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D A Krueger

Publications and source records attributed to D A Krueger.

11 recordsLinked to original sources
PubMed2000

Involvement of thyrotroph embryonic factor in calcium-mediated regulation of gene expression.

In the present study, we used an expression cloning strategy to identify transcription factors that bind specifically to a limited region of the inducible cAMP early repressor (ICER) promoter and regulate transcription. Murine thyrotroph embryonic factor (mTEF) was isolated and was shown to bind to a site located at nucleotides -117 to -108 from the transcriptional start site. Transient expression of reporter constructs containing either a consensus TEFRE or the icerTEF binding site demonstrated that TEF-dependent transcription correlated with relative binding affinities, i.e. the consensus TEFRE bound TEF more tightly and was more responsive to TEF than the icerTEFRE. Because the icerTEFRE overlapped a cAMP response element, the responsiveness of these sequences to either cAMP or Ca(2+) was tested. Although TEF expression had no effect on the cAMP-regulated transcriptional response of the ICER promoter, TEF did confer calcium responsiveness to these sequences. Calcium also modestly increased the TEF-mediated transcription from a consensus TEFRE. Additional studies using Ca(2+)-activated kinases indicate that Ca(2+)/TEF/TEFRE-regulated transcription may be mediated through Ca(2+)/calmodulin-dependent kinase (CaMK) IV. Moreover, studies with the icerTEFRE in a CaMK IV-deficient cell line demonstrated that these cells were transcriptionally unresponsive to thapsigargin; however, responsiveness was restored by co-expression of the active CaMK IV. These studies are the first to demonstrate that TEF is a calcium-responsive transcription factor, and they suggest that there are two classes of TEF-regulated genes. One class, represented by a consensus TEFRE, is regulated by TEF in the resting cell; the second class, represented by icerTEFRE, is regulated by TEF in the calcium-activated cell.

Amino Acid Sequence↗
PubMed1999

Functional analysis of the mouse ICER (Inducible cAMP Early Repressor) promoter: evidence for a protein that blocks calcium responsiveness of the CAREs (cAMP autoregulatory elements).

Although Ca2+ and cAMP mediate their effects through distinct pathways, both signals converge upon the phosphorylation of the cAMP response element (CRE) binding protein, CREB, thereby activating transcription of CRE-regulated genes. In WEHI7.2 thymocytes, cAMP increases the expression of the inducible cAMP early repressor (ICER) gene through CRE-like elements, known as cAMP autoregulatory elements (CAREs). Because Ca2+ -and cAMP-mediated transcription converge in WEHI7.2 thymocytes, we examined the effect of Ca2+ fluxes on the expression of the ICER gene in these cells. Despite the presence of multiple CAREs within its promoter, ICER gene transcription was not activated by Ca2+. Moreover, Ca2+ attenuated the stimulatory effect of cAMP on ICER expression. Transient expression of reporter constructs demonstrated that when these CAREs were placed in a different DNA promoter context, the elements became responsive to Ca2+. Detailed studies using chimeric promoter constructs to map the region responsible for blocking the transcriptional response to Ca2+ indicated that a small portion of the ICER promoter was necessary for the effect. Southwestern blot analysis identified a 83-kDa nuclear protein that bound specifically to that region. The relative binding activity of the factor to the ICER promoter and mutant promoter sequences correlated with an inhibition of Ca2+ -activated gene expression in WEHI7.2 cells. These data suggest that the factor functions as a putative Ca2+ -activated repressor of CREB/CRE-mediated transcription. Thus, depending on the surrounding context in which the CRE is located, CREs of individual genes can be regulated separately by Ca2+ and cAMP despite the convergence of these two signaling pathways.

Animals↗
PubMed1992

Comparison of three immunoassays for the rapid detection of bovine respiratory syncytial virus.

Three enzyme-linked immunosorbent assays (EIA) designed for the detection of human respiratory syncytial virus (RSV) were evaluated for the detection of bovine respiratory syncytial virus (BRSV) in bovine lungs and the results were compared with those obtained by a direct fluorescent antibody assay (DFA). The EIA tests used were Directigen EIA, Kallestad Pathfinder EIA, and Abbott RSV EIA. Homogenates of lung tissues obtained from 64 cattle that had died of respiratory disease were used; 32 were positive by DFA and 32 were negative. All EIA's varied in the amount of labor and time involved but their relative sensitivities were similar ranging between 59 and 66% when compared with DFA. The specificity of Pathfinder EIA was lower than those of the Directigen and Abbott tests. The overall agreement between the three EIA's and the DFA was 66-77% indicating that DFA is still the test of choice for detecting BRSV infection in lung tissues of cattle.

Animals↗
PubMed1991

Functional characterization of the human hypoxanthine phosphoribosyltransferase gene promoter: evidence for a negative regulatory element.

The enzyme hypoxanthine phosphoribosyltransferase (HPRT) catalyzes the metabolic salvage of the purine bases hypoxanthine and guanine. We previously characterized the genomic structure of the human HPRT gene and described its promoter sequence. In this report, we identify cis-acting transcriptional control regions of the human HPRT gene by linking various 5'-flanking sequences to the bacterial chloramphenicol acetyltransferase gene. The sequence from positions -219 to -122 relative to the translation initiation site is required for maximal expression of this gene, and it functions equally in both normal and reverse orientations. In addition, a cis-acting negative element is present in the region spanning from positions -570 to -388. This negative element can also repress promoters of heterologous genes, such as those of adenosine deaminase and dihydrofolate reductase, which are structurally and functionally similar to the human HPRT promoter. Furthermore, this repressor element functions independently of its orientation but appears to be distance dependent. In vivo competition assays demonstrated that the trans-acting factor(s) that binds to this negative element specifically inhibits human HPRT promoter activity. Taken together, these data localize cis-acting sequences important in the regulation of human HPRT gene expression and should allow the study of protein-DNA interactions which modulate the transcription of this gene.

Animals↗
PubMed1989

Characterization of gamma interferon-mediated cytotoxicity to chlamydia-infected fibroblasts.

Addition of murine recombinant gamma interferon (IFN-gamma) to mouse fibroblast cultures infected with Chlamydia psittaci was found to induce a cytotoxic response that was dependent on the concentration of IFN-gamma added and the multiplicity of infection given. No cytotoxicity was observed for uninfected cells treated with IFN-gamma, nor did infection alone elicit cytotoxicity. Cytotoxicity was detected only if IFN-gamma was present for at least the first 18 h of a 30-h incubation period. Cytotoxic activity was not observed when infected cells were treated with 50 micrograms of chloramphenicol per ml, a drug which inhibits differentiation of infectious elementary bodies to noninfectious reticulate bodies. Cytotoxic activity was restored if addition of chloramphenicol was delayed until 18 h postinfection. Addition of 100 U of penicillin per ml to infected host cells reduced but did not abolish cytotoxic activity. Treatment of host cells with as little as 0.2 microgram of cycloheximide per ml inhibited cytotoxicity without interfering with chlamydial growth. When addition of cycloheximide was delayed until 12 h after infection and IFN-gamma treatment, cytotoxicity was restored. These data indicate that IFN-gamma functions as a cytotoxic cytokine against chlamydia-infected fibroblasts. Cytotoxicity was found to be dependent on chlamydial multiplicity of infection, differentiation of chlamydiae to the metabolically active form, and host cell protein synthesis.

Animals↗
PubMed1985

In vitro expression of factor-mediated cytotoxic activity generated during the immune response to Chlamydia in the mouse.

Supernatant fluid (SF) prepared by mitogen incubation of spleen cells from A/J mice previously immunized against lethal challenge by the 6BC strain of Chlamydia psittaci was cytotoxic for mouse fibroblasts (L cells) infected with 6BC, as detected by the [3H]thymidine release assay and the trypan blue exclusion test. In contrast, SF prepared from spleen cells taken from unimmunized animals (controls) was not cytotoxic when added to infected L cells. No cytotoxicity was observed when SF was added to uninfected L cells. Maximal levels of cytotoxicity were observed only from cells infected with 6BC for at least 26 hr and exposed to SF for greater than 20 hr. Furthermore, the degree of cytotoxicity was dependent on both the dose of Chlamydia administered and the concentration of SF in the medium. We conclude that the capacity to secrete a spleen cell cytotoxic factor is an aspect of the immune response against the obligate intracellular prokaryotic pathogen Chlamydia. Our results indicate that SF-mediated cytotoxicity is induced subsequent to immunization with Chlamydia, and is significantly more pronounced against infected as opposed to uninfected L cells.

Animals↗
PubMed1983

Lymphokine-mediated inhibition of Chlamydia replication in mouse fibroblasts is neutralized by anti-gamma interferon immunoglobulin.

Experiments were carried out to characterize immunologically mediated chlamydial persistence in cell culture. Mouse fibroblasts were activated to restrict Chlamydia psittaci 6BC replication by including mitogen (concanavalin A)-induced spleen cell supernatant fluids from immunized animals in the growth medium. When mouse fibroblasts were incubated with lymphokine for 24 h before infection and then with growth medium after infection (preinfection treatment), chlamydial replication was delayed but eventually detected. No substantial chlamydial growth occurred, even with extended incubation times when mouse fibroblasts were continuously exposed to lymphokine before and after infection. Low levels of infectious chlamydiae were produced in preinfection-treated mouse fibroblasts but not in mouse fibroblasts subjected to continuous lymphokine exposure. Incubation of lymphokine with anti-murine gamma interferon immunoglobulin neutralized the observed lymphokine-mediated activity, but incubation in the presence of anti-murine alpha plus beta interferon serum did not alter lymphokine activity. We conclude that the lymphokine components responsible for activating fibroblasts to restrict C. psittaci replication exhibits properties similar to gamma interferon.

Animals↗
PubMedDate not supplied

Isotopic composition of carbon in vinegars.

Measurements of delta 13C and 14C-activity were performed on vinegars from various known sources. Natural vinegar can be distinguished from petrochemical acetic acid by 14C-analysis: Natural vinegar currently gives values of greater than 112% of modern activity; petrochemical acetic acid yields values of 0% of modern activity. Apple cider vinegar can be distinguished from corn-derived vinegar by delta 13C-analysis: Cider vinegar gives delta 13C-values near -26%; corn-derived vinegars yield delta 13C-values near -10%. delta 13C-Analysis also can be applied, with some restrictions, to wine vinegars. These techniques are applied to a series of retail vinegars.

Acetates↗
PubMedDate not supplied

Sample preparation bias in carbon stable isotope ratio analysis of fruit juices and sweeteners.

Two sample preparation methods are commonly used for carbon stable isotope ratio analysis (SIRA). One involves combustion of the sample with oxygen at 850 degrees C; the other involves combustion of the sample with CuO in an evacuated glass tube at 550 degrees C. I observed in our laboratory that these 2 methods yield different results for sugar-based products such as fruit juices, sweeteners, and vanillin. The CuO method yields results approximately 1%. more positive than the oxygen combustion method. This bias is also observed in other laboratories, as shown in an analysis of the results of the AOAC collaborative studies of carbon SIRA of maple syrup, orange juice, honey, and honey protein. The oxygen combustion method is the AOAC method for honey, apple juice, and orange juice; both methods are incorporated into the AOAC method for maple syrup. I recommend that data generated by the CuO combustion method be appropriately corrected to yield results concordant with the official oxygen combustion method.

Beverages↗