Biology of the Cell, 1999

Rho family GTPases have been implicated in the regulation of the actin cytoskeleton in response t... more Rho family GTPases have been implicated in the regulation of the actin cytoskeleton in response to extracellular cues and in the transduction of signals from the membrane to the nucleus. Their role in development and cell differentiation, however, is little understood. Here we show that the transient expression of constitutively active Rac1 and Cdc42 in unestablished avian myoblasts is sufficient to cause inhibition of myogenin expression and block of the transition to the myocyte compartment, whereas activated RhoA affects myogenic differentiation only marginally. Activation of c-Jun N-terminal kinase (JNK) appears not to be essential for block of differentiation because, although Rac1 and Cdc42 GTPases modestly activate JNK in quail myoblasts, a Rac1 mutant defective for JNK activation can still inhibit myogenic differentiation. Stable expression of active Rac1, attained by infection with a recombinant retrovirus, is permissive for terminal differentiation, but the resulting myotu...

Research paper thumbnail of Inhibition of ErbB-2 Mitogenic and Transforming Activity by RALT, a Mitogen-Induced Signal Transducer Which Binds to the ErbB-2 Kinase Domain

Molecular and Cellular Biology, 2000

The product of rat gene 33 was identified as an ErbB-2-interacting protein in a two-hybrid screen... more The product of rat gene 33 was identified as an ErbB-2-interacting protein in a two-hybrid screen employing the ErbB-2 juxtamembrane and kinase domains as bait. This interaction was reproduced in vitro with a glutathione S -transferase fusion protein spanning positions 282 to 395 of the 459-residue gene 33 protein. Activation of ErbB-2 catalytic function was required for ErbB-2–gene 33 physical interaction in living cells, whereas ErbB-2 autophosphorylation was dispensable. Expression of gene 33 protein was absent in growth-arrested NIH 3T3 fibroblasts but was induced within 60 to 90 min of serum stimulation or activation of the ErbB-2 kinase and decreased sharply upon entry into S phase. New differentiation factor stimulation of mitogen-deprived mammary epithelial cells also caused accumulation of gene 33 protein, which could be found in a complex with ErbB-2. Overexpression of gene 33 protein in mouse fibroblasts inhibited (i) cell proliferation driven by ErbB-2 but not by serum, ...

Research paper thumbnail of X-ray absorption near edge structure (XANES) determination of calcium sites of troponin C and parvalbumin

Journal of Molecular Biology, 1983

Using synchrotron radiation at the Frascati storage ring ADONE, the X-ray Absorption Near Edge St... more Using synchrotron radiation at the Frascati storage ring ADONE, the X-ray Absorption Near Edge Structure (XANES) has been applied to determine homologies and modifications of the local structure of the caicium binding sites of troponin C. In all four calcium binding sites, Ca 2+ appears to be coordinated to earboxyl and carbony! groups in a characteristic configuration. No structural difference has been found between high and low-affinity sites. A distortion of the Ca 2+ site geometry by binding of Mg 2+ has been observed. The XANES of parvalbumin has been measured and found to be different from troponin C. A tentative identification of the characteristic XANES spectra of the two different Ca 2 ÷ sites in this protein is reported.

Research paper thumbnail of IKKα Is a p63 Transcriptional Target Involved in the Pathogenesis of Ectodermal Dysplasias

Journal of Investigative Dermatology, 2009

The transcription factor p63 plays a pivotal role in the development and differentiation of the e... more The transcription factor p63 plays a pivotal role in the development and differentiation of the epidermis and epithelial appendages. Indeed, mutations in p63 are associated with a group of ectodermal dysplasias characterized by skin, limb, and craniofacial defects. It was hypothesized that p63 exerts its functions by activating specific genes during epidermal development, which in turn regulate epidermal stratification and differentiation. We have identified I-kappaB kinase alpha (IKKa) as a direct transcriptional target of p63 that is induced at early phases of terminal differentiation of primary keratinocytes. We show that the DNp63 isoform is required for IKKa expression in differentiating keratinocytes and that mutant p63 proteins expressed in ectodermal dysplasia patients exhibit defects in inducing IKKa. Furthermore, we observed reduced IKKa expression in the epidermis of an ankyloblepharon ectodermal dysplasia clefting patient. Our data demonstrate that a failure to properly express IKKa may play a role in the development of ectodermal dysplasias.

Research paper thumbnail of Functional Properties of Neuronal Nicotinic Acetylcholine Receptor Channels Expressed in Transfected Human Cells

European Journal of Neuroscience, 1997

Within the chick central nervous system, expression of the ␤3 nicotinic acetylcholine receptor ge... more Within the chick central nervous system, expression of the ␤3 nicotinic acetylcholine receptor gene is restricted to a subset of retinal neurons, the majority of which are ganglion cells. Transient transfection in retinal neurons and in neural and non-neural cells from other regions of the chick embryo allowed the identification of the cis-regulatory domain of the ␤3 gene. Within this domain, a 75-base pair fragment located immediately upstream of the transcription start site suffices to reproduce the neuron-specific expression pattern of ␤3. This fragment encompasses an E-box and a CAAT box, both of which are shown to be key positive regulatory elements of the ␤3 promoter. Co-transfection experiments into retinal, telencephalic, and tectal neurons with plasmid reporters of ␤3 promoter activity and a number of vectors expressing different neuronal (ASH-1, NeuroM, NeuroD, CTF-4) and non-neuronal (MyoD) basic helix-loop-helix transcription factors indicate that the cis-regulatory domain of ␤3 has the remarkable property of discriminating accurately between related members of the basic helix-loop-helix protein family. The sequence located immediately 3 of the E-box participates in this selection, and the E-box acts in concert with the nearby CAAT box.

Research paper thumbnail of Identification of a Determinant of Acetylcholine Receptor Gating Kinetics in the Extracellular Portion of the γ Subunit

European Journal of Neuroscience, 1996

A large body of structure‐function studies has identified many of the functional motifs underlyin... more A large body of structure‐function studies has identified many of the functional motifs underlying ion permeation through acetylcholine receptor (AChR) channels. The structural basis of channel gating kinetics is, however, incompletely understood. We have previously identified a novel shorter form of the AChR γ subunit, which lacks the 52 amino acids within the extracellular amino‐terminal half, encoded by exon 5. To define the contribution of the missing domain to AChR channel function, we have transiently coexpressed the mouse short γ subunit (γs) with α, β and δ subunits in human cells and recorded single‐channel currents from the resulting AChRs. Our findings show that replacement of the γ by the γs subunit confers a long duration characteristic to AChR channel openings without altering unitary conductance sizes or receptor affinity for the transmitter. We also show that βγδSδ AChR channels exhibit a peculiar voltage sensitivity characterized by a short opening duration when the...

Research paper thumbnail of The neuronal α6 subunit forms functional heteromeric acetylcholine receptors in human transfected cells

European Journal of Neuroscience, 1998

We examine some of the biological and physiological properties of the avian α6 neuronal nicotinic... more We examine some of the biological and physiological properties of the avian α6 neuronal nicotinic acetylcholine receptor (nAChR) subunit. We show here that, beginning at embryonic day 5, α6 mRNA is abundantly expressed in the developing chick neuroretina, where it coexists with other nicotinic receptor subunit mRNAs such as α3, β2 and β4. In contrast, α6 mRNA is absent from the optic tectum and from the peripheral ganglia. Despite numerous efforts, the α6 subunit has long failed the critical test of functional reconstitution. Here we use patch‐clamp techniques and confocal laser microscopy to measure ACh‐activated currents and nicotine‐elicited Ca2+ transients in human BOSC 23 cells transfected with chick α6 in combination with other chick nAChR neuronal subunits. Heterologously expressed α6 and β4 subunits form functional heteromeric nAChRs, which are permeable to Ca2+ ions and blocked by the nicotinic antagonist methyllycaconitine (10 μm). Likewise, ACh elicits measurable currents...

Research paper thumbnail of Role of cell division in differentiation of myoblasts infected with a temperature-sensitive mutant of Rous sarcoma virus

The EMBO Journal, 1984

The relationship between a potential requirement for cell DNA synthesis and the expression of dif... more The relationship between a potential requirement for cell DNA synthesis and the expression of differentiated muscle cell functions was investigated using primary chicken embryo myoblasts infected with a temperature-sensitive mutant of Rous sarcoma virus (RSV). Under optimized conditions, transformed myoblasts growing at the permissive temperature could differentiate into multinucleated myotubes, express muscle-specific myosin, desmin and acetylcholine receptors in the absence of DNA synthesis and cell division following a shift to the non-permissive temperature. Furthermore, the experiments demonstrate that individual RSV-infected myoblasts have two options: either to divide and express the transformed phenotype or to withdraw from the cell cycle and differentiate into myotubes. The choice between these options appears to depend on the protein-kinase activity of pp6(c, the src gene product. Key words: cell division/mitomycin C/myogenic differentiation/pp6Osrc/Rous sarcoma virus 1975a; Boettiger et at., 1977; Pacifici et at., 1977), it is not clear whether transformed cells constitute lineage compartments distinct from or similar to those of normal cells (Holtzer et al., 1980a, 1980b). Hence, the quantal cell cycle model has also been applied to the transition from transformed myoblasts to multinucleated myotubes of cells transformed by ts-RSV and shifted to the restrictive temperature (Fiszman and Fuchs, 1975; Holtzer et al., 1975a; Moss, et al., 1979). Moss et al. (1979) found that >95% of the nuclei incorporated into myotubes could be labelled with [3H]thymidine after temperature shift up, whereas Holtzer et al. (1975a), in a similar experiment, found 75% of labelled nuclei in myotubes. The presence of unlabelled nuclei could be explained by the finding that some of the RSV-transformed myoblasts can spontaneously differentiate at the permissive temperature (Tato et al., 1983). Both reports suggested that a cell cycle separates the transformed myoblast state from the differentiated myotubes; however, these experiments did not demonstrate whether a cell cycle was necessary for the transition to occur. In this paper we have addressed two intriguing questions that are raised by the above-mentioned observations: (i) does a cell cycle obligatorily separate ts-transformed myoblasts from temperature shift-induced myotubes, and (ii) is RSVinduced block of differentiation the cause or the consequence of transformation-associated altered proliferation?

Research paper thumbnail of Erratum: p120 catenin is required for growth factor-dependent cell motility and scattering in epithelial cells (Molecular Biology of the Cell (2003) 14 (1964-1977))

Molecular Biology of the Cell, 2003

Research paper thumbnail of Mechanisms underlying the block of differentiation in transformed quail myoblasts

Cell Biology International Reports, 1990

The early development of the ventral nervous system in the Drosophila embryo is characterized by ... more The early development of the ventral nervous system in the Drosophila embryo is characterized by a fundamental cell fate decision. Approximately one fourth of the cells in the ventral ~_~ ~~ _ ~~_ ~_ .~~

Research paper thumbnail of Acetylcholine in Intercostal Muscle from Myasthenia Gravis Patients and in Rat Diaphragm after Blockade of Acetylcholine Receptors

Progress in Brain Research, 1979

Publisher Summary This chapter discusses whether the reduction in the number of AChRs in MG is ac... more Publisher Summary This chapter discusses whether the reduction in the number of AChRs in MG is accompanied by changes in the synthesis and release of ACh. It also investigates rat diaphragms, whose AChRs were blocked in vitro by a-bungarotoxin, or by immunization with AChR purified from Torpedo electroplax. ACh was extracted from the intercostal muscles from patients with acquired and congenital myasthenia gravis (MG) and from control patients with no clinical signs of muscle disease. ACh was estimated by mass fragmentography. It was found that the concentration of ACh was about two times higher in muscle from acquired MG than from congenital MG or control patients. Muscle from acquired MG and control patients was also incubated in Ringer with 50 mM KCl in order to stimulate the release of ACh. During the first 15 min of incubation with KCl more ACh was released from myasthenic than from control muscle, but this difference was not sustained on prolonged incubation. It was found that tetrodotoxin depressed the amount of ACh released into the medium in the presence of 50 mM KCI. Diaphragms from normal rats were treated with a-bungarotoxin, or taken from the animals that had been immunized against the ACh receptor protein from Torpedo marmorata. It was found that from these preparations KCl released about twice as much ACh as from control diaphragms.

Research paper thumbnail of Properties of end-plate channels in rats immunized against acetylcholine receptors

The Journal of Physiology, 1981

S. ALEMA AND OTHERS channel complexes is reduced without modification of single channel propertie... more S. ALEMA AND OTHERS channel complexes is reduced without modification of single channel properties. In this respect the immunized rat end-plate is a good model for myasthenia gravis affected human end-plates.

Research paper thumbnail of X-ray absorption spectroscopy (EXAFS and XANES) a structural tool for calcium binding proteins: effect of Mg2+ on Ca2+ binding sites of troponin-C

Progress in clinical and biological research, 1982

Research paper thumbnail of Proceedings: Studies on the interaction of nerve growth factor with tubulin and actin

Hoppe-Seyler's Zeitschrift für physiologische Chemie, 1975

Research paper thumbnail of Effects of retroviral oncogenes on myogenesis

Cell Biology International Reports, 1990

Expression of the proto-oncogene c-fos by ovine conceptus was analyzed by Northern and slot blots... more Expression of the proto-oncogene c-fos by ovine conceptus was analyzed by Northern and slot blots in relation with the expression of an interferon a-II (IFN a-II), named ovine Trophoblastin Protein (oTP). This IFN is produced only by the trophectoderm during the peri-implantation period. The proto-oncogene

Research paper thumbnail of Neuronal differentiation of P19 embryonal cells exhibits cell-specific regulation of neurotrophin receptors

NeuroReport, 1995

During development, the regulation of expression of the Trk family of tyrosine kinase receptors p... more During development, the regulation of expression of the Trk family of tyrosine kinase receptors plays an important role in defining the cellular responses to neurotrophin action. We report here that neurotrophin receptors are differentially expressed in distinct populations of retinoic acid-differentiated P19 cells. TrkB, the tyrosine kinase receptor for brain-derived neurotrophic factor, and LNGFR, the low-affinity receptor for all neutrophins, are preferentially expressed in P19-derived neurones. In contrast, retinoic acid induces the expression of TrkA, the high-affinity receptor for nerve growth factor, and of a non-catalytic form of TrkB, in non-neural subsets of differentiated cells. We propose P19 cells as a model system to study the mechanisms controlling the expression of neurotrophin receptors and the responsiveness of developing neurones to a specific neurotrophin.

Research paper thumbnail of Distinctive effects of the viral oncogenes myc, erb, fps, and src on the differentiation program of quail myogenic cells

Proceedings of the National Academy of Sciences, 1985

The relationship between susceptibility to transformation in vitro by different oncogenes and ter... more The relationship between susceptibility to transformation in vitro by different oncogenes and terminal differentiation was analyzed in embryonic quail myogenic cells. Infection with Rous sarcoma virus (RSV), Fujinami sarcoma virus (FSV), avian erythroblastosis virus (AEV), and the avian myelocytomatosis virus MC29 led to rapid and massive transformation. Transformed cells had distinctive morphological alterations, increased proliferation rates, and the ability to grow in agar suspension. Furthermore, homogeneously transformed cultures failed to fuse into multinucleated myotubes and to express muscle-specific genes. However, cloned populations of RSV-, FSV-, and AEV-transformed myogenic cells could, under appropriate culture conditions, partially differentiate into atypical "revertant" myotubes. In contrast, competence for terminal differentiation was completely and irreversibly suppressed on transformation by MC29. The specificity of action of a given oncogenic sequence on...

Research paper thumbnail of Black widow spider toxin-induced calcium fluxes and transmitter release in a neurosecretory cell line

Research paper thumbnail of Differentiation of PC12 phaeochromocytoma cells induced by v-src oncogene

Nature, 1985

PC12 rat phaeochromocytoma cells are a model system that can be used to study both neuronal diffe... more PC12 rat phaeochromocytoma cells are a model system that can be used to study both neuronal differentiation and the mechanism of action of nerve growth factor (NGF). PC12 cells respond to NGF protein by shifting from a chromaffin-cell-like phenotype to a neurite-bearing sympathetic neurone-like phenotype. Here we present data on the effect of infection of PC12 cells with retroviruses carrying the src oncogene of Rous sarcoma virus. Previous studies have demonstrated that the expression of src severely affects the synthesis and accumulation of differentiated cell products in a variety of cell types. We show that in the PC12 cell system, expression of v-src appears to have an inductive effect on differentiation that resembles the action of a 'physiological' growth factor.

Research paper thumbnail of The v-myc oncogene is sufficient to induce growth transformation of chick neuroretina cells

Nature, 1987

A number of studies have shown that full transformation of non-established rodent fibroblasts can... more A number of studies have shown that full transformation of non-established rodent fibroblasts can be efficiently achieved in vitro by the concerted action of two oncogenes belonging to different complementation groups. Extension of the two-genes carcinogenesis model to other differentiated cell types, presumably endowed with different controls of growth, is desirable for a better understanding of questions such as the host cell selectivity of oncogene action. A recent report claimed that cooperation between two oncogenes, v-myc and v-mil, is required to achieve transformation of chicken embryo neuroretina cells, which are characterized by a limited growth capacity in monolayer culture. Here we present evidence that the v-myc oncogene alone is sufficient to induce growth transformation of glial and neuronal precursor cell types from chick neuroretina. We also report that induction of transformation by v-myc is accompanied by faithful preservation of some of the differentiated functions of the chick cells.

Research paper thumbnail of Distinct Effects of Rac1 on Differentiation of Primary Avian Myoblasts

Molecular Biology of the Cell, 1999

Rho family GTPases have been implicated in the regulation of the actin cytoskeleton in response t... more Rho family GTPases have been implicated in the regulation of the actin cytoskeleton in response to extracellular cues and in the transduction of signals from the membrane to the nucleus. Their role in development and cell differentiation, however, is little understood. Here we show that the transient expression of constitutively active Rac1 and Cdc42 in unestablished avian myoblasts is sufficient to cause inhibition of myogenin expression and block of the transition to the myocyte compartment, whereas activated RhoA affects myogenic differentiation only marginally. Activation of c-Jun N-terminal kinase (JNK) appears not to be essential for block of differentiation because, although Rac1 and Cdc42 GTPases modestly activate JNK in quail myoblasts, a Rac1 mutant defective for JNK activation can still inhibit myogenic differentiation. Stable expression of active Rac1, attained by infection with a recombinant retrovirus, is permissive for terminal differentiation, but the resulting myotu...

Research paper thumbnail of Inhibition of ErbB-2 Mitogenic and Transforming Activity by RALT, a Mitogen-Induced Signal Transducer Which Binds to the ErbB-2 Kinase Domain

Molecular and Cellular Biology, 2000

The product of rat gene 33 was identified as an ErbB-2-interacting protein in a two-hybrid screen... more The product of rat gene 33 was identified as an ErbB-2-interacting protein in a two-hybrid screen employing the ErbB-2 juxtamembrane and kinase domains as bait. This interaction was reproduced in vitro with a glutathione S -transferase fusion protein spanning positions 282 to 395 of the 459-residue gene 33 protein. Activation of ErbB-2 catalytic function was required for ErbB-2–gene 33 physical interaction in living cells, whereas ErbB-2 autophosphorylation was dispensable. Expression of gene 33 protein was absent in growth-arrested NIH 3T3 fibroblasts but was induced within 60 to 90 min of serum stimulation or activation of the ErbB-2 kinase and decreased sharply upon entry into S phase. New differentiation factor stimulation of mitogen-deprived mammary epithelial cells also caused accumulation of gene 33 protein, which could be found in a complex with ErbB-2. Overexpression of gene 33 protein in mouse fibroblasts inhibited (i) cell proliferation driven by ErbB-2 but not by serum, ...

Research paper thumbnail of X-ray absorption near edge structure (XANES) determination of calcium sites of troponin C and parvalbumin

Journal of Molecular Biology, 1983

Using synchrotron radiation at the Frascati storage ring ADONE, the X-ray Absorption Near Edge St... more Using synchrotron radiation at the Frascati storage ring ADONE, the X-ray Absorption Near Edge Structure (XANES) has been applied to determine homologies and modifications of the local structure of the caicium binding sites of troponin C. In all four calcium binding sites, Ca 2+ appears to be coordinated to earboxyl and carbony! groups in a characteristic configuration. No structural difference has been found between high and low-affinity sites. A distortion of the Ca 2+ site geometry by binding of Mg 2+ has been observed. The XANES of parvalbumin has been measured and found to be different from troponin C. A tentative identification of the characteristic XANES spectra of the two different Ca 2 ÷ sites in this protein is reported.

Research paper thumbnail of IKKα Is a p63 Transcriptional Target Involved in the Pathogenesis of Ectodermal Dysplasias

Journal of Investigative Dermatology, 2009

The transcription factor p63 plays a pivotal role in the development and differentiation of the e... more The transcription factor p63 plays a pivotal role in the development and differentiation of the epidermis and epithelial appendages. Indeed, mutations in p63 are associated with a group of ectodermal dysplasias characterized by skin, limb, and craniofacial defects. It was hypothesized that p63 exerts its functions by activating specific genes during epidermal development, which in turn regulate epidermal stratification and differentiation. We have identified I-kappaB kinase alpha (IKKa) as a direct transcriptional target of p63 that is induced at early phases of terminal differentiation of primary keratinocytes. We show that the DNp63 isoform is required for IKKa expression in differentiating keratinocytes and that mutant p63 proteins expressed in ectodermal dysplasia patients exhibit defects in inducing IKKa. Furthermore, we observed reduced IKKa expression in the epidermis of an ankyloblepharon ectodermal dysplasia clefting patient. Our data demonstrate that a failure to properly express IKKa may play a role in the development of ectodermal dysplasias.

Research paper thumbnail of Functional Properties of Neuronal Nicotinic Acetylcholine Receptor Channels Expressed in Transfected Human Cells

European Journal of Neuroscience, 1997

Within the chick central nervous system, expression of the ␤3 nicotinic acetylcholine receptor ge... more Within the chick central nervous system, expression of the ␤3 nicotinic acetylcholine receptor gene is restricted to a subset of retinal neurons, the majority of which are ganglion cells. Transient transfection in retinal neurons and in neural and non-neural cells from other regions of the chick embryo allowed the identification of the cis-regulatory domain of the ␤3 gene. Within this domain, a 75-base pair fragment located immediately upstream of the transcription start site suffices to reproduce the neuron-specific expression pattern of ␤3. This fragment encompasses an E-box and a CAAT box, both of which are shown to be key positive regulatory elements of the ␤3 promoter. Co-transfection experiments into retinal, telencephalic, and tectal neurons with plasmid reporters of ␤3 promoter activity and a number of vectors expressing different neuronal (ASH-1, NeuroM, NeuroD, CTF-4) and non-neuronal (MyoD) basic helix-loop-helix transcription factors indicate that the cis-regulatory domain of ␤3 has the remarkable property of discriminating accurately between related members of the basic helix-loop-helix protein family. The sequence located immediately 3 of the E-box participates in this selection, and the E-box acts in concert with the nearby CAAT box.

Research paper thumbnail of Identification of a Determinant of Acetylcholine Receptor Gating Kinetics in the Extracellular Portion of the γ Subunit

European Journal of Neuroscience, 1996

A large body of structure‐function studies has identified many of the functional motifs underlyin... more A large body of structure‐function studies has identified many of the functional motifs underlying ion permeation through acetylcholine receptor (AChR) channels. The structural basis of channel gating kinetics is, however, incompletely understood. We have previously identified a novel shorter form of the AChR γ subunit, which lacks the 52 amino acids within the extracellular amino‐terminal half, encoded by exon 5. To define the contribution of the missing domain to AChR channel function, we have transiently coexpressed the mouse short γ subunit (γs) with α, β and δ subunits in human cells and recorded single‐channel currents from the resulting AChRs. Our findings show that replacement of the γ by the γs subunit confers a long duration characteristic to AChR channel openings without altering unitary conductance sizes or receptor affinity for the transmitter. We also show that βγδSδ AChR channels exhibit a peculiar voltage sensitivity characterized by a short opening duration when the...

Research paper thumbnail of The neuronal α6 subunit forms functional heteromeric acetylcholine receptors in human transfected cells

European Journal of Neuroscience, 1998

We examine some of the biological and physiological properties of the avian α6 neuronal nicotinic... more We examine some of the biological and physiological properties of the avian α6 neuronal nicotinic acetylcholine receptor (nAChR) subunit. We show here that, beginning at embryonic day 5, α6 mRNA is abundantly expressed in the developing chick neuroretina, where it coexists with other nicotinic receptor subunit mRNAs such as α3, β2 and β4. In contrast, α6 mRNA is absent from the optic tectum and from the peripheral ganglia. Despite numerous efforts, the α6 subunit has long failed the critical test of functional reconstitution. Here we use patch‐clamp techniques and confocal laser microscopy to measure ACh‐activated currents and nicotine‐elicited Ca2+ transients in human BOSC 23 cells transfected with chick α6 in combination with other chick nAChR neuronal subunits. Heterologously expressed α6 and β4 subunits form functional heteromeric nAChRs, which are permeable to Ca2+ ions and blocked by the nicotinic antagonist methyllycaconitine (10 μm). Likewise, ACh elicits measurable currents...