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1.
Sci Rep ; 4: 5942, 2014 Aug 06.
Article in English | MEDLINE | ID: mdl-25096726

ABSTRACT

Quantification of basic cell functions is a preliminary step to understand complex cellular mechanisms, for e.g., to test compatibility of biomaterials, to assess the effectiveness of drugs and siRNAs, and to control cell behavior. However, commonly used quantification methods are label-dependent, and end-point assays. As an alternative, using our lensfree video microscopy platform to perform high-throughput real-time monitoring of cell culture, we introduce specifically devised metrics that are capable of non-invasive quantification of cell functions such as cell-substrate adhesion, cell spreading, cell division, cell division orientation and cell death. Unlike existing methods, our platform and associated metrics embrace entire population of thousands of cells whilst monitoring the fate of every single cell within the population. This results in a high content description of cell functions that typically contains 25,000 - 900,000 measurements per experiment depending on cell density and period of observation. As proof of concept, we monitored cell-substrate adhesion and spreading kinetics of human Mesenchymal Stem Cells (hMSCs) and primary human fibroblasts, we determined the cell division orientation of hMSCs, and we observed the effect of transfection of siCellDeath (siRNA known to induce cell death) on hMSCs and human Osteo Sarcoma (U2OS) Cells.


Subject(s)
Fibroblasts/physiology , Mesenchymal Stem Cells/physiology , Microscopy, Video/methods , Osteoblasts/metabolism , Video Recording/methods , Cell Adhesion , Cell Count , Cell Death/genetics , Cell Division , Cell Line, Tumor , Fibroblasts/cytology , Humans , Mesenchymal Stem Cells/cytology , Microscopy, Video/instrumentation , Osteoblasts/pathology , Primary Cell Culture , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Video Recording/instrumentation
2.
J Physiol ; 535(Pt 3): 741-55, 2001 Sep 15.
Article in English | MEDLINE | ID: mdl-11559772

ABSTRACT

1. Two ligand binding alpha subunits, alpha1 and alpha2, of the human (H) glycine receptor (GlyR) are involved at inhibitory synapses in the adult and neonatal spinal cord, respectively. The ability of homomeric alphaH1 and alphaH2 GlyRs to be activated by glycine, taurine and GABA was studied in Xenopus oocytes or in the human embryonic kidney HEK-293 cell line. 2. In outside-out patches from HEK cells, glycine, taurine and GABA activated both GlyRs with the same main unitary conductance, i.e. 85 +/- 3 pS (n = 6) for alphaH1, and 95 +/- 5 pS (n = 4) for alphaH2. 3. The sensitivity of both alphaH1 and alphaH2 GlyRs to glycine was highly variable. In Xenopus oocytes the EC50 for glycine (EC50gly) was between 25 and 280 microM for alphaH1 (n = 44) and between 46 and 541 microM for alphaH2 (n = 52). For both receptors, the highest EC50gly values were found on cells with low maximal glycine responses. 4. The actions of taurine and GABA were dependent on the EC50gly: (i) their EC50 values were linearly correlated to EC50gly, with EC50tau approximately 10 EC50gly and EC50GABA approximately 500-800 EC50gly; (ii) they could act either as full or weak agonists depending on the EC50gly. 5. The Hill coefficient (n(H)) of glycine remained stable regardless of the EC50gly whereas n(H) for taurine decreased with increasing EC50tau. 6. The degree of desensitization, evaluated by fast application of saturating concentrations of agonist on outside-out patches from Xenopus oocytes, was similar for glycine and taurine on both GlyRs and did not exceed 50 %. 7. Our data concerning the variations of EC50gly and the subsequent behaviour of taurine and GABA could be qualitatively described by the simple del Castillo-Katz scheme, assuming that the agonist gating constant varies whereas the binding constants are stable. However, the stability of the Hill coefficient for glycine was not explained by this model, suggesting that other mechanisms are involved in the modulation of EC50.


Subject(s)
Receptors, Glycine/agonists , Taurine/pharmacology , gamma-Aminobutyric Acid/pharmacology , Algorithms , Animals , DNA, Complementary/genetics , Electrophysiology , Glycine/pharmacology , Humans , Kinetics , Oocytes/drug effects , Oocytes/metabolism , Patch-Clamp Techniques , Receptors, Glycine/genetics , Xenopus
3.
Gene ; 271(2): 239-45, 2001 Jun 27.
Article in English | MEDLINE | ID: mdl-11418245

ABSTRACT

Gephyrin was first described as a peripheral membrane protein of 93 kDa anchoring the glycine receptor (GlyR) to subsynaptic microtubules and cytoskeleton. Analysis of knock-out mice demonstrated that gephyrin has additional functions in GABA(A) receptor localization at the synapse and in the biosynthetic pathway of the molybdenum cofactor (Moco). Here we describe a human non-neuronal gephyrin cDNA and the exon/intron organization of the human gephyrin gene. We found the coding region to consist of 27 exons and to span approximately 800 kb on the long arm of chromosome 14. This structure is almost identical to that of the mouse gephyrin gene except that sequences corresponding to three exons described in rat and mouse could not be identified in human. Mutations of the GlyR subunits and of gephyrin lead to severe neuromotor phenotypes in human and mouse. Hyperekplexia involves most frequently a mutation in the GlyR alpha1 subunit in humans. However, inactivation of the Moco biosynthesis pathway results in very similar symptomatology. The recent characterization of a deletion of two exons of the gephyrin gene in a patient with symptoms typical of Moco deficiency confirmed that the involvement of gephyrin in these pathologies cannot be excluded. The precise localization of the gephyrin gene allowed us to exclude it from being a candidate for the autosomal dominant spastic paraplegia, the locus of which maps to 14q between markers D14S259 and D14S1018. A description of its structure and exon boundaries should lay the groundwork for further analysis of its expression in humans.


Subject(s)
Carrier Proteins/genetics , Membrane Proteins/genetics , Animals , Base Sequence , Cell Line , Chromosome Mapping , Chromosomes, Human, Pair 14/genetics , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , Exons , Female , Gene Expression , Genes/genetics , Humans , Introns , Kidney/cytology , Kidney/metabolism , Male , Mice , Molecular Sequence Data , Neurons/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Sequence Analysis, DNA , Spinal Cord/cytology , Spinal Cord/metabolism , Tissue Distribution
4.
Mech Dev ; 90(1): 89-94, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10585565

ABSTRACT

The gene underlying X chromosome-linked Kallmann syndrome, KAL-1, has been identified for several years, yet its role in development is still poorly understood. In order to take advantage of the zebrafish as a model in developmental genetics, we isolated the two KAL-1 orthologues, kal1.1 and kal1.2, in this species. Comparison of deduced protein sequences with the human one shows 75.5 and 66.5% overall homology, respectively. The most conserved domains are the whey acidic protein-like domain and the first of four fibronectin-like type III repeats. However, kal1.2 putative protein lacks the basic C-terminal domain (20 residues) found in kal1.1 and KAL-1. The expressions of kal1.1 and kal1.2 were studied in the embryo between 6 and 96 hours post fertilization using whole-mount in situ hybridization. Although a few structures express both genes, kal1.1 and kal1.2 expression patterns are largely non-overlapping. Taken together, these patterns match fairly well those previously reported for human KAL-1 and chicken kal1. As regards the olfactory system, kal1.1 is expressed, from 37 h.p.f. onward, in the presumptive olfactory bulbs, whereas kal1.2 transcript is only detected, from 48 h.p.f., in the epithelium of the nasal cavity. The relevance of the zebrafish as an animal model for studying both the function of KAL-1 in normal development and the developmental failure leading to the olfactory defect in Kallmann syndrome, is discussed.


Subject(s)
Extracellular Matrix Proteins , Kallmann Syndrome/genetics , Nerve Tissue Proteins/genetics , X Chromosome , Zebrafish Proteins/genetics , Zebrafish/genetics , Amino Acid Sequence , Animals , Chromosome Mapping , Gene Expression Regulation, Developmental , Humans , Molecular Sequence Data , Sequence Alignment , Sequence Analysis , Zebrafish/embryology
5.
Neuropharmacology ; 38(6): 785-92, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10465682

ABSTRACT

The alpha subunit (alphaZ1) of the zebrafish glycine receptor (GlyR) has been N-terminus fused with green fluorescent protein (GFP). We found that both pharmacological and electrophysiological properties of this chimeric alphaZ1-GFP are indistinguishable from those of the wild-type receptor when expressed in Xenopus oocytes and cell lines. The apparent affinities of this receptor for agonists (glycine, taurine and GABA), and the antagonist (strychnine) are unchanged, and single channel kinetics are not altered. In the same expression systems, alphaZ1-GFP was visualized using fluorescence microscopy. Fluorescence was distributed anisotropically across cellular membranes. In addition to the Golgi apparatus and endoplasmic reticulum, its presence was also detected on the plasmalemma, localized at discrete hot-spots which were identified as sites of high membrane turnover. Overall, the preservation in alphaZ1-GFPs of the wild type receptor functional properties makes it a promising new tool for further in situ investigations of GlyR expression, distribution and function.


Subject(s)
Luminescent Proteins/chemistry , Receptors, Glycine/chemistry , Animals , Green Fluorescent Proteins , Indicators and Reagents , Microscopy, Fluorescence , Zebrafish
6.
J Physiol ; 517 ( Pt 2): 369-83, 1999 Jun 01.
Article in English | MEDLINE | ID: mdl-10332088

ABSTRACT

1. Glycine and GABA can be co-released from the same presynaptic terminals and in lower vertebrates they can activate the same glycine receptors (GlyRs). Thus we examined the effects of these two inhibitory transmitters on the homomeric GlyRs formed by the alphaZ1 subunit, of the zebrafish using two expression systems: Xenopus oocytes and the human BOSC 23 cell line. 2. The apparent affinity (EC50) of alphaZ1 for these neurotransmitters was highly variable. In Xenopus oocytes the EC50 ranged from 37 to 360 microM (mean +/- s. d. EC50 116 +/- 75 microM, n = 83) for glycine and from 8 to 120 mM (mean EC50 40 +/- 30 mM, n = 37) for GABA. 3. In BOSC cells the EC50 varied from 9 to 92 microM (mean EC50 33 +/- 17 microM, n = 19) and from 0.7 to 19.1 mM (mean EC50 4.9 +/- 4.7 mM, n = 29) for glycine and GABA, respectively. 4. GABA activated alphaZ1 GlyRs either as a weak or full agonist: its efficacy (defined as Imax,GABA/Imax,Gly) was related to EC50 by an exponential relationship. A linear relationship was observed between EC50 values for GABA and glycine. 5. In outside-out patches, GABA and glycine activated alphaZ1 with identical single-channel conductances (85-100 pS), but with different kinetics and marked effect of concentration on burst duration for glycine only. 6. In outside-out patches deactivation time constants were concentration dependent for glycine, but not for GABA. 7. Our data demonstrate that the kinetics of glycine and GABA interactions with alphaZ1 are different and that they determine the properties of these neurotransmitter actions on the GlyR.


Subject(s)
Glycine/pharmacology , Receptors, Glycine/drug effects , gamma-Aminobutyric Acid/pharmacology , Animals , Cell Line , Electric Conductivity , Female , Humans , Kinetics , Oocytes/metabolism , Receptors, Glycine/physiology , Xenopus laevis , Zebrafish
7.
Neuroscience ; 90(1): 303-17, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10188956

ABSTRACT

The glycine receptor is a ligand-gated anion channel protein, providing inhibitory drive within the nervous system. We report here the isolation and functional characterization of a novel alpha subunit (alphaZ1) of the glycine receptor from adult zebrafish (Danio rerio) brain. The predicted amino acid sequence is 86%, 81% and 77% identical to mammalian isoforms alpha1, alpha3 and alpha2, respectively. AlphaZ1 exhibits many of the molecular features of mammalian alpha1, but the sequence patterns in the M4 and C-terminal domains are more similar to alpha2/alpha3. Phylogenetic analysis indicates that alphaZ1 is more closely related to the mammalian alpha1 subunits, being positioned, however, on a distinct branch. The alphaZ1 messenger RNA is 9.5 kb, similar to that described previously for alpha1 messenger RNAs. When expressed in Xenopus oocytes or a human cell line (BOSC 23), alphaZ1 forms a homomeric receptor which is activated by glycine and antagonized by strychnine. This receptor demonstrates unexpectedly high sensitivity to taurine and can also be activated by GABA. These results are consistent with physiological findings in lamprey and goldfish, and they suggest that this teleost fish glycine receptor displays a lower selectivity to neurotransmitters than that reported for glycine mammalian receptors.


Subject(s)
Cloning, Molecular , Receptors, Glycine/genetics , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , Electrophysiology , Gene Expression , Molecular Sequence Data , Polymerase Chain Reaction , Zebrafish
8.
Cell Death Differ ; 3(1): 37-46, 1996 Jan.
Article in English | MEDLINE | ID: mdl-17180053

ABSTRACT

The Q10 gene is a member of the major histocompatibility complex of the mouse that is expressed in the liver and kidney of the adult. Using transient expression assays, we found that the Q10 promoter was activated by retinoic acid (RA) and exogenous RARs and/or RXRs in a cell type-dependent manner. In addition, the basal activity of the Q10 promoter in HepG2 cells is lowered by expressing a dominant negative form of RARalpha. Incidentally, we have identified two cis-elements which consist of sequences related to retinoic acid response elements (RAREs) and a putative cAMP responsive element (CRE) the sequence of which overlaps one of the RAREs. RAR, RXR, CREB-ATF, and COUP-TF factors bind these elements and/or affect their activity. We also demonstrate that the CRE mediates part of the stimulation induced by activation of the cAMP pathway on the Q10 promoter, the residual activation being mediated by RARs. Our results suggest that Q10 expression in liver depends upon RA and the interaction between nuclear receptors that are expressed in this organ. The overlapping of the CRE with one of the RAREs together with the results of PKA activation also suggest that RA and cAMP signalling pathways are linked.

9.
Immunol Today ; 11(8): 286-92, 1990 Aug.
Article in English | MEDLINE | ID: mdl-1698378

ABSTRACT

The expression of mouse MHC class I genes and their products in vivo reveals complex patterns of regulation. Different promoter elements, which are required for gene activation or modulation in response to various external stimuli, have now been characterized as well as the proteins that bind to them. As described here by Brigitte David-Watine and colleagues, the picture that has gradually emerged from these in vitro studies is of an intricate interplay of transacting factors that ultimately lead to the fine tuning of MHC class I expression in vivo.


Subject(s)
Gene Expression Regulation , Genes, MHC Class I , Transcriptional Activation , Adenoviridae/genetics , Adenoviridae/physiology , Animals , Base Sequence , Cell Transformation, Viral , Cyclic AMP/physiology , H-2 Antigens/biosynthesis , H-2 Antigens/genetics , Histocompatibility Antigens Class I/biosynthesis , Histocompatibility Antigens Class I/genetics , Humans , Interferons/pharmacology , Mice , Molecular Sequence Data , Organ Specificity , Rats , Regulatory Sequences, Nucleic Acid , Transcription Factors/physiology
10.
Int Immunol ; 2(10): 981-93, 1990.
Article in English | MEDLINE | ID: mdl-2078524

ABSTRACT

The murine MHC genes code for the classical H-2K, D, and L transplantation antigens, and for other class I-like proteins called Qa and TIa molecules. Most of the latter have a restricted tissue distribution whereas classical transplantation antigens are virtually expressed by all somatic cells of the adult organism. Q10 is a Qa region gene, which was found to be expressed in liver and yolk sac, a regulatory pattern more evocative of the expression of a large set of serum proteins secreted by the liver than of a classical class I antigen. First, we have characterized several regions in the promoter of Q10 which bind factors present in liver nuclear extracts. Our most striking observation is that one of these factors, which we named TA-f, binds in the TATA box region of Q10 and Kb and displays tissue-specific expression, in that we found the activity only in liver and kidney. Secondly, we have performed a comparative analysis of the 5' upstream sequences of Q10 with those of H-2Kb, Ld, and other Qa genes. We have shown that most of the regulatory elements involved in the ubiquitous expression of H-2Kb are punctually altered and not functional in Q10. Similarly, most of the binding sequences for liver factors in the Q10 promoter, except the TA region, do not exist in the other H-2 class I genes which, however, have conserved most of the functional regions defined in H-2Kb and Ld. These results suggest that the tissue-specific expression of Q10 is associated with both alteration of the sequences conferring ubiquitous expression to other class I genes and/or creation of new sequences able to bind liver-specific regulatory factors. However, our observations also suggest that a unique sequence, the TATA box, may confer differential regulation in different tissues, since it binds a factor whose expression is restricted to the liver and kidney.


Subject(s)
Carrier Proteins/genetics , Genes, MHC Class I , Histocompatibility Antigens Class I/genetics , Animals , Base Sequence , DNA/genetics , Enhancer Elements, Genetic , Genes, Regulator , H-2 Antigens/genetics , Liver/immunology , Mice , Molecular Sequence Data , Promoter Regions, Genetic , Sequence Homology, Nucleic Acid , TATA Box/genetics
11.
J Exp Med ; 166(2): 341-61, 1987 Aug 01.
Article in English | MEDLINE | ID: mdl-3036997

ABSTRACT

We have previously described the isolation of pH-2d-37, a cDNA clone that encodes a so far unknown, poorly polymorphic, class I surface molecule. We report here the isolation of the corresponding gene, its nucleotide sequence, and its localization in the Tla region of the murine MHC. Using a RNase mapping assay, we have confirmed that the second domain coding region of the 37 gene displays very limited polymorphism, and that the gene is transcribed in a broad variety of cell types, in contrast to the genes encoding the known Qa and TL antigens. Possible functions are discussed.


Subject(s)
Genes, MHC Class II , H-2 Antigens/genetics , Mice, Inbred BALB C/genetics , Animals , Base Sequence , Chromosome Mapping , DNA Restriction Enzymes , Mice , Polymorphism, Genetic , RNA, Messenger/analysis , Tissue Distribution
12.
Gene ; 61(2): 145-54, 1987.
Article in English | MEDLINE | ID: mdl-3443306

ABSTRACT

We have studied the pattern of expression of the Q10 gene, a H-2 class-I gene located in the major histocompatibility complex which encodes a soluble class-I molecule, in the mid-gestation mouse embryo, and compared it to those of two other class-I genes, namely Kd and 37, the latter gene located in the thymus leukemia region. We found that the steady-state amount of these different mRNAs gradually increased from day 13 to day 18. By comparison with the level of expression of these genes in adult liver, the increase during gestation was fairly more marked for Q10 mRNA than for the others. Furthermore, we found that the Q10 gene is transiently expressed in the endoderm layer of the visceral yolk sac and in the fetal heart. Expression in the latter tissue decreases abruptly while increasing in the liver. It has been proposed that the Q10 protein is involved in immune tolerance. However, the time course of expression of Q10 mRNA and its tissue distribution during embryogenesis suggest that the Q10 protein could play a role in the differentiation of hematopoietic stem cells.


Subject(s)
Genes, MHC Class I , H-2 Antigens/genetics , Mice/genetics , Animals , Gene Expression Regulation , Liver/embryology , Mice/embryology , RNA, Messenger/genetics , Tissue Distribution , Yolk Sac/physiology
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