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1.
Antimicrob Agents Chemother ; 56(10): 5149-56, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22825118

ABSTRACT

Type III phosphatidylinositol-4-kinase beta (PI4KIIIß) was previously implicated in hepatitis C virus (HCV) replication by small interfering RNA (siRNA) depletion and was therefore proposed as a novel cellular target for the treatment of hepatitis C. Medicinal chemistry efforts identified highly selective PI4KIIIß inhibitors that potently inhibited the replication of genotype 1a and 1b HCV replicons and genotype 2a virus in vitro. Replicon cells required more than 5 weeks to reach low levels of 3- to 5-fold resistance, suggesting a high resistance barrier to these cellular targets. Extensive in vitro profiling of the compounds revealed a role of PI4KIIIß in lymphocyte proliferation. Previously proposed functions of PI4KIIIß in insulin secretion and the regulation of several ion channels were not perturbed with these inhibitors. Moreover, PI4KIIIß inhibitors were not generally cytotoxic as demonstrated across hundreds of cell lines and primary cells. However, an unexpected antiproliferative effect in lymphocytes precluded their further development for the treatment of hepatitis C.


Subject(s)
1-Phosphatidylinositol 4-Kinase/antagonists & inhibitors , Antiviral Agents/pharmacology , Hepacivirus/drug effects , Animals , Antiviral Agents/adverse effects , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Humans , Male , Mice , Rats , Rats, Sprague-Dawley
2.
J Biol Chem ; 276(25): 22258-64, 2001 Jun 22.
Article in English | MEDLINE | ID: mdl-11301334

ABSTRACT

The ZIP superfamily of transporters plays important roles in metal ion uptake in diverse organisms. There are 12 ZIP-encoding genes in humans, and we hypothesize that many of these proteins are zinc transporters. In this study, we addressed the role of one human ZIP gene, hZIP1, in zinc transport. First, we examined (65)Zn uptake activity in K562 erythroleukemia cells overexpressing hZIP1. These cells accumulated more zinc than control cells because of increased zinc influx. Moreover, consistent with its role in zinc uptake, hZIP1 protein was localized to the plasma membrane. Our results also demonstrated that hZIP1 is responsible for the endogenous zinc uptake activity in K562 cells. hZIP1 is expressed in untransfected K562 cells, and the increase in mRNA levels found in hZIP1-overexpressing cells correlated with the increased zinc uptake activity. Furthermore, hZIP1-dependent (65)Zn uptake was biochemically indistinguishable from the endogenous activity. Finally, inhibition of endogenous hZIP1 expression with antisense oligonucleotides caused a marked decrease in endogenous (65)Zn uptake activity. The observation that hZIP1 is the major zinc transporter in K562 cells, coupled with its expression in many normal cell types, indicates that hZIP1 plays an important role in zinc uptake in human tissues.


Subject(s)
Carrier Proteins/metabolism , Zinc/metabolism , Base Sequence , Carrier Proteins/genetics , Cation Transport Proteins , DNA Primers , Humans , K562 Cells , RNA, Messenger/genetics
3.
Biometals ; 14(3-4): 251-70, 2001.
Article in English | MEDLINE | ID: mdl-11831460

ABSTRACT

The last ten years have witnessed major advances in our understanding of zinc transporters and their regulation in eukaryotic organisms. Two families of transporters, the ZIP (Zrt-, Irt-like Protein) and CDF (Cation Diffusion Facilitator) families, have been found to play a number of important roles in zinc transport. These are ancient gene families that span all phylogenetic levels. The characterized members of each group have been implicated in the transport of metal ions, frequently zinc, across lipid bilayer membranes. This remarkable conservation of function suggests that other, as yet uncharacterized members of the family, will also be involved in metal ion transport. Many of the ZIP family transporters are involved in cellular zinc uptake and at least one member, the Zrt3 transporter of S. cerevisiae, transports stored zinc out of an intracellular compartment during adaptation to zinc deficiency. In contrast, CDF family members mediate zinc efflux out of cells or facilitate zinc transport into intracellular compartments for detoxification and/or storage. The activity of many of these transporters is regulated in response to zinc through transcriptional and post-transcriptional mechanisms to maintain zinc homeostasis at both the cellular and organismal levels.


Subject(s)
Carrier Proteins/genetics , Carrier Proteins/metabolism , Zinc/metabolism , Amino Acid Sequence , Animals , Carrier Proteins/chemistry , Cation Transport Proteins , Conserved Sequence , Eukaryotic Cells , Humans , Ion Transport , Mammals , Models, Biological , Models, Molecular , Molecular Sequence Data , Phylogeny , Plants/genetics , Plants/metabolism , Protein Processing, Post-Translational , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Sequence Homology, Amino Acid , Transcription, Genetic
4.
Proc Natl Acad Sci U S A ; 97(14): 7957-62, 2000 Jul 05.
Article in English | MEDLINE | ID: mdl-10884426

ABSTRACT

The Zap1p transcription factor senses cellular zinc status and increases expression of its target genes in response to zinc deficiency. Previously known Zap1p-regulated genes encode the Zrt1p, Zrt2p, and Zrt3p zinc transporter genes and Zap1p itself. To allow the characterization of additional genes in yeast important for zinc homeostasis, a systematic study of gene expression on the genome-wide scale was used to identify other Zap1p target genes. Using a combination of DNA microarrays and a computer-assisted analysis of shared motifs in the promoters of similarly regulated genes, we identified 46 genes that are potentially regulated by Zap1p. Zap1p-regulated expression of seven of these newly identified target genes was confirmed independently by using lacZ reporter fusions, suggesting that many of the remaining candidate genes are also Zap1p targets. Our studies demonstrate the efficacy of this combined approach to define the regulon of a specific eukaryotic transcription factor.


Subject(s)
Genome, Fungal , Regulon , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Trans-Activators/metabolism , Zinc/pharmacology , Gene Expression Profiling , Gene Expression Regulation, Fungal , Genes, Fungal , Homeostasis/genetics , Oligonucleotide Array Sequence Analysis , Transcription Factors , Transcription, Genetic
5.
EMBO J ; 19(12): 2845-55, 2000 Jun 15.
Article in English | MEDLINE | ID: mdl-10856230

ABSTRACT

All cells regulate their intracellular zinc levels. In yeast, zinc uptake is mediated by Zrt1p and Zrt2p, which belong to the ZIP family of metal transporters. Under zinc limitation, ZRT1 and ZRT2 transcription is induced by the Zap1p transcriptional activator. We describe here a new component of zinc homeostasis, vacuolar zinc storage, that is also regulated by Zap1p. Zinc-replete cells accumulate zinc in the vacuole via the Zrc1p and Cot1p transporters. Our results indicate that another zinc transporter, Zrt3p, mobilizes this stored zinc in zinc-limited cells. ZRT3 is a Zap1p-regulated gene whose transcription increases in low zinc. Zrt3p is also a member of the ZIP family and it localizes to the vacuolar membrane. The effects of ZRT3 mutation and overexpression on cell growth, cellular zinc accumulation and intracellular labile zinc pools are all consistent with its proposed role. Furthermore, we demonstrate that zrt3 mutants inefficiently mobilize stored zinc to offset deficiency. Thus, our studies define a system of zinc influx and efflux transporters in the vacuole that play important roles in zinc homeostasis.


Subject(s)
Carrier Proteins/metabolism , Cation Transport Proteins , Fungal Proteins/metabolism , Membrane Proteins/metabolism , Saccharomyces cerevisiae Proteins , Vacuoles/metabolism , Zinc/metabolism , Amino Acid Sequence , Biological Transport , Carrier Proteins/genetics , Epistasis, Genetic , Fungal Proteins/genetics , Genes, Fungal , Homeostasis , Membrane Proteins/genetics , Membrane Transport Proteins , Molecular Sequence Data , Multigene Family , Saccharomyces cerevisiae/physiology , Sequence Homology, Amino Acid
6.
J Biol Chem ; 275(8): 5560-4, 2000 Feb 25.
Article in English | MEDLINE | ID: mdl-10681536

ABSTRACT

Zinc is an essential nutrient for humans, yet we know little about how this metal ion is taken up by mammalian cells. In this report, we describe the characterization of hZip2, a human zinc transporter identified by its similarity to zinc transporters recently characterized in fungi and plants. hZip2 is a member of the ZIP family of eukaryotic metal ion transporters that includes two other human genes, hZIP1 and hZIP3, and genes in mice and rats. To test whether hZip2 is a zinc transporter, we examined (65)Zn uptake activity in transfected K562 erythroleukemia cells expressing hZip2 from the CMV promoter. hZip2-expressing cells accumulated more zinc than control cells because of an increased initial zinc uptake rate. This activity was time-, temperature-, and concentration-dependent and saturable with an apparent K(m) of 3 microM. hZip2 zinc uptake activity was inhibited by several other transition metals, suggesting that this protein may transport other substrates as well. hZip2 activity was not energy-dependent, nor did it require K(+) or Na(+) gradients. Zinc uptake by hZip2 was stimulated by HCO(3)(-) treatment, suggesting a Zn(2+)-HCO(3)(-) cotransport mechanism. Finally, hZip2 was exclusively localized in the plasma membrane. These results indicate that hZip2 is a zinc transporter, and its identification provides one of the first molecular tools to study zinc uptake in mammalian cells.


Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , Zinc Fingers , Amino Acid Sequence , Bicarbonates/pharmacology , Carrier Proteins/genetics , Cell Membrane/metabolism , Dose-Response Relationship, Drug , Fluorescent Antibody Technique, Indirect , Gene Expression , Humans , Hydrogen-Ion Concentration , K562 Cells , Kinetics , Molecular Sequence Data , Multigene Family , Sequence Homology, Amino Acid , Substrate Specificity , Time Factors , Zinc/pharmacokinetics
7.
J Comp Neurol ; 374(4): 523-34, 1996 Oct 28.
Article in English | MEDLINE | ID: mdl-8910733

ABSTRACT

The goal of this study was to characterize the synaptic organization of the normal human adult striatum for comparison with other species and with the diseased human striatum. Samples of striatal tissue from the Maryland Brain Collection obtained at autopsy with postmortem intervals of less than 4 hours were prepared for electron microscopic analysis according to standard techniques. The caudate nucleus and the putamen were similar in terms of the proportions of synaptic subtypes, the lengths of synaptic subtypes, and the area of most types of axon terminals. The proportions of major striatal synaptic subdivisions, such as axospinous synapses (83.5%) and asymmetric synapses (77.5%), were similar to that of the monkey (82% and 77%, respectively) but slightly lower than found in the rat (90% and 89%, respectively). Interestingly, the proportion of synapses with perforated postsynaptic densities (23%), a type of synapse thought to represent synaptic plasticity, was much higher in humans than in rats (5-8%). The lengths of asymmetric synapses (0.697 micron) were significantly longer than that of symmetric synapses (0.423 microns), a relationship found in other mammals. Also, the areas of terminals forming asymmetric synapses (0.707 micron2) were larger than those forming symmetric synapses (0.401 micron2), also consistent with data from other species. The length of axospinous synapses (0.656 micron) and the area of the terminals forming them (0.611 micron2) were not significantly different from the length of axodendritic synapses (0.523 micron) or the area of terminals forming them (0.602 micron2). This study is the first quantitative study on synaptic organization in human postmortem striatum. The results indicate that the synaptic organization of the human striatum is similar, but not identical, to that of other mammalian species.


Subject(s)
Neostriatum/ultrastructure , Synapses/ultrastructure , Adult , Aged , Basal Ganglia/ultrastructure , Caudate Nucleus/ultrastructure , Female , Humans , Male , Microscopy, Electron , Middle Aged , Presynaptic Terminals/physiology , Presynaptic Terminals/ultrastructure , Putamen/ultrastructure , Reference Values
8.
Synapse ; 20(3): 234-43, 1995 Jul.
Article in English | MEDLINE | ID: mdl-7570355

ABSTRACT

Neuroleptics given chronically to rats induce behavioral sequelae which mimic tardive dyskinesia in some respects. The intent of this study was to investigate the ultrastructural correlates of oral dyskinesias (vacuous chewing movements [VCMs]), induced by chronic haloperidol treatment. After 6 months of treatment, rats were divided into low or high VCM groups. Rats in the high VCM group were either sacrificed on drug or were withdrawn from drug for 4 weeks. Ultrastructural analyses of the striatum indicated that synaptic density: 1) was significantly decreased in both the low and high VCM groups compared to normal controls; 2) was more profoundly decreased in the high VCM group as compared to the low VCM group; and 3) recovered to normal following drug withdrawal. Compared to controls, the density of asymmetric synapses was reduced by a similar magnitude in both the low and high VCM groups, suggesting that this change is a result of haloperidol treatment and independent of VCMs. Conversely, the density of symmetric synapses was reduced compared to normal, only in the high VCM group, suggesting that this change is specifically related to the expression of VCMs. In addition, mitochondrial profiles were hypertrophied and less frequent in the high VCM group in comparison to controls; size, but not number, recovered following drug withdrawal. These results identify distinct ultrastructural correlates of chronic haloperidol treatment that are unique to rats that develop VCMs and suggest that these ultrastructural features may play a role in the pathophysiology of oral dyskinesias in rats.


Subject(s)
Antipsychotic Agents/pharmacology , Dyskinesia, Drug-Induced/pathology , Haloperidol/pharmacology , Neostriatum/pathology , Animals , Behavior, Animal/drug effects , Male , Microscopy, Electron , Mitochondria/drug effects , Mitochondria/ultrastructure , Neostriatum/ultrastructure , Rats , Rats, Sprague-Dawley , Synapses/drug effects , Synapses/ultrastructure
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