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J Dittmer

Publications and source records attributed to J Dittmer.

At least 37 records · Page 2Linked to original sources
PubMed1995

An Ets site in the whey acidic protein gene promoter mediates transcriptional activation in the mammary gland of pregnant mice but is dispensable during lactation.

The whey acidic protein (WAP) gene is specifically expressed in mammary tissue, and its transcription is induced several thousand-fold during pregnancy and remains high throughout lactation. A purine-rich sequence (PRS) located around -110 of the WAP gene promoter is conserved between mice, rats, and rabbits, suggesting that it features a regulatory element. This PRS contains an invariant GGAA/T core motif characteristic of the binding site for Ets transcription factors. Electromobility shift assays demonstrate that Ets1 binds specifically to the PRS. Experiments in transgenic mice further demonstrate that this PRS/Ets site plays a critical role in the activation of WAP transgenes during pregnancy, but that its presence is not required for high expression throughout lactation. Transgenes with an intact PRS/Ets site are expressed at high levels at day 13 of pregnancy, with little further increase during late pregnancy and lactation. In contrast, WAP transgenes with a mutation in the PRS/Ets site, which abrogates the binding of Ets1, are not expressed at midpregnancy, but their transcriptional activity is not affected during lactation. These results demonstrate that Ets-signaling pathways can function as stage-specific transcriptional activators of milk protein genes in the developing mammary gland. In addition, this work extends earlier findings that gene activation during pregnancy and lactation is mediated, in part, by different mechanisms.

Animals↗
PubMed1994

Regulation of parathyroid hormone-related protein (PTHrP) gene expression. Sp1 binds through an inverted CACCC motif and regulates promoter activity in cooperation with Ets1.

We have previously shown that mutations in the GGAA core motif of the Ets1 binding site, EBSI, or deletion of EBSI, reduced basal and Tax1 transactivation of the PTHrP P2 promoter. Here we demonstrate that, in addition to EBSI, a CACCC-like motif located between -53 and -58 is required for full basal activity of this promoter in Jurkat T-cells. Site-specific mutations in the CACCC motif decreased promoter activity approximately 5-fold. In an effort to identify transcription factors that bind to the CACCC element, we found that purified human Sp1, as well as Sp1 in HeLa nuclear extract, can specifically bind to a DNA probe that corresponds to the PTHrP-specific sequence between -94 and -34. Gel shift competition studies and DNase I footprinting analyses revealed that Sp1 specifically interacts with the CACCC motif. In the presence of Ets1, the mobility of the Sp1-specific gel shift complex with the PTHrP DNA decreased. DNase I footprint analysis of this gel shift complex showed an extended footprint over both the Sp1 and the Ets1 binding site, demonstrating that Sp1 and Ets1 form a ternary complex with the PTHrP DNA. Cotransfection of an Ets1 and Sp1 expression vector into Drosophila Schneider cells demonstrated that Sp1 can functionally cooperate with Ets1 to transactivate the PTHrP promoter. We conclude from these data that Ets1 and Sp1 can cooperatively regulate PTHrP P2 promoter activity.

Base Sequence↗
PubMed1994

Involvement of transcription factor YB-1 in human T-cell lymphotropic virus type I basal gene expression.

Sequences which control basal human T-cell lymphotropic virus type I (HTLV-I) transcription likely play an important role in initiation and maintenance of virus replication. We previously identified and analyzed a 45-nucleotide sequence (downstream regulatory element 1 [DRE 1]), +195 to +240, at the boundary of the R/U5 region of the long terminal repeat which is required for HTLV-I basal transcription. We identified a protein, p37, which specifically bound to DRE 1. An affinity column fraction, containing p37, stimulated HTLV-I transcription approximately 12-fold in vitro. We now report the identification of a cDNA clone (15B-7), from a Jurkat expression library, that binds specifically to the DRE 1 regulatory sequence. Binding of the cDNA fusion protein, similarly to the results obtained with purified Jurkat protein, was decreased by introduction of site-specific mutations in the DRE 1 regulatory sequence. In vitro transcription and translation of 15B-7 cDNA produced a fusion protein which bound specifically to the HTLV-I +195 to +240 oligonucleotide. The partial cDNA encodes a protein which is homologous to the C-terminal 196 amino acids of the 36-kDa transcription factor, YB-1. Cotransfection of a YB-1 expression plasmid increases HTLV-I basal transcription approximately 14-fold in Jurkat T lymphocytes. On the basis of the molecular weight, DNA-binding characteristics, and in vivo transactivation activity, we suggest that the previously identified DRE 1-binding protein, p37, is YB-1.

Amino Acid Sequence↗
PubMed1993

Transactivation of the P2 promoter of parathyroid hormone-related protein by human T-cell lymphotropic virus type I Tax1: evidence for the involvement of transcription factor Ets1.

Expression of the parathyroid hormone-related protein (PTHrP), a protein that plays a primary role in the development of the humoral hypercalcemia of malignancy, is regulated by two distinct promoters, P1 and P2. PTHrP is overexpressed in lymphocytes from adult T-cell leukemia patients. We now demonstrate that in the human T-cell lymphotropic virus type I-transformed cell line MT-2, RNA synthesis is initiated primarily at the P2 promoter. Furthermore, in cotransfection experiments, Tax1 transactivates the P2 promoter 10- to 12-fold. By using deletion and site-specific point mutations, we have identified a promoter-proximal sequence (positions -72 to -40) which is important for Tax1 transactivation. The PTHrP promoter-proximal element contains two potential overlapping Ets1 binding sites, EBS I and EBS II. Gel shift analysis demonstrated that Ets1 binds specifically to both EBS I and EBS II. Mutation of the consensus GGAA core motif in EBS I abolished binding and Tax1 transactivation in Jurkat T lymphocytes. In Ets1-deficient cells, cotransfection of Tax1 and Ets1 expression plasmids stimulates PTHrP promoter activity. In the absence of Ets1, minimal transactivation of the PTHrP promoter is observed. These data suggest that Ets1 binds to EBS I and cooperates with Tax1 to transactivate the PTHrP P2 promoter.

Base Sequence↗
PubMed1993

Transcriptional activation of the human T-lymphotropic virus type I long terminal repeat by functional interaction of Tax1 and Ets1.

Transcription regulation of the oncogenic retrovirus human T-lymphotropic virus type I (HTLV-I) involves the composite activity of both viral and cellular transcription factors. The HTLV-I transforming protein, Tax1, modulates the activity of several cellular transcription factors, upregulating the level of viral gene expression. In addition, cellular transcription factors, such as Ets1, independently bind to the viral long terminal repeat in a sequence-specific manner and activate transcription. It was of interest to analyze the possible interaction of Tax1 and Ets1 in viral gene regulation. We now report that Tax1 and Ets1 increase expression from the HTLV-I promoter in a cooperative manner. The level of expression was increased 5- to 10-fold above the combined individual effect of Tax1 and Ets1. S1 nuclease analysis demonstrated that the cooperative effect was due to an increase in the levels of steady-state RNA. The functional interaction between Tax1 and Ets1 required the presence of the Tax1-responsive 21-bp repeat element TRE-1 and the Ets1-responsive element ERR-1. These results suggested the possible interaction of Ets1 with transcriptional regulatory proteins that bind to the 21-bp repeats. This interaction is demonstrated by decreased electrophoretic mobility of specific 21-bp repeat gel shift complexes in the presence of Ets1. Furthermore, interaction of Ets1 with the 21-bp repeat-binding proteins enhances the relative efficiency of binding to the DNA. This cooperative interaction between Ets1 and proteins which bind to the Tax1-responsive 21-bp repeats suggests a possible role for Ets1 in the regulation of viral gene expression.

Animals↗
PubMed1993

Stabilization of wild-type p53 in human T-lymphocytes transformed by HTLV-I.

Adult T-cell leukemia/lymphoma is an aggressive malignancy associated with infection by the human T-lymphotropic virus type-I (HTLV-I). We now demonstrate that p53 expression is elevated in the HTLV-I-transformed T-lymphocyte lines C81, MT-2, MT-4 and HUT 102. In pulse-chase experiments, the p53 protein demonstrated a prolonged half-life of 2 to 8 h in HTLV-I-transformed cells compared with 0.5 to 1.0 h for wild-type p53 in primary human and murine fibroblasts, or human peripheral blood lymphocytes. In cell lines C81 and HUT 102, which exhibited the longest p53 protein half-life, the wild-type-related PAb1620 epitope was detected at reduced levels. The PAb240 mutant-related p53 epitope was not detected in any of the transformed cell lines. By direct sequence analysis of RT-PCR products, the entire p53 cDNA coding sequence was determined to be wild-type in all four cell lines. Stabilization of wild-type p53 may represent its functional inactivation and contribute to lymphocyte transformation by HTLV-I.

Base Sequence↗
PubMed1992

Inhibitory effect of zinc on stimulated erythropoietin synthesis in HepG2 cells.

The effect of zinc on erythropoietin (EPO) synthesis in HepG2 cells was investigated. The increase in EPO synthesis induced by Co2+ (50 microM), Ni2+ (300 microM) or oxygen (1% O2) was inhibited by the presence of ZnCl2 (50-150 microM) in the tissue-culture medium, whereas basal EPO synthesis was unaffected. The effect was reflected by corresponding changes in the EPO mRNA level. These effects of zinc on EPO synthesis could not be mimicked by CdCl2 (less than or equal to 2 microM). Addition of FeCl3 to the medium appeared to decrease the inhibitory effect of zinc on hypoxia-induced EPO synthesis, implying that zinc may interfere with an iron-dependent step in EPO regulation.

Blotting, Northern↗
PubMed1992

Intravascular circulation and distribution of human 51Cr-DBBF stroma-free hemoglobin.

Male B6C3HF1 mice were infused with 51Cr-labeled DBBF (bis 3,5-dibromosalicyl fumarate) crosslinked stroma-free hemoglobin (SFH). The intravascular halftime (T50) of the DBBF-SFH, determined from plasma hemoglobin levels, was 0.5 hours in the first 10 minutes and 4.3 hours during the next 50 minutes. At 24 hours, less than 5% of the DBBF-SFH remained. Elution of 51Cr was reflected in a lower T50 determined from the radioactivity levels: during the first 10 minutes the T50 was 0.3 hours; in the next 50 minutes it was 1 hour. The radioactivity sequestered in each organ in the first hour following DBBF-SFH infusion was as follows: 11.2% of the infused radioactivity was in the skin, 11.4% in muscle, 9.1% in the skeleton, and 5% in the liver. After 24 hours, the percentages in skin, muscle, skeleton and liver were 15.4, 10.3, 16.6 and 6.7% respectively. The percentage of infused radioactivity in the gastrointestinal tract and kidney at 1 hour and 24 hours ranged from 3.5 to 5.5%. Less than 0.4% was found in the spleen and lung. At 24 hours, 25% of the radioactivity was recovered in urine and 3% in feces.

Animals↗
PubMed1990

Expression of rat liver Na+/L-alanine co-transport in Xenopus laevis oocytes. Effect of glucagon in vivo.

Poly(A)+ RNA (mRNA) isolated from rat liver was injected into Xenopus laevis oocytes, and expression of Na+/L-alanine transport was assayed by measuring Na(+)-dependent uptake of L-[3H]alanine. Expression of Na+/L-alanine transport was detected 3-7 days after mRNA injection, and was due to an increment of the Na(+)-dependent component. After injection of 40 ng of total mRNA, Na(+)-dependent uptake of L-alanine was 2.5-fold higher than in water-injected oocytes. In contrast with Na+/L-alanine transport by water-injected oocytes, expressed Na+/L-alanine transport was inhibited by N-methylaminoisobutyric acid, was inhibited by an extracellular pH of 6.5 and was saturated at approx. 1 mM-L-alanine. After sucrose-density-gradient fractionation, highest expression of Na+/L-alanine uptake was observed with mRNA of 1.9-2.5 kb in length. Compared with mRNA isolated from control rats, mRNA isolated from glucagon-treated rats showed a approx. 2-fold higher expression of Na+/L-alanine transport. The results demonstrate that both liver Na+/L-alanine transport systems (A and ASC) can be expressed in X. laevis oocytes. Furthermore, the data obtained with mRNA isolated from glucagon-treated rats suggest that glucagon regulates liver Na+/L-alanine transport (at least in part) via the availability of the corresponding mRNA.

Alanine↗
PubMed1990

Decline of erythropoietin formation at continuous hypoxia is not due to feedback inhibition.

Serum erythropoietin (EPO) levels in response to hypoxia are known to decline before an increase in blood oxygen carrying capacity. To define the possible mechanisms underlying this phenomenon, we have investigated 1) how renal EPO mRNA content and EPO production rate underlying the early kinetics of serum EPO levels change under different degrees of normobaric hypoxia, and 2) if a feedback inhibition of either EPO formation or EPO survival in the circulation exists by the hormone itself. We found that serum immunoreactive EPO levels in rats peaked after 12-h exposure to 7.5 or 9% oxygen (2,949 +/- 600 and 756 +/- 108 mU/ml, respectively, mean +/- SE) and declined to 29 and 64% of peak levels, respectively, after 36 h of hypoxia. EPO levels in response to 11.5% oxygen showed no consistent change between 12 (122 +/- 21 mU/ml, mean +/- SE) and 36 h (182 +/- 35 mU/ml) of hypoxia. The decline in EPO levels under severe hypoxia (7.5% O2) was paralleled by a marked reduction in renal EPO mRNA content, indicating that it was primarily a result of diminished hormone production. The observed reductions in serum EPO after 36 h corresponded to preceding declines of calculated EPO production rates from 163- to 62-fold (7.5% O2) and 36- to 25-fold (9% O2) basal values. Application of 50 IU recombinant human EPO to rats 12 h, 6 h, or immediately before hypoxic exposure to mimic the early increase in EPO levels did not affect endogenous EPO formation during a subsequent hypoxic exposure of 12 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

A native, affinity-based protein blot for the analysis of streptavidin heterogeneity: consequences for the specificity of streptavidin mediated binding assays.

Commercial preparations of streptavidin, a bacterial biotin-binding protein, were analyzed by isoelectric focusing combined with an affinity-based protein blot using biotinylated, protein-saturated nitrocellulose. The colorimetrical detection of streptavidin with biotinylated alkaline phosphatase allows the selective visualization of streptavidin molecules with at least two active biotin-binding sites. Dependent on the preparation, seven to sixteen streptavidin forms were found with isoelectric points ranging from 5 to 8. Molecular weight analysis of the subunits of streptavidin showed that the observed heterogeneity was mainly due to limited proteolysis, which does not destroy the biotin-binding activity. The preparations differed also in the nonspecific reactivity of streptavidin with single-stranded DNA, bovine serum albumin and Tween 20. No relationship was observed between heterogeneity and non-specific binding activity. Data obtained from protein blots onto nitrocellulose saturated with single-stranded DNA showed that it cannot be excluded that streptavidin with only a single active biotin-binding site is mainly responsible for the nonspecific reactivity of some streptavidin preparations.

Bacterial Proteins↗
PubMed1986

Mechanisms of in situ nick translation of chromosomes using restriction endonucleases.

In situ nick translation of mammalian chromosomes by restriction endonuclease treatment to nick the chromosomal DNA, and 'translation' in the presence of DNA polymerase I and biotinylated dUTP, results in a distinct banding pattern. Further experiments have elucidated the mechanisms producing these bands. The hypothesis is presented that differences in the local conformation of the DNA-protein complex, rather than the DNA sequence itself, lead to the nick translation bands. The different DNase I sensitivity along the chromosomes suggests that the bands, which were clearly evident, reflect morphological units closely related to biological functions.

Animals↗
PubMed1986

An improved method for in situ nick translation of human chromosomes with biotin 11-labelled dUTP detected by biotinylated alkaline phosphatase.

Nick translation of the DNA of conventionally prepared human metaphase chromosomes using DNase I and biotin dUTP combined with streptavidin-phosphatase-detection assay produced a banding-like appearance. This pattern seems to be due to differences in DNase I sensitivity along the chromosomes. The Y chromosome could be clearly distinguished from the other chromosomes because of its intensely dark labelled heterochromatic region. In addition to DNase I concentration, hypotonic treatment seems to be an important methodological factor influencing band resolution. Together with recently published similar methods these results indicate that in situ nick translation using biotinylated nucleotides may develop into a useful technique to overcome several problems of human cytogenetics.

Alkaline Phosphatase↗
PubMed1983

Immunological studies of transplantation tolerance in irradiated rats repopulated with syngeneic hemopoietic cells.

Adult Lewis (LEW) rats that are lethally irradiated, grafted with allogeneic Wistar Furth (WF) hearts and repopulated with syngeneic bone marrow (LEW) become specifically and permanently tolerant to the allografts. In vivo transfer of spleen cells from tolerant animals to sublethally irradiated LEW rats was capable of preventing the rejection of WF cardiac allografts in 16 of 25 animals, suggesting the possibility of suppressor cells. For further characterization of this putative suppressor cell, mixed lymphocyte reactions (MLR) and cell mediated lympholysis (CML) assays were performed with spleen cells from tolerant and normal LEW rats. In 24 of 52 cases, spleen cells from rats bearing intact WF grafts proliferated in response to the tolerated WF alloantigens, and in 27 of 49 cases they were unable to generate effector cells against WF targets, which indicates that many of these animals were competent to respond to donor antigens as represented in bulk MLR. No suppression was found when spleen cells from nonresponsive recipients were mixed with normal LEW spleen cells in vitro either at the sensitization (MLR) or at the effector (CML) phase of the assay. Neither was there consistent in vitro suppression at the sensitization or effector level, with serum from LEW rats bearing long-term WF cardiac allografts. We suggest that the unresponsiveness observed in vivo is mediated by suppressor cells that interfere with the generation of mature effector cells from immature precursors. It is conceivable that suppressor cells that are present in vivo cannot act on mature effector cells generated in vitro. Additionally, or alternatively, the failure to detect suppression in vitro may result from the presence of stimulator cells in vitro that are not representative of stimulator cells seen by the tolerant animals in vivo.

Animals↗
PubMed1978

Successful cardiac allografts in syngeneic radiation chimeras.

Successful cardiac allografts were accomplished across the major histocompatibility complex of rats. LEW and F344 (Ag-B2) rats were lethally irradiated and grafted with WF (Ag-B1) hearts on day 0. Either on day 0 or day 2, the hosts were repopulated with syngeneic hemopoietic cells. The best results were obtained (86%) when a mixture of 3.0 x10(7) non-adherent syngeneic bone marrow and thymus cells were used to repopulate the recipients. In contrast, all of the WF to LEW heart grafts were rejected within 30 days if syngeneic thoracic duct and bone marrow cells were used to repopulate the host. Tolerant rats bearing a functioning WF heart graft were able to mount a normal antibody response to SRBC and a proliferative response to Con A. They accepted a second WF heart or a WF kidney graft but rejected WF skin and bone marrow grafts as well as "third-party" ACl or BN hearts. The lymphocytes of tolerant rats had a reduced response to WF antigens as assayed by local or systemic graft-versus-host reactions in (LEW x WF)F1 recipients. Tolerance to the WF hearts was resistant to a large innoculum of normal spleen and lymph node cells. The unresponsive state could be transferred to unirradiated LEW rats with a mixture of spleen, thymus, lymph node and bone marrow cells.

Animals↗