Biomedical subjects

B J Lee

Publications and source records attributed to B J Lee.

At least 109 records · Page 6Linked to original sources

PubMed1998

Overproduction of selenocysteine tRNA in Chinese hamster ovary cells following transfection of the mouse tRNA[Ser]Sec gene.

Selenocysteine insertion during selenoprotein biosynthesis begins with the aminoacylation of selenocysteine tRNA[ser]sec with serine, the conversion of the serine moiety to selenocysteine, and the recognition of specific UGA codons within the mRNA. Selenocysteine tRNA[ser]sec exists as two major forms, differing by methylation of the ribose portion of the nucleotide at the wobble position of the anticodon. The levels and relative distribution of these two forms of the tRNA are influenced by selenium in mammalian cells and tissues. We have generated Chinese hamster ovary cells that exhibit increased levels of tRNA[ser]sec following transfection of the mouse tRNA[ser]sec gene. The levels of selenocysteine tRNA[ser]sec in transfectants increased proportionally to the number of stably integrated copies of the tRNA[ser]sec gene. Although we were able to generate transfectants overproducing tRNA[ser]sec by as much as tenfold, the additional tRNA was principally retained in the unmethylated form. Selenium supplementation could not significantly affect the relative distributions of the two major selenocysteine tRNA[ser]sec isoacceptors. In addition, increased levels of tRNA[ser]sec did not result in measurable alterations in the levels of selenoproteins, including glutathione peroxidase.

Amino Acyl-tRNA Synthetases↗

PubMed1998

Novel binding sites for regulatory factors in the human papillomavirus type 18 enhancer and promoter identified by in vivo footprinting.

The E6 and E7 genes of human papillomaviruses (HPVs) associated with anogenital cancers are largely responsible for the oncogenic activity of these viruses, and regulation of these genes has been intensively studied. Transcription of the E6 and E7 genes is controlled by the viral upstream regulatory region (URR). We have used in vivo footprinting to examine the occupancy by regulatory factors of the HPV type 18 (HPV18) URR enhancer and promoter in the cervical carcinoma cell lines HeLa and C4-II. While corroborating occupancy in vivo of all of the elements previously implicated in the transcriptional control of the HPV18 E6 and E7 genes by in vitro DNase I footprinting, gel retardation assays, and transfection studies, we also detect occupancy in vivo of several enhancer and promoter sequences which have not been previously identified as HPV18 URR regulatory elements. Our data suggest that the HPV18 enhancer and promoter are more densely occupied by DNA-binding proteins than previously thought and raise the possibility that additional, possibly novel factors contribute to transcription of the HPV18 early genes.

Base Sequence↗

PubMed1997

Colocalization of dopamine D1 and D2 receptor mRNAs in rat placenta.

Dopamine is present in the human placenta. The major function of dopamine is the inhibition of human placental lactogen (hPL) release from human trophoblastic cells. This effect is mediated by cAMP through dopamine D2 receptors. However, studies on the effects of cAMP in the control of hPL release have yielded conflicting results. The purpose of this study is to explore the distribution of dopamine receptors in the rat placenta. Dopamine D1 and D2 receptor mRNAs were colocalized in the rat placenta by in situ hybridization histochemistry using radiolabeled cRNA probes. Dopamine D1 and D2 receptor mRNAs were detected in large cells of the endometrium of the uterus on day 10 of gestation. On days 12-16 of gestation, hybridization signals were localized mainly in the spongiotrophoblast and giant cells of the junctional zone of the placenta. With the development of the placenta, signals were moving from the junctional zone to the labyrinth zone. Pit-1 mRNA was detected in the placental lactotrophs and was also colocalized in neighboring placental sections. Our results clearly showed that dopamine D1 and D2 receptor mRNAs were coexpressed in the placental lactotrophs that express Pit-1 mRNA.

Animals↗

PubMed1997

Estradiol enhances prostaglandin E2 receptor gene expression in luteinizing hormone-releasing hormone (LHRH) neurons and facilitates the LHRH response to PGE2 by activating a glia-to-neuron signaling pathway.

Prostaglandin E2 (PGE2) mediates the stimulatory effect of norepinephrine (NE) on the secretion of luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling reproductive function. In rodents, this facilitatory effect requires previous exposure to estradiol, suggesting that the steroid affects downstream components in the cascade that leads to PGE2-induced LHRH release. Because astroglia are the predominant cell type contacting LHRH-secreting nerve terminals, we investigated the involvement of hypothalamic astrocytes in the estradiol facilitation of PGE2-induced LHRH release. A subpopulation of LHRH neurons was found to express the mRNA encoding the PGE2 receptor subtype EP1-R, which is coupled to calcium mobilization. The LHRH-producing cell line GT1-1 also contains EP1-R mRNA and, to a lesser extent, the three alternatively spliced forms of EP3-R mRNA (alpha, beta, and gamma) that encode receptors linked to inhibition and stimulation of cAMP formation. Hypothalamic astrocytes treated with estradiol produced a conditioned medium that when applied to GT1-1 cells resulted in a selective upregulation of EP1-R and EP3gamma-R mRNAs. The conditioned medium also enhanced the LHRH response to EP1-R and EP3-R agonists and the cAMP response to EP3-R activation. Thus, one mechanism by which estradiol facilitates the effect of neurotransmitters acting via PGE2 to stimulate LHRH release is by enhancing the glial production of substances that upregulate PGE2 receptors on LHRH neurons. The existence of such a mechanism underscores the emerging importance of glial-neuronal communication in the control of brain neurosecretory activity.

Animals↗

PubMed1997

Prolonged ethanol intake increases D2 dopamine receptor expression in the rat brain.

Dopamine via interaction with its receptor is known to be involved in the behavioral and endocrine actions in the mammalian brain. Behavioral effects produced by ethanol appear to be due to its actions on the dopaminergic system. In the present study using in situ hybridization histochemistry and RNase protection assay, the effect of prolonged ethanol intake on the expression of D2 dopamine receptor mRNA was examined in the rat brain. Specific D1 and D2 receptor mRNA signals were detected in the caudate putamen, nucleus accumbens, olfactory tubercle, hippocampus, dentate gyrus, and amygdaloid complex of the rat brain. Within the hypothalamus, the level of receptor mRNA was low in most nuclei with a somewhat higher level in the arcuate nucleus. Only the supurachiasmatic nucleus showed moderate to dense dopamine receptor mRNAs. Prefrontal cortex showed hybridization signals but their intensity was very low. A considerable amount of D2 mRNA was localized in the substantia nigra but D1 mRNA was not. Ethanol (10%) intake for 5 weeks increased both the density of hybridization signal and number of cells expressing D2 dopamine receptor mRNA in the caudate putamen, and nucleus accumbens, but not in the olfactory tubercle. RNase protection assay revealed about a 1.5-fold increase in the D2 dopamine receptor mRNA level in the corpus striatum. These results provide a basis for the involvement of dopamine D2 receptor expression in alcoholism.

Alcohol Drinking↗

PubMed1997

Replenishment of selenium deficient rats with selenium results in redistribution of the selenocysteine tRNA population in a tissue specific manner.

We reported previously that the selenium status of rats influences both the steady-state levels and distributions of two selenocysteine tRNA isoacceptors and that these isoacceptors differ by a single methyl group attached to the ribosyl moiety at position 34. In this study, we demonstrate that repletion of selenium-deficient rats results in a gradual, tissue-dependent shift in the distribution of these isoacceptors. Rats fed a selenium-deficient diet possess a greater abundance of the species unmethylated on the ribosyl moiety at position 34 compared to the form methylated at this position. A redistribution of the Sec-tRNA isoacceptors occurred in tissues of selenium-supplemented rats whereby the unmethylated form gradually shifted toward the methylated form. This was true in each of four tissues examined, muscle, kidney, liver and heart, although the rate of redistribution was tissue-specific. Muscle manifested a predominance of two minor serine isoacceptors under conditions of extreme selenium-deficiency which also appeared to respond to selenium. Ribosomal binding studies revealed that one of the two additional isoacceptors decodes the serine codeword, AGU, and the second decodes the serine codeword, UCU. Interestingly, muscle and heart were the slower tissues to return to a 'selenium adequate' tRNA distribution pattern.

Animals↗

PubMed1997

Selenium supplementation suppresses tumor necrosis factor alpha-induced human immunodeficiency virus type 1 replication in vitro.

Selenium is a nutritionally essential trace element that is important for optimal function of the immune system. It is incorporated into selenoproteins as the amino acid selenocysteine and it is known to inhibit the expression of some viruses. In this study, we show that selenium supplementation for 3 days prior to exposure to tumor necrosis factor alpha (TNF-alpha) partially suppresses the induction of human immunodeficiency virus type 1 (HIV-1) replication in both chronically infected T lymphocytic and monocytic cell lines. In acute HIV-1 infection of T lymphocytes and monocytes in the absence of exogenous TNF-alpha, the suppressive effect of selenium supplementation was not observed. However, selenium supplementation did suppress the enhancing effect of TNF-alpha on HIV-1 replication in vitro in acutely infected human monocytes, but not in T lymphocytes. Selenium supplementation also increased the activities of the selenoproteins, glutathione peroxidase (GPx) and thioredoxin reductase (TR), which serve as cellular antioxidants. Taken together, these results suggest that selenium supplementation may prove beneficial as an adjuvant therapy for AIDS through reinforcement of endogenous antioxidative systems.

Cells, Cultured↗

PubMed1997

Induction of T cell clonal anergy results in resistance, whereas CD28-mediated costimulation primes for susceptibility to Fas- and Bax-mediated programmed cell death.

Since TCR-mediated stimulation induces T cells to become sensitive to Fas-mediated activation-induced cell death (Fas-AICD), we examined whether anergized and CD28-costimulated T cell clones were equally sensitive to Fas-AICD. Here, we show that TCR signal in the presence or absence of CD28 costimulation induced equivalent expression of Fas and Fas ligand. Although anergized cells expressed Fas and Fas ligand, they were resistant to Fas-AICD. Induction of anergy resulted in up-regulation and persistent expression of moderate amounts of bcl-xL and bax and absence of induction of bad. In contrast, CD28-costimulated cells that also expressed Fas and Fas ligand were initially resistant to Fas-AICD but became susceptible after 72 h of culture. Although Fas-mediated apoptosis was the major mechanism of AICD, the IL-1beta-converting enzyme-like protease inhibitor zVAD-FMK totally abrogated DNA fragmentation but not cell death, suggesting that additional Fas-independent apoptotic mechanisms were also operative. Resistance to apoptotic cell death was temporally associated with a dramatic increase of bcl-xL and the presence of bcl-xL:bax heterodimers. Subsequent sensitivity to AICD was associated with down-regulation of bcl-xL, induction of bad, and the displacement of bax from bcl-xL:bax heterodimers. Although induced following CD28 costimulation, bcl-2 did not protect against AICD. Therefore, besides its role in promotion of viability, prevention of anergy, and clonal expansion, CD28 costimulation also has a central role in the induction of subsequent AICD by up-regulating apoptotic mediators.

3T3 Cells↗

PubMed1997

Differential mode of TBP utilization in transcription of the tRNA[Ser]Sec gene and TATA-less class III genes.

The Xenopus laevis selenocysteine tRNA[Ser]Sec gene utilizes the TATA box binding protein (TBP) for its transcription in a manner more like TATA-dependent class II genes than TATA-less class III tRNA genes, even though this gene is transcribed by RNA polymerase III (Pol III). Addition of TBP increased in vitro transcription of the tRNA[Ser]Sec gene and a RNA polymerase II-(Pol II-) dependent template, while it decreased TATA-independent tRNA(Met) gene transcription, in a dose-dependent manner. Addition of wild-type TBP, truncated TBP containing the highly conserved COOH-terminal domain or a mutant TBP defective in TATA-independent Pol III transcription to TBP-depleted extracts restored tRNA[Ser]Sec transcription, while addition of a mutant TBP defective in Pol II transcription did not. These studies provide evidence that common surfaces of TBP may be used in transcription from TATA-dependent promoters of the tRNA[Ser]Sec gene and class II genes. Further, we show that distinct chromatographic fractions of TBP complexes are required for tRNA[Ser]Sec gene transcription and TATA-less class III gene transcription.

Animals↗

PubMed1997

Selenocysteine tRNA[Ser]Sec levels and selenium-dependent glutathione peroxidase activity in mouse embryonic stem cells heterozygous for a targeted mutation in the tRNA[Ser]Sec gene.

To investigate the effect of a reduced level of selenocysteine (Sec) tRNA[Ser]Sec in selenoprotein biosynthesis, two mouse embryonic stem (ES) cell lines heterozygous for the corresponding gene were generated by homologous recombination of the host genome with targeting vectors encoding a deleted or a disrupted tRNA[Ser]Sec gene. The presence of a single functional gene in ES cells afforded us an opportunity to determine directly in the cell line the effect of reduced gene dosage on (1) the levels of the Sec tRNA[Ser]Sec population, (2) the distributions of the isoacceptors within the Sec tRNA population, and (3) selenoprotein biosynthesis. We therefore determined the amounts and distributions of the two major tRNA[Ser]Sec isoacceptors, designated mcm5U and mcm5Um, within the Sec tRNA population and determined the activity of the anti-oxidant, selenium-containing glutathione peroxidase (GPx) in the heterozygotes and in wild type cells grown in media with and without added selenium. The level of the Sec tRNA[Ser]Sec population in the heterozygotes was approximately 60% of that of wild type cells grown in media under normal conditions, while the ratio of the mcmU isoacceptor in wild type vs mutant cells was approximately 2:1 and of the mcmUm isoacceptor approximately 1:1. In the presence of media supplemented with selenium, the Sec tRNA[Ser]Sec population increased about 20% in wild type cells and virtually not all in heterozygous cells, and the level of the Sec tRNA[Ser]Sec population was, therefore, approximately 50% of that of wild type cells. GPx activity was indistinguishable among these cell lines in either selenium-supplemented or unsupplemented media, indicating that the resultant changes in tRNA[Ser]Sec levels did not have a measurable effect on GPx biosynthesis.

Animals↗

PubMed1997

Cross-competition for TATA-binding protein between TATA boxes of the selenocysteine tRNA[Ser]Sec promoter and RNA polymerase II promoters.

In this study we show that the TATA-binding protein (TBP) interacts with the selenocysteine tRNA[Ser]Sec TATA element in a fashion analogous to the TBP-TATA interaction in RNA polymerase (Pol) II-transcribed genes even though the gene is transcribed by Pol III. Recombinant TBPs expressed in Escherichia coli bound to the tRNA[Ser]Sec TATA element. A factor was detected in Xenopus oocyte extracts which contain TBP and bind to the TATA boxes of the tRNA[Ser]Sec gene and various class II genes. Transcription of the microinjected tRNA[Ser]Sec gene was inhibited in Xenopus oocytes by coinjection with the TATA box of the adenovirus major late promoter (AdMLP). Transcription of a 5S gene was not affected under these conditions. These results suggest that the tRNA[Ser]Sec gene recruits TBP in a manner similar to that of TATA-dependent Pol II-transcribed genes and differently from that of Pol III-transcribed genes lacking a TATA box.

Animals↗

PubMed1997

Neuropathology of bovine herpesvirus type 5 (BHV-5) meningo-encephalitis in a rabbit seizure model.

The suitability of a rabbit seizure model for studying the neuropathogenesis of bovine herpesvirus type 5 (BHV-5) encephalitis was evaluated. Intranasal administration of BHV-5 (strain TX89) together with intramuscular administration of dexamethasone produced seizures in 70% of rabbits tested and meningo-encephalitis in 100%. Infectious BHV-5 was consistently isolated from the following sites: olfactory bulb; anterior cortex, containing the frontal cortex, olfactory tract and anterior portion of the olfactory cortex; posterior cortex, containing the temporal, parietal, piriform, entorhinal and occipital cortices; amygdala; hippocampus. Less frequently, BHV-5 was isolated from the midbrain and diencephalon, the pons and medulla, the cerebellum, and the trigeminal ganglia. Rabbits similarly infected with the Cooper strain of bovine herpesvirus type 1 showed no neurological signs or meningo-encephalitis, and virus was not recovered from the brain. The brains of BHV-5-infected rabbits showed neuronal degeneration, leptomeningitis, gliosis and perivascular cuffing, predominantly in the olfactory cortex (piriform and entorhinal cortices), amygdala and hippocampus. Mild lymphocytic meningitis was seen in the olfactory bulb and focal lymphocytic infiltration was sometimes present in the medulla and cerebellum. BHV-5, specific antigens and nucleic acids were detected in the olfactory cortex, amygdala and hippocampus by immunohistochemical methods and in-situ hybridization. The results suggested that, after intranasal BHV-5 inoculation, the virus spread to the central nervous system via the olfactory and trigeminal pathways. The olfactory pathway was more susceptible than the trigeminal pathway to neuropathogenic effects.

Animals↗

PubMed1997

Selenocysteine tRNAs as central components of selenoprotein biosynthesis in eukaryotes.

Selenocysteine (Sec) tRNAs serve as carrier molecules for the biosynthesis of Sec from serine and to donate Sec to protein in response to specific UGA codons. In this study, we describe the current status of Sec tRNAs in higher animals and further we examine: (i) the Sec tRNA population in Drosophila; (ii) transcription of the Sec tRNA in vivo (in Xenopus oocytes) and in vitro (in Xenopus oocyte extracts); (iii) the effect of selenium on the Sec tRNA population in various rat tissues following replenishment of extremely selenium deficient rats with this element; and (iv) the biosynthesis of the modified bases on Sec tRNA in Xenopus oocytes.

Animals↗

PubMed1996

Analysis of the selenocysteine tRNA[Ser]Sec gene transcription in vitro using Xenopus oocyte extracts.

An in vitro transcription system was devised using a Xenopus oocyte S150 extract for analyzing expression of the Sec tRNA[Ser]Sec gene. The activator element, located at about -200 that is required for maximal expression of the tRNA[Ser]Sec gene in vivo, had no effect on tRNA[Ser]Sec transcription in vitro. In vitro transcription of this gene even tolerated mutations within or deletion of the PSE, showing marked contrast with the requirements observed in vivo. However, the TATA box was indispensable for basal level expression of the gene both in vivo and in vitro. The region spanning from the TATA box to the transcription start point (-33 to -1) was sufficient for Pol III to recognize the tRNA[Ser]Sec gene promoter in vitro.

Animals↗

PubMed1996

Differential association of protein tyrosine kinases with the T cell receptor is linked to the induction of anergy and its prevention by B7 family-mediated costimulation.

When stimulated through their antigen receptor, without costimulation, T cells enter a state of antigen-specific unresponsiveness, termed anergy. B7-mediated costimulation, signaling via CD28, is sufficient to prevent the induction of anergy. Here we show that ligation of T cell receptor (TCR) by alloantigen alone, which results in anergy, activates tyrosine phosphorylation of TCR zeta and its association with fyn. In contrast, TCR ligation in the presence of B7 costimulation, which results in productive immunity, activates tyrosine phosphorylation of TCR zeta and CD3 chains, which associate with activated lck and zeta-associated protein (ZAP) 70. Under these conditions, CD28 associates with activated lck and TCR zeta. These data suggest that the induction of anergy is an active signaling process characterized by the association of TCR zeta and fyn. In addition, CD28-mediated costimulation may prevent the induction of anergy by facilitating the effective association of TCR zeta and CD3 epsilon with the critical protein tyrosine kinase lck, and the subsequent recruitment of ZAP-70. Strategies to inhibit or activate TCR-associated, specific protein tyrosine kinase-mediated pathways may provide a basis for drug development with potential applications in the fields of transplantation, autoimmunity, and tumor immunity.

3T3 Cells↗

PubMed1996

Local expression of a POU family transcription factor, Pit-1, in the rat placenta.

A pituitary-specific trans-acting factor, Pit-1 regulates transcriptional activity of growth hormone (GH) and prolactin (PRL) genes. Pit-1 can bind and activate the promoters of human chorionic somatomammotropin (hCS-A) and placental GH variants (hGH-V) as well. However, expression of Pit-1 in the rat placenta has not yet been elucidated. The present study aims to determine whether the Pit-1 gene is locally expressed in the rat placenta using reverse transcription-polymerase chain reaction (RT-PCR), Northern blot and Western blot hybridization, in situ hybridization and immunohistochemistry. PCR products were further analyzed by Southern hybridization and DNA sequencing. The estimated size of Pit-1 mRNA in placenta was very similar to that in anterior pituitary (AP). PCR products from placenta were exactly the same size with that from AP and confirmed as Pit-1-specific by Southern hybridization. The Pit-1 specific sequence was also confirmed by sequencing of partial amplification fragments. Immunoreactive 33 kDa Pit-1 was present in the placenta as well as in AP. Pit-1 specific mRNA and protein were localized in the trophoblast cells of placenta. These data suggest that Pit-1 is locally synthesized in the rat placenta and may be involved in the regulation of GH- and/or PRL-like gene expression in the placenta.

Animals↗

PubMed1996

Production and characterisation of a monoclonal antibody to Serpulina hyodysenteriae.

A monoclonal antibody (mAb) directed against Serpulina hyodysenteriae, the causative agent of swine dysentery, was produced and characterised. The mAb (BJL/SH1) reacted in Western blots with a protein with a molecular mass of about 30 kDa in outer membrane preparations from a range of S. hyodysenteriae isolates of different serotypes. It did not react with preparations made from a variety of non-S. hyodysenteriae intestinal spirochaetes. Immunogold labelling was used to confirm the location of the reactive epitope on the cell outer membrane. The mAb agglutinated and produced fluorescence only with strains of S. hyodysenteriae, and should prove to be a useful reagent for identification of S. hyodysenteriae.

Animals↗

PubMed1996

Purification and characterization of mitochondrial ribonuclease P from Aspergillus nidulans.

Mitochondrial ribonuclease (RNase) P from Aspergillus nidulans was purified to near homogeneity using whole-cell extract as the starting material. A 4400-fold purification with a yield of 5.2% was achieved by ammonium sulfate fractionation, heat treatment, and five types of column chromatography, including tRNA-affinity column chromatography. This enzyme, which has a molecular mass of 232 kDa determined by glycerol gradient sedimentation analysis, appears to be composed of seven polypeptides and an RNA moiety. These seven polypeptides consistently copurified with the RNase P activity through two ion-exchange chromatography columns and in a glycerol gradient. As judged by nuclease sensitivity, the enzyme requires an RNA component for its activity. The 3'-end-labeled RNAs that copurified with the enzyme displayed identical sequences but had variable lengths for the 5' end, indicating that they originated from a common RNA molecule, the putative RNA component of RNase P. The purified enzyme cleaved mitochondrial precursor tRNAHis, resulting in an 8-bp acceptor stem. This implies that the purified RNase P is a mitochondrial enzyme and that an additional guanylate residue (at position -1) of tRNAHis in A. nidulans mitochondria is generated by a mode that is analogous to the generation of their counterparts in prokaryotes and chloroplasts.

Aspergillus nidulans↗