Mary Loeken - Academia.edu (original) (raw)

Papers by Mary Loeken

Research paper thumbnail of The adenovirus EIIA enhancer. Analysis of regulatory sequences and changes in binding activity of ATF and EIIF following adenovirus infection

Journal of Biological Chemistry

Adenovirus EIIA upstream sequences which contain the binding sites for proteins ATF and EIIF act ... more Adenovirus EIIA upstream sequences which contain the binding sites for proteins ATF and EIIF act as an enhancer and can be trans-activated by both E1A and SV40 T/t-antigens. Specific mutation of either the ATF or EIIF binding site demonstrates that both act as positive regulators, decreasing transcription greater than 10-fold. Mutation of both the ATF and EIIF binding sites inhibited the EIIA enhancer 200-fold. Analysis of insertion mutations suggests that the spatial alignment of the upstream ATF and EIIF binding sites with respect to the downstream EIIF binding site on the DNA helix is important. Consistent with previous findings, using gel shift analysis we demonstrate that the binding activity of EIIF is increased following wild-type adenovirus infection. In contrast, using identical gel shift conditions, the binding activity of ATF is decreased by viral infection.

Research paper thumbnail of Effects of mutation of the CREB binding site of the somatostatin promoter on cyclic AMP responsiveness in CV-1 cells

Research paper thumbnail of Simian virus 40 small t antigen trans activates the adenovirus E2A promoter by using mechanisms distinct from those used by adenovirus E1A

Journal of Virology

As reported previously for simian virus 40 small t antigen, polyomavirus small t antigen stimulat... more As reported previously for simian virus 40 small t antigen, polyomavirus small t antigen stimulates transcription directed by the adenovirus E2A and VA-I promoters during transient transfection assays. To determine whether papovaviral small t antigens might employ biochemical mechanisms during transcription activation that are either similar to or distinct from other viral trans activators, I compared the abilities of simian virus 40 small t antigen and adenovirus ElA to regulate the E2A promoter during transient transfection assays. I determined that, whereas activation of the E2A promoter by E1A involves the transcription factors ATF and EIIF, activation by small t antigen involves only EIIF. The effects of cotransfecting maximal concentrations of plasmids encoding small t antigen with ELA suggested that they activate the E2A promoter by different mechanisms. To determine whether small t antigen employs a mechanism different from that encoded in ElA domain H, domain m, or both, I compared the effects of transfecting plasmids expressing small t antigen, the 12S product of EIA, or the 13S product with a mutation in domain H on trans activation of the E2A promoter in two cellular backgrounds. On the basis of these comparisons, it appears that small t antigen does not activate transcription by a mechanism similar to either of the activities encoded in ElA. This suggests that papovavirus small t antigens belong to a distinct class of trans-acting proteins.

Research paper thumbnail of Multiple, distinct trans-activation func-tions are encoded by the simian virus 40 large T and small t antigens, only some of which require the 82-residue amino-terminal common domain

Journal of Virology

ABSTRACT

Research paper thumbnail of Analysis of maternal IgG subpopulations which are transported into the chicken oocyte

Immunology

Chicken serum and oocyte IgG were compared. Purified intact IgG and mercuripapain-produced Fc fra... more Chicken serum and oocyte IgG were compared. Purified intact IgG and mercuripapain-produced Fc fragments of yolk and serum IgG were analysed by isoelectric focusing. All IgG bands were identical, indicating that all subpopulations of serum IgG were present in the yolk. Upon papain hydrolysis of both serum and yolk IgG, four identical Fc bands were produced from all serum and yolk samples. Sialic acid measurements showed that there was no significant difference in sialic acid content between serum and oocyte IgG. From these results we conclude that: (i) ovarian IgG receptor(s) selectively transports all subpopulations of maternal IgG; (ii) there is no selective destruction of IgG during transport; and (iii) yolk IgG has the same amount of sialic acid as the serum IgG.

Research paper thumbnail of Stimulation of the adenovirus E2 promoter by simian virus 40 T antigen or E1A occurs by different mechanisms

Molecular and cellular biology, 1986

We have examined the ability of simian virus 40 T antigen to stimulate transcription from the ade... more We have examined the ability of simian virus 40 T antigen to stimulate transcription from the adenovirus E2 promoter. T antigen, produced from a cotransfected plasmid, stimulated chloramphenicol acetyltransferase enzyme and mRNA production from an E2 promoter-chloramphenicol acetyltransferase fusion plasmid (pEC113) in monkey kidney CV-1 cells. The level of stimulation of E2 transcription by simian virus 40 T antigen was equal to that observed in cotransfections of pEC113 and the adenovirus E1A gene product. Deletion mutations from the 5' end of the E2 promoter were examined for their ability to express basal, T-antigen, or E1A trans-activated promoter activity. In each case, deletion of upstream promoter sequences to -70 base pairs reduced chloramphenicol acetyltransferase expression to approximately 30% of the level observed with the intact E2 promoter. Deletion to -59 base pairs resulted in chloramphenicol acetyltransferase expression that was 3 to 5% of that observed with th...

Research paper thumbnail of Opportunities and challenges for repair of macrovascular disease using circulating blood-derived progenitor cells

Diabetes/metabolism research and reviews, 2014

There are currently few solutions for diabetic vascular disease that involve repair of damaged ti... more There are currently few solutions for diabetic vascular disease that involve repair of damaged tissues. The manuscript by Porat et al. in this issue, suggests a possible method to use a patient's own circulating blood cells to provide progenitors to repair damaged vascular tissues.

Research paper thumbnail of Free radicals and birth defects

The journal of maternal-fetal & neonatal medicine : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstetricians, 2004

Maternal diabetes significantly increases the risk for birth defects. Studies using animal models... more Maternal diabetes significantly increases the risk for birth defects. Studies using animal models indicate that oxidative stress may play a causative role. Oxidative stress can result from exposure to certain drugs, ionizing radiation and folic acid deficiency. Therefore, study of the mechanisms by which maternal diabetes affects embryogenesis may provide insight into general processes by which birth defects occur. Study of embryonic gene expression has demonstrated that maternal diabetes causes birth defects by disturbing expression of genes that control essential developmental processes, and that oxidative stress is involved. A model in which oxidative stress-induced deficient gene expression leads to congenital defects involving p53-dependent apoptosis is discussed.

Research paper thumbnail of Oxidant regulation of gene expression and neural tube development: Insights gained from diabetic pregnancy on molecular causes of neural tube defects

Diabetologia, 2003

Maternal diabetes increases oxidative stress in embryos. Maternal diabetes also inhibits expressi... more Maternal diabetes increases oxidative stress in embryos. Maternal diabetes also inhibits expression of embryonic genes, most notably, Pax-3, which is required for neural tube closure. Here we tested the hypothesis that oxidative stress inhibits expression of Pax-3, thereby providing a molecular basis for neural tube defects induced by diabetic pregnancy. Maternal diabetes-induced oxidative stress was blocked with alpha-tocopherol (vitamin E), and oxidative stress was induced with the complex III electron transport inhibitor, antimycin A, using pregnant diabetic or non-diabetic mice, primary cultures of neurulating mouse embryo tissues, or differentiating P19 embryonal carcinoma cells. Pax-3 expression was assayed by quantitative RT-PCR, and neural tube defects were scored by visual inspection. Oxidation-induced DNA fragmentation in P19 cells was assayed by electrophoretic analysis. Maternal diabetes inhibited Pax-3 expression and increased neural tube defects, and alpha-tocopherol b...

Research paper thumbnail of Genotoxicity and Diabetic Embryopathy: Impaired Expression of Developmental Control Genes as a Cause of Defective Morphogenesis

Seminars in Reproductive Medicine, 1999

Since the advent of insulin therapy for diabetes mellitus, the survival of mothers with diabetes ... more Since the advent of insulin therapy for diabetes mellitus, the survival of mothers with diabetes prior to pregnancy and their offspring has greatly improved. Nevertheless, the observation that the earliest stages of organogenesis can be impaired in the offspring of women with diabetes raises the question of how abnormal fuel metabolism disturbs embryogenesis. Research into this process has been made possible in recent years by advances in molecular biology which makes it possible to study gene expression in early embryos, and by the availability of genetically engineered mutant mouse strains. Using these approaches, a model is emerging in which elevated glucose, by disturbing expression of genes which regulate embryonic development and cell cycle progression, causes premature cell death of emerging organ structures, thereby causing defective morphogenesis. Investigation into the signaling mechanisms by which excess glucose metabolism exhibits toxic effects on embryo gene expression will explain how diabetic embryopathy occurs on a molecular and cellular level, as well as increase our understanding of the role of metabolic homeostasis in proper embryonic development.

Research paper thumbnail of Advances in Understanding the Molecular Causes of Diabetes-Induced Birth Defects

Journal of the Society for Gynecologic Investigation, 2006

Research paper thumbnail of Interaction between two transcriptional control sequences required for tumor-antigen-mediated simian virus 40 late gene expression

Proceedings of the National Academy of Sciences, 1985

Transcriptional control signals required for tumor (T)-antigen trans-activation ofthe simian viru... more Transcriptional control signals required for tumor (T)-antigen trans-activation ofthe simian virus 40 (SV40) late promoter include T-antigen binding sites I and II and the SV40 72-base-pair (bp) repeats. We have used in vivo competition studies to examine how these signals function in relationship to one another. In vivo competition with recombinant plasmids containing the entire SV40 late regulatory region and promoter sequences [map position (mp) 5171-272] results in quantitative removal of limiting trans-acting factor(s) required for late gene expression in COS-1 cells. Deletion of either the T-antigen binding sites (mp 5171-5243) or the 72-bp tandem repeat (mp 128-272) from the competitor plasmid results in markedly less efficient binding of the transacting factor, as judged by the loss of competition. Cotransfection of two separate plasmids, one containing the T-antigen binding sites I and II and the other containing the 72-bp repeats, fails to compete for the trans-acting factors. Insertion of increasing lengths of DNA sequences between the T-antigen binding sites and the enhancer sequences also dramatically reduces the efficiency of competition. These results suggest that efficient binding of trans-acting factors requires the presence, in cis, of at least two SV40 regulatory domains. Our studies further suggest that the distance separating these two transcriptional signals is important.

Research paper thumbnail of Intersection of complex genetic traits affecting maternal metabolism, fetal metabolism, and neural tube defect risk: Looking for needles in multiple haystacks

Molecular Genetics and Metabolism, 2014

Research paper thumbnail of Evidence that elevated glucose causes altered gene expression, apoptosis, and neural tube defects in a mouse model of diabetic pregnancy

Diabetes, 1999

Congenital malformations, including neural tube defects (NTDs), are significantly increased in th... more Congenital malformations, including neural tube defects (NTDs), are significantly increased in the offspring of diabetic mothers. We previously reported that in the embryos of a mouse model of diabetic pregnancy, NTDs are associated with reduced expression of the gene P a x -3, which encodes a transcription factor that regulates neural tube development, and that reduced expression of P a x -3 leads to neuroepithelial apoptosis. In this study, we used three approaches to test whether glucose alone could be responsible for these adverse e ffects of diabetes on embryonic development. First, primary culture of embryo tissue in medium containing 15 mmol/l glucose inhibited P a x -3 expression compared with culture in medium containing 5 mmol/l glucose. Second, inducing hyperglycemia in pregnant mice by subcutaneous glucose administration significantly inhibited P a x -3 expression (P < 0.05), as demonstrated by quantitative reverse transcription-polymerase chain reaction assay of Pax-3 mRNA, and also increased neural tube apoptosis (P < 0.05). NTDs were significantly increased in glucose-injected pregnancies when blood glucose levels were >250 mg/dl (P < 0.002) but not in moderately hyperglycemic pregnancies (150-250 mg/dl, P = 0.37). Third, phlorizin administration to pregnant diabetic mice reduced blood glucose levels and the rate of NTDs. As seen with glucose-injected pregnancies, the rate of NTDs in phlorizin-treated diabetic pregnancies was related to the severity of hyperglycemia, since NTDs were significantly increased in severely hyperglycemic (>250 mg/dl) diabetic pregnancies (P < 0.001) but not in moderately hyperglycemic pregnancies (150-250 mg/dl, P = 0.35). These two findings, that elevated glucose alone can cause the changes in P a x -3 expression observed during diabetic pregnancy and that the NTD rate rises with significant increases in blood glucose levels, suggest that congenital malformations associated with diabetic pregnancy are caused by disruption of regulatory gene expression in the embryo in response to elevated glucose.

Research paper thumbnail of Challenges in Understanding Diabetic Embryopathy

Research paper thumbnail of Understanding diabetic teratogenesis: Where are we now and where are we going?

Birth Defects Research Part A: Clinical and Molecular Teratology, 2010

Research paper thumbnail of Activation of the hexosamine pathway causes oxidative stress and abnormal embryo gene expression: Involvement in diabetic teratogenesis

Birth Defects Research Part A: Clinical and Molecular Teratology, 2004

Oxidative stress is critical to the teratogenic effects of diabetic pregnancy, yet the specific b... more Oxidative stress is critical to the teratogenic effects of diabetic pregnancy, yet the specific biochemical pathways responsible for oxidative stress have not been fully elucidated. The hexosamine pathway is activated in many tissues during diabetes and could contribute to oxidative stress by inhibiting the pentose shunt pathway, thereby diminishing production of the cellular antioxidant, reduced glutathione (GSH). METHODS: To test the hypothesis that activation of the hexosamine pathway might contribute to the teratogenic effects of diabetic pregnancy, pregnant mice were injected with glucose, to induce hyperglycemia, or glucosamine, to directly activate the hexosamine pathway. Embryo tissue fragments were also cultured in physiological glucose, high glucose, or physiological glucose plus glucosamine, to test effects on oxidative stress and embryo gene expression. RESULTS: Glucosamine increased hexosamine synthesis and inhibited pentose shunt activity. There was a trend for transient hyperglycemia to have the same effects, but they did not reach statistical significance. However, both glucose and glucosamine significantly decreased GSH, and increased oxidative stress, as indicated by 2Ј,7Ј-dichloro-dihydrofluorescein fluorescence. Glucose and glucosamine inhibited expression of Pax-3, a gene required for neural tube closure both in vivo and in vitro, and increased neural tube defects (NTDs) in vivo; these effects were prevented by GSH ethyl ester. High glucose and glucosamine inhibited Pax-3 expression by embryo culture, but culture in glutamine-free media to block the hexosamine pathway prevented the inhibition of Pax-3 expression by high glucose. CONCLUSIONS: Activation of the hexosamine pathway causes oxidative stress through depletion of GSH and consequent disruption of embryo gene expression. Activation of this pathway may contribute to diabetic teratogenesis.

Research paper thumbnail of Involvement of simian virus 40 (SV40) small t antigen in trans activation of SV40 early and late promoters

Journal of Virology

We have previously found that simian virus 40 (SV40) small t antigen (small t) can trans activate... more We have previously found that simian virus 40 (SV40) small t antigen (small t) can trans activate the E2A and VA-I genes of adenovirus in plasmid DNA-transfected cells (M. R. Loeken, I. Bikel, D. M. Livingston, and J. Brady, Cell 55:1171-1177. To determine whether trans activation by small t might be involved in the SV40 productive infection cycle, we examined the effects of cotransfecting plasmids encoding small t with plasmids containing the chloramphenicol acetyltransferase (CAT) gene linked to the SV40 early or late promoter. Small t increased threeto fivefold the expression of a CAT plasmid linked to the SV40 early promoter and enhancer. Small t expression had no effect by itself on CAT activity directed by the SV40 late promoter, but small t enhanced the effect of a suboptimal concentration of a plasmid expressing large T up to 10-fold. When the concentration of the plasmid expressing large T was increased to a level at which large T alone stimulated the late promoter ninefold, the enhancement by small t was only twofold. The effects of small t on both the SV40 early and late promoters depended on sequences within the small t-unique domain, since a plasmid expressing only the first 82 amino acids common to both large T and small t was inactive. The effects of small t on earlyand late-promoter-directed CAT enzyme activity was reflected in increased CAT mRNA as measured by S1 analysis. These results suggest that SV40 small t may play a role in viral infection by increasing transcription from the early promoter and from the late promoter at times when large T levels are low.

Research paper thumbnail of Identification of a new binding motif for the paired domain of Pax-3 and unusual characteristics of spacing of bipartite recognition elements on binding and transcription activation

Journal of Biological Chemistry

Pax-3, a transcription factor that is required for development of the embryonic neural tube, neur... more Pax-3, a transcription factor that is required for development of the embryonic neural tube, neural crest, and somitic derivatives, contains two DNA-binding domains, a paired domain, and a paired-type homeodomain. Although Pax-3 binds to sequences related to the e5 element of the Drosophila even-skipped gene, the sequence requirements of an optimal Pax-3 response element have not been well characterized. Using both DNAbinding domains and a pool of random oligonucleotides, we identified a new paired box consensus motif, "GT-TAT," which was located 1, 4, 5, 8, or 13 base pairs downstream of the homeobox binding motif, "ATTA." Binding analysis of these sequences demonstrated that the distance between recognition elements for the homeodomain and the paired domain affects affinity. Specifically, spacing elements 1 or 13 base pairs apart from each other conferred low affinity Pax-3 binding, whereas intermediate spacing (5 or 8 base pairs) conferred high affinity binding. Contrary to previous reports, oligonucleotides deleted for either the ATTA or the GTTAT could also be bound by Pax-3, although both sites were necessary for maximal affinity. Finally, transient transfections demonstrated that Pax-3 trans-activation correlated with binding affinity. Because the Pax-3-responsive genes identified to date contain almost exclusively low affinity binding sequences, our analysis indicates that they may be responsive to Pax-3 only when cellular levels are high.

Research paper thumbnail of Mouse embryonic stem cells established in physiological-glucose media express the high KM Glut2 glucose transporter expressed by normal embryos

Stem cells translational medicine, 2013

Glut2 is one of the facilitative glucose transporters expressed by preimplantation and early post... more Glut2 is one of the facilitative glucose transporters expressed by preimplantation and early postimplantation embryos. Glut2 is important for survival before embryonic day 10.5. The Glut2 KM (∼16 mmol/liter) is significantly higher than physiologic glucose concentrations (∼5.5 mmol/liter), suggesting that Glut2 normally performs some essential function other than glucose transport. Nevertheless, Glut2 efficiently transports glucose when extracellular glucose concentrations are above the Glut2 KM. Media containing 25 mmol/liter glucose are widely used to establish and propagate embryonic stem cells (ESCs). Glut2-mediated glucose uptake by embryos induces oxidative stress and can cause embryo cell death. Here we tested the hypothesis that low-glucose embryonic stem cells (LG-ESCs) isolated in physiological-glucose (5.5 mmol/liter) media express a functional Glut2 glucose transporter. LG-ESCs were compared with conventional D3 ESCs that had been cultured only in high-glucose media. LG-...

Research paper thumbnail of The adenovirus EIIA enhancer. Analysis of regulatory sequences and changes in binding activity of ATF and EIIF following adenovirus infection

Journal of Biological Chemistry

Adenovirus EIIA upstream sequences which contain the binding sites for proteins ATF and EIIF act ... more Adenovirus EIIA upstream sequences which contain the binding sites for proteins ATF and EIIF act as an enhancer and can be trans-activated by both E1A and SV40 T/t-antigens. Specific mutation of either the ATF or EIIF binding site demonstrates that both act as positive regulators, decreasing transcription greater than 10-fold. Mutation of both the ATF and EIIF binding sites inhibited the EIIA enhancer 200-fold. Analysis of insertion mutations suggests that the spatial alignment of the upstream ATF and EIIF binding sites with respect to the downstream EIIF binding site on the DNA helix is important. Consistent with previous findings, using gel shift analysis we demonstrate that the binding activity of EIIF is increased following wild-type adenovirus infection. In contrast, using identical gel shift conditions, the binding activity of ATF is decreased by viral infection.

Research paper thumbnail of Effects of mutation of the CREB binding site of the somatostatin promoter on cyclic AMP responsiveness in CV-1 cells

Research paper thumbnail of Simian virus 40 small t antigen trans activates the adenovirus E2A promoter by using mechanisms distinct from those used by adenovirus E1A

Journal of Virology

As reported previously for simian virus 40 small t antigen, polyomavirus small t antigen stimulat... more As reported previously for simian virus 40 small t antigen, polyomavirus small t antigen stimulates transcription directed by the adenovirus E2A and VA-I promoters during transient transfection assays. To determine whether papovaviral small t antigens might employ biochemical mechanisms during transcription activation that are either similar to or distinct from other viral trans activators, I compared the abilities of simian virus 40 small t antigen and adenovirus ElA to regulate the E2A promoter during transient transfection assays. I determined that, whereas activation of the E2A promoter by E1A involves the transcription factors ATF and EIIF, activation by small t antigen involves only EIIF. The effects of cotransfecting maximal concentrations of plasmids encoding small t antigen with ELA suggested that they activate the E2A promoter by different mechanisms. To determine whether small t antigen employs a mechanism different from that encoded in ElA domain H, domain m, or both, I compared the effects of transfecting plasmids expressing small t antigen, the 12S product of EIA, or the 13S product with a mutation in domain H on trans activation of the E2A promoter in two cellular backgrounds. On the basis of these comparisons, it appears that small t antigen does not activate transcription by a mechanism similar to either of the activities encoded in ElA. This suggests that papovavirus small t antigens belong to a distinct class of trans-acting proteins.

Research paper thumbnail of Multiple, distinct trans-activation func-tions are encoded by the simian virus 40 large T and small t antigens, only some of which require the 82-residue amino-terminal common domain

Journal of Virology

ABSTRACT

Research paper thumbnail of Analysis of maternal IgG subpopulations which are transported into the chicken oocyte

Immunology

Chicken serum and oocyte IgG were compared. Purified intact IgG and mercuripapain-produced Fc fra... more Chicken serum and oocyte IgG were compared. Purified intact IgG and mercuripapain-produced Fc fragments of yolk and serum IgG were analysed by isoelectric focusing. All IgG bands were identical, indicating that all subpopulations of serum IgG were present in the yolk. Upon papain hydrolysis of both serum and yolk IgG, four identical Fc bands were produced from all serum and yolk samples. Sialic acid measurements showed that there was no significant difference in sialic acid content between serum and oocyte IgG. From these results we conclude that: (i) ovarian IgG receptor(s) selectively transports all subpopulations of maternal IgG; (ii) there is no selective destruction of IgG during transport; and (iii) yolk IgG has the same amount of sialic acid as the serum IgG.

Research paper thumbnail of Stimulation of the adenovirus E2 promoter by simian virus 40 T antigen or E1A occurs by different mechanisms

Molecular and cellular biology, 1986

We have examined the ability of simian virus 40 T antigen to stimulate transcription from the ade... more We have examined the ability of simian virus 40 T antigen to stimulate transcription from the adenovirus E2 promoter. T antigen, produced from a cotransfected plasmid, stimulated chloramphenicol acetyltransferase enzyme and mRNA production from an E2 promoter-chloramphenicol acetyltransferase fusion plasmid (pEC113) in monkey kidney CV-1 cells. The level of stimulation of E2 transcription by simian virus 40 T antigen was equal to that observed in cotransfections of pEC113 and the adenovirus E1A gene product. Deletion mutations from the 5' end of the E2 promoter were examined for their ability to express basal, T-antigen, or E1A trans-activated promoter activity. In each case, deletion of upstream promoter sequences to -70 base pairs reduced chloramphenicol acetyltransferase expression to approximately 30% of the level observed with the intact E2 promoter. Deletion to -59 base pairs resulted in chloramphenicol acetyltransferase expression that was 3 to 5% of that observed with th...

Research paper thumbnail of Opportunities and challenges for repair of macrovascular disease using circulating blood-derived progenitor cells

Diabetes/metabolism research and reviews, 2014

There are currently few solutions for diabetic vascular disease that involve repair of damaged ti... more There are currently few solutions for diabetic vascular disease that involve repair of damaged tissues. The manuscript by Porat et al. in this issue, suggests a possible method to use a patient's own circulating blood cells to provide progenitors to repair damaged vascular tissues.

Research paper thumbnail of Free radicals and birth defects

The journal of maternal-fetal & neonatal medicine : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstetricians, 2004

Maternal diabetes significantly increases the risk for birth defects. Studies using animal models... more Maternal diabetes significantly increases the risk for birth defects. Studies using animal models indicate that oxidative stress may play a causative role. Oxidative stress can result from exposure to certain drugs, ionizing radiation and folic acid deficiency. Therefore, study of the mechanisms by which maternal diabetes affects embryogenesis may provide insight into general processes by which birth defects occur. Study of embryonic gene expression has demonstrated that maternal diabetes causes birth defects by disturbing expression of genes that control essential developmental processes, and that oxidative stress is involved. A model in which oxidative stress-induced deficient gene expression leads to congenital defects involving p53-dependent apoptosis is discussed.

Research paper thumbnail of Oxidant regulation of gene expression and neural tube development: Insights gained from diabetic pregnancy on molecular causes of neural tube defects

Diabetologia, 2003

Maternal diabetes increases oxidative stress in embryos. Maternal diabetes also inhibits expressi... more Maternal diabetes increases oxidative stress in embryos. Maternal diabetes also inhibits expression of embryonic genes, most notably, Pax-3, which is required for neural tube closure. Here we tested the hypothesis that oxidative stress inhibits expression of Pax-3, thereby providing a molecular basis for neural tube defects induced by diabetic pregnancy. Maternal diabetes-induced oxidative stress was blocked with alpha-tocopherol (vitamin E), and oxidative stress was induced with the complex III electron transport inhibitor, antimycin A, using pregnant diabetic or non-diabetic mice, primary cultures of neurulating mouse embryo tissues, or differentiating P19 embryonal carcinoma cells. Pax-3 expression was assayed by quantitative RT-PCR, and neural tube defects were scored by visual inspection. Oxidation-induced DNA fragmentation in P19 cells was assayed by electrophoretic analysis. Maternal diabetes inhibited Pax-3 expression and increased neural tube defects, and alpha-tocopherol b...

Research paper thumbnail of Genotoxicity and Diabetic Embryopathy: Impaired Expression of Developmental Control Genes as a Cause of Defective Morphogenesis

Seminars in Reproductive Medicine, 1999

Since the advent of insulin therapy for diabetes mellitus, the survival of mothers with diabetes ... more Since the advent of insulin therapy for diabetes mellitus, the survival of mothers with diabetes prior to pregnancy and their offspring has greatly improved. Nevertheless, the observation that the earliest stages of organogenesis can be impaired in the offspring of women with diabetes raises the question of how abnormal fuel metabolism disturbs embryogenesis. Research into this process has been made possible in recent years by advances in molecular biology which makes it possible to study gene expression in early embryos, and by the availability of genetically engineered mutant mouse strains. Using these approaches, a model is emerging in which elevated glucose, by disturbing expression of genes which regulate embryonic development and cell cycle progression, causes premature cell death of emerging organ structures, thereby causing defective morphogenesis. Investigation into the signaling mechanisms by which excess glucose metabolism exhibits toxic effects on embryo gene expression will explain how diabetic embryopathy occurs on a molecular and cellular level, as well as increase our understanding of the role of metabolic homeostasis in proper embryonic development.

Research paper thumbnail of Advances in Understanding the Molecular Causes of Diabetes-Induced Birth Defects

Journal of the Society for Gynecologic Investigation, 2006

Research paper thumbnail of Interaction between two transcriptional control sequences required for tumor-antigen-mediated simian virus 40 late gene expression

Proceedings of the National Academy of Sciences, 1985

Transcriptional control signals required for tumor (T)-antigen trans-activation ofthe simian viru... more Transcriptional control signals required for tumor (T)-antigen trans-activation ofthe simian virus 40 (SV40) late promoter include T-antigen binding sites I and II and the SV40 72-base-pair (bp) repeats. We have used in vivo competition studies to examine how these signals function in relationship to one another. In vivo competition with recombinant plasmids containing the entire SV40 late regulatory region and promoter sequences [map position (mp) 5171-272] results in quantitative removal of limiting trans-acting factor(s) required for late gene expression in COS-1 cells. Deletion of either the T-antigen binding sites (mp 5171-5243) or the 72-bp tandem repeat (mp 128-272) from the competitor plasmid results in markedly less efficient binding of the transacting factor, as judged by the loss of competition. Cotransfection of two separate plasmids, one containing the T-antigen binding sites I and II and the other containing the 72-bp repeats, fails to compete for the trans-acting factors. Insertion of increasing lengths of DNA sequences between the T-antigen binding sites and the enhancer sequences also dramatically reduces the efficiency of competition. These results suggest that efficient binding of trans-acting factors requires the presence, in cis, of at least two SV40 regulatory domains. Our studies further suggest that the distance separating these two transcriptional signals is important.

Research paper thumbnail of Intersection of complex genetic traits affecting maternal metabolism, fetal metabolism, and neural tube defect risk: Looking for needles in multiple haystacks

Molecular Genetics and Metabolism, 2014

Research paper thumbnail of Evidence that elevated glucose causes altered gene expression, apoptosis, and neural tube defects in a mouse model of diabetic pregnancy

Diabetes, 1999

Congenital malformations, including neural tube defects (NTDs), are significantly increased in th... more Congenital malformations, including neural tube defects (NTDs), are significantly increased in the offspring of diabetic mothers. We previously reported that in the embryos of a mouse model of diabetic pregnancy, NTDs are associated with reduced expression of the gene P a x -3, which encodes a transcription factor that regulates neural tube development, and that reduced expression of P a x -3 leads to neuroepithelial apoptosis. In this study, we used three approaches to test whether glucose alone could be responsible for these adverse e ffects of diabetes on embryonic development. First, primary culture of embryo tissue in medium containing 15 mmol/l glucose inhibited P a x -3 expression compared with culture in medium containing 5 mmol/l glucose. Second, inducing hyperglycemia in pregnant mice by subcutaneous glucose administration significantly inhibited P a x -3 expression (P < 0.05), as demonstrated by quantitative reverse transcription-polymerase chain reaction assay of Pax-3 mRNA, and also increased neural tube apoptosis (P < 0.05). NTDs were significantly increased in glucose-injected pregnancies when blood glucose levels were >250 mg/dl (P < 0.002) but not in moderately hyperglycemic pregnancies (150-250 mg/dl, P = 0.37). Third, phlorizin administration to pregnant diabetic mice reduced blood glucose levels and the rate of NTDs. As seen with glucose-injected pregnancies, the rate of NTDs in phlorizin-treated diabetic pregnancies was related to the severity of hyperglycemia, since NTDs were significantly increased in severely hyperglycemic (>250 mg/dl) diabetic pregnancies (P < 0.001) but not in moderately hyperglycemic pregnancies (150-250 mg/dl, P = 0.35). These two findings, that elevated glucose alone can cause the changes in P a x -3 expression observed during diabetic pregnancy and that the NTD rate rises with significant increases in blood glucose levels, suggest that congenital malformations associated with diabetic pregnancy are caused by disruption of regulatory gene expression in the embryo in response to elevated glucose.

Research paper thumbnail of Challenges in Understanding Diabetic Embryopathy

Research paper thumbnail of Understanding diabetic teratogenesis: Where are we now and where are we going?

Birth Defects Research Part A: Clinical and Molecular Teratology, 2010

Research paper thumbnail of Activation of the hexosamine pathway causes oxidative stress and abnormal embryo gene expression: Involvement in diabetic teratogenesis

Birth Defects Research Part A: Clinical and Molecular Teratology, 2004

Oxidative stress is critical to the teratogenic effects of diabetic pregnancy, yet the specific b... more Oxidative stress is critical to the teratogenic effects of diabetic pregnancy, yet the specific biochemical pathways responsible for oxidative stress have not been fully elucidated. The hexosamine pathway is activated in many tissues during diabetes and could contribute to oxidative stress by inhibiting the pentose shunt pathway, thereby diminishing production of the cellular antioxidant, reduced glutathione (GSH). METHODS: To test the hypothesis that activation of the hexosamine pathway might contribute to the teratogenic effects of diabetic pregnancy, pregnant mice were injected with glucose, to induce hyperglycemia, or glucosamine, to directly activate the hexosamine pathway. Embryo tissue fragments were also cultured in physiological glucose, high glucose, or physiological glucose plus glucosamine, to test effects on oxidative stress and embryo gene expression. RESULTS: Glucosamine increased hexosamine synthesis and inhibited pentose shunt activity. There was a trend for transient hyperglycemia to have the same effects, but they did not reach statistical significance. However, both glucose and glucosamine significantly decreased GSH, and increased oxidative stress, as indicated by 2Ј,7Ј-dichloro-dihydrofluorescein fluorescence. Glucose and glucosamine inhibited expression of Pax-3, a gene required for neural tube closure both in vivo and in vitro, and increased neural tube defects (NTDs) in vivo; these effects were prevented by GSH ethyl ester. High glucose and glucosamine inhibited Pax-3 expression by embryo culture, but culture in glutamine-free media to block the hexosamine pathway prevented the inhibition of Pax-3 expression by high glucose. CONCLUSIONS: Activation of the hexosamine pathway causes oxidative stress through depletion of GSH and consequent disruption of embryo gene expression. Activation of this pathway may contribute to diabetic teratogenesis.

Research paper thumbnail of Involvement of simian virus 40 (SV40) small t antigen in trans activation of SV40 early and late promoters

Journal of Virology

We have previously found that simian virus 40 (SV40) small t antigen (small t) can trans activate... more We have previously found that simian virus 40 (SV40) small t antigen (small t) can trans activate the E2A and VA-I genes of adenovirus in plasmid DNA-transfected cells (M. R. Loeken, I. Bikel, D. M. Livingston, and J. Brady, Cell 55:1171-1177. To determine whether trans activation by small t might be involved in the SV40 productive infection cycle, we examined the effects of cotransfecting plasmids encoding small t with plasmids containing the chloramphenicol acetyltransferase (CAT) gene linked to the SV40 early or late promoter. Small t increased threeto fivefold the expression of a CAT plasmid linked to the SV40 early promoter and enhancer. Small t expression had no effect by itself on CAT activity directed by the SV40 late promoter, but small t enhanced the effect of a suboptimal concentration of a plasmid expressing large T up to 10-fold. When the concentration of the plasmid expressing large T was increased to a level at which large T alone stimulated the late promoter ninefold, the enhancement by small t was only twofold. The effects of small t on both the SV40 early and late promoters depended on sequences within the small t-unique domain, since a plasmid expressing only the first 82 amino acids common to both large T and small t was inactive. The effects of small t on earlyand late-promoter-directed CAT enzyme activity was reflected in increased CAT mRNA as measured by S1 analysis. These results suggest that SV40 small t may play a role in viral infection by increasing transcription from the early promoter and from the late promoter at times when large T levels are low.

Research paper thumbnail of Identification of a new binding motif for the paired domain of Pax-3 and unusual characteristics of spacing of bipartite recognition elements on binding and transcription activation

Journal of Biological Chemistry

Pax-3, a transcription factor that is required for development of the embryonic neural tube, neur... more Pax-3, a transcription factor that is required for development of the embryonic neural tube, neural crest, and somitic derivatives, contains two DNA-binding domains, a paired domain, and a paired-type homeodomain. Although Pax-3 binds to sequences related to the e5 element of the Drosophila even-skipped gene, the sequence requirements of an optimal Pax-3 response element have not been well characterized. Using both DNAbinding domains and a pool of random oligonucleotides, we identified a new paired box consensus motif, "GT-TAT," which was located 1, 4, 5, 8, or 13 base pairs downstream of the homeobox binding motif, "ATTA." Binding analysis of these sequences demonstrated that the distance between recognition elements for the homeodomain and the paired domain affects affinity. Specifically, spacing elements 1 or 13 base pairs apart from each other conferred low affinity Pax-3 binding, whereas intermediate spacing (5 or 8 base pairs) conferred high affinity binding. Contrary to previous reports, oligonucleotides deleted for either the ATTA or the GTTAT could also be bound by Pax-3, although both sites were necessary for maximal affinity. Finally, transient transfections demonstrated that Pax-3 trans-activation correlated with binding affinity. Because the Pax-3-responsive genes identified to date contain almost exclusively low affinity binding sequences, our analysis indicates that they may be responsive to Pax-3 only when cellular levels are high.

Research paper thumbnail of Mouse embryonic stem cells established in physiological-glucose media express the high KM Glut2 glucose transporter expressed by normal embryos

Stem cells translational medicine, 2013

Glut2 is one of the facilitative glucose transporters expressed by preimplantation and early post... more Glut2 is one of the facilitative glucose transporters expressed by preimplantation and early postimplantation embryos. Glut2 is important for survival before embryonic day 10.5. The Glut2 KM (∼16 mmol/liter) is significantly higher than physiologic glucose concentrations (∼5.5 mmol/liter), suggesting that Glut2 normally performs some essential function other than glucose transport. Nevertheless, Glut2 efficiently transports glucose when extracellular glucose concentrations are above the Glut2 KM. Media containing 25 mmol/liter glucose are widely used to establish and propagate embryonic stem cells (ESCs). Glut2-mediated glucose uptake by embryos induces oxidative stress and can cause embryo cell death. Here we tested the hypothesis that low-glucose embryonic stem cells (LG-ESCs) isolated in physiological-glucose (5.5 mmol/liter) media express a functional Glut2 glucose transporter. LG-ESCs were compared with conventional D3 ESCs that had been cultured only in high-glucose media. LG-...