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1.
FEMS Microbiol Lett ; 348(1): 66-73, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24024613

ABSTRACT

Among the species of the Mycobacterium genus, more than 50 have been recognized as human pathogens. In spite of the different diseases caused by mycobacteria, the interspecies genetic similarity ranges from 94% to 100%, and for some species, this value is higher than in other bacteria. Consequently, it is important to understand the relationships existing among mycobacterial species. In this context, the possibility to use Mycobacterium tuberculosis dprE1 gene as new phylogenetic/taxonomic marker has been explored. The dprE1 gene codes for the target of benzothiazinones, belonging to a very promising class of antitubercular drugs. Mutations in cysteine 387 of DprE1 are responsible for benzothiazinone resistance. The DprE1 tree, obtained with 73 amino acid sequences of mycobacterial species, revealed that concerning the benzothiazinone sensitivity/resistance, it is possible to discriminate two clusters. To validate it, a concatamer obtained from the amino acid sequences of nine mycobacterial housekeeping genes was performed. The concatamer revealed that there is no separation between the benzothiazinone-susceptible and benzothiazinone-resistant species; consequently, this parameter is not linked to the phylogeny. DprE1 tree might represent a good taxonomic marker for the assignment of a mycobacterial isolate to a species. Moreover, the concatamer represents a good reference phylogeny for the Mycobacterium genus.


Subject(s)
Bacterial Proteins/genetics , Mycobacterium/classification , Mycobacterium/genetics , Oxidoreductases/genetics , Alcohol Oxidoreductases , Cluster Analysis , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Molecular Sequence Data , Phylogeny , Sequence Analysis, DNA , Sequence Homology
3.
J Neurosci ; 31(7): 2675-87, 2011 Feb 16.
Article in English | MEDLINE | ID: mdl-21325536

ABSTRACT

During brain development, neurogenesis, migration, and differentiation of neural progenitor cells are regulated by an interplay between intrinsic genetic programs and extrinsic cues. The Dlx homeogene transcription factors have been proposed to directly control the genesis and maturation of GABAergic interneurons of the olfactory bulb (OB), subpallium, and cortex. Here we provide evidence that Dlx genes promote differentiation of olfactory interneurons via the signaling molecule Wnt5a. Dlx2 and Dlx5 interact with homeodomain binding sequences within the Wnt5a locus and activate its transcription. Exogenously provided Wnt5a promotes GABAergic differentiation in dissociated OB neurons and in organ-type brain cultures. Finally, we show that the Dlx-mutant environment is unfavorable for GABA differentiation, in vivo and in vitro. We conclude that Dlx genes favor interneuron differentiation also in a non-cell-autonomous fashion, via expression of Wnt5a.


Subject(s)
Cell Differentiation/physiology , Gene Expression Regulation, Developmental/physiology , Interneurons/physiology , Neural Stem Cells/physiology , Wnt Proteins/metabolism , Animals , Animals, Newborn , Brain/cytology , Cell Differentiation/drug effects , Cells, Cultured , Chlorocebus aethiops , Chromatin Immunoprecipitation/methods , Coculture Techniques , Embryo, Mammalian , Epidermal Growth Factor/pharmacology , Fibroblast Growth Factor 2/pharmacology , Gene Expression Regulation, Developmental/genetics , Glutamate Decarboxylase/metabolism , Green Fluorescent Proteins/genetics , Homeodomain Proteins/genetics , MAP Kinase Kinase 4/genetics , MAP Kinase Kinase 4/metabolism , Mice , Mice, Transgenic , Models, Biological , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neural Stem Cells/drug effects , Olfactory Bulb/cytology , Protein Array Analysis/methods , Time Factors , Transfection/methods , Wnt Proteins/genetics , Wnt-5a Protein , beta Catenin/genetics , beta Catenin/metabolism , gamma-Aminobutyric Acid/metabolism
4.
J Biotechnol ; 150(1): 11-21, 2010 Oct 01.
Article in English | MEDLINE | ID: mdl-20688113

ABSTRACT

We describe the use of DNA transposons as tools for carrying out functional screenings in murine embryonic stem (ES) cell-derived neural stem (NS) cells. NS cells are a new type of stem cells featuring radial glial properties, that undergoes symmetric cell division for an indefinite number of passages, expanding as a monolayer. In this model, the previously unreported Sleeping Beauty transposase M3A achieves an optimal blend of clone generation efficiency and low redundancy of integrations per clone, compared to the SB100X Sleeping Beauty variant and to the piggyBac transposon. The technology described here makes it possible to randomly trap genes in the NS cell genome and modify their expression or tag them with fluorescent markers and selectable genes, allowing recombinant cells to be isolated and expanded clonally. This approach will facilitate the identification of novel determinants of stem cell biology and neural cell fate specification in NS cells.


Subject(s)
DNA Transposable Elements/genetics , Models, Genetic , Mutagenesis, Insertional/methods , Neural Stem Cells/physiology , Transposases/genetics , Animals , Cells, Cultured , Computer Simulation , Humans , Mice , Neomycin , Transposases/metabolism
5.
BMC Neurosci ; 11: 7, 2010 Jan 19.
Article in English | MEDLINE | ID: mdl-20085655

ABSTRACT

BACKGROUND: There is an urgent need of neuronal cell models to be applied to high-throughput screening settings while recapitulating physiological and/or pathological events occurring in the Central Nervous System (CNS). Stem cells offer a great opportunity in this direction since their self renewal capacity allows for large scale expansion. Protocols for directed differentiation also promise to generate populations of biochemically homogenous neuronal progenies. NS (Neural Stem) cells are a novel population of stem cells that undergo symmetric cell division in monolayer and chemically defined media, while remaining highly neurogenic. RESULTS: We report the full adaptation of the NS cell systems for their growth and neuronal differentiation to 96- and 384-well microplates. This optimized system has also been exploited in homogeneous and high-content assays. CONCLUSIONS: Our results show that these mouse NS cells may be suitable for a series of applications in high-throughput format.


Subject(s)
Adult Stem Cells/physiology , Cell Culture Techniques/instrumentation , Cell Culture Techniques/methods , High-Throughput Screening Assays/instrumentation , High-Throughput Screening Assays/methods , Neurons/physiology , Animals , Apoptosis , Cell Differentiation , Cell Line , Cell Survival , Cyclic AMP/metabolism , Mice , Mice, Inbred Strains , Neurogenesis , Oxidative Stress/physiology , Receptors, GABA-A/metabolism , Stem Cell Niche/physiology , Time Factors
6.
Mol Cell Neurosci ; 41(1): 74-84, 2009 May.
Article in English | MEDLINE | ID: mdl-19386228

ABSTRACT

SHC genes codify for a family of adaptor molecules comprising four genes. Previous data have implicated the Shc(s) molecules in stem cell division and differentiation. Specifically, the p66(ShcA) isoform has been found to contribute to longevity and resistance from oxidative stress. Here we report that p66(ShcA) is up-regulated during in vitro neural induction in embryonic stem cells. p66(ShcA) over-expression in ES cells reduces GSK-3beta kinase activation and increases beta-catenin stabilization and its transcriptional activity. p66(ShcA) over-expression results in ES cells undergoing an anticipated neural induction and accelerated neuronal differentiation. Similar effects are obtained in human ES cells over-expressing p66(ShcA). This study reveals a role for p66(ShcA) in the modulation of Wnt/beta-catenin pathway and in ES cell neuralization which is consistent between mouse and human.


Subject(s)
Cell Differentiation/physiology , Embryonic Induction/physiology , Embryonic Stem Cells/physiology , Neurons/physiology , Protein Isoforms/metabolism , Shc Signaling Adaptor Proteins/metabolism , Animals , Cells, Cultured , Embryonic Stem Cells/cytology , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Mice , Neurons/cytology , Protein Isoforms/genetics , Shc Signaling Adaptor Proteins/genetics , Signal Transduction/physiology , Src Homology 2 Domain-Containing, Transforming Protein 1 , Wnt Proteins/genetics , Wnt Proteins/metabolism
7.
Development ; 131(17): 4239-49, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15280216

ABSTRACT

The subcortical telencephalon is the major source of GABAergic interneurons that, during development, tangentially migrate to the cerebral cortex, where they modulate the glutamatergic excitatory action of pyramidal cells. The transcription factor Vax1, an intracellular mediator of both Shh and Fgf signaling, is expressed at high levels in the medial and lateral ganglionic eminences (MGE and LGE, respectively), in the septal area (SA), in the anterior entopeduncular area (AEP) and in the preoptic area (POA). We show that Vax1 expression in the neuroepithelium is graded: low in the ventricular zone (VZ) and high in the subventricular zone (SVZ), in a pattern that closely reproduces that of several members of the Dlx and Gsh family of homeobox transcription factors. We provide evidence that Vax1 plays an important role in proliferation and differentiation of MGE, POA/AEP and septum, and that the last structure is completely absent in Vax1-/- mice. We show that the absence of Vax1 causes a severe depletion of GABAergic neurons in the neocortex, ranging from 30% to 44%, depending on the cortical areas considered. Taken together, our data indicate that a loss of function mutation in the Vax1 gene generates abnormalities in basal ganglia subventricular zone development and that it prevents the formation of the septum, impairing GABAergic interneuron generation.


Subject(s)
GABA Modulators/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/physiology , Interneurons/pathology , Neuropeptides/genetics , Neuropeptides/physiology , Animals , Brain/metabolism , Brain/pathology , Bromodeoxyuridine/pharmacology , Cell Division , Cerebral Cortex/metabolism , Coloring Agents/pharmacology , Epithelium/metabolism , Genotype , Homeodomain Proteins/metabolism , Immunohistochemistry , In Situ Hybridization , Interneurons/metabolism , Mice , Mice, Transgenic , Mutation , Prosencephalon/embryology , Pyramidal Cells/metabolism , RNA/metabolism , Telencephalon/metabolism , Time Factors , Transcription Factors
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