Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Many receptors stimulate proliferation of NIH 3T3 cells in a ligand dependent fashion. Based on this observation, we developed a high throughput assay of cloned receptor pharmacology. In this assay, receptors are transiently co-expressed with the marker enzyme beta-galactosidase. Receptors that induce cellular proliferation select and amplify the cells that also express the marker, thus the ability of ligands to alter receptor activity are reported as changes in enzyme activity. In the present study, we used this assay to evaluate the ability of agonist ligands to stimulate four cloned receptors. The agonists phenylephrine, carbachol, substance P and nerve growth factor selectively stimulated cells transfected with the alpha-1b adrenergic, m4 muscarinic, NK1 neurokinin and trkA neurotrophin receptors, respectively. These data demonstrate that a high throughput colorimetric assay performed in 96 well plates can be used to evaluate the pharmacology of ligands for cloned receptors belonging to a wide range of functional and pharmacological classes.
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PMID:High throughput assays of cloned adrenergic, muscarinic, neurokinin, and neurotrophin receptors in living mammalian cells. 756 80

Gene therapy may be a useful means of delivering substances to the brain that are capable of preventing neuronal degeneration. In the present experiment, we determined whether intraparenchymal transplants of primary autologous cells genetically modified to produce nerve growth factor (NGF) would prevent injury-induced degeneration of cholinergic neurons. Cultured primary monkey fibroblasts were genetically modified to produce human NGF, and secreted 13.2 ng NGF/10(6) cells/h in vitro. Adult monkeys then underwent fornix transections to induce degeneration of basal forebrain cholinergic neurons, and received autologous grafts of either NGF-producing or control, beta-galactosidase-producing fibroblasts directly into the basal forebrain region. One month later, 61.7 +/- 8.9% of cholinergic neurons remained indentifiable in NGF-graft recipients compared to 26.2 +/- 5.0% in control graft recipients (P < 0.02). Neuronal protection correlated with the accuracy of graft placement: up to 92% protection from neuronal degeneration occurred when NGF-secreting grafts were accurately placed immediately adjacent to injured neurons. Thus, intraparenchymal NGF delivery to the adult primate brain by gene transfer can prevent the degeneration of basal forebrain cholinergic neurons. Gene therapy can target intraparenchymal brain sites for regionally specific neurotrophin delivery, thereby avoiding limitations imposed by diffusion of substances across the blood-brain barrier and through CNS parenchyma, while avoiding adverse effects of neurotrophic factors delivered in a non-directed manner to the central nervous system. The delivery of NGF by gene transfer to the brain merits further study as a means of preventing cholinergic neuronal degeneration in human disorders such as Alzheimer's disease.
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PMID:Gene therapy in the adult primate brain: intraparenchymal grafts of cells genetically modified to produce nerve growth factor prevent cholinergic neuronal degeneration. 873 62

The neurotrophins are a family of growth factors that play an important role in the development and maintenance of the nervous system. Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family that appears to participate in the maturation and function of mammalian auditory neurons. Forms of deafness due to varied injurious stimuli that are amenable to treatment with implantable prosthetic devices require the survival of these BDNF-responsive auditory neurons for effective outcome. To evaluate the feasibility of developing a gene therapy for deafness that may be used in conjunction with a prosthetic device, we constructed replication-defective herpes simplex virus (HSV) amplicon vectors that carry the human BDNF cDNA. Using these vectors, HSVbdnf and HSVbdnflac (expresses BDNF and Escherichia coli beta-galactosidase), we evaluated the expression and biological activity in established cell lines and explant cultures prepared from spiral ganglia of the murine ear. Gene transfer with HSVbdnf resulted in the efficient expression of human BDNF mRNA in murine fibroblasts. Using two BDNF-responsive cell lines, PC12trkB and MG87trkB, we demonstrate efficient secretion of biologically active BDNF. Finally, transduction of explanted spiral ganglia with HSVbdnflac elicited robust neuritic process outgrowth comparable to exogenously added BDNF. Overall, these data demonstrate that HSV vectors can efficiently transfer and express the BDNF gene in many cell types, including auditory neurons. Moreover, they suggest that similar vectors may be used to express the neurotrophin in auditory neurons in vivo and perhaps as adjunctive gene therapy for deafness.
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PMID:Defective HSV-1 vector expressing BDNF in auditory ganglia elicits neurite outgrowth: model for treatment of neuron loss following cochlear degeneration. 878 68

Little spontaneous regeneration of axons occurs after acute and chronic injury to the CNS. Previously we have shown that the continuous local delivery of neurotrophic factors to the acutely injured spinal cord induces robust growth of spinal and supraspinal axons. In the present study we examined whether chronically injured axons also demonstrate significant neurotrophin responsiveness. Adult rats underwent bilateral dorsal hemisection lesions that axotomize descending supraspinal pathways, including the corticospinal, rubrospinal, and cerulospinal tracts, and ascending dorsal spinal sensory projections. One to three months later, injured rats received grafts of syngenic fibroblasts genetically modified to produce nerve growth factor (NGF). Control subjects received unmodified cell grafts or cells transduced to express the reporter gene beta-galactosidase. Three to five months after grafting, animals that received NGF-secreting grafts showed dense growth of putative cerulospinal axons and primary sensory axons of the dorsolateral fasciculus into the grafted lesion site. Growth from corticospinal, raphaespinal, and local motor axons was not detected. Thus, robust growth of defined populations of supraspinal and spinal axons can be elicited in chronic stages after spinal cord injury by localized, continuous transgenic delivery of neurotrophic factors.
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PMID:Robust growth of chronically injured spinal cord axons induced by grafts of genetically modified NGF-secreting cells. 941 24

Functional loss after spinal cord injury (SCI) is caused, in part, by demyelination of axons surviving the trauma. Neurotrophins have been shown to induce oligodendrogliagenesis in vitro, but stimulation of oligodendrocyte proliferation and myelination by these factors in vivo has not been examined. We sought to determine whether neurotrophins can induce the formation of new oligodendrocytes and myelination of regenerating axons after SCI in adult rats. In this study, fibroblasts producing neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor, nerve growth factor, basic fibroblast growth factor, or beta-galactosidase (control grafts) were transplanted subacutely into the contused adult rat spinal cord. At 10 weeks after injury, all transplants contained axons. NT-3 and BDNF grafts, however, contained significantly more axons than control or other growth factor-producing grafts. In addition, significantly more myelin basic protein-positive profiles were detected in NT-3 and BDNF transplants, suggesting enhanced myelination of ingrowing axons within these neurotrophin-producing grafts. To determine whether augmented myelinogenesis was associated with increased proliferation of oligodendrocyte lineage cells, bromodeoxyuridine (BrdU) was used to label dividing cells. NT-3 and BDNF grafts contained significantly more BrdU-positive oligodendrocytes than controls. The association of these new oligodendrocytes with ingrowing myelinated axons suggests that NT-3- and BDNF-induced myelinogenesis resulted, at least in part, from expansion of oligodendrocyte lineage cells, most likely the endogenous oligodendrocyte progenitors. These findings may have significant implications for chronic demyelinating diseases or CNS injuries.
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PMID:Neurotrophin-3 and brain-derived neurotrophic factor induce oligodendrocyte proliferation and myelination of regenerating axons in the contused adult rat spinal cord. 965 Dec 18

Analysis of complex signalisation networks involving distinct cell types is required to understand most developmental processes. Differentiation of male germ cells in adult mammals involves such a cross-talk between Sertoli cells, the somatic component which supports and controls germinal differentiation, and germ cells at their successive maturation stages. We developed a gene trapping strategy to identify genes, which, in Sertoli cells, are either up- or down-regulated by signals emitted by the germinal component. A library of approximately 2,000 clones was constituted from colonies independently selected from the Sertoli line 15P-1 by growth in drug-containing medium after random integration of a promoter-less (beta)geo transgene (neo(r)-lacZ fusion), which will be expressed as a fusion transcript from a 'trapped' cellular promoter, different in each clone. A first screen conducted on 700 events identified six clones in which beta-galactosidase activity was increased and one in which it was repressed upon addition of germ cells. The targeted loci were identified by cloning and sequencing the genomic region 5' of the insert. One of them was identified as the gene encoding Fra1, a component of the AP1 transcription regulatory complex. Accumulation of Fra1 mRNA was induced, both in 15P-1 and in freshly explanted Sertoli cells, by addition of either round spermatids or nerve growth factor (NGF). The effect of NGF was mediated by the TrkA receptor and the ERK1-ERK2 kinase kinase pathway. Fos and Fra1 transcription were induced within the first hour after addition of the neurotrophin, but, unlike what is observed after serum induction in the same cells, a second wave of transcription of Fra1, but not of Fos, started 16 hours later and peaked at higher levels at about 20 hours. These results suggest that AP1 activation may be an important relay in the Sertoli-germ cell cross-talk, and validate the gene trapping approach as a tool for the identification of target genes in cell culture systems.
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PMID:Gene trap analysis of germ cell signaling to Sertoli cells: NGF-TrkA mediated induction of Fra1 and Fos by post-meiotic germ cells. 3109 34

Lesioned axons within the dorsal roots fail to regenerate through the peripheral nerve transition zone and into the spinal cord. This regenerative failure leads to a persistent loss of sensory function. To induce axonal growth across this barrier, we used recombinant adenovirus to express fibroblast growth factor-2 (FGF2), nerve growth factor (NGF), L1 cell adhesion molecule (L1), or beta-galactosidase (LacZ) within the endogenous glia of the dorsal spinal cord 16 d after injury. Expression of either FGF2 or NGF, but not L1 or LacZ, induced robust axonal regeneration into normal as well as ectopic locations within the dorsal spinal cord. This regeneration led to near-normal recovery of thermal sensory function. Functional recovery and the majority of regenerating axons within the dorsal horn disappeared with recutting of the sensory roots. Injections of adenovirus encoding NGF, but not FGF2, also resulted in extensive sprouting of noninjured sensory axons, which we previously demonstrated could cause hyperalgesia and chronic pain. Thus, neurotrophic factor gene therapy administered as late as 16 d after injury may serve as a useful treatment to elicit recovery after dorsal root avulsion; however, the choice of neurotrophin is important to induce selective regeneration of damaged axons.
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PMID:Functional regeneration of chronically injured sensory afferents into adult spinal cord after neurotrophin gene therapy. 1160 29

Ototoxicity is a major dose-limiting side effect of cisplatin chemotherapy for cancer patients. We previously demonstrated in vitro that herpes simplex type 1 (HSV-1) amplicon-mediated delivery of a neurotrophin-3 (NT-3)/myc chimera protects spiral ganglion neurons (SGNs) in murine cochlear cultures from cisplatin-induced ototoxicity. To extend these findings, a newly constructed amplicon vector (HSVnt-3myc/SV40lac) that expresses the NT-3myc chimera and the Escherichia coli beta-galactosidase (lacZ) reporter gene under separate transcriptional control was initially tested in vitro and then was delivered to the cochlea of aged mice that were subsequently treated with cisplatin. Successful transduction with the new amplicon was observed in vitro as determined by its capacity to infect SGNs and to express NT-3myc mRNA and protein. To determine whether amplicon-directed NT-3myc overexpression could abrogate the ototoxicity in vivo, two groups of aged mice (CBA) were inoculated with HSVnt-3myc/SV40lac or control vector, HSVSV40lac, preceding administration of cisplatin. Cochleas inoculated with HSVnt-3myc/SV40lac harbored significantly greater numbers of surviving SGNs and showed lower incidence of cisplatin-induced apoptosis or necrosis than those injected with the control virus. These data demonstrate that HSV amplicon-mediated NT-3 delivery can attenuate the ototoxic actions of cisplatin in the peripheral auditory system of the aged mouse. The potency of NT-3 in SGN neuroprotection suggests that in vivo neurotrophin-based gene therapy is a promising preventative treatment for chemical-induced hearing disorders, and potentially for hearing degeneration due to normal aging.
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PMID:Neurotrophin-3 transduction attenuates cisplatin spiral ganglion neuron ototoxicity in the cochlea. 1209 98

Some mammalian neurons undergo apoptosis after neurotrophin deprivation. We studied neuronally differentiated PC6-3 pheochromocytoma cells, which are highly dependent on nerve growth factor for survival. We found that transient transfection with green fluorescent protein or beta-galactosidase protected cells from apoptosis induced by nerve growth factor deprivation. Individual transfection reagent components did not produce increased viability of nerve growth factor-deprived cells. This apparent neuroprotective effect from transient transfection was specific to neurotrophin deprivation, as cells treated with H(2)O(2) or staurosporine were not protected. To determine the mechanism of neuroprotection after transfection, the transfection status of identified groups of cells was assessed both before and after nerve growth factor deprivation. The results were consistent with a model whereby cells that are transfected but not yet expressing the transfected protein are relatively protected from nerve growth factor deprivation. We suggest that apoptosis induced by neurotrophin deprivation may interact with processes of transient transfection and expression of foreign genes in neuronal cells. Not only should these interactions be considered in transient transfection studies of neurotrophin-deprived neurons, but also their elucidation could lead to novel methods for achieving neuroprotection.
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PMID:Transient transfection protects PC6-3 cells from apoptosis induced by nerve growth factor deprivation. 1253 34

Brain-derived neurotrophic factor (BDNF), one of the members of the nerve growth factor family of neurotrophins, is expressed in developing gustatory papillae and is thought to be the neurotrophin that supports gustatory innervation during development. BDNF expression does not cease after development but continues in some taste cells of adult mice. To determine which types of taste cells produce BDNF, we undertook an immunohistochemical study of taste cells in BDNF(LacZ) gene targeted "knock-in" adult mice. In these mice, beta-galactosidase (beta-gal) immunoreactivity is an indicator of cells that produce BDNF transcripts. In the tongues of adult BDNF(LacZ) mice, beta-gal (BDNF) is present in long slender taste cells, as well as pyriform taste cells. Bromodeoxyuridine labeling experiments in BDNF(LacZ) mice indicate that BDNF is not present in taste cells that are younger than 3 days postmitotic. BDNF mainly colocalizes with markers of type II and type III taste cells: ubiquitin carboxyl terminal hydrolase (PGP 9.5), serotonin (5-HT), neural cell adhesion molecule (N-CAM), synaptic associated protein of 25 kDa (SNAP-25), and to a lesser extent with alpha-gustducin. beta-Gal immunoreactivity is not associated with blood group H antigen, a marker of type I taste cells. We conclude that BDNF is absent from basal cells and type I (blood group H antigen immunoreactive) taste cells but is present in differentiated type II and type III taste cells. The presence of SNAP-25 in BDNF-expressing cells suggests a role for BDNF in synaptic formation and transmission.
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PMID:Brain-derived neurotrophic factor is present in adult mouse taste cells with synapses. 1262 64


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