Differences in benzo(a)pyrene metabolism between rodent liver microsomes and embryonic cells.
Differences in benzo(a)pyrene metabolite pattern have been shown by rodent liver microsomes (Sprague-Dawley) and rodent embryo cells from Syrian hamsters and NIH Swiss mice. Rodent liver induced by methylcholanthrene shows marked quantitative variation between species. Additional pattern changes were found in mouse and hamster embryo secondary cultures with a reduction of the K-region metabolites and a marked increase in 9-hydroxybenzo(a)-pyrene. These results are indicative of a region-specific attack on the carcinogen by the cell monooxygenases which is distinct from the liver attack of microsomal enzymes on benzo(a)pyrene. These results suggest that activation and detoxification of benzo(a)pyrene may be species and tissue variable, and susceptibility and resistence to malignant transformation may be predicted on induction of a fortuitous combination of intermediate metabolic steps. (+info)
A molecular pathway revealing a genetic basis for human cardiac and craniofacial defects.
Microdeletions of chromosome 22q11 are the most common genetic defects associated with cardiac and craniofacial anomalies in humans. A screen for mouse genes dependent on dHAND, a transcription factor implicated in neural crest development, identified Ufd1, which maps to human 22q11 and encodes a protein involved in degradation of ubiquitinated proteins. Mouse Ufd1 was specifically expressed in most tissues affected in patients with 22q11 deletion syndrome. The human UFD1L gene was deleted in all 182 patients studied with 22q11 deletion, and a smaller deletion of approximately 20 kilobases that removed exons 1 to 3 of UFD1L was found in one individual with features typical of 22q11 deletion syndrome. These data suggest that UFD1L haploinsufficiency contributes to the congenital heart and craniofacial defects seen in 22q11 deletion. (+info)
Regulation of neurotrophin-3 expression by epithelial-mesenchymal interactions: the role of Wnt factors.
Neurotrophins regulate survival, axonal growth, and target innervation of sensory and other neurons. Neurotrophin-3 (NT-3) is expressed specifically in cells adjacent to extending axons of dorsal root ganglia neurons, and its absence results in loss of most of these neurons before their axons reach their targets. However, axons are not required for NT-3 expression in limbs; instead, local signals from ectoderm induce NT-3 expression in adjacent mesenchyme. Wnt factors expressed in limb ectoderm induce NT-3 in the underlying mesenchyme. Thus, epithelial-mesenchymal interactions mediated by Wnt factors control NT-3 expression and may regulate axonal growth and guidance. (+info)
Low resting potential and postnatal upregulation of NMDA receptors may cause Cajal-Retzius cell death.
Using in situ patch-clamp techniques in rat telencephalic slices, we have followed resting potential (RP) properties and the functional expression of NMDA receptors in neocortical Cajal-Retzius (CR) cells from embryonic day 18 to postnatal day 13, the time around which these cells normally disappear. We find that throughout their lives CR cells have a relatively depolarized RP (approximately -50 mV), which can be made more hyperpolarized (approximately -70 mV) by stimulation of the Na/K pump with intracellular ATP. The NMDA receptors of CR cells are subjected to intense postnatal upregulation, but their similar properties (EC50, Hill number, sensitivity to antagonists, conductance, and kinetics) throughout development suggest that their subunit composition remains relatively homogeneous. The low RP of CR cells is within a range that allows for the relief of NMDA channels from Mg2+ blockade. Our findings are consistent with the hypothesis that CR cells may degenerate and die subsequent to uncontrolled overload of intracellular Ca2+ via NMDA receptor activation by ambient glutamate. In support of this hypothesis we have obtained evidence showing the protection of CR cells via in vivo blockade of NMDA receptors with dizocilpine. (+info)
The amyloid precursor protein interacts with Go heterotrimeric protein within a cell compartment specialized in signal transduction.
The function of the beta-amyloid protein precursor (betaAPP), a transmembrane molecule involved in Alzheimer pathologies, is poorly understood. We recently reported the presence of a fraction of betaAPP in cholesterol and sphingoglycolipid-enriched microdomains (CSEM), a caveolae-like compartment specialized in signal transduction. To investigate whether betaAPP actually interferes with cell signaling, we reexamined the interaction between betaAPP and Go GTPase. In strong contrast with results obtained with reconstituted phospholipid vesicles (Okamoto et al., 1995), we find that incubating total neuronal membranes with 22C11, an antibody that recognizes an N-terminal betaAPP epitope, reduces high-affinity Go GTPase activity. This inhibition is specific of Galphao and is reproduced, in the absence of 22C11, by the addition of the betaAPP C-terminal domain but not by two distinct mutated betaAPP C-terminal domains that do not bind Galphao. This inhibition of Galphao GTPase activity by either 22C11 or wild-type betaAPP cytoplasmic domain suggests that intracellular interactions between betaAPP and Galphao could be regulated by extracellular signals. To verify whether this interaction is preserved in CSEM, we first used biochemical, immunocytochemical, and ultrastructural techniques to unambiguously confirm the colocalization of Galphao and betaAPP in CSEM. We show that inhibition of basal Galphao GTPase activity also occurs within CSEM and correlates with the coimmunoprecipitation of Galphao and betaAPP. The regulation of Galphao GTPase activity by betaAPP in a compartment specialized in signaling may have important consequences for our understanding of the physiopathological functions of betaAPP. (+info)
Identification of the Kv2.1 K+ channel as a major component of the delayed rectifier K+ current in rat hippocampal neurons.
Molecular cloning studies have revealed the existence of a large family of voltage-gated K+ channel genes expressed in mammalian brain. This molecular diversity underlies the vast repertoire of neuronal K+ channels that regulate action potential conduction and neurotransmitter release and that are essential to the control of neuronal excitability. However, the specific contribution of individual K+ channel gene products to these neuronal K+ currents is poorly understood. We have shown previously, using an antibody, "KC, " specific for the Kv2.1 K+ channel alpha-subunit, the high-level expression of Kv2.1 protein in hippocampal neurons in situ and in culture. Here we show that KC is a potent blocker of K+ currents expressed in cells transfected with the Kv2.1 cDNA, but not of currents expressed in cells transfected with other highly related K+ channel alpha-subunit cDNAs. KC also blocks the majority of the slowly inactivating outward current in cultured hippocampal neurons, although antibodies to two other K+ channel alpha-subunits known to be expressed in these cells did not exhibit blocking effects. In all cases the blocking effects of KC were eliminated by previous incubation with a recombinant fusion protein containing the KC antigenic sequence. Together these studies show that Kv2.1, which is expressed at high levels in most mammalian central neurons, is a major contributor to the delayed rectifier K+ current in hippocampal neurons and that the KC antibody is a powerful tool for the elucidation of the role of the Kv2.1 K+ channel in regulating neuronal excitability. (+info)
Differential regulation of the human nidogen gene promoter region by a novel cell-type-specific silencer element.
Transfection analyses of the human nidogen promoter region in nidogen-producing fibroblasts from adult skin revealed multiple positive and negative cis-acting elements controlling nidogen gene expression. Characterization of the positive regulatory domains by gel mobility-shift assays and co-transfection studies in Drosophila SL2 cells unequivocally demonstrated that Sp1-like transcription factors are essential for a high expression of the human nidogen gene. Analysis of the negative regulatory domains identified a novel silencer element between nt -1333 and -1322, which is bound by a distinct nuclear factor, by using extracts from adult but not from embryonal fibroblasts. In embryonal fibroblasts, which express significantly higher amounts of nidogen mRNA as compared with adult fibroblasts, this inhibitory nidogen promoter region did not affect nidogen and SV40 promoter activities. The silencer element seems to be active only in nidogen-producing cells. Therefore this regulatory element might function in vivo to limit nidogen gene expression in response to external stimuli. However, none of the identified regulatory elements, including the silencer, contribute significantly to cell-specific expression of the human nidogen gene. Instead we provide evidence that gene expression in epidermal keratinocytes that are not producing nidogen is repressed by methylation-specific and chromatin-dependent mechanisms. (+info)
Onset of nucleolar and extranucleolar transcription and expression of fibrillarin in macaque embryos developing in vitro.
Specific aims were to characterize the onset of nucleolar and extranucleolar transcription and expression of the nucleolar protein fibrillarin during preimplantation development in vitro in macaque embryos using autoradiographic and immunocytochemical techniques. Autoradiography was performed on whole embryos cultured with [3H]uridine for assessment of nucleolar (rRNA) and extranucleolar (mRNA) transcription. Expression of fibrillarin was immunocytochemically assessed in whole embryos using a primary antibody against fibrillarin and a fluorescein isothiocyanate-conjugated secondary antibody. Extranucleolar incorporation of [3H]uridine was first detected in 2-cell embryos cultured 6-10 h with [3H]uridine. Culture with alpha-amanitin prevented incorporation of label in 2-cell embryos, and treatment with ribonuclease reduced the signal to background levels, indicating that [3H]uridine was incorporated into mRNA and not rRNA or DNA. Nucleolar incorporation of [3H]uridine was not evident in pronucleate-stage or 2- to 5-cell embryos, but it was detected in one 6-cell embryo and in all 8-cell to blastocyst-stage embryos. Fibrillarin was first expressed in some 6- to 7-cell embryos, but it was consistently expressed in all 8-cell embryos. Fibrillarin was localized to the perimeter of the nucleolar precursor bodies, forming a ring that completely encapsulated these structures. Fibrillarin was not expressed in 8- to 16-cell embryos cultured with alpha-amanitin, indicating that it is transcribed, rather than recruited, at the 8-cell stage. In conclusion, in in vitro-fertilized macaque embryos developing in vitro, extranucleolar synthesis of mRNA is initiated at the 2-cell stage while the onset of nucleolar transcription occurs at the 6- to 8-cell stage, coincident with expression of fibrillarin. (+info)