Elizabeth Dudenhausen - Academia.edu (original) (raw)

Papers by Elizabeth Dudenhausen

The Journal of biological chemistry, Jan 3, 2004

The Journal of Biological Chemistry, Mar 25, 1989

In the liver, System A-mediated uptake of neutral amino acids may play a key role in metabolic co... more In the liver, System A-mediated uptake of neutral amino acids may play a key role in metabolic control. Knowing the properties of the solubilized and reconstituted System A activity is important for future studies on the purification of the carrier protein. Solubilization of System A activity by the combination of 2.5% cholate and 4 M urea resulted in greater than 85% extraction of the activity. Previous removal of easily extracted plasma membrane proteins with 1% cholate alone followed by solubilization of the transporter with cholate/urea resulted in a 2-fold enrichment in transport activity. Based on the observation that the carrier protein aggregates in the presence of low detergent concentrations, a selective polyethylene glycol precipitation procedure was developed resulting in recovery of more than 70% of the initial transport activity and less than 10% of the total protein. A concomitant 10-fold enrichment in carrier activity was achieved. The precipitated carrier could be resuspended in buffer containing Triton X-100, asolectin, and glycerol. Transporter activity in this buffer was stable for up to 5 days when maintained at -20 "C or for 2 days at 4 "C. The general applicability of the devised reconstitution is illustrated by the presence of Systems N and Gly in the reconstituted proteolipommes at specific activities greater than those in the native vesicles.

The Biochemical journal, Jan 15, 2008

The Biochemical journal, 2005

The Journal of biological chemistry, Jan 2, 2004

CCAAT/enhancer-binding protein beta (C/EBPbeta) is a member of the bZIP family of transcription f... more CCAAT/enhancer-binding protein beta (C/EBPbeta) is a member of the bZIP family of transcription factors that contribute to the regulation of a wide range of important cellular processes. The data in the present study document that transcription from the human C/EBPbeta gene is induced in response to endoplasmic reticulum stress, such as glucose deprivation, or treatment of cells with tunicamycin or thapsigargin. Transient transfection of C/EBPbeta genomic fragments linked to a luciferase reporter gene demonstrated that the C/EBPbeta promoter plays no major regulatory role. Instead, by deletion analysis it was discovered that a 46-bp region, located at a genomic site that corresponds to the 3'-untranslated region of the C/EBPbeta mRNA, harbored an element that was required for the stress response. Mutagenesis demonstrated that a cis-regulatory element located at nt +1614-1621 (5'-TGACGCAA-3') is responsible for activation of the C/EBPbeta gene. Electrophoresis mobility sh...

Gamete Research, 1982

ABSTRACT

Biochimica et biophysica acta, Jan 16, 2014

Amino acid (AA) deprivation in mammalian cells activates a collection of signaling cascades known... more Amino acid (AA) deprivation in mammalian cells activates a collection of signaling cascades known as the AA response (AAR), which is characterized by transcriptional induction of stress-related genes, including FBJ murine osteosarcoma viral oncogene homolog (cFOS). The present study established that the signaling mechanism underlying the AA-dependent transcriptional regulation of the cFOS gene in HepG2 human hepatocellular carcinoma cells is independent of the classic GCN2-eIF2-ATF4 pathway. Instead, a RAS-RAF-MEK-ERK cascade mediates AAR signaling to the cFOS gene. Increased cFOS transcription is observed from 4-24h after AAR-activation, exhibiting little or no overlap with the rapid and transient increase triggered by the well-known serum response. Furthermore, serum is not required for the AA-responsiveness of the cFOS gene and no phosphorylation of promoter-bound serum response factor (SRF) is observed. The ERK-phosphorylated transcription factor E-twenty six-like (p-ELK1) is in...

Nucleic Acids Research, 2008

It is unclear whether Mediator complex in yeast is necessary for all RNA polymerase II (Pol II) t... more It is unclear whether Mediator complex in yeast is necessary for all RNA polymerase II (Pol II) transcription or if it is limited to genes activated by environmental stress. In mammals, amino acid limitation induces SNAT2 transcription through ATF4 binding at an amino acid response element. ATF4 is the functional counterpart to the yeast amino acid-dependent regulator GCN4 and GCN4 recruits Mediator during transcriptional activation. Consistent with enhanced SNAT2 transcription activity, the present data demonstrate that amino acid limitation increased SNAT2 promoter association of the general transcription factors that make up the preinitiation complex, including Pol II, but there was no increase in Mediator recruitment. Furthermore, siRNA knockdown of eight Mediator subunits caused no significant decrease in SNAT2 transcription. The estrogen-dependent pS2 gene was used as a positive control for both the ChIP and the siRNA approaches and the data demonstrated the requirement for Mediator recruitment. These results document that activation of the SNAT2 gene by the mammalian amino acid response pathway occurs independently of enhanced Mediator recruitment.

Journal of Biological Chemistry, 2004

Expression of human asparagine synthetase (ASNS), which catalyzes asparagine and glutamate biosyn... more Expression of human asparagine synthetase (ASNS), which catalyzes asparagine and glutamate biosynthesis, is transcriptionally induced following amino acid deprivation. Previous overexpression and electrophoresis mobility shift analysis showed the involvement of the transcription factors ATF4, C/EBP␤, and ATF3-FL through the nutrient-sensing response element-1 (NSRE-1) within the ASNS promoter. Amino acid deprivation caused an elevated mRNA level for ATF4, C/EBP␤, and ATF3-FL, and the present study established that the nuclear protein content for ATF4 and ATF3-FL were increased during amino acid limitation, whereas C/EBP␤-LIP declined slightly. The total amount of C/EBP␤-LAP protein was unchanged, but changes in the distribution among multiple C/EBP␤-LAP forms were observed. Overexpression studies established that ATF4, ATF3-FL, and C/EBP␤-LAP could coordinately modulate the transcription from the human ASNS promoter. Chromatin immunoprecipitation demonstrated that amino acid deprivation increased ATF3-FL, ATF4, and C/EBP␤ binding to the ASNS promoter and enhanced promoter association of RNA polymerase II, TATA-binding protein, and TFIIB of the general transcription machinery. A time course revealed a markedly different temporal order of interaction between these transcription factors and the ASNS promoter. During the initial 2 h, there was a 20-fold increase in ATF4 binding and a rapid increase in histone H3 and H4 acetylation, which closely paralleled the increased transcription rate of the ASNS gene, whereas the increase in ATF3-FL and C/EBP␤ binding was considerably slower and more closely correlated with the decline in transcription rate between 2 and 6 h. The data suggest that ATF3-FL and C/EBP␤ act as transcriptional suppressors for the ASNS gene to counterbalance the transcription rate activated by ATF4 following amino acid deprivation.

Journal of Biological Chemistry, 2011

Mammalian cells respond to protein or amino acid (AA) limitation by activating a number of signal... more Mammalian cells respond to protein or amino acid (AA) limitation by activating a number of signaling pathways, collectively referred to as the AA response (AAR), that modulate a range of cellular functions, including transcriptional induction of target genes. The present report demonstrates that in hepatocellular carcinoma cells expression of cJUN, JUN-B, cFOS, and FOS-B was induced by the AAR, whereas JUN-D, FRA-1, and FRA-2 were not. Of the four activated FOS/JUN members, cJUN made the largest contribution to the induction of several known AAR target genes. For several human liver, prostate, and ovarian cell lines, the AAR-induced increase in cJUN expression was greater in transformed cells compared to non-transformed counterparts, an effect independent of cell growth rate. Thus far, the best characterized AA-responsive genes are all transcriptionally activated by ATF4, but the AAR-dependent induction of cJUN transcription was ATF4independent. The increased expression of cJUN was dependent on ATF2 and on activation of the MEK-ERK and JNK arms of the MAPK signaling pathways. Formation of cJUN-ATF2 activated heterodimers was increased after AA limitation and cJUN or ATF2 knockdown suppressed the induction of cJUN and other AAR target genes. AA deprivation triggers a feed-forward process that involves phosphorylation of existing cJUN protein by JNK and subsequent auto-activation of the cJUN gene by recruitment of cJUN and ATF2 to two AP-1 sites within the proximal promoter. The results document the novel observation that AP-1 sequences within the cJUN gene can function as transcriptional amino acid response elements.

Journal of Biological Chemistry, 2004

CCAAT/enhancer-binding protein ␤ (C/EBP␤) is a member of the bZIP family of transcription factors... more CCAAT/enhancer-binding protein ␤ (C/EBP␤) is a member of the bZIP family of transcription factors that contribute to the regulation of a wide range of important cellular processes. The data in the present study document that transcription from the human C/EBP␤ gene is induced in response to endoplasmic reticulum stress, such as glucose deprivation, or treatment of cells with tunicamycin or thapsigargin. Transient transfection of C/EBP␤ genomic fragments linked to a luciferase reporter gene demonstrated that the C/EBP␤ promoter plays no major regulatory role. Instead, by deletion analysis it was discovered that a 46-bp region, located at a genomic site that corresponds to the 3-untranslated region of the C/EBP␤ mRNA, harbored an element that was required for the stress response. Mutagenesis demonstrated that a cis-regulatory element located at nt ؉1614 -1621 (5-TGACGCAA-3) is responsible for activation of the C/EBP␤ gene. Electrophoresis mobility shift analysis revealed that proteins are bound to this element and that the amount of binding is increased following glucose deprivation. This element is homologous to a previously reported mammalian unfolded protein response element that binds XBP-1. Consistent with those data, overexpression of XBP-1 caused an increase in transcription that was mediated by the C/EBP␤ mammalian unfolded protein response element.

Biochemical Journal, 2008

A nutrient stress signalling pathway is triggered in response to protein or amino acid deprivatio... more A nutrient stress signalling pathway is triggered in response to protein or amino acid deprivation, namely the AAR (amino acid response), and previous studies have shown that C/EBPbeta (CCAAT/enhancer-binding protein beta) expression is up-regulated following activation of the AAR. DNA-binding studies, both in vitro and in vivo, have revealed increased C/EBPbeta association with AARE (AAR element) sequences in AAR target genes, but its role is still unresolved. The present results show that in HepG2 human hepatoma cells, the total amount of C/EBPbeta protein, both the activating [LAP* and LAP (liver-enriched activating protein)] and inhibitory [LIP (liver-enriched inhibitory)] isoforms, was increased in histidine-deprived cells. Immunoblotting of subcellular fractions and immunostaining revealed that most of the C/EBPbeta was located in the nucleus. Consistent with these observations, amino acid limitation caused an increase in C/EBPbeta DNA-binding activity in nuclear extracts and chromatin immunoprecipitation revealed an increase in C/EBPbeta binding to the AARE region in vivo, but at a time when transcription from the target gene was declining. A constant fraction of the basal and increased C/EBPbeta protein was phosphorylated on Thr(235) and the phospho-C/EBPbeta did bind to an AARE. Induction of AARE-enhanced transcription was slightly greater in C/EBPbeta-deficient MEFs (mouse embryonic fibroblasts) or C/EBPbeta siRNA (small interfering RNA)-treated HepG2 cells compared with the corresponding control cells. Transient expression of LAP*, LAP or LIP in C/EBPbeta-deficient fibroblasts caused suppression of increased transcription from an AARE-driven reporter gene. Collectively, the results demonstrate that C/EBPbeta is not required for transcriptional activation by the AAR pathway but, when present, acts in concert with ATF3 (activating transcription factor 3) to suppress transcription during the latter stages of the response.

Biochemical Journal, 2005

For animals, dietary protein is critical for the nutrition of the organism and, at the cellular l... more For animals, dietary protein is critical for the nutrition of the organism and, at the cellular level, protein nutrition translates into amino acid availability. Amino acid deprivation triggers the AAR (amino acid response) pathway, which causes enhanced transcription from specific target genes. The present results show that C/EBPβ (CCAAT/enhancer-binding protein β) mRNA and protein content were increased following the deprivation of HepG2 human hepatoma cells of a single amino acid. Although there was a modest increase in mRNA half-life following histidine limitation, the primary mechanism for the elevated steady-state mRNA was increased transcription. Transient transfection documented that C/EBPβ genomic fragments containing the 8451 bp 5 upstream of the transcription start site did not contain aminoacid-responsive elements. However, deletion analysis of the genomic region located 3 downstream of the protein coding sequence revealed that a 93 bp fragment contained an amino-acidresponsive activity that functioned as an enhancer. Exogenous expression of ATF4 (activating transcription factor 4), known to activate other genes through amino acid response elements, caused increased transcription from reporter constructs containing the C/EBPβ enhancer in cells maintained in complete amino acid medium. Chromatin immunoprecipitation demonstrated that RNA polymerase II is bound at the C/EBPβ promoter and at the 93 bp regulatory region in vivo, whereas ATF4 binds to the enhancer region only. Immediately following amino acid removal, the kinetics of binding for ATF4, ATF3, and C/EBPβ itself to the 93 bp regulatory region were similar to those observed for the amino-acid-responsive asparagine synthetase gene. Collectively the findings show that expression of C/EBPβ, which contributes to the regulation of amino-acid-responsive genes, is itself controlled by amino acid availability through transcription.

Canadian Journal of Zoology, 1983

DUDENHAUSEN, E. E., and P. TALBOT. 1983. An ultrastructural comparison of soft and hardened sperm... more DUDENHAUSEN, E. E., and P. TALBOT. 1983. An ultrastructural comparison of soft and hardened spermatophores from the crayfish Pacifastacus leniusculus . The spermatophore of the crayfish Pacifastacus leniusculus consists of two main parts: a sperm mass composed of sperm embedded in adense fibrillar matrix and an acellular wall which surrounds the sperm mass and is formed from secretions produced in the vas deferens. Following extrusion from the male, the spermatophore wall, which is initially soft and sticky, undergoes a hardening process. In this study, the structure of the spermatophore walls of unextruded (soft) and hardened spermatophores were compared using light and electron microscopy. The wall of the unextruded spermatophore is composed of three concentric layers: a thin primary spermatophore layer which directly surrounds the sperm mass; a thick middle layer composed primarily of electron-dense, spherical granules; and a thick outer layer formed from a dense globular secretion. The primary spermatophore layer and outer globular layer are positive for carbohydrate with the periodic acid -Schiff method. Following extrusion and hardening, the walls of spermatophores showed several structural changes. These are (i) division of the middle granular layer into a compact inner region and a highly reticulated outer region; (ii) the loss of the outer globular layer; and (iii) the formation of a thickened ridge along one side of the spermatophore wall. The thickened ridge is fibrillar in structure and is believed to be derived from a structural modification of the outer globular layer. No structural modifications in the primary spermatophore layer were observed. We interpret these observations to indicate that the outer globular layer functions in attachment of the spermatophore to the female and the middle layer is involved in spermatophore hardening and sperm protection during storage.

The Journal of biological chemistry, Jan 3, 2004

The Journal of Biological Chemistry, Mar 25, 1989

The Biochemical journal, Jan 15, 2008

The Biochemical journal, 2005

The Journal of biological chemistry, Jan 2, 2004

Gamete Research, 1982

ABSTRACT

Biochimica et biophysica acta, Jan 16, 2014

Nucleic Acids Research, 2008

Journal of Biological Chemistry, 2004

Journal of Biological Chemistry, 2011

Journal of Biological Chemistry, 2004

Biochemical Journal, 2008

Biochemical Journal, 2005

Canadian Journal of Zoology, 1983