23 results on '"Albert VR"'
Search Results
2. Human placenta-derived adherent cells induce tolerogenic immune responses.
- Author
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Liu W, Morschauser A, Zhang X, Lu X, Gleason J, He S, Chen HJ, Jankovic V, Ye Q, Labazzo K, Herzberg U, Albert VR, Abbot SE, Liang B, and Hariri R
- Abstract
Human placenta-derived adherent cells (PDAC cells) are a culture expanded, undifferentiated mesenchymal-like population derived from full-term placental tissue, with immunomodulatory and anti-inflammatory properties. PDA-001 (cenplacel-L), an intravenous formulation of PDAC cells, is in clinical development for the treatment of autoimmune and inflammatory diseases. To elucidate the mechanisms underlying the immunoregulatory properties of PDAC cells, we investigated their effects on immune cell populations, including T cells and dendritic cells (DC) in vitro and in vivo. PDAC cells suppressed T-cell proliferation in an OT-II T-cell adoptive transfer model, reduced the severity of myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis and ameliorated inflammation in a delayed type hypersensitivity response model. In vitro, PDAC cells suppressed T-cell proliferation and inhibited Th1 and Th17 differentiation. Analysis of tissues derived from PDAC cell-treated animals revealed diminished CD86 expression on splenic DC, suggesting that they can also modulate DC populations. Furthermore, PDAC cells modulate the differentiation and maturation of mouse bone marrow-derived DC. Similarly, human DC differentiated from CD14(+) monocytes in the presence of PDAC cells acquired a tolerogenic phenotype. These tolerogenic DC failed to induce allogeneic T-cell proliferation and differentiation toward Th1, but skewed T-cell differentiation toward Th2. Inhibition of cyclo-oxygenase-2 activity resulted in a significant, but not complete, abrogation of PDAC cells' effects on DC phenotype and function, implying a role for prostaglandin E2 in PDAC-mediated immunomodulation. This study identifies modulation of DC differentiation toward immune tolerance as a key mechanism underlying the immunomodulatory activities of PDAC cells.
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- 2014
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3. The remarkable flexibility of the human antibody repertoire; isolation of over one thousand different antibodies to a single protein, BLyS.
- Author
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Edwards BM, Barash SC, Main SH, Choi GH, Minter R, Ullrich S, Williams E, Du Fou L, Wilton J, Albert VR, Ruben SM, and Vaughan TJ
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- Amino Acid Sequence, B-Cell Activating Factor, Complementarity Determining Regions, Humans, Immunoglobulin Heavy Chains, Immunoglobulin Light Chains, Molecular Sequence Data, Peptide Library, Phylogeny, Point Mutation, Antibodies chemistry, Antibodies classification, Antibodies genetics, Antibodies immunology, Membrane Proteins immunology, Tumor Necrosis Factor-alpha immunology
- Abstract
It is well established that the humoral immune response can generate antibodies to many different antigens. The antibody diversity required to achieve this is believed to be substantial. However, the extent to which the immune repertoire can generate structural diversity against a single target antigen has never been addressed. Here, we have used phage display to demonstrate the extraordinary capacity of the human antibody repertoire. Over 1000 antibodies, all different in amino acid sequence, were generated to a single protein, B-lymphocyte stimulator (BLyS protein). This is a highly diverse panel of antibodies as exemplified by the extensive heavy and light chain germline usage: 42/49 functional heavy chain germlines and 19/33 V(lambda) and 13/35 V(kappa) light chain germlines were all represented in the panel of antibodies. Moreover, a high level of sequence diversity was observed in the V(H) CDR3 domains of these antibodies, with 568 different amino acid sequences identified. Thus we have demonstrated that specific recognition of a single antigen can be achieved from many different VDJ combinations, illustrating the remarkable problem-solving ability of the human immune repertoire. When studied in a biochemical assay, around 500 (40%) of these antibodies inhibited the binding of BLyS to its receptors on B-cell lines. The most potent antibodies inhibited BLyS binding with sub-nanomolar IC(50) values and with sub-nanomolar affinities. Such antibodies provide excellent choices as candidates for the treatment of BLyS-associated autoimmune diseases.
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- 2003
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4. Generation and characterization of LymphoStat-B, a human monoclonal antibody that antagonizes the bioactivities of B lymphocyte stimulator.
- Author
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Baker KP, Edwards BM, Main SH, Choi GH, Wager RE, Halpern WG, Lappin PB, Riccobene T, Abramian D, Sekut L, Sturm B, Poortman C, Minter RR, Dobson CL, Williams E, Carmen S, Smith R, Roschke V, Hilbert DM, Vaughan TJ, and Albert VR
- Subjects
- Animals, Antibodies, Monoclonal administration & dosage, Antibodies, Monoclonal immunology, B-Cell Activation Factor Receptor, B-Cell Maturation Antigen, B-Lymphocytes drug effects, B-Lymphocytes immunology, Cell Division drug effects, Dose-Response Relationship, Drug, Female, Humans, Injections, Intravenous, Leukocytes, Mononuclear drug effects, Lymph Nodes cytology, Lymph Nodes drug effects, Macaca fascicularis, Male, Mice, Mutagenesis, Site-Directed, Neutralization Tests, Receptors, Tumor Necrosis Factor administration & dosage, Receptors, Tumor Necrosis Factor immunology, Spleen cytology, Spleen drug effects, Transmembrane Activator and CAML Interactor Protein, Antibodies, Monoclonal biosynthesis, B-Lymphocytes metabolism, Membrane Proteins, Receptors, Tumor Necrosis Factor metabolism
- Abstract
Objective: To identify and characterize a fully human antibody directed against B lymphocyte stimulator (BLyS), a tumor necrosis factor-related cytokine that plays a critical role in the regulation of B cell maturation and development. Elevated levels of BLyS have been implicated in the pathogenesis of autoimmune diseases., Methods: A human phage display library was screened for antibodies against human BLyS. A human monoclonal antibody, LymphoStat-B, specific for human BLyS was obtained from the library screening and subsequent affinity optimization mutagenesis. The antibody was tested for inhibition of human BLyS in vitro and in an in vivo murine model. Additionally, the consequences of BLyS inhibition were tested in vivo by administration of LymphoStat-B to cynomolgus monkeys., Results: LymphoStat-B bound with high affinity to human BLyS and inhibited the binding of BLyS to its 3 receptors, TACI, BCMA, and BLyS receptor 3/BAFF-R. LymphoStat-B potently inhibited BLyS-induced proliferation of B cells in vitro, and administration of LymphoStat-B to mice prevented human BLyS-induced increases in splenic B cell numbers and IgA titers. In cynomolgus monkeys, administration of LymphoStat-B resulted in decreased B cell representation in both spleen and mesenteric lymph nodes., Conclusion: A fully human monoclonal antibody has been isolated that binds to BLyS with high affinity and neutralizes human BLyS bioactivity in vitro and in vivo. Administration of this antibody to cynomolgus monkeys resulted in B cell depletion in spleen and lymph node. This antibody may prove therapeutically useful in the treatment of autoimmune diseases in humans.
- Published
- 2003
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5. Tissue-specific expression of the nonneuronal promoter of the aromatic L-amino acid decarboxylase gene is regulated by hepatocyte nuclear factor 1.
- Author
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Aguanno A, Afar R, and Albert VR
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- Animals, Antibodies pharmacology, Base Sequence, Cell Line, Hepatocyte Nuclear Factor 1, Hepatocyte Nuclear Factor 1-alpha, Hepatocyte Nuclear Factor 1-beta, Humans, Kidney, Luciferases biosynthesis, Molecular Sequence Data, Neurons, Oligodeoxyribonucleotides, Organ Specificity, Plasmids, Polymerase Chain Reaction, Rats, Recombinant Proteins biosynthesis, Restriction Mapping, Sequence Homology, Nucleic Acid, Transcription Factors immunology, Transcription, Genetic, Transfection, Aromatic-L-Amino-Acid Decarboxylases biosynthesis, Aromatic-L-Amino-Acid Decarboxylases genetics, DNA-Binding Proteins, Gene Expression Regulation, Enzymologic drug effects, Nuclear Proteins, Promoter Regions, Genetic, Transcription Factors physiology
- Abstract
The rat aromatic l-amino acid decarboxylase (AADC) gene contains alternative promoters which direct expression of neuronal and nonneuronal mRNAs that differ only in their 5'-untranslated regions (UTRs). We have analyzed the expression of the nonneuronal promoter of the rat AADC gene in the kidney epithelial cell line LLC-PK1 and in cells which do not express the nonneuronal form of AADC by transient transfection. These studies revealed that the first 1.1 kilobases of the nonneuronal promoter, including the nonneuronal-specific 5'-UTR (Exon 1), contains sufficient information to direct tissue-specific expression. Serial deletions of this promoter localized the cis-active element to a region between -52 and -28 base pairs upstream of the nonneuronal transcription start site. An A/T-rich sequence, within this region which we have termed KL-1, was found to bind a kidney and liver-specific factor by DNase footprint analysis and was capable of directing tissue-specific expression from a heterologous promoter. Moreover, when the KL-1 sequence was mutated in the context of the entire promoter sequence, all transcriptional activity was abolished. DNA sequence comparison revealed that the KL-1 fragment is highly homologous to the binding site for hepatocyte nuclear factor-1 (HNF-1). Mobility shift studies utilizing an antibody to HNF-1 demonstrated binding of HNF-1 to the KL-1 fragment and cotransfection of HNF-1 cDNA into cells which do not express the nonneuronal form of AADC resulted in activation of transfected AADC nonneuronal promoter constructs. These results strongly suggest that the transcription factor which regulates the tissue-specific expression of the nonneuronal form of AADC mRNA is HNF-1.
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- 1996
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6. Positive and negative elements contribute to the cell-specific expression of the rat dopamine beta-hydroxylase gene.
- Author
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Afar R, Silverman R, Aguanno A, and Albert VR
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- Analysis of Variance, Animals, Base Sequence, Cloning, Molecular, Genome, Humans, Molecular Sequence Data, Organ Specificity, PC12 Cells, RNA, Messenger biosynthesis, Rats, Transfection, Tumor Cells, Cultured, Dopamine beta-Hydroxylase genetics, Gene Expression Regulation physiology, Promoter Regions, Genetic
- Abstract
Dopamine beta-hydroxylase catalyzes the final step in noradrenaline synthesis and is expressed exclusively in noradrenergic and adrenergic cells. In order to identify elements within the dopamine beta-hydroxylase (DBH) gene which contribute to the regulation of tissue-specific expression, we have analyzed the expression of the rat DBH promoter by transient transfection in both DBH-expressing and non-expressing cell lines. We have found that 1 kilobase of the DBH promoter can direct expression of the luciferase reporter gene in the DBH-expressing PC12, CATH.a, and SK-N-SH cell lines, but not in the non-DBH-expressing C6 glioma or CA77 cell lines. This activity was localized to a region between -133 and -173 upstream of the transcription start site. This element, however, also directed expression in non-DBH-expressing cell lines, but was inhibited when sequences between -212 and -388 were included. This inhibitory region contains sequences homologous to a silencer element recently identified in the human DBH gene, and shares homology with other previously identified silencer elements. Gel retardation experiments demonstrate that the rat DBH inhibitory region and the silencer elements found in the rat sodium type II channel and SCG10 genes bind a similar factor. The region between -133 and -173, which contains a consensus cyclic AMP response element (CRE), was also found to be responsive to cAMP in both DBH-expressing and non-expressing cells. Inclusion of sequences between -173 and -190 diminished the cAMP induction in PC12 cells, and nearly abolished the induction in C6 and CA77 cells, suggesting the presence of an additional negative element which inhibits cAMP induction in non-DBH expressing cells. DNA binding assays using antibodies to CRE binding protein-related transcription factors identified ATF-1 binding to the rat DBH-CRE, and further suggest that inhibition of cAMP regulation may be due to inhibition of ATF-1 binding by an additional factor, which binds to the DBH promoter immediately upstream of the CRE. These results demonstrate the importance of both positive and negative regulatory elements in the regulation of tissue-specific expression of the rat DBH gene.
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- 1996
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7. Analysis of the neuronal promoter of the rat aromatic L-amino acid decarboxylase gene.
- Author
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Aguanno A, Lee MR, Marden CM, Rattray M, Gault A, and Albert VR
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- Animals, Base Sequence, DNA Footprinting, Deoxyribonuclease I, In Situ Hybridization, Molecular Probes genetics, Molecular Sequence Data, PC12 Cells, RNA, Messenger analysis, Rats, Ribonucleases, Aromatic-L-Amino-Acid Decarboxylases genetics, Genes, Neurons physiology, Promoter Regions, Genetic
- Abstract
The rat aromatic L-amino acid decarboxylase (AADC) gene contains alternative promoters directing expression of neuronal and nonneuronal mRNAs that differ only in their 5' untranslated regions (UTRs). We have analyzed the expression of the neuronal promoter of the AADC gene in cells synthesizing catecholamines and serotonin, as well as in non-AADC-expressing cells. We demonstrate the use of the neuronal-specific UTR in individual dopamine-, norepinephrine-, and serotonin-containing neurons. Transfection analyses show that the rat AADC neuronal promoter, containing 2,400 bp upstream of the transcription start site and including the 68-bp untranslated exon 2, can activate transcription from a reporter gene in both catecholaminergic and serotonergic cell lines. These analyses identified several positive and negative cis-active elements within this region. Unexpectedly, we observed that this promoter, when removed from its native context within the AADC gene, can also direct expression of a reporter gene in cells that do not normally express AADC mRNA. These results suggest that tissue-specific expression of the neuronal promoter may not be controlled by cis-active elements within the first 2,400 bp of the promoter. Additional information may be required to restrict neuronal promoter expression to appropriate cell types. This regulatory information could reside elsewhere within the promoter, within introns, or may be provided by interactions between the two AADC promoters.
- Published
- 1995
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8. Distinct promoters direct neuronal and nonneuronal expression of rat aromatic L-amino acid decarboxylase.
- Author
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Albert VR, Lee MR, Bolden AH, Wurzburger RJ, and Aguanno A
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- Animals, Base Sequence, Cloning, Molecular, Gene Expression Regulation, Genes, Molecular Sequence Data, Oligodeoxyribonucleotides chemistry, RNA Splicing, RNA, Messenger genetics, Rats, Sequence Alignment, Transcription, Genetic, Aromatic-L-Amino-Acid Decarboxylases genetics, Neurons physiology, Promoter Regions, Genetic
- Abstract
Aromatic L-amino acid decarboxylase (AADC, EC 4.1.1.28) catalyzes the decarboxylation of L-dopa to dopamine in catecholamine cells and 5-hydroxytryptophan to serotonin in serotonin-producing neurons. This enzyme is also expressed in relatively large quantities in nonneuronal tissues such as liver and kidney, where its function is unknown. Neuronal and nonneuronal tissues express AADC mRNAs with distinct 5' untranslated regions. To understand how this is accomplished at the genomic level, we have isolated rat genomic DNA encoding AADC. The organization of the AADC gene suggests that there are two separate promoters specific for the transcription of neuronal and nonneuronal forms of the AADC message. A small exon containing 68 bases of the neuronal-specific 5' end is located approximately 9.5 kilobases upstream of the translation start site, which is contained in the third exon. Approximately 7 kilobases upstream from the neuron-specific promoter is another small exon containing 71 bases of the 5' end of the nonneuronal AADC message. These data suggest that transcription initiating at distinct promoters, followed by alternative splicing, is responsible for the expression of the neuronal and nonneuronal forms of the AADC message.
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- 1992
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9. Autoregulation of pit-1 gene expression mediated by two cis-active promoter elements.
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Chen RP, Ingraham HA, Treacy MN, Albert VR, Wilson L, and Rosenfeld MG
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- Animals, Base Sequence, Cell Line, Cell Nucleus metabolism, Cyclic AMP Response Element-Binding Protein, DNA-Binding Proteins metabolism, Kinetics, Molecular Sequence Data, Oligonucleotide Probes, Rats, Transcription Factor Pit-1, Transcription Factors metabolism, Transcription, Genetic, DNA-Binding Proteins genetics, Gene Expression Regulation, Multigene Family, Promoter Regions, Genetic, Transcription Factors genetics
- Abstract
The pit-1 gene is a member of a large family of genes that encode proteins which are involved in development and which contain a highly homologous region, referred to as the POU domain. Pit-1, a pituitary-specific transcription factor, can activate the transcription of the growth hormone and prolactin promoters. It is expressed in mature thyrotroph, somatotroph and lactotroph cell types of the anterior pituitary which arise sequentially during development; somatotrophs and lactotrophs, which secrete growth hormone and prolactin, respectively, are the last to arise. Intriguingly, during ontogeny, pit-1 transcripts are observed in the rat neural tube and neural plate (embryonic day 10-11) and disappear thereafter (day 13), only to reappear exclusively in the anterior lobe of the pituitary gland (day 15) just before activation of prolactin and growth hormone. This biphasic pattern suggests a complex mechanism of initial activation of pit-1 gene expression. Transcription and transfection analyses in vitro using wild-type and mutated promoters indicate that Pit-1 can positively autoregulate the expression of the pit-1 promoter as a consequence of binding to two Pit-1-binding elements. Mutation of the 5' Pit-1-binding site abolished positive autoregulation, whereas mutation of the element located immediately 3' of the cap site markedly increased expression of the pit-1 promoter. These data are consistent with a positive, attenuated autoregulatory loop that seems to function in maintaining pit-1 gene expression.
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- 1990
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10. The POU-specific domain of Pit-1 is essential for sequence-specific, high affinity DNA binding and DNA-dependent Pit-1-Pit-1 interactions.
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Ingraham HA, Flynn SE, Voss JW, Albert VR, Kapiloff MS, Wilson L, and Rosenfeld MG
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- Algorithms, Animals, Base Sequence, Binding Sites, DNA-Binding Proteins genetics, Kinetics, Molecular Sequence Data, Mutation, Oligonucleotide Probes chemical synthesis, Plasmids, Polymerase Chain Reaction, Protein Binding, Protein Conformation, Restriction Mapping, Transcription Factor Pit-1, Transcription Factors genetics, Transcriptional Activation, Transfection, DNA metabolism, DNA-Binding Proteins metabolism, Oligodeoxyribonucleotides metabolism, Transcription Factors metabolism
- Abstract
Pit-1 is a member of a family of transcription factors sharing two regions of homology: a highly conserved POU-specific (POUS) domain and a more divergent homeodomain (POUHD). Analysis of mutant Pit-1 proteins suggests that, while the POUHD is required and sufficient for low affinity DNA binding, the POUS domain is necessary for high affinity binding and accurate recognition of natural Pit-1 response elements. Pit-1 is monomeric in solution but associates as a dimer on its DNA response element, exhibiting DNA-dependent protein-protein interactions requiring the POUS domain. Analysis of alpha-helical domains and conserved structures in Pit-1 suggests that POU domain proteins interact with their DNA recognition sites differently than classic homeodomain proteins, with both the POUHD and the POUS domain contacting DNA. Transcriptional activity of Pit-1 on enhancer elements is conferred primarily by a Ser- and Thr-rich N-terminal region unrelated to other known transcription-activating motifs.
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- 1990
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11. A family of POU-domain and Pit-1 tissue-specific transcription factors in pituitary and neuroendocrine development.
- Author
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Ingraham HA, Albert VR, Chen RP, Crenshaw 3d EB, Elsholtz HP, He X, Kapiloff MS, Mangalam HJ, Swanson LW, and Treacy MN
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- Animals, Brain growth & development, Gene Expression Regulation, Genes, Regulator, Growth Hormone genetics, Growth Hormone metabolism, Neurons metabolism, Pituitary Gland, Anterior cytology, Pituitary Gland, Anterior metabolism, Prolactin genetics, Thyrotropin metabolism, Transcription Factors metabolism, Transcription, Genetic, Transcriptional Activation, Neurosecretory Systems growth & development, Pituitary Gland growth & development, Transcription Factors physiology
- Abstract
The anterior pituitary gland provides a model for investigating the molecular basis for the appearance of phenotypically distinct cell types, within an organ, a central question in development. The rat prolactin and growth hormone genes are selectively expressed in distinct cell types (lactotrophs and somatotrophs) of the anterior pituitary gland, which reflect differential mechanisms of gene activation or restriction because of interactions of multiple factors binding to these genes. We find that the pituitary-specific 33,000 dalton transcription factor, Pit-1, normally expressed in somatotrophs, lactotrophs, and thyrotrophs, can bind to and activate both growth hormone and prolactin promoters in vitro at levels even tenfold lower than those normally present in pituitary cells. In the case of the prolactin gene, high levels of expression in transgenic animals required two cis-active regions; a distal enhancer (-1.8 to -1.5 kb) and a proximal region (-422 to +33 bp). Each of these regions alone can direct low levels of fusion gene expression to prolactin-producing cell types in transgenic mice, but a synergistic interaction between these regions is necessary for high levels of expression. The initial appearance of the prolactin transgene expression closely follows the appearance of high levels of Pit-1, but later increases in expression coincident with appearance of mature lactotrophs suggest the operation of additional, critical positive factor(s). Unexpectedly, transgenes containing the distal enhancer removed from its normal context are expressed in both the prolactin-producing lactotrophs and the TSH-producing thyrotrophs, thereby suggesting that sequences flanking this enhancer are necessary to restrict expression to the correct cell type within the pituitary. These data indicate that distinct processes of gene activation and restriction are necessary for the fidelity of cell-type specific expression within an organ. Consistent with this model, we find that lactotroph cell lines that cannot express the growth hormone gene contain high levels of functional Pit-1. We suggest a large, highly related POU-domain gene family, potentially exceeding 100 members, has been conserved and expanded in evolution to meet the increasing requirements for more intricate patterns of cell phenotypes. The POU-domain subgroup of the homeodomain gene family, in concert with other homeodomain proteins and with other classes of transcription factors, is likely to contribute to the establishment of the mammalian neuroendocrine system.
- Published
- 1990
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12. A two-base change in a POU factor-binding site switches pituitary-specific to lymphoid-specific gene expression.
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Elsholtz HP, Albert VR, Treacy MN, and Rosenfeld MG
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- Animals, Base Sequence, Binding Sites, Binding, Competitive, Cell Line, Genes, Immunoglobulin, Molecular Sequence Data, Mutation, Prolactin genetics, Rats, Transcription, Genetic, Transfection, B-Lymphocytes metabolism, Gene Expression Regulation, Pituitary Gland metabolism, Promoter Regions, Genetic, Transcription Factors metabolism
- Abstract
The structurally related POU homeo domain proteins Pit-1 and Oct-2 activate pituitary- and lymphoid-specific transcription, respectively, by binding to similar AT-rich motifs in their target genes. In this study we identify bases critical for recognition and activation by Pit-1 and examine how small differences in Pit-1 and Oct-2-binding sites can impart differential transcriptional responses in pituitary and B-lymphoid cells. Scanning mutagenesis of Pit-1 response elements in both the rat prolactin and growth hormone genes reveals a critical binding motif recognized in an identical manner by the native Pit-1 protein and cloned Pit-1 gene product. This motif, ATTATTCCAT, differs by only two bases from the octamer element, ATTTGCAT, required for Oct-2-dependent activation of immunoglobulin genes. Cross recognition of Pit-1 and Oct-2 sites by both factors can be demonstrated in competitive binding assays, in which an oligometric Pit-1 site from the prolactin gene is converted to an Oct-2 site by a double point mutation. In contrast to the binding data, no cross activation of transcription is detectable in cultured cell lines. When inserted immediately 5' to a prolactin TATA box, the wild-type prolactin element enhances transcription strongly in pituitary cells but is inactive in B cells, whereas the octamer variant of the prolactin site activates expression in B cells but is silent in pituitary lines. Both elements are nonfunctional in heterologous cell lines that lack Pit-1 and Oct-2.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1990
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13. A pituitary POU domain protein, Pit-1, activates both growth hormone and prolactin promoters transcriptionally.
- Author
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Mangalam HJ, Albert VR, Ingraham HA, Kapiloff M, Wilson L, Nelson C, Elsholtz H, and Rosenfeld MG
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- Animals, Cell Line, Chromatography, Affinity, Deoxyribonuclease I metabolism, Gene Expression Regulation, Genetic Vectors, HeLa Cells, Humans, Nucleic Acid Hybridization, Phenotype, Rats, Simian virus 40 genetics, Transcription, Genetic, Transcriptional Activation, Transfection, Growth Hormone genetics, Prolactin genetics, Promoter Regions, Genetic, Transcription Factors genetics
- Abstract
The anterior pituitary gland provides a model for investigating the molecular basis for the appearance of phenotypically distinct cell types within an organ, a central question in development. The rat prolactin and growth hormone genes are expressed selectively in distinct cell types (lactotrophs and somatotrophs, respectively) of the anterior pituitary gland, reflecting differential mechanisms of gene activation or restriction, as a result of the interactions of multiple factors binding to these genes. We find that when the pituitary-specific 33-kD transcription factor Pit-1, expressed normally in both lactotrophs and somatotrophs, is expressed in either the heterologous HeLa cell line or in bacteria, it binds to and activates transcription from both growth hormone and prolactin promoters in vitro at levels even 10-fold lower than those normally present in pituitary cells. This suggests that a single factor, Pit-1, may be capable of activating the expression of two genes that define different anterior pituitary cell phenotypes. Because a putative lactotroph cell line (235-1) that does not express the growth hormone gene, but only the prolactin gene, appears to contain high levels of functional Pit-1, a mechanism selectively preventing growth hormone gene expression may, in part, account for the lactotroph phenotype.
- Published
- 1989
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14. Discrete cis-active genomic sequences dictate the pituitary cell type-specific expression of rat prolactin and growth hormone genes.
- Author
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Nelson C, Crenshaw EB 3rd, Franco R, Lira SA, Albert VR, Evans RM, and Rosenfeld MG
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- Animals, Base Sequence, Genes, Rats, Gene Expression Regulation, Growth Hormone genetics, Pituitary Gland cytology, Prolactin genetics
- Abstract
The anterior pituitary gland, which is derived from a common primordium originating in Rathke's pouch, contains phenotypically distinct cell types, each of which express discrete trophic hormones: adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH), prolactin, growth hormone, and follicle stimulating hormone (FSH)/luteinizing hormone (LH). The structurally related prolactin and growth hormone genes, which are evolutionarily derived from a single primordial gene, are expressed in discrete cell types--lactotrophs and somatotrophs, respectively--with their expression virtually limited to the pituitary gland. The pituitary hormones exhibit a temporal pattern of developmental expression with rat growth hormone and prolactin characteristically being the last hormones expressed. The reported co-expression of these two structurally related neuroendocrine genes within single cells prior to the appearance of mature lactotrophs, in a subpopulation of mature anterior pituitary cells, and in many pituitary adenomas raises the possibility that the prolactin and growth hormone genes are developmentally controlled by a common factor(s). We now report the identification and characterization of nucleotide sequences in the 5'-flanking regions of the rat prolactin and growth hormone genes, respectively, which act in a position- and orientation-independent fashion to transfer cell-specific expression to heterologous genes. At least one putative trans-acting factor required for the growth hormone genomic sequence to exert its effects is apparently different from those modulating the corresponding enhancer element(s) of the prolactin gene because a pituitary 'lactotroph' cell line producing prolactin but not growth hormone selectively fails to express fusion genes containing the growth hormone enhancer sequence.
- Published
- 1986
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15. A c-erb-A binding site in rat growth hormone gene mediates trans-activation by thyroid hormone.
- Author
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Glass CK, Franco R, Weinberger C, Albert VR, Evans RM, and Rosenfeld MG
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- Animals, Binding Sites, DNA, Recombinant, DNA-Binding Proteins physiology, Enhancer Elements, Genetic, Rats, Transcription, Genetic, Gene Expression Regulation, Genes, Regulator, Growth Hormone genetics, Proto-Oncogene Proteins genetics, Receptors, Thyroid Hormone physiology, Regulatory Sequences, Nucleic Acid, Triiodothyronine physiology
- Abstract
The substance 3,5,3-triiodothyronine (T3) stimulates growth hormone gene transcription in rat pituitary tumour cells. This stimulation is thought to be mediated by the binding of nuclear T3 receptors to regulatory elements 5' to the transcriptional start site. Understanding of the mechanism by which thyroid hormone activates gene transcription has been limited by failure to purify nuclear T3 receptors because of their low abundance, and by the absence of defined T3 receptor-DNA binding sites affecting T3 regulation. Recently, human and avian c-erb-A gene products have been shown to bind thyroid hormone with high affinity and to have a molecular weight and nuclear association characteristic of the thyroid hormone receptor. In the present report, we describe the development of an avidin-biotin complex DNA-binding assay which can detect specific, high-affinity binding of rat pituitary cell T3 receptors to the sequence 5'CAGGGACGTGACCGCA3', located 164 base pairs 5' to the transcriptional start site of the rat growth hormone gene. An oligonucleotide containing this sequence transferred T3 regulation to the herpes simplex virus thymidine kinase promoter in transfected rat pituitary GC2 cells, and specifically bound an in vitro translation product of the human placental c-erb-A gene. The data provide supporting evidence that the human c-erb-A gene product mediates the transcriptional effects of T3 and also that GC2 cell nuclear extracts contain additional factors that modify the binding of pituitary T3 receptors to the rat growth hormone gene T3 response element.
- Published
- 1987
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16. Some neurons of the rat central nervous system contain aromatic-L-amino-acid decarboxylase but not monoamines.
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Jaeger CB, Teitelman G, Joh TH, Albert VR, Park DH, and Reis DJ
- Subjects
- Animals, Neurons enzymology, Neurotransmitter Agents biosynthesis, Rats, Aromatic-L-Amino-Acid Decarboxylases metabolism, Biogenic Amines metabolism, Brain metabolism, Spinal Cord metabolism
- Abstract
Neurons containing the enzyme aromatic-L-amino-acid decarboxylase (AADC) but lacking either tyrosine hydroxylase or serotonin were found in the spinal cord of neonatal and adult rats by light and electron microscopic immunocytochemistry. The majority of these neurons localized to area X of Rexed contact ependyma. Thus, spinal AADC neurons have the enzymatic capacity to catalyze directly the conversion of the amino acids tyrosine, tryptophan, or phenylalanine to their respective amines tyramine, tryptamine, or phenylethylamine. These amines normally present in the central nervous system may be of potential clinical significance as endogenous psychotomimetics.
- Published
- 1983
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17. A single gene codes for aromatic L-amino acid decarboxylase in both neuronal and non-neuronal tissues.
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Albert VR, Allen JM, and Joh TH
- Subjects
- Adrenal Medulla enzymology, Animals, Cattle, DNA analysis, Electrophoresis, Polyacrylamide Gel, Kidney enzymology, Liver enzymology, Rats, Aromatic-L-Amino-Acid Decarboxylases genetics, Neurons enzymology
- Abstract
We have sought to determine whether aromatic L-amino acid decarboxylase which functions as a neurotransmitter biosynthetic enzyme in neuronal cells can be distinguished from an enzyme with similar activity found in peripheral tissues where no neurotransmitters are synthesized. Aromatic L-amino acid decarboxylase was purified to electrophoretic homogeneity from bovine adrenal medulla, and highly specific antibodies were produced. In addition, a DNA clone complementary to aromatic L-amino acid decarboxylase mRNA was isolated by immunological screening of a lambda gt11 cDNA expression library. We have used these antibodies and cDNA probes for biochemical, immunochemical, and molecular analyses. A single form of aromatic L-amino acid decarboxylase is detected in rat and bovine tissue. Specifically, aromatic L-amino acid decarboxylase protein is biochemically and immunochemically indistinguishable in brain, liver, kidney, and adrenal medulla. Hybridization to aromatic L-amino acid decarboxylase cDNA identifies a single mRNA species of 2.3 kilobase pairs in rat tissue. Furthermore, Southern blot analysis reveals that a single gene codes for aromatic L-amino acid decarboxylase.
- Published
- 1987
18. Aromatic L-amino acid decarboxylase in the rat brain: immunocytochemical localization in neurons of the brain stem.
- Author
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Jaeger CB, Ruggiero DA, Albert VR, Park DH, Joh TH, and Reis DJ
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- Animals, Female, Histocytochemistry, Immunologic Techniques, Male, Medulla Oblongata enzymology, Mesencephalon enzymology, Neurons enzymology, Pons enzymology, Rats, Rats, Inbred Strains, Tissue Distribution, Aromatic-L-Amino-Acid Decarboxylases analysis, Brain enzymology, Brain Stem enzymology
- Abstract
Neurons containing the enzyme aromatic L-amino acid decarboxylase were immunocytochemically localized in the brain stem of the rat. The enzyme occurred as expected in previously well characterized monoaminergic cell groups, and in addition in some nuclei with unknown neurotransmitters. Major aggregates of neurons that were immunoreactive for aromatic L-amino acid decarboxylase but contained neither tyrosine hydroxylase nor serotonin, were found in the pretectal nuclei, the lateral parabrachial nucleus, and the dorsolateral subdivision of the nucleus tractus solitarius. Aromatic L-amino acid decarboxylase was also present in serotonin neurons and the majority of catecholamine cell groups. Dopamine, noradrenaline, and adrenaline cells exhibited characteristic staining intensities to anti-aromatic L-amino acid decarboxylase reflective of relative enzyme levels in the different groups. Some cells in the dorsal motor nucleus of the vagus that were previously classified as dopaminergic lacked immunoreactivity to aromatic L-amino acid decarboxylase.
- Published
- 1984
- Full Text
- View/download PDF
19. Two different cis-active elements transfer the transcriptional effects of both EGF and phorbol esters.
- Author
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Elsholtz HP, Mangalam HJ, Potter E, Albert VR, Supowit S, Evans RM, and Rosenfeld MG
- Subjects
- Animals, Base Sequence, Enhancer Elements, Genetic, Genes, Regulator, Promoter Regions, Genetic, Rats, Epidermal Growth Factor pharmacology, Genes, Viral, Moloney murine leukemia virus genetics, Prolactin genetics, Tetradecanoylphorbol Acetate pharmacology, Transcription, Genetic drug effects
- Abstract
Short cis-active sequences of the rat prolactin or Moloney murine leukemia virus genes transfer transcriptional regulation by both epidermal growth factor and phorbol esters to fusion genes. These sequences act in a position- and orientation-independent manner. Competitive binding analyses with nuclear extracts from stimulated and unstimulated cells suggest that different trans-acting factors associate with the regulatory sequence of each gene. A model is proposed suggesting that both epidermal growth factor and phorbol esters stimulate the transcription of responsive genes via discrete classes of hormone-dependent, enhancer-like elements that bind different trans-acting factors, even in the absence of hormone stimulation.
- Published
- 1986
- Full Text
- View/download PDF
20. Partial expression of catecholaminergic traits in cholinergic chick ciliary ganglia: studies in vivo and in vitro.
- Author
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Iacovitti L, Joh TH, Albert VR, Park DH, Reis DJ, and Teitelman G
- Subjects
- Animals, Chick Embryo metabolism, Choline O-Acetyltransferase metabolism, Culture Techniques, Ganglia, Parasympathetic physiology, Neurons enzymology, Tyrosine 3-Monooxygenase metabolism, Catecholamines physiology, Chick Embryo physiology, Ganglia, Parasympathetic embryology
- Abstract
We have previously demonstrated that at embryonic Day (E) 8, some cells of the chick ciliary ganglion (CG) contain the catecholaminergic (CA) enzyme tyrosine hydroxylase (TH), but not phenylethanolamine-N-methyltransferase (PNMT); and that in culture essentially all cells express both enzymes. In the present study, we sought to determine, first, whether the expression of adrenergic traits in the CG in vivo is transient or permanent in the CG. To do so, CGs were removed from E5 to postnatal Day 5, fixed, and processed for the immunocytochemical localization of the CA enzymes: TH, L-amino acid decarboxylase (AADC), and PNMT. At all stages examined, some CG neurons expressed TH immunoreactivity (TH-IR) and all contained AADC-IR. However, none stained with PNMT antibodies, indicating that these cells stably express some, but not all, of the CA enzymes. Second, we examined whether CG neurons in culture expressed other CA markers. CG neurons did not contain detectable levels of TH enzyme activity nor did they transport and store exogenously supplied monoamines. These results indicate that some but not all traits necessary for adrenergic function are present in CG neurons in vitro. Third, we sought to establish whether CA expression in CG neurons is affected by modification in culture conditions. Cultures of CG neurons continued to express TH-IR even when grown in the presence of either 50% HCM or 20 mM KCl for 5 days. Finally, the expression of the cholinergic enzyme, choline acetyltransferase (CAT) was assessed in CG cultures by biochemical assay. CAT activity increased five-fold between 5 and 17 days in vitro, irrespective of the presence of TH-IR in 100% of the CG neurons of sister cultures. These data suggest that at least a subpopulation of CG neurons express both TH and CAT in culture. We conclude that the postmitotic neurons of the CG are able to express some but not all of the traits characteristic of a CA phenotype while maintaining cholinergic expression. These findings suggest that (1) the appearance of the full complement of adrenergic properties is not coordinated and may be regulated by different environmental cues and (2) parasympathetic neurons can express both adrenergic and cholinergic traits simultaneously.
- Published
- 1985
- Full Text
- View/download PDF
21. Aromatic L-amino acid decarboxylase in the rat brain: coexistence with vasopressin in small neurons of the suprachiasmatic nucleus.
- Author
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Jaeger CB, Albert VR, Joh TH, and Reis DJ
- Subjects
- Animals, Brain Mapping, Female, Homozygote, Male, Rats, Rats, Brattleboro, Rats, Inbred Strains, Species Specificity, Suprachiasmatic Nucleus cytology, Arginine Vasopressin metabolism, Aromatic-L-Amino-Acid Decarboxylases metabolism, Suprachiasmatic Nucleus metabolism
- Abstract
We demonstrated the coexistence of aromatic L-amino acid decarboxylase (AADC) and arginine-vasopressin in neurons of the hypothalamic suprachiasmatic nucleus of Sprague-Dawley rats. Neurons that lacked monoamines but expressed immunoreactivity to the enzyme AADC occupied the rostral and caudal poles of the suprachiasmatic nucleus and mediodorsal and dorsolateral positions along the entire extent of the nucleus. AADC was also localized in similar neurons of the suprachiasmatic nucleus of rats from other strains including the homozygous Brattleboro rat.
- Published
- 1983
- Full Text
- View/download PDF
22. Activation of cell-specific expression of rat growth hormone and prolactin genes by a common transcription factor.
- Author
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Nelson C, Albert VR, Elsholtz HP, Lu LI, and Rosenfeld MG
- Subjects
- Animals, Avian Sarcoma Viruses genetics, Binding, Competitive, Cell Line, DNA, Recombinant, Enhancer Elements, Genetic, Phenotype, Photochemistry, Pituitary Gland, Anterior metabolism, Promoter Regions, Genetic, Rats, Regulatory Sequences, Nucleic Acid, Transcription, Genetic, Gene Expression Regulation, Growth Hormone genetics, Prolactin genetics, Transcription Factors physiology
- Abstract
In the anterior pituitary gland, there are five phenotypically distinct cell types, including cells that produce either prolactin (lactotrophs) or growth hormone (somatotrophs). Multiple, related cis-active elements that exhibit synergistic interactions appear to be the critical determinants of the transcriptional activation of the rat prolactin and growth hormone genes. A common positive tissue-specific transcription factor, referred to as Pit-1, appears to bind to all the cell-specific elements in each gene and to be required for the activation of both the prolactin and growth hormone genes. The data suggest that, in the course of development, a single tissue-specific factor activates sets of genes that ultimately exhibit restricted cell-specific expression and define cellular phenotype.
- Published
- 1988
- Full Text
- View/download PDF
23. Different forms of adrenal phenylethanolamine N-methyltransferase: species-specific posttranslational modification.
- Author
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Park DH, Baetge EE, Kaplan BB, Albert VR, Reis DJ, and Joh TH
- Subjects
- Animals, Cattle, Cross Reactions, Electrophoresis, Polyacrylamide Gel, Female, Isoenzymes biosynthesis, Phenylethanolamine N-Methyltransferase biosynthesis, Poly A analysis, Polyribosomes analysis, Protein Biosynthesis, RNA, Messenger analysis, Rats, Species Specificity, Adrenal Glands enzymology, Isoenzymes isolation & purification, Phenylethanolamine N-Methyltransferase isolation & purification
- Abstract
Phenylethanolamine N-methyltransferase was purified from rat and cow adrenal glands. The enzymes from the two species have the same molecular weight of 31,000, but differ in electrophoretic mobility. During polyacrylamide gel electrophoresis, the rat form migrates faster than the bovine form. Antibodies to bovine enzyme precipitated equally well the rat and cow form of the enzyme, but antibodies against rat enzyme precipitated poorly the bovine form. In contrast, both antibodies recognized a similar protein in the in vitro translation products of poly(A+)mRNA isolated from cow adrenal glands. The results suggest that the primary protein structure of rat and bovine enzyme is similar and that differences in electrophoretic mobility are due to posttranslational modification of the enzyme molecule.
- Published
- 1982
- Full Text
- View/download PDF
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