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J A Goodrich

Publications and source records attributed to J A Goodrich.

At least 19 recordsLinked to original sources
PubMed2001

Kinetic and mechanistic analysis of the RNA polymerase II transcrption reaction at the human interleukin-2 promoter.

Interleukin-2 (IL-2) is a cytokine critical for the proper stimulation of T-cells during the mammalian immune response. Shortly after T-cell stimulation, transcription of the IL-2 gene is upregulated. Here, we studied the kinetic mechanism of basal transcription at the IL-2 promoter using a human in vitro RNA polymerase II transcription system. We experimentally divided the transcription reaction into discrete steps, including preinitiation complex formation, initiation, escape commitment, and promoter escape. Using pre-steady state approaches, we measured the rate at which each of these steps occurs. We found that the rate of functional preinitiation complex formation limits the overall rate of transcription at the IL-2 promoter under the conditions described here. Furthermore, we found that the recruitment of TFIIF and RNA polymerase II to a TFIID/TFIIA/TFIIB/promoter complex dictates the rate of preinitiation complex formation. The rate of synthesis of 28 nt RNA from preinitiation complexes was rapid compared to the rate of preinitiation complex formation. Moreover, we found that the synthesis of a four nucleotide RNA was necessary and sufficient to rapidly complete the escape commitment step of transcription at the IL-2 promoter. Comparative experiments with the adenovirus major late promoter revealed that, while the overall mechanism of transcription is the same at the two promoters, promoter sequence and/or architecture dictate the rate of promoter escape. We present a kinetic model for a single round of basal transcription at the IL-2 promoter that provides insight into mechanisms by which the IL-2 gene is transcriptionally regulated.

Adenoviridae↗
PubMed2001

Expression and purification of recombinant human c-Fos/c-Jun that is highly active in DNA binding and transcriptional activation in vitro.

c-Fos and c-Jun are members of the AP-1 family of transcriptional activators that regulate the expression of genes during cell proliferation. To facilitate in vitro studies of mechanisms of transcriptional activation by c-Jun and c-Fos we developed a method for obtaining recombinant c-Fos/c-Jun that is highly active in DNA binding and transcriptional activation in vitro. Full-length human c-Fos and c-Jun were expressed in Escherichia coli. The expression of c-Fos was dependent on a helper plasmid that encodes rare (Arg)tRNAs. Both over-expressed c-Fos and c-Jun were recovered from inclusion bodies. A c-Fos/c-Jun complex was generated by co-renaturation and purified via a His-tag on the full-length human c-Fos. The resulting c-Fos/c-Jun bound DNA with high affinity and specificity, and activated transcription in a reconstituted human RNA polymerase II transcription system. The availability of active recombinant human c-Fos/c-Jun will allow future biochemical studies of these important transcriptional activators.

DNA Footprinting↗
PubMed2001

Increased prostacyclin synthesis by atherosclerotic arteries from estrogen-treated monkeys.

We hypothesized that the atheroinhibitory and cardioprotective effects of estrogen may be mediated in part through increased prostacyclin formation by the artery wall. Atherosclerotic abdominal aorta was collected at necropsy from ovariectomized female monkeys fed an atherogenic diet alone or with added Premarin. Basal and arachidonate-stimulated prostacyclin and thromboxane synthesis by artery segments was measured by radioimmunoassay. In contrast to no observed differences in basal release of prostacyclin by the control and estrogen-treated arteries, there was a marked increase (approximately 165%) in arachidonate-stimulated formation of prostacyclin by estrogen-treated arteries, and prostacyclin synthesis was inversely correlated with plaque size. No differences were observed in basal or arachidonate-stimulated thromboxane synthesis by the control and estrogen-treated arteries. In light of known antiatherogenic and vasodilatory effects of estrogen, increased prostacyclin synthesis by estrogen-treated arteries may, in part, explain estrogen's beneficial effects on the artery wall.

Animals↗
PubMed2001

c-Jun binds the N terminus of human TAF(II)250 to derepress RNA polymerase II transcription in vitro.

c-Jun is an oncoprotein that activates transcription of many genes involved in cell growth and proliferation. We studied the mechanism of transcriptional activation by human c-Jun in a human RNA polymerase II transcription system composed of highly purified recombinant and native transcription factors. Transcriptional activation by c-Jun depends on the TATA-binding protein (TBP)-associated factor (TAF) subunits of transcription factor IID (TFIID). Protein-protein interaction assays revealed that c-Jun binds with high specificity to the largest subunit of human TFIID, TAF(II)250. The region of TAF(II)250 bound by c-Jun lies in the N-terminal 163 amino acids. This same region of TAF(II)250 binds to TBP and represses its interaction with TATA boxes, thereby decreasing DNA binding by TFIID. We hypothesized that c-Jun is capable of derepressing the effect of the TAF(II)250 N terminus on TFIID-driven transcription. In support of this hypothesis, we found that c-Jun increased levels of TFIID-driven transcription in vitro when added at high concentrations to a DNA template lacking activator protein 1 (AP-1) sites. Moreover, c-Jun blocked the repression of TBP DNA binding caused by the N terminus of TAF(II)250. In addition to revealing a mechanism by which c-Jun activates transcription, our studies provide the first evidence that an activator can bind directly to the N terminus of TAF(II)250 to derepress RNA polymerase II transcription in vitro.

Chromatography, Affinity↗
PubMed2001

Human Taf(II)130 is a coactivator for NFATp.

NFATp is one member of a family of transcriptional activators that regulate the expression of cytokine genes. To study mechanisms of NFATp transcriptional activation, we established a reconstituted transcription system consisting of human components that is responsive to activation by full-length NFATp. The TATA-associated factor (TAF(II)) subunits of the TFIID complex were required for NFATp-mediated activation in this transcription system, since TATA-binding protein (TBP) alone was insufficient in supporting activated transcription. In vitro interaction assays revealed that human TAF(II)130 (hTAF(II)130) and its Drosophila melanogaster homolog dTAF(II)110 bound specifically and reproducibly to immobilized NFATp. Sequences contained in the C-terminal domain of NFATp (amino acids 688 to 921) were necessary and sufficient for hTAF(II)130 binding. A partial TFIID complex assembled from recombinant hTBP, hTAF(II)250, and hTAF(II)130 supported NFATp-activated transcription, demonstrating the ability of hTAF(II)130 to serve as a coactivator for NFATp in vitro. Overexpression of hTAF(II)130 in Cos-1 cells inhibited NFATp activation of a luciferase reporter. These studies demonstrate that hTAF(II)130 is a coactivator for NFATp and represent the first biochemical characterization of the mechanism of transcriptional activation by the NFAT family of activators.

Animals↗
PubMed2001

Non-invasive measurement of blood pressures in the Yucatan micropig (Sus scrofa domestica), with and without midazolam-induced sedation.

Current literature suggests that the effects of midazolam, a water-soluble benzodiazepine, on blood pressure in swine are minimal. The hypothesis of the study reported here was that a light sedative dose would induce a decrease in blood pressure in this species. Healthy female Yucatan Micropigs (n = 20), 16 to 30 (mean, 22) kg, aged four six months, were individually placed in a humane restraint sling and allowed to acclimate. Systolic (SBP), diastolic (DBP), and mean (MBP) blood pressures (mmHg) and heart rate (HR; beats per min [bpm]) were measured by use of oscillometry. The pressure cuff was placed at the base of the tail, and five sets of values were recorded at five-min intervals, beginning at 10 and ending 30 min after cuff placement. Following a three- to four-day rest period, this procedure was repeated with the addition of a dose of 0.5 mg of midazolam HCl/kg of body weight given intramuscularly at the time of cuff placement. A paired one-way Student's t-test was used to compare the means of the five measures between control and midazolam treatment. Mean (+/- SD) differences for SBP, DBP, MBP, and HR were 18.9 (+/- 3.97), 17.8 (+/- 5.27), and 18.6 (+/- 5.09) mmHg and 20.7 (+/- 3.73) bpm, respectively. All four parameters were significantly reduced in the midazolam-sedated group (P < 0.001). The maximal decrease in SBP, DBP, and MBP occurred at 15 and 20 min after dosing. Mean values based on the means of the five measures were 128 (+/- 12.6), 80 (+/- 9.4), and 99 (+/- 9.2) mmHg and 135 (+/- 17.4) bpm, and 109 (+/- 15.4), 63 (+/- 12.6), and 80 (+/- 13.6) mmHg and 115 (+/- 15.5) bpm for SBP, DBP, MBP, and HR in the control (n = 20) and midazolam (n = 20) groups, respectively. The control values can serve as normal oscillometric values for this age, sex, and breed of Micropig. We conclude that midazolam, given intramuscularly at a sedative dosage, negatively affects cardiovascular parameters measured by use of a blood pressure cuff, in sexually mature female Micropigs, compared with values in untreated pigs, which is similar to reports for humans.

Animals↗
PubMed2000

A kinetic model for the early steps of RNA synthesis by human RNA polymerase II.

Eukaryotic mRNA synthesis is a highly regulated process involving numerous proteins acting in concert with RNA polymerase II to set levels of transcription from individual promoters. The transcription reaction consists of multiple steps beginning with preinitiation complex formation and ending in the production of a full-length primary transcript. We used pre-steady-state approaches to study the steps of human mRNA transcription at the adenovirus major late promoter in a minimal in vitro transcription system. These kinetic studies revealed an early transition in RNA polymerase II transcription, termed escape commitment, that occurs after initiation and prior to promoter escape. Escape commitment is rapid and is characterized by sensitivity to competitor DNA. Upon completion of escape commitment, ternary complexes are resistant to challenge by competitor DNA and slowly proceed forward through promoter escape. Escape commitment is stimulated by transcription factors TFIIE and TFIIH. We measured forward and reverse rate constants for discrete steps in transcription and present a kinetic model for the mechanism of RNA polymerase II transcription that describes five distinct steps (preinitiation complex formation, initiation, escape commitment, promoter escape, and transcript elongation) and clearly shows promoter escape is rate-limiting in this system.

Animals↗
PubMed2000

Characterization of DNA binding, transcriptional activation, and regulated nuclear association of recombinant human NFATp.

BACKGROUND: NFATp is one member of a family of transcriptional activators whose nuclear accumulation and hence transcriptional activity is regulated in mammalian cells. Human NFATp exists as a phosphoprotein in the cytoplasm of naive T cells. Upon antigen stimulation, NFATp is dephosphorylated, accumulates in nuclei, and functions to regulate transcription of genes including those encoding cytokines. While the properties of the DNA binding domain of NFATp have been investigated in detail, biochemical studies of the transcriptional activation and regulated association with nuclei have remained unexplored because of a lack of full length, purified recombinant NFATp. RESULTS: We developed methods for expressing and purifying full length recombinant human NFATp that has all of the properties known to be associated with native NFATp. The recombinant NFATp binds DNA on its own and cooperatively with AP-1 proteins, activates transcription in vitro, is phosphorylated, can be dephosphorylated by calcineurin, and exhibits regulated association with nuclei in vitro. Importantly, activation by recombinant NFATp in a reconstituted transcription system required regions of the protein outside of the central DNA binding domain. CONCLUSIONS: We conclude that NFATp is a bona fide transcriptional activator. Moreover, the reagents and methods that we developed will facilitate future studies on the mechanisms of transcriptional activation and nuclear accumulation by NFATp, a member of an important family of transcriptional regulatory proteins.

Animals↗
PubMed2000

Acetyl coenzyme A stimulates RNA polymerase II transcription and promoter binding by transcription factor IID in the absence of histones.

Protein acetylation has emerged as a means of controlling levels of mRNA synthesis in eukaryotic cells. Here we report that acetyl coenzyme A (acetyl-CoA) stimulates RNA polymerase II transcription in vitro in the absence of histones. The effect of acetyl-CoA on basal and activated transcription was studied in a human RNA polymerase II transcription system reconstituted from recombinant and highly purified transcription factors. Both basal and activated transcription were stimulated by the addition of acetyl-CoA to transcription reaction mixtures. By varying the concentrations of general transcription factors in the reaction mixtures, we found that acetyl-CoA decreased the concentration of TFIID required to observe transcription. Electrophoretic mobility shift assays and DNase I footprinting revealed that acetyl-CoA increased the affinity of the general transcription factor TFIID for promoter DNA in a TBP-associated factor (TAF)-dependent manner. Interestingly, acetyl-CoA also caused a conformational change in the TFIID-TFIIA-promoter complex as assessed by DNase I footprinting. These results show that acetyl-CoA alters the DNA binding activity of TFIID and indicate that this biologically important cofactor functions at multiple levels to control gene expression.

Acetyl Coenzyme A↗
PubMed1998

Promoter escape limits the rate of RNA polymerase II transcription and is enhanced by TFIIE, TFIIH, and ATP on negatively supercoiled DNA.

To measure rate constants for discrete steps of single-round transcription (preinitiation complex formation, promoter escape, and transcript elongation), kinetic studies were performed in a well defined human RNA polymerase II transcription system. These experiments revealed that promoter escape limits the rate of transcription from the adenovirus major late promoter (AdMLP) contained on negatively supercoiled DNA. TFIIE and TFIIH were found to significantly increase fractional template usage during a single round of transcription in an ATP-dependent reaction. The observed rate constant for promoter escape, however, was not greatly affected by TFIIE and TFIIH. Our results are explained by a model in which transcription branches into at least two pathways: one that results in functional promoter escape and full-length RNA synthesis, and another in which preinitiation complexes abort during promoter escape and do not produce full-length RNA transcripts. These results with negatively supercoiled templates agree with our earlier conclusion that TFIIE, TFIIH, and ATP direct promoter escape and support a model in which the TFIIH helicases stimulate promoter escape in an ATP-dependent reaction.

Adenosine Triphosphate↗
PubMed1998

Use of an internal fixator device to treat comminuted fractures of the distal radius: report of a technique.

An internal fixator technique for stabilizing comminuted Colles fractures has been developed in the anatomy laboratory and used in 35 clinical cases. The Colles Fracture Plate (Biomet, Inc, Warsaw, Indiana) can be used to treat any comminuted Colles fracture for which an external fixator is considered proper management. We have determined, based on our surgical experience with both the internal and external fixator techniques, that internal fixation using the Colles Fracture Plate is technically just as simple as external fixation. In addition to requiring a significantly less expensive device, internal fixation using this technique offers the advantages of better patient acceptance and fewer complications. This report will be followed by a more comprehensive analysis of the technical outcome of this procedure to further substantiate the initial results presented here. The process of compiling and analyzing these data is under way.

Bone Plates↗
PubMed1997

Repression of p53-mediated transcription by MDM2: a dual mechanism.

The oncoprotein MDM2 binds to the activation domain of the tumor suppressor p53 and inhibits its ability to stimulate transcription. This same region of p53 is able to bind several basal transcription factors that appear to be important for the transactivation function of p53. It has therefore been suggested that MDM2 acts to inhibit p53 by concealing its activation domain from the basal machinery. Here we present data suggesting that MDM2 possesses an additional inhibitory function. Our experiments reveal that in addition to a p53-binding domain, MDM2 also contains an inhibitory domain that can directly repress basal transcription in the absence of p53. By fusing portions of MDM2 to a heterologous DNA-binding domain to allow p53-independent promoter recruitment, we have localized this inhibitory domain to a region encompassing amino acids 50-222 of MDM2. Furthermore, the function of this inhibitory domain does not require the presence of either TFIIA or the TAFs. Of the remaining basal factors, both the small subunit of TFIIE and monomeric TBP are bound by the MDM2 inhibitory domain. It is possible that MDM2 inhibits the ability of the preinitiation complex to synthesize RNA through one of these interactions. Our results are consistent with a model in which MDM2 represses p53-dependent transcription by a dual mechanism: a masking of the activation domain of p53 through a protein-protein interaction that additionally serves to recruit MDM2 to the promoter where it directly interferes with the basal transcription machinery.

Cell-Free System↗
PubMed1997

Sacral fixation technique in lumbosacral fusion.

STUDY DESIGN: Servohydraulic load displacement testing was used to study the biomechanical properties of sacral fixation in human cadaveric specimens. OBJECTIVES: To evaluate a modification of standard sacral fixation that uses the first dorsal sacral foramina as an adjunct location for the placement of a sacral hook in addition to S1 pedicle screws. BACKGROUND DATA: The stiffness or rigidity of an instrumentation construct governs the amount of relative movement allowed between motion segments undergoing fusion. This property provides the greatest influence over the mechanical conditions necessary for fusion to occur. METHODS: Sixteen human cadaveric specimens were divided into two groups with similar bone density assessed by quantitative computed tomography scan. All were instrumented with pedicle screws at L4 and S1. One group also had downgoing offset hooks in the first sacral foramina distracted against the S1 pedicle screw. Instron servohydraulic testing was performed in anterior compressive flexion, and load displacement curves were recorded. RESULTS: The bending stiffness of the specimens instrumented with screw and hook was significantly higher than in those instrumented with pedicle screws alone. The ultimate strength and energy absorbed did not differ between the two groups. CONCLUSIONS: The addition of sacral foraminal hooks to standard pedicle screw instrumentation constructs across the lumbosacral junction provides more rigid stabilization of the lumbosacral motion segment in this model.

Bone Density↗
PubMed1997

Experimental infection of cynomolgus monkeys with simian parvovirus.

Simian parvovirus is a recently discovered parvovirus that was first isolated from cynomolgus monkeys. It is similar to human B19 parvovirus in terms of virus genome, tropism for erythroid cells, and characteristic pathology in natural infections. Cynomolgus monkeys were infected with simian parvovirus to investigate their potential usefulness as an animal model of human B19 parvovirus. Six adult female cynomolgus monkeys were inoculated with purified simian parvovirus by the intravenous or intranasal route and monitored for evidence of clinical abnormalities; this included the preparation of complete hematological profiles. Viremia and simian parvovirus-specific antibody were determined in infected monkeys by dot blot and Western blot assays, respectively. Bone marrow was examined at necropsy 6, 10, or 15 days postinfection. All of the monkeys developed a smoldering, low-grade viremia that peaked approximately 10 to 12 days after inoculation. Peak viremia coincided with the appearance of specific antibody and was followed by sudden clearance of the virus and complete, but transient, absence of reticulocytes from the peripheral blood. Clinical signs were mild and involved mainly anorexia and slight weight loss. Infection was associated with a mild decrease in hemoglobin, hematocrit, and erythrocyte numbers. Bone marrow showed marked destruction of erythroid cells coincident with peak viremia. Our findings indicate that infection of healthy monkeys by simian parvovirus is self-limited and mild, with transient cessation of erythropoiesis. Our study has reproduced Koch's postulates and further shown that simian parvovirus infection of monkeys is almost identical to human B19 parvovirus infection of humans. Accordingly, this animal model may prove valuable in the study of the pathogenesis of B19 virus infection.

Animals↗
PubMed1995

Molecular cloning and expression of the 32-kDa subunit of human TFIID reveals interactions with VP16 and TFIIB that mediate transcriptional activation.

Transcription factor TFIID consists of TATA binding protein (TBP) and at least eight TBP-associated factors (TAFs). As TAFs are required for activated but not basal transcription, we have proposed that TAFs act as coactivators to mediate signals between activators and the basal transcription machinery. Here we report the cloning, expression, and biochemical characterization of the 32-kDa subunit of human (h) TFIID, termed hTAFII32. We find that hTAFII32 is the human homologue of Drosophila TAFII40. In vitro protein-protein interaction assays reveal that as observed with Drosophila TAFII40, hTAFII32 interacts with the C-terminal 39-amino acid activation domain of the acidic transactivator viral protein 16 (VP16) as well as with the general transcription factor TFIIB. Moreover, a partial recombinant TFIID complex containing hTAFII32 was capable of mediating in vitro transcriptional activation by the VP16 activation domain. These findings indicate that specific activator-coactivator interactions have been conserved between human and Drosophila and provide additional support for the function of these interactions in mediating transcriptional activation.

Amino Acid Sequence↗
PubMed1995

Tobramycin gender-related nephrotoxicity in Fischer but not Sprague-Dawley rats.

Published reports suggest a gender-related difference in susceptibility to the nephrotoxicity of gentamicin which may also be strain-related in rats. However, certain ambiguities exist in the results obtained with the functional and morphologic determinants of nephrotoxicity used in these studies. Within the same experimental protocol we examined the potential gender-related differences in tobramycin nephrotoxicity in age-matched male and female rats of both the Sprague-Dawley (SD) and Fischer (F344) strains. Equal numbers of both genders were dosed at 30 mg/kg (F344) and 90 mg/kg (SD) twice daily for 9 days. Results of BUN, serum creatinine, whole body weight change and histologic score comparisons (P < 0.05) indicate that male F344 rats are more sensitive to tobramycin nephrotoxicity than F344 females but this gender-related susceptibility was not observed in SD rats.

Animals↗
PubMed1995

Normal serum biochemical and hematological values of the Sulawesi macaques.

Sulawesi macaque (SM) species are currently of interest to primatologists for genetic and behavioral studies world wide. However, there are no published reference hematological and serum biochemical profile values for several of the seven SM species. Twenty-five clinically healthy, outdoor housed, ketamine sedated Macaca tonkeana and Macaca maurus were serially sampled (two to four times) over a 16-month period (N = 68). Normal ranges were defined to include two standard deviations and 2.5-97.5 percentile. Significant differences in single sample data comparisons (P < 0.05) of species (serum Ca, MCHC), age (BUN, BUN/creatinine, alkaline phosphatase, phosphorus), gender (total protein, RBC, hemoglobin, HCT) and pregnancy (phosphorus and neutrophil count) groups were found. This normative data facilitates clinical evaluation of SM species and allows comparison to other macaque species.

Aging↗