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Biomedical subjects

G Cossu

Publications and source records attributed to G Cossu.

At least 91 records · Page 5Linked to original sources
PubMed1995

Isradipine inhibits nicotine intravenous self-administration in drug-naive mice.

The effect of isradipine, a dihydropyridine calcium antagonist, on intravenous self-administration of nicotine in naive mice has been investigated. When nicotine injections were made contingent upon nose-poke response by naive mice, they increased their rate of nose poking with respect to animals receiving contingent saline injections or yoked control animals receiving noncontingent nicotine injections. Pretreatment of mice with mecamylamine (2.4 mg/kg) inhibited self-administration of nicotine contingent upon a nose-poke response. The same effect was observed with isradipine (0.5-1.0 mg/kg) in a dose-related manner and stereospecifically. These data suggest that isradipine suppresses the reinforcing properties of nicotine and might be useful for treatment of nicotine abuse.

Animals↗
PubMed1995

Myosin light chain 3F regulatory sequences confer regionalized cardiac and skeletal muscle expression in transgenic mice.

The myosin light chain IF/3F locus contains two independent promoters, MLC1F and MLC3F, which are differentially activated during skeletal muscle development. Transcription at this locus is regulated by a 3' skeletal muscle enhancer element, which directs correct temporal and tissue-specific expression from the MLC1F promoter in transgenic mice. To investigate the role of this enhancer in regulation of the MLC3F promoter in vivo, we have analyzed reporter gene expression in transgenic mice containing lacZ under transcriptional control of the mouse MLC3F promoter and 3' enhancer element. Our results show that these regulatory elements direct strong expression of lacZ in skeletal muscle; the transgene, however, is activated 4-5 d before the endogenous MLC3F promoter, at the time of initiation of MLC1F transcription. In adult mice, transgene activity is downregulated in muscles that have reduced contributions of type IIB fibers (soleus and diaphragm). The rostrocaudal positional gradient of transgene expression documented for MLC1F transgenic mice (Donoghue, M., J. P. Merlie, N. Rosenthal, and J. R. Sanes. 1991. Proc. Natl. Acad. Sci. USA. 88:5847-5851) is not seen in MLC3F transgenic mice. Although MLC3F was previously thought to be restricted to skeletal striated muscle, the MLC3F-lacZ transgene is expressed in cardiac muscle from 7.5 d of development in a spatially restricted manner in the atria and left ventricular compartments, suggesting that transcriptional differences exist between cardiomyocytes in left and right compartments of the heart. We show here that transgene-directed expression of the MLC3F promoter reflects low level expression of endogenous MLC3F transcripts in the mouse heart.

Animals↗
PubMed1995

A retroviral vector containing a muscle-specific enhancer drives gene expression only in differentiated muscle fibers.

Genetically modified myogenic cells have a number of potentially relevant applications for gene therapy of genetic defects. Retroviral vectors proved to be a safe and efficient tool to transfer and express genes into satellite cells and their differentiated progeny, although muscle-specific regulation of the transferred gene is very difficult to achieve in a conventional vector framework. We modified a Moloney murine leukemia virus (MoMLV)-derived retroviral vector containing a bacterial beta-galactosidase (beta-Gal) reporter gene by inserting a muscle creatinine kinase (MCK) enhancer element into the U3 region of the viral long terminal repeat (LTR). The resulting vector (mLBSN) was transferred into cells of different histological origin, including undifferentiated murine and human myogenic cells, which were unable to express the transgene at detectable levels. Instead, gene expression from the modified LTR was obtained in a mouse myogenic cell line and in human primary satellite cells upon induction of differentiation into myotubes in culture, and correlated with the activation of the muscle differentiation program. beta-Gal-negative, mLBSN-transduced human satellite cells were also transplanted into the quadricep muscle of immunodeficient mice, where activation of the transgene expression was observed in vivo after differentiation and fusion into muscle fibers. These results show that retroviral vectors carrying LTRs modified in the enhancer sequences may be used to target tissue- and differentiation-specific gene expression into the muscle. For practical purposes, satellite cells engineered by muscle-specific retroviral vectors might represent an effective tool to deliver expression of a given gene product specifically into the muscle tissue, avoiding undesired protein accumulation in mononucleated cells. More generally, this type of vector might be useful whenever regulated expression of a transferred gene is necessary in a target cell or tissue.

Animals↗
PubMed1994

A population of myogenic cells derived from the mouse neural tube.

Embryonic mouse neural tubes produce a variety of terminally differentiated cells in vitro, mostly neurons and glia. We report here that some of these cells differentiate into skeletal muscle cells. The possibility of mesoderm contamination was ruled out as follows. First, Dil+ muscle cells were present in cultures from a Dil-labeled neuroepithelium. Second, a small fraction of cultured neural tube cells coexpressed muscle myosin and neuronal beta III tubulin within the same cell. Third, embryos generated from embryonic stem cells in which nlacZ was targeted into the myogenic gene myf-5 expressed nlacZ in a localized region of the neural tube. These myf-5+ cells coexpress neuronal and muscle markers in culture. The developmental significance of this phenomenon is discussed in the context of overlapping regulatory networks between myogenesis and neurogenesis.

Animals↗
PubMed1994

Differential response of embryonic and fetal myoblasts to TGF beta: a possible regulatory mechanism of skeletal muscle histogenesis.

Embryonic and fetal skeletal myoblasts were grown in culture in the presence of TGF beta. Under the conditions employed, TGF beta inhibited differentiation of fetal but not of embryonic myoblasts. To investigate the possible relevance of these data to skeletal muscle histogenesis in vivo, we studied the proliferation/differentiation state of mesodermal cells in the proximal region of the limb bud at the time of primary fiber formation. BrdU labeling and immunostaining for myosin heavy chains revealed that very few mesodermal cells enter the S phase of the cycle when differentiated primary fibers first appear. However, a few hours later, many cells in S phase surround newly formed muscle fibers, suggesting that the latter may be a source of mitogens for undifferentiated myoblasts. Co-culture experiments supported this hypothesis, showing that medium conditioned by fiber-containing explants can stimulate myoblast proliferation. Taken together these data suggested a possible mechanism for the regulation of muscle fiber formation. The model assumes that fibers form in the proximal region of the limb bud, where TGF beta is known to be present, and BrdU labeling experiments did not reveal cells in S phase. It is conceivable that non-dividing embryonic myoblasts (which do not respond to TGF beta) can undergo differentiation, while fetal myoblasts are inhibited by TGF beta. Once formed, primary fibers may stimulate a new wave of proliferation in fetal myoblasts, in order to expand the pool of cells needed to form secondary fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Genetic studies on the Senegal population. II. Polymorphisms of the plasma proteins F13A, F13B, ORM1, AHSG, C6, C7, and APOC2.

Using isoelectric focusing and immunoblotting techniques, we tested 270 plasma samples from 3 populations of Senegal (Wolof, Peul, Tukulor) to determine genetic variation at 7 protein loci (F13A, F13B, ORM1, AHSG, C6, C7, APOC2). Four of the seven systems (F13A, ORM1, AHSG, C6) have not been studied previously in sub-Saharan Africa, and one system (C7) has never been examined in any population of African ancestry. The assumption that F13B*6, F13B*23, and APOC2*2 represent African marker alleles is supported by this study. At the AHSG locus we observed a four-allele polymorphism rather than the two-allele polymorphism commonly seen in other ethnic groups. At the C6 locus, in addition to the two common alleles C6*A and C6*B, we observed three other alleles, one of which (C6*A3), found at polymorphic frequencies, seems to be another example of a unique African allele. The C7 locus was found to be monomorphic in the Peul but polymorphic in the Wolof and the Tukulor. At the F13A and ORM1 loci, Senegalese have allele frequencies similar to those reported for American blacks. All three Senegalese samples display typical African features, such as a high frequency of the F13B*2 allele and the presence of the APOC2*2 allele at a polymorphic level. However, some differences in allele frequencies have been found between the three groups, and this could have implications for reconstructing their remote history.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed1993

Paracrine stimulation of senescent satellite cell proliferation by factors released by muscle or myotubes from young mice.

The proliferative potential of satellite cells undergoes a dramatic decrease in the early postnatal period and a more modest but continuous decrease throughout the life span of the animal. To address the problem of the mechanism regulating this phenomenon and to understand whether it is causally linked to senile muscle atrophy, we studied the response of aged satellite cells to serum and to different growth factors. The data reported indicate a generalised reduction in the response to all mitogens tested, which could not be compensated for by increased concentrations of serum or growth factors. On the other hand, conditioned medium of differentiated myotubes from young mice exhibited a strong mitogenic action on aged satellite cells, while conditioned media of myotubes from old mice or from a variety of non-muscle cells were ineffective. Furthermore, saline extracts from muscle of young mice are also able to exert this mitogenic action. Saline extracts of muscle from old mice were poorly mitogenic for satellite cells from young mice, and not at all for satellite cells from old mice. These data indicate that paracrine interactions operate inside the muscle tissue and are probably required for the normal replicative behaviour of satellite cells. The failure of such interactions may be among the causes leading to age-related muscle hypotrophy.

Animals↗
PubMed1993

Hemoglobin Ozieri: a new alpha-chain variant (alpha 71(E20)Ala-->Val). Characterization using FAB- and electrospray-mass spectrometric techniques.

A new silent hemoglobin variant, Hb Ozieri (alpha 71(E20)Ala-->Val), was observed in five apparently unrelated newborn babies during a screening for hemoglobinopathies on the island of Sardinia. This asymptomatic variant was detected by means of isoelectric focusing (IEF), isolated using IEF in an immobilized ultranarrow pH-gradient and characterized at the structural level using FAB- and electrospray-mass spectrometric techniques. A Val for Ala substitution was unambiguously detected at position 71 of the alpha-globin chain. This substitution indicates that a C to T transition occurred in the GCG codon for Ala which contains one of the 35 unmethylated CpG dinucleotides of the alpha-globin gene. This observation brings the number of variants due to a mutation in the alpha-globin gene CpGs (the third instance of a silent mutation) to 13 and raises the possibility that unmethylated CpGs might be hotspots for mutations as the methylated ones.

Alanine↗
PubMed1993

Pro-opiomelanocortin gene is expressed in post-implantation mouse embryos and enhances growth potential of myogenic cells.

We previously showed that the adrenocorticotropin hormone (ACTH) is a mitogen for myoblasts and is present in post-implantation mammalian embryos (Cossu et al. [1989] Dev. Biol. 131:331-336; De Angelis et al. [1992] Dev. Biol. 151:446-458). In this paper, we investigated the expression of the corresponding gene, pro-opiomelanocortin (POMC), by in situ hybridization and polymerase chain reaction. In situ analysis revealed low level expression in the basal layer of 10.5 d.p.c. neural tube and in several discrete areas around the dorsal aorta. By more sensitive Reverse-transcription polymerase chain reaction (RT-PCR) analysis, expression was detected also in developing limb buds and in cultured myogenic cells, but not in fibroblasts. To investigate the possible role of POMC gene expression in myogenesis, we induced its over-expression in proliferating myoblasts. Upon sub-optimal growth conditions, over-expressing cells were found to give rise to clones larger than control cells. The differentiation potential of POMC over-expressing myogenic cells was unchanged.

Animals↗
PubMed1993

TPA-induced differentiation of human rhabdomyosarcoma cells: expression of the myogenic regulatory factors.

RD cells (a cell line derived from a human rhabdomyosarcoma) undergo a very limited myogenic differentiation despite the fact that they express several myogenic determination genes. Since we have previously shown (Aguanno et al., Cancer Res. 50, 3377, 1990) that the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces myogenic differentiation in these cells, in this paper we investigate the mechanism by which TPA interferes with the expression and/or function of the myogenic determination genes. Northern blot analysis revealed that RD cells express the myf3 (the human analog of MyoD) and myf4 (the human analog of myogenin) transcripts, but not myf5 or myf6 transcripts. The myf3 and the myf4 gene products are correctly translated and accumulated in the nuclei as shown by immunofluorescence analysis. The tumor promoter (TPA) does not modify the pattern of expression of the myf factors while it induces the accumulation of muscle-specific transcripts, such as alpha-actin and fast myosin light chain 1, and their corresponding proteins. On the other hand, within 1 day of treatment, TPA inhibits the expression of the Id gene, which is a negative regulator of MyoD activity. However, while the TPA-induced inhibition of Id message accumulation correlates with differentiation, cell confluence also causes a reduction in Id message accumulation, without inducing differentiation. Under our experimental conditions, overexpression of any of the myf cDNAs in RD cells does induce spontaneous differentiation but enhances the effect of TPA treatment independently from the level of the expressed message. These data suggest that differentiation of RD cells is likely to depend upon the activity of complexes containing the various members of the MyoD family, which can be regulated by proteins affecting MyoD dimerization such as Id, but also by other mechanisms induced by TPA, such as phosphorylation.

Cell Differentiation↗
PubMed1993

When reading is acquired but phonemic awareness is not: a study of literacy in Down's syndrome.

The concept of phonological awareness (PA) has loomed large in recent discussions of the acquisition of literacy in alphabetic orthographies (Bryant & Goswami, 1987). The term is usually taken to imply overt knowledge of how spoken words can be analysed into their constituent sounds ("phones"). This awareness is assessed by such tasks as requiring the child to produce (or recognize) rhymes, to indicate how many sounds there are in a particular word, or to delete a constituent (phone or syllable) of a word and pronounce the remainder. It has been asserted that these skills play a causal role in the development of reading ability (Bradley & Bryant, 1983). We provide evidence against the position that such skills are essential prerequisites for reading (and any other hypothesis that claims necessary causal links between reading and PA). It is shown that some children with Down's syndrome can learn to read despite their failure on a set of tasks conventionally employed to assess PA. The pedagogic implication is that reading should be taught by teaching reading skills (including letter-sound correspondences), not phonological awareness skills.

Adolescent↗
PubMed1993

Retroviral vector-mediated gene transfer into human primary myogenic cells leads to expression in muscle fibers in vivo.

Primary human myogenic cells isolated from fetal and adult muscle were infected with a high-titer, Moloney murine leukemia virus (MoMLV)-derived retroviral vector expressing a bacterial beta-galactosidase (beta-gal) gene under long terminal repeat (LTR) control. Gene transfer efficiency averaged 50% in both fetal myoblasts and adult satellite cells, as revealed by beta-gal staining. The reporter gene was stably integrated, faithfully inherited, and expressed at significant levels in myogenic cells for at least 10 generations under clonal growth conditions, and throughout the culture life span upon differentiation into myotubes. Comparable gene transfer efficiency was obtained in myogenic cells from muscle biopsies of patients affected by a number of genetic or acquired myopathies, including Duchenne muscular dystrophy. Transduced normal human satellite cells were injected into regenerating muscle of immunodeficient mice, where they formed new muscle fibers in which the product of the reporter gene was detectable for 2 months after injection. These results show that retroviral vectors can be used to transfer foreign genes with high efficiency into normal or abnormal primary human myogenic cells, leading to stable expression into mature muscle. Satellite cells engineered in this way might represent an effective tool for gene therapy of muscular dystrophies as well as for systemic delivery of recombinant gene products for correction of inherited and acquired disorders. The human-mouse model described here will allow in vivo testing of such gene therapy approaches.

Adolescent↗
PubMed1992

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.

Base Sequence↗
PubMed1992

ACTH-like peptides in postimplantation mouse embryos: a possible role in myoblast proliferation and muscle histogenesis.

ACTH and related peptides are mitogens for certain mesodermal cell types such as adrenocortical cells, T-lymphocytes, and skeletal myoblasts. In order to postulate a possible physiological role for these peptides in skeletal muscle histogenesis, it is necessary to establish whether they are present in muscle forming anlagens of postimplantation mouse embryos. By radioimmunoassay and immunofluorescence with antibodies specific for ACTH, we have detected these peptides in many areas of mouse embryos including neural tube, limb buds, eye lens, and myotomal muscles. During fetal development, immunoreactivity decreased in muscle tissue and appeared in visceral ganglia. Furthermore, primary myotubes or C2C12 myotubes, but not muscle or 3T3 fibroblasts, release significant levels of ACTH immunoreactive peptides into the culture medium. Using a microassay for mitogen production, primary myotubes or C2C12 myotubes, but not other mesodermal cells (with the exception of dermal fibroblasts) were shown to release factors into the medium which support myoblast proliferation. Neutralizing antibodies against ACTH inhibit myoblast but not fibroblast proliferation in a dose-dependent fashion. Based on these results, we propose that myotube-derived mitogens (including ACTH-like peptides) promote the proliferation of surrounding myoblast during muscle histogenesis in vivo.

Adrenocorticotropic Hormone↗
PubMed1992

Induction of Calbindin-D28K by 1,25-dihydroxyvitamin D3 in cultured chicken intestinal cells.

Intestinal cells from chicken embryos were grown in chemically defined, serum-free medium. The majority of cultured cells exhibits an epithelial-like morphology. As demonstrated by indirect immunofluorescence, the epithelial cells, and not the contaminating fibroblasts, express Calbindin-D28K only after 1,25-dihydroxyvitamin D3, the hormonally active form of vitamin D, is added to the culture medium. The highly sensitive reverse transcriptase-polymerase chain reaction shows that both Calbindin-D28K mRNA and the corresponding primary unprocessed transcripts (pre-mRNA) are dramatically increased in cultured intestinal cells treated with 1,25-dihydroxyvitamin D3, thus indicating that Calbindin-D28K is induced by the increased rate of transcription of the corresponding gene.

Animals↗
PubMed1992

The effects of AZT and DDI on pre- and postimplantation mammalian embryos: an in vivo and in vitro study.

This study reports the effects of the nucleoside analogs dideoxyinosine (DDI) and 3'-azido-3'-deoxythymidine (AZT) on mammalian embryonic development. When administered to pregnant mice (at concentrations ranging from 10 to 300 mg/kg/day), through all or part of gestation, AZT and DDI did not result in any visible effect on mouse embryos nor did they cause any obvious malformation or defect at birth or during postnatal growth. Similarly, when embryonic or fetal mouse or human cells (from brain, limb buds, or different organ rudiments) were exposed to AZT or DDI in vitro, cytotoxicity was observed only in the mM range, with AZT showing slightly higher cytotoxicity and brain cells appearing slightly more sensitive to both nucleosides. However, even in cultures treated with very high concentrations of AZT or DDI, the reduction in the number of terminally differentiated skeletal myotubes, cardiocytes, neurons, and chondrocytes was similar to the reduction in the total number of cells, indicating that AZT and DDI did not selectively inhibit differentiation of any of the above-mentioned cell types. Finally, preimplantation mouse embryos (at the 2-cell or 4-cell stage), treated in vitro with micromolar concentrations of AZT, were arrested at the 4-cell stage. DDI or other nucleoside analogs tested did not have this effect.

Animals↗