Biomedical subjects

T A Libermann

Publications and source records attributed to T A Libermann.

At least 19 recordsLinked to original sources

PubMed2002

Insertion of an exogenous domain in the adenovirus type 2 fiber globular region.

Adenoviruses have been used for gene therapy or immunization due to their ability to efficiently infect a broad range of cells and tissues. These applications as well as specificity could be improved further by redirecting binding of the virus to specific cell types. In this regard, modification of viral genes encoding coat proteins is an option to achieve improvement in retargeting. In this report, we describe a substitution in the adenovirus type 2 fiber globular region by the 44 amino acid C4 domain of human immunodeficiency virus type 1 gp120. In vitro translation analysis and immunoprecipitation assays show that the incorporation of the C4 domain into the fiber protein does not ablate its trimerization property and demonstrates the availability of the C4 epitope for interaction with monoclonal anti-C4 antibody. The recombinant adenovirus containing this modified fiber was also characterized by immunoprecipitation with the same antibody, showing the viability of such kind of modification.

Adenoviridae↗

PubMed2002

CHIP TUNER: a web tool for evidence-based noise reduction in gene discovery.

The potential for gene discovery, fueled by DNA microchip technology and the sequencing of hundreds of genomes, is unprecedented. In this context, trying to discover genes that are actually of significance rather than merely appearing so due to noise is of utmost importance. We present a web application, CHIP TUNER, which assists in this gene discovery process. Our system uses evidence-based noise reduction to help delineate candidate target genes of biological importance. Specifically, CHIP TUNER learns from redundant experiments an "identity mask" that defines a region of noise inherent to biological sampling and DNA microarray processing; it then takes this into account during actual sample comparisons. The goal of CHIP TUNER is to improve the chances that newly discovered "important" genes are actually of importance before large amounts of time and resources are invested.

Computational Biology↗

PubMed2001

Epstein-Barr Virus and its glycoprotein-350 upregulate IL-6 in human B-lymphocytes via CD21, involving activation of NF-kappaB and different signaling pathways.

Epstein-Barr virus (EBV) is a ubiquitous and highly immunotropic gamma herpesvirus that infects more than 90 % of humans worldwide. Its pathogenicity leads to a number of diseases including tumors that result from EBV's ability to readily transform B-lymphocytes and, to a lesser extent, epithelial cells. EBV utilizes CD21/CR2 as its receptor on B cells to initiate the infection process. EBV binds to CR2 through its major envelope glycoprotein-350 (gp350) and is also a remarkable immunomodulating agent. We had previously shown that EBV is capable of modulating the synthesis of a number of cytokines. We now show that while both purified recombinant gp350 (rgp350) and EBV upregulate IL-6 mRNA synthesis in B cells, EBV-induced IL-6 gene activation occurs for a significantly longer period of time (i.e. 12 hours for EBV as compared to 6 hours for rgp350). Moreover, the half-life of EBV-induced IL-6 mRNA was also significantly longer (10 hours) than that of mRNA induced by rgp350 (about 6 hours). Both EBV and gp350 enhance the binding of the NF-kappaB transcription factor, as determined by band-shift and augment NF-kappaB-mediated activation of a CAT reporter plasmid. Furthermore, we demonstrate that while the activation of IL-6 gene expression by gp350 is mediated primarily by the protein kinase C pathway, EBV can mediate its effects through multiple signaling pathways. To our knowledge this is the first report showing that the binding of a herpesvirus envelope glycoprotein to CR2 on human B cells results in the activation of the NF-kappaB transcription factor leading to the upregulation of IL-6 gene expression in these lymphocytes.

B-Lymphocytes↗

PubMed2001

Elf-1 is a transcriptional regulator of the Tie2 gene during vascular development.

Vascular development requires the tightly coordinated expression of several growth factors and their receptors. Among these are the Tie1 and Tie2 receptors, which are almost exclusively endothelial cell-specific. The critical transcriptional regulators of vascular-specific gene expression remain largely unknown. The Ets factors are a family of evolutionarily conserved transcription factors that regulate genes involved in cellular growth and differentiation. We have recently shown that the Ets factor NERF is a strong transactivator of the Tie1 and Tie2 genes. To extend these studies, we have begun to identify the Ets factors that are expressed in developing blood vessels of the chicken chorioallantoic membrane (CAM), a highly vascular embryonic network. RNA was extracted from microdissected CAM blood vessels, and reverse transcriptase-polymerase chain reaction was performed using oligonucleotides encoding conserved amino acids within the Ets domain. One of the polymerase chain reaction fragments was subcloned and identified as the chicken homologue of the Ets factor ELF-1, cELF-1. ELF-1 is most closely related to the Ets factor NERF. In situ hybridization and immunohistochemistry demonstrate that cELF-1 is enriched in developing chicken blood vessels. cELF-1 is also a strong transactivator of the Tie1 and Tie2 genes and can bind to conserved Ets sites within the promoters of these genes. A complex of similar size forms when gel shifts are performed with cellular extracts derived from the CAM blood vessels, which is recognized by an antibody against cELF-1. In summary, ELF-1 belongs to a subset of Ets factors that regulate vascular-specific gene expression during blood vessel development.

Allantois↗

PubMed2001

On reporting fold differences.

As we enter an age in which genomics and bioinformatics make possible the discovery of new knowledge about the biological characteristics of an organism, it is critical that we attempt to report newly discovered "significant" phenotypes only when they are actually of significance. With the relative youth of genome-scale gene expression technologies, how to make such distinctions has yet to be better defined. We present a "mask technology" by which to filter out those levels of gene expression that fall within the noise of the experimental techniques being employed. Conversely, our technique can lend validation to significant fold differences in expression level even when the fold value may appear quite small (e.g. 1.3). Given array-organized expression level results from a pair of identical experiments, our ID Mask Tool enables the automated creation of a two-dimensional "region of insignificance" that can then be used with subsequent data analyses. Fundamentally, this should enable researchers to report on findings that are more likely to be in nature truly meaningful. Moreover, this can prevent major investments of time, energy, and biological resources into the pursuit of candidate genes that represent false positives.

Computational Biology↗

PubMed2000

Tel-2 is a novel transcriptional repressor related to the Ets factor Tel/ETV-6.

We report here the isolation of Tel-2, a novel member of the Ets transcription factor family, with high homology to Tel/ETV-6. Tel-2 is the second mammalian member of the Tel Ets family subclass whose prototype Tel is involved in various chromosomal translocations in human cancers. Six differentially expressed alternative splice products of Tel-2 were characterized encoding different Tel-2 isoforms which either contain or lack the amino-terminal Pointed domain and also vary at the carboxyl terminus. In contrast to Tel, which is highly expressed in several different cell types and tissues, Tel-2 is only weakly expressed in a variety of tissues and cell types, including placenta, prostate, spleen, liver, and lung. Tel-2 binds to functionally relevant Ets-binding sites of several genes and only the Tel-2 isoform containing the Pointed domain and the DNA-binding domain acts as a strong repressor of transcription. The retinoic acid receptor alpha and bone morphogenetic protein-6B (BMP-6) genes are specifically repressed by Tel-2 indicating a function for Tel-2 as an inhibitor of differentiation. Due to the important involvement of Tel in human cancer and the location of Tel-2 within the MHC cluster region, Tel-2 might be involved in chromosomal translocations in human cancer as well.

Amino Acid Sequence↗

PubMed2000

ESE-1 is a novel transcriptional mediator of inflammation that interacts with NF-kappa B to regulate the inducible nitric-oxide synthase gene.

Inflammation is a hallmark of several vascular diseases. The nuclear factor kappaB (NF-kappaB) transcription factors are dimeric proteins involved in the activation of a large number of genes in response to inflammatory stimuli. We report the involvement of a novel member of the ETS transcription factor, ESE-1, in mediating vascular inflammation. ESE-1 is induced in response to inflammatory cytokines and lipopolysaccharide in vascular smooth muscle cells, endothelial cells, and cells of the monocyte-macrophage lineage. This induction occurs within hours of stimulation and is mediated by NF-kappaB transactivation of the ESE-1 promoter. We have identified the inducible form of nitric-oxide synthase (NOS2) as a putative target for ESE-1. ESE-1 can bind to the p50 subunit of NF-kappaB, and cotransfection of ESE-1 with the p50 and p65 subunits of NF-kappaB synergistically enhances transactivation of the NOS2 promoter by ESE-1. An ESE-1-binding site within the NOS2 promoter has been identified, the site-directed mutagenesis of which completely abolishes the ability of ESE-1 to transactivate the NOS2 promoter. Finally, in a mouse model of endotoxemia, associated with acute vascular inflammation, ESE-1 is strongly expressed in vascular endothelium and smooth muscle cells. In summary, ESE-1 represents a novel mediator of vascular inflammation.

Binding Sites↗

PubMed2000

Dual function of the epithelial specific ets transcription factor, ELF3, in modulating differentiation.

The ets family of transcription factors comprises many members which contribute to diverse cellular functions that vary depending upon the cell- and tissue-type context. Recently, different groups have identified a novel member of the ets family that is epithelial-specific. Variably called ESE-1, ERT, jen, ESX, this gene is designated currently as ELF3. In order to understand transcriptional regulatory mechanisms mediated by ELF3, we investigated its effect on the human keratin 4 gene promoter based upon the role of keratin 4 in early differentiation of the esophageal squamous epithelium. Interestingly, ELF3 suppressed basal keratin 4 promoter activity in both esophageal and cervical epithelial cancer cell lines, a novel result, while simultaneously activating the late-differentiation linked SPRR2A promoter. Furthermore, serial deletion constructs of the keratin 4 promoter continued to be suppressed by ELF3, a phenomenon that was only partially rescued by ELF3 ets domain mutants, but completely abrogated by deletion of the ELF3 pointed domain. These results suggest that ELF3 may have dual functions in the transcriptional regulation of genes involved in squamous epithelial differentiation. One of these functions may not be exclusively mediated through DNA binding in the context of transcriptional suppression of the keratin 4 promoter.

Amino Acid Sequence↗

PubMed2000

ESE-3, a novel member of an epithelium-specific ets transcription factor subfamily, demonstrates different target gene specificity from ESE-1.

Most cancers originate as a result of aberrant gene expression in mainly glandular epithelial tissues leading to defects in epithelial cell differentiation. The latter is governed by distinct sets of transcriptional regulators. Here we report the characterization of epithelium-specific Ets factor, family member 3 (ESE-3), a novel member of the ESE subfamily of Ets transcription factors. ESE-3 shows highest homology to two other epithelium restricted Ets factors, ESE-1 and ESE-2. ESE-3, like ESE-1 and ESE-2, is exclusively expressed in a subset of epithelial cells with highest expression in glandular epithelium such as prostate, pancreas, salivary gland, and trachea. A potential role in branching morphogenesis is suggested, since ESE-3 transactivates the c-MET promoter via three high affinity binding sites. Additionally, ESE-3 binding to DNA sequences in the promoters of several glandular epithelium-specific genes suggests a role for ESE-3 in later stages of glandular epithelium differentiation. Although ESE-3 and ESE-1 bind with similar affinity to various Ets binding sites, ESE-3 and ESE-1 differ significantly in their ability to transactivate the promoters containing these sites. Our results support the notion that ESE-1, ESE-2, and ESE-3 represent a unique epithelium-specific subfamily of Ets factors that have critical but distinct functions in epithelial cell differentiation and proliferation.

Adaptor Proteins, Vesicular Transport↗

PubMed2000

PDEF, a novel prostate epithelium-specific ets transcription factor, interacts with the androgen receptor and activates prostate-specific antigen gene expression.

Prostate cancer, the most frequent solid cancer in older men, is a leading cause of cancer deaths. Although proliferation and differentiation of normal prostate epithelia and the initial growth of prostate cancer cells are androgen-dependent, prostate cancers ultimately become androgen-independent and refractory to hormone therapy. The prostate-specific antigen (PSA) gene has been widely used as a diagnostic indicator for androgen-dependent and -independent prostate cancer. Androgen-induced and prostate epithelium-specific PSA expression is regulated by a proximal promoter and an upstream enhancer via several androgen receptor binding sites. However, little progress has been made in identifying androgen-independent regulatory elements involved in PSA gene regulation. We report the isolation of a novel, prostate epithelium-specific Ets transcription factor, PDEF (prostate-derived Ets factor), that among the Ets family uniquely prefers binding to a GGAT rather than a GGAA core. PDEF acts as an androgen-independent transcriptional activator of the PSA promoter. PDEF also directly interacts with the DNA binding domain of androgen receptor and enhances androgen-mediated activation of the PSA promoter. Our results, as well as the critical roles of other Ets factors in cellular differentiation and tumorigenesis, strongly suggest that PDEF is an important regulator of prostate gland and/or prostate cancer development.

Amino Acid Sequence↗

PubMed2000

Adenovirus vector-induced expression of the C-X-C chemokine IP-10 is mediated through capsid-dependent activation of NF-kappaB.

The use of adenovirus vectors for gene therapy has been limited by well-defined cellular and humoral immune responses. We have previously shown that adenovirus vectors rapidly induce the expression of the C-X-C chemokine, interferon-inducible protein 10 (IP-10), in vivo. Various first-generation, type 5 adenovirus vectors, including adCMVbetagal and UV-psoralen-inactivated adenovirus, equally induced the expression of IP-10 mRNA as early as 3 h following infection in mouse renal epithelial cells (REC). Luciferase reporter experiments using deletional mutants of the murine IP-10 5'-flanking region revealed that transcriptional activation of the IP-10 promoter by adCMVbetagal was dependent on the -161- to -96-bp region upstream of the transcription start site. In electrophoretic mobility shift assays, adCMVbetagal, adCMV-GFP, FG140, and transcription-defective adenovirus induced protein binding to oligonucleotides containing a consensus sequence for NF-kappaB at position -113 of the IP-10 promoter. Supershift assays confirmed an increase in binding activity of NF-kappaB p65 but not p50 or cRel in REC cells infected with various replication-deficient adenoviruses. Coinfection of REC cells with adCMVbetagal and an adenoviral vector expressing IkappaBalpha resulted in suppression of adCMVbetagal-induced expression of IP-10 at 6 and 16 h, further strengthening the conclusion that adenovirus-induced activation of IP-10 is dependent on NF-kappaB. The induction of IP-10 appeared to be direct because infection with adenovirus vectors failed to induce the expression of the potent IP-10 stimulators, interferon gamma and tumor necrosis factor alpha. Together, these findings demonstrate that adenovirus vectors directly induce the expression of IP-10 through capsid dependent activation of NF-kappaB.

Adenoviruses, Human↗

PubMed1999

Characterization of ESE-2, a novel ESE-1-related Ets transcription factor that is restricted to glandular epithelium and differentiated keratinocytes.

Epithelial cell differentiation is tightly controlled by distinct sets of transcription factors that regulate the expression of stage-specific genes. We recently isolated the first epithelium-specific Ets transcription factor (ESE-1). Here we describe the characterization of ESE-2, a second epithelium-restricted ESE-1-related Ets factor. Like ESE-1, ESE-2 is induced during keratinocyte differentiation. However, whereas ESE-1 is expressed in the majority of epithelial cell types, ESE-2 expression is restricted to differentiated keratinocytes and glandular epithelium such as salivary gland, prostate, mammary gland, and kidney. In contrast to ESE-1, full-length ESE-2 binds poorly to DNA due to the presence of a negative regulatory domain at the amino terminus. Furthermore, although ESE-1 and the amino-terminally deleted ESE-2 bind with similar affinity to the canonical E74 Ets site, ESE-2 and ESE-1 differ strikingly in their relative affinity toward binding sites in the c-MET and PSMA promoters. Similarly, ESE-1 and ESE-2 drastically differ in their ability to transactivate epithelium-specific promoters. Thus, ESE-2, but not ESE-1, transactivates the parotid gland-specific PSP promoter and the prostate-specific PSA promoter. In contrast, ESE-1 transactivates the keratinocyte-specific SPRR2A promoter Ets site and the prostate-specific PSMA promoter significantly better than ESE-2. Our results demonstrate the existence of a unique class of related epithelium-specific Ets factors with distinct functions in epithelial cell gene regulation.

Alternative Splicing↗

PubMed1999

AML1 (CBFalpha2) cooperates with B cell-specific activating protein (BSAP/PAX5) in activation of the B cell-specific BLK gene promoter.

AML1 plays a critical role during hematopoiesis and chromosomal translocations involving AML1 are commonly associated with different forms of leukemia, including pre-B acute lymphoblastic leukemia. To understand the function of AML1 during B cell differentiation, we analyzed regulatory regions of B cell-specific genes for potential AML1-binding sites and have identified a putative AML1-binding site in the promoter of the B cell-specific tyrosine kinase gene, blk. Gel mobility shift assays and transient transfection assays demonstrate that AML1 binds specifically to this site in the blk promoter and this binding site is important for blk promoter activity. Furthermore, in vitro binding analysis revealed that the AML1 runt DNA-binding domain physically interacts with the paired DNA-binding domain of BSAP, a B cell-specific transcription factor. BSAP has been shown previously to be important for B cell-specific regulation of the blk gene. Physical interaction of AML1 with BSAP correlates with functional cooperativity in transfection studies where AML1 and BSAP synergistically activate blk promoter transcription by more than 50-fold. These results demonstrate physical and functional interactions between AML1 and BSAP and suggest that AML1 is an important factor for regulating a critical B cell-specific gene, blk.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Role of the Ets transcription factors in the regulation of the vascular-specific Tie2 gene.

The Tie2 gene encodes a vascular endothelium-specific receptor tyrosine kinase that is required for normal vascular development and is also upregulated during angiogenesis. The regulatory regions of the Tie2 gene that are required for endothelium-specific gene expression in vivo have been identified. However, the transcription factors required for Tie2 gene expression remain largely unknown. We have identified highly conserved binding sites for Ets transcription factors in the Tie2 promoter. Mutations in 2 particular binding sites lead to a 50% reduction in the endothelium-specific activity of the promoter. We have compared the ability of several members of the Ets family to transactivate the Tie2 promoter. Our results demonstrate that 1 of 3 distinct isoforms of the novel Ets transcription factor NERF, NERF2, is expressed in endothelial cells and can strongly transactivate the regulatory regions of the Tie2 gene in comparison to other Ets factors, which have little or no effect. NERF2 can bind to the Tie2 promoter Ets sites in electrophoretic mobility shift assays. These studies support a role for Ets factors in the regulation of vascular-specific gene expression and suggest that the novel Ets factor NERF2 may be a critical transcription factor in specifying the expression of the Tie2 gene in vascular endothelial cells.

Base Sequence↗

PubMed1999

Adenoviral gene therapy leads to rapid induction of multiple chemokines and acute neutrophil-dependent hepatic injury in vivo.

Replication-deficient adenoviruses are known to induce acute injury and inflammation of infected tissues, thus limiting their use for human gene therapy. However, molecular mechanisms triggering this response have not been fully defined. To characterize this response, chemokine expression was evaluated in DBA/2 mice following the intravenous administration of various adenoviral vectors. Administration of adCMVbeta gal, adCMV-GFP, or FG140 intravenously rapidly induced a consistent pattern of C-X-C and C-C chemokine expression in mouse liver in a dose-dependent fashion. One hour following infection with 10(10) PFU of adCMVbeta gal, hepatic levels of MIP-2 mRNA were increased >60-fold over baseline. MCP-1 and IP-10 mRNA levels were also increased immediately following infection with various adenoviral vectors, peaking at 6 hr with >25- and >100-fold expression, respectively. Early induction of RANTES and MIP-1beta mRNA by adenoviral vectors also occurred, but to a lesser degree. The induction of chemokines occurred independently of viral gene expression since psoralen-inactivated adenoviral particles produced an identical pattern of chemokine gene transcription within the first 16 hr of administration. The expression of chemokines correlated as expected with the influx of neutrophils and CD11b+ cells into the livers of infected animals. At high titers, all adenoviral vectors caused significant hepatic necrosis and apoptosis following systemic administration to DBA/2 mice. To investigate the role of neutrophils in this adenovirus-induced hepatic injury, animals were pretreated with neutralizing anti-MIP-2 antibodies or depleted of neutrophils. MIP-2 antagonism and neutrophil depletion both resulted in reduced serum ALT/AST levels and attenuation of the adenovirus-induced hepatic injury histologically, confirming that this early injury is largely due to chemokine production and neutrophil recruitment. Our findings further clarify the early immune response against replication-deficient adenoviral vectors and suggest a strategy to prevent adenovirus-mediated inflammation and tissue injury by interfering with chemokine or neutrophil function.

Adenoviridae↗

PubMed1999

Genomic organization of the human ELF3 (ESE-1/ESX) gene, a member of the Ets transcription factor family, and identification of a functional promoter.

We recently isolated a novel member of the Ets transcription factor/oncogene family, ESE-1/ESX/ELF3, with features distinct from any other Ets-related factor. ELF3 is the prototype of a new subclass of Ets factors, contains two DNA-binding domains, and, in contrast to any known Ets factor, is expressed exclusively in epithelial cells. ELF3 expression is induced during differentiation of the epidermis, indicating a role in the regulation of terminal differentiation genes in the epidermis. Due to the important role that other Ets factors play in cellular differentiation, ELF3 is expected to be a critical regulator of epithelial gene expression. We report here the cloning and the structural organization of the human ELF3 gene. The human ELF3 gene contains nine exons, which span approximately 5.8 kb of genomic DNA. Intron/exon borders and number of exons are almost identical to those in the mouse ELF3 gene. Comparison of the immediate promoter regions of the human and mouse ELF3 genes demonstrates the presence of TATA and CCAAT boxes as well as potential binding sites for Ets factors and NF-kappaB. Transfection experiments demonstrate that a 1.5-kb fragment of the 5' upstream region acts as a strong promoter in two epithelial cell lines.

Animals↗

PubMed1999

Bradykinin induces interleukin-6 expression in astrocytes through activation of nuclear factor-kappaB.

Bradykinin, a mediator of inflammation, is produced in the brain during trauma and stroke. It is thought to open the blood-brain barrier, although the mechanism is unclear. We have investigated, therefore, the effect of bradykinin on the expression of interleukin-6 (IL-6), a putative modulator of the blood-brain barrier, in astrocytes. IL-6 gene transcription was evaluated by transient transfection of the human IL-6 promoter linked to the luciferase gene. In murine astrocytes, bradykinin stimulated IL-6 secretion and gene transcription. The effect of bradykinin was blocked by KN-93, an inhibitor of Ca2+/calmodulin-dependent protein kinases, and by bisindolylmaleimide I, an inhibitor of protein kinase C, suggesting the involvement of these protein kinases. Mutations in the multiple response element and the binding site for nuclear factor-kappaB (NF-kappaB), but not in other known elements of the IL-6 promoter, interfered with induction of IL-6 transcription. The involvement of NF-kappaB was supported further by the finding that overexpression of nmIkappaB alpha, a stable inhibitor of NF-kappaB, inhibited the induction of IL-6 by bradykinin. Bradykinin activated NF-kappaB in primary astrocytes as shown by increased DNA binding of NF-kappaB. These data demonstrate that bradykinin stimulates IL-6 expression through activation of NF-kappaB, which may explain several inflammatory effects of bradykinin.

Animals↗

PubMed1998

Cloning and characterization of GETS-1, a goldfish Ets family member that functions as a transcriptional repressor in muscle.

An Ets transcription factor family member, GETS-1, was cloned from a goldfish retina cDNA library. GETS-1 contains a conserved Ets DNA-binding domain at its N-terminus and is most similar to ternary complex factor (TCF) serum-response-factor protein-1a (SAP-1a). GETS-1 is expressed in many tissues, but is enriched in retina and brain. As with the TCFs SAP-1a and ets-related protein (ERP), overexpression of the GETS-1 promoter suppresses nicotinic acetylcholine receptor epsilon-subunit gene expression in cultured muscle cells. A consensus Ets binding site sequence in the promoter of the epsilon-subunit gene is required for GETS-1-mediated repression. GETS-1 repressor activity is abrogated by overexpression of an activated Ras/mitogen-activated protein kinase (MAP kinase) or by mutation of Ser-405, a MAP kinase phosphorylation site in GETS-1. Fusion proteins created between GETS-1 and the Gal4 DNA-binding domain show that, like other TCFs, GETS-1 contains a C-terminal activation domain that is activated by a Ras/MAP kinase signalling cascade. Interestingly, mutation of Ser-405 located within this activation domain abrogated transcriptional activation of the fusion protein.

Amino Acid Sequence↗