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B J Lee

Publications and source records attributed to B J Lee.

At least 163 records · Page 9Linked to original sources
PubMed1991

Studies on secretion of catecholamine evoked by caffeine from the isolated perfused rat adrenal gland.

The influence of caffeine on secretion of catecholamines (CA) was examined in the isolated perfused rat adrenal gland. Caffeine (0.3 mM) perfused into an adrenal vein of the gland produced a marked increase in secretion of CA. This secretory effect of CA evoked by perfusion of caffeine for one minute was considerably prolonged, lasting for more than 90 minutes. The tachyphylaxis to releasing effect of CA induced by caffeine was observed by repeated perfusion of this drug. The caffeine-evoked CA secretion was markedly inhibited by pretreatment with ouabain, trifluoperazine, TMB-8 and perfusion with calcium-free Krebs solution containing 5 mM EGTA, but was not affected by perfusion of calcium-free Krebs solution without other addition. CA secretion evoked by caffeine was not reduced significantly by pretreatment with chlorisondamine but after the first collection of perfusate for 3 min was clearly inhibited. Interestingly, the caffeine-evoked CA secretion was considerably potentiated by pretreatment with atropine or pirenzepine, but after the first collection for 3 min it was markedly decreased. These experimental results suggest that caffeine causes a marked increase in secretion of CA from the isolated perfused rat adrenal gland by an extracellular calcium-independent exocytotic mechanism. The secretory effect of caffeine may be mainly due to mobilization of calcium from an intracellular calcium pool in the rat chromaffin cells and partly due to stimulation of both muscarinic and nicotinic receptors.

Adrenal Glands↗
PubMed1991

Selenocysteyl-tRNA occurs in the diatom Thalassiosira and in the ciliate Tetrahymena.

Selenocysteyl-tRNAs that decode UGA were identified previously in animal and bacterial cells and the genes for these tRNAs have been shown to be widespread in animals and eubacteria. In the present study, we identify a selenocysteyl-tRNA that codes for UGA in Thalassiosira pseudonana, which is a diatom, and in Tetrahymena borealis, which is a ciliate. The fact that these very diverse unicellular organisms also contain a selenocysteyl-tRNA suggests that selenocysteine-containing proteins and the use of UGA as a codon for selenocysteine are widespread, if not ubiquitous, in nature.

Animals↗
PubMed1991

Adrenergic mediation of the naloxone-induced GnRH release from hypothalami of ovariectomized, steroid-treated immature rats.

The present study examines the effect of naloxone on GnRH release in vitro under different steroid milieus. Naloxone (6.1 mumol/kg) administered 30 min before decapitation was highly effective in evoking GnRH release from superfused hypothalamic tissues derived from ovariectomized, estradiol- and progesterone-treated immature rats, while ineffective in altering GnRH release from intact, ovariectomized and vehicle- or estradiol-treated rats. To further explore the possible involvement of catecholamines in the naloxone-stimulated GnRH release, diethyldithiocarbamic acid (2.9 mmol/kg), an inhibitor of noradrenalin synthesis, was administered ip 30 min before naloxone injection into ovariectomized, estradiol- and progesterone-treated rats. Diethyldithiocarbamic acid markedly reduced the naloxone-evoked GnRH release, although it was ineffective in modifying the spontaneous release of GnRH. A blockade of alpha-adrenergic receptor with phenoxybenzamine significantly suppressed the naloxone-stimulated GnRH release, whereas treatment with propranolol, a beta-adrenergic receptor blocker, failed to alter GnRH release. The present data suggest that the endogenous opioid peptide may participate in the regulation of GnRH release under a particular steroid milieu, and the inhibitory action of endogenous opioid peptide seems to require the mediation of adrenergic neurotransmission, presumably through alpha-adrenergic receptor.

Adrenergic Fibers↗
PubMed1990

Activation of intracellular pathways with forskolin and phorbol ester increases LHRH mRNA level in the rat hypothalamus superfused in vitro.

Two intracellular signal transduction mechanisms such as cAMP-protein kinase a and phosphatidylinositol (PI) turnover-protein kinase c are known to be dually involved in the regulation of luteinizing hormone-releasing hormone (LHRH) release. However, it is not yet evident that the activation of two intracellular pathways affects the LHRH gene expression. The present study aims, therefore, to determine whether the activation of two intracellular pathways affects changes in LHRH mRNA. To this end, we took advantage of an in vitro superfusion system, where rat hypothalamic tissues were superfused with media containing forskolin (FKN) and/or phorbol-12-myristate-13-acetate (PMA). Superfusates were collected at 10-min intervals and LHRH release was determined by radioimmunoassay. After a 2-h superfusion period, the post-superfusion hypothalami were recovered and poly (A) RNA fractions were isolated. Alterations in LHRH mRNA in response to FKN and/or PMA were determined by an RNA-blot hybridization assay using a 32P-end-labeled LHRH oligonucleotide (29-mer) probe. In vitro perfusion of hypothalamic fragments with PMA and/or FKN stimulated LHRH release as well as LHRH mRNA. The combined infusion of FKN and PMA did not produce an additive effect on the LHRH mRNA levels, but it was effective in synergistically increasing LHRH secretion in vitro. These data clearly demonstrate that the biosynthetic machinery of LHRH is influenced by activation of two intracellular pathways, both cAMP-protein kinase a and phosphatidyl-inositol turnover-protein kinase c, indicating the transsynaptic regulation of hypothalamic LHRH gene expression.

Animals↗
PubMed1990

A nuclear magnetic resonance study of the cyclic AMP receptor protein (CRP): assignments of the NH protons of histidine and tryptophan residues and the effect of binding of cAMP to CRP.

Seven well-resolved signals could be observed in the field lower than 9.5 ppm in the 1H-NMR spectrum of the H2O solution of cyclic AMP receptor protein (CRP). The signals of the tryptophan and histidine residues were identified on the basis of the CPMG spin echo spectra, the intra-residue NOE, 15N labeling, deuterium labeling, and the results of pH titration. The assignments of peaks to specific tryptophan and histidine residues are discussed in relation to the amino acid sequence and X-ray crystallographic data, and were confirmed by experiments involving partial subtilisin digestion. The four signals E (11.25 ppm), F (11.15 ppm), G (10.75 ppm), and H (10.65 ppm) were tentatively assigned to the resonances of the histidine residue at position 159, the arginine residue at position 82, and the tryptophan residues at positions 85 and 13, respectively. On the addition of cAMP and cGMP, signals F and G shifted up- and downfield respectively and conformational changes in the structure of CRP could be detected. The conformational transition mostly occurs when one cAMP molecule binds to one of the dimer subunits, but is completed only when both cAMP binding sites are saturated.

Carrier Proteins↗
PubMed1990

Selenocysteine tRNA[Ser]Sec gene is ubiquitous within the animal kingdom.

Recently, a mammalian tRNA which was previously designated as an opal suppressor seryl-tRNA and phosphoseryl-tRNA was shown to be a selenocysteyl-tRNA (B. J. Lee, P. J. Worland, J. N. Davis, T. C. Stadtman, and D. Hatfield, J. Biol. Chem. 264:9724-9727, 1989). Hence, this tRNA is now designated as selenocysteyl-tRNA[Ser]Sec, and its function is twofold, to serve as (i) a carrier molecule upon which selenocysteine is biosynthesized and (ii) as a donor of selenocysteine, which is the 21st naturally occurring amino acid of protein, to the nascent polypeptide chain in response to specific UGA codons. In the present study, the selenocysteine tRNA gene was sequenced from Xenopus laevis, Drosophila melanogaster, and Caenorhabditis elegans. The tRNA product of this gene was also identified within the seryl-tRNA population of a number of higher and lower animals, and the human tRNA[Ser]Sec gene was used as a probe to identify homologous sequences within genomic DNAs of organisms throughout the animal kingdom. The studies showed that the tRNA[Ser]Sec gene has undergone evolutionary change and that it is ubiquitous in the animal kingdom. Further, we conclude that selenocysteine-containing proteins, as well as the use of UGA as a codon for selenocysteine, are far more widespread in nature than previously thought.

Animals↗
PubMed1990

Treatment results with an aggressive chemotherapeutic regimen (MACOP-B) for intermediate- and some high-grade non-Hodgkin's lymphomas.

Seventy previously untreated patients with stage II, III, and IV intermediate- or high-grade lymphoma were treated with methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, and bleomycin (MACOP-B) between September 1985 and November 1987. Forty-nine of these patients had diffuse large-cell lymphoma (DLCL), and eight of these patients were human immunodeficiency virus (HIV)-positive. Complete responses were achieved in 54% of all patients and 52% of those with DLCL. With follow-up extending to 36 months, 45% of all DLCL patients are alive, and 50% are still living, if the HIV-positive patients are excluded from the analysis. Chemotherapy was quite toxic. Seventy-five percent of patients had severe mucositis, 42% had peripheral neuropathy, 50% required hospitalization, and 54% experienced leukopenia with a WBC count below 1,000/microL. Seven percent (five patients) died of toxicity related to the chemotherapy. Our analysis of prognostic parameters indicated that B symptoms, a performance status below 80, and, to a lesser extent, elevation of serum lactic acid dehydrogenase (LDH) (in HIV-negative DLCL patients) were associated with an inferior survival. Advanced age, sex, and bulky disease were not found to have a statistically significant effect on survival. Our preliminary results indicate that MACOP-B chemotherapy is an effective regimen for high- and intermediate-grade lymphomas. However, the survival for patients with DLCL treated with MACOP-B is no different than that achieved with previous regimens at our institution.

Adolescent↗
PubMed1989

Identification of a selenocysteyl-tRNA(Ser) in mammalian cells that recognizes the nonsense codon, UGA.

The presence of a unique opal suppressor seryl-tRNA in higher vertebrates which is converted to phosphoseryl-tRNA has been known for several years, but its function has been uncertain (see Hatfield, D. (1985) Trends Biochem. Sci. 10, 201-204 for review). In the present study, we demonstrate that this tRNA species also occurs in vivo as selenocysteyl-tRNA(Ser) suggesting that it functions both as a carrier molecule upon which selenocysteine is synthesized and as a direct selenocysteine donor to a growing polypeptide chain in response to specific UGA codons. [75Se]Seleno[3H]cysteyl-tRNA(Ser) formed by administering 75Se and [3H]serine to rat mammary tumor cells (TMT-081-MS) in culture was isolated from the cell extract. The amino acid attached to the tRNA was identified as selenocysteine following its deacylation and reaction with iodoacetate and 3-bromopropionate. The resulting alkyl derivatives co-chromatographed on an amino acid analyzer with authentic carboxymethylselenocysteine and carboxyethylselenocysteine. Seryl-tRNA(Ser) and phosphoseryl-tRNA(Ser) (Hatfield, D., Diamond, A., and Dudock, B. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6215-6219), which co-migrate on a reverse phase chromatographic column with selenocysteyl-tRNA(Ser), were also identified in extracts of TMT-018-MS cells. Hence, we propose that a metabolic pathway for selenocysteine synthesis in mammalian cells is the conversion of seryl-tRNA(Ser) via phosphoseryl-tRNA(Ser) to selenocysteyl-tRNA(Ser). In a ribosomal binding assay selenocysteyl-tRNA(Ser) recognizes UGA but not any of the serine codons. Selenocysteyl-tRNA(Ser) is deacylated more readily than seryl-tRNA(Ser) (i.e. 58% deacylation during 15 min at pH 8.0 and 37 degrees C as compared to 41%).

Animals↗
PubMed1989

Transcription of Xenopus selenocysteine tRNA Ser (formerly designated opal suppressor phosphoserine tRNA) gene is directed by multiple 5'-extragenic regulatory elements.

A tRNA gene whose product is aminoacylated with serine and the serine moiety is then phosphorylated to form phosphoseryl-tRNA (see Hatfield, D. (1985) Trends Biochem. Sci. 10, 201-204 for review) has now been shown to form selenocysteyl-tRNA; hence the corresponding gene is designated as selenocysteine tRNA Ser (B. J. Lee, P. J. Worland, J. N. Davis, T. C. Stadtman, and D. Hatfield (1989) J. Biol. Chem. 264, in press). In the present study, we show that the expression of this unique tRNA gene is governed by at least three upstream regulatory elements. In initial studies, the relative efficiencies of transcription of the human, rabbit, chicken, and Xenopus selenocysteine tRNA genes were compared in vivo in Xenopus oocytes and in vitro in HeLa cell extracts. The Xenopus gene was severalfold more actively expressed, both in vivo and in vitro, than the human and rabbit genes, whereas the chicken gene was poorly expressed. Exchange of the 5'-flanking regions of the Xenopus and chicken genes, which have identical gene sequences, reversed their levels of transcription, demonstrating that a regulatory site or sites exist upstream of these genes. Deletion-substitution mutants in the Xenopus gene and its 5'-flanking sequence show in in vitro assays that 1) the level of transcription is reduced substantially when a GC-rich stretch that is immediately upstream of a TATA box in the -30 region is removed; 2) the level of transcription is virtually abolished when the TATA box is removed; and 3) deletions up to and further upstream of the GC-rich region do not affect the level of transcription. The same deletions, when used in in vivo assays, demonstrate a step-down in expression with the deletion removing the GC-rich region, a further step-down in expression with the deletion removing the TATA box, but the most pronounced reduction in expression was observed with a deletion removing an AT-rich region between nucleotides -62 and -76. Thus, a regulatory site was identified in vivo which was not detected in vitro, and transcription of the selenocysteine tRNA Ser gene is determined by multiple upstream regulatory elements.

Amino Acid Sequence↗
PubMed1989

Chromatographic analysis of the aminoacyl-tRNAs which are required for translation of codons at and around the ribosomal frameshift sites of HIV, HTLV-1, and BLV.

An examination of the frameshift signals or proposed signals within published sequences of retroviruses and other genetic elements from higher animals shows that each site utilizes a tRNA which normally contains Wybutoxine (Wye) base or Queuine (Q) base in the anticodon loop. We find experimentally that most of the Phe-tRNA present in HIV-1 infected cells lacks the highly modified Wye base in its anticodon loop and most of the Asn-tRNA in HTLV-1 and BLV infected cells lacks the highly modified Q base in its anticodon loop. Interestingly, Phe-tRNA translates a UUU codon within the ribosomal frameshift signal in HIV and Asn-tRNA translates a AAC codon within the proposed frameshift signals in HTLV-1 and BLV. Thus, the lack of a highly modified base in the anticodon loop of tRNAs in retroviral infected cells is correlated with the participation of these undermodified tRNAs in the corresponding frameshift event. This suggests that the "shifty" tRNAs proposed by Jacks et al. (Cell 55, 447-458, 1988) to carry out frameshifting may be hypomodified isoacceptors.

Animals↗
PubMed1989

Progesterone increases messenger ribonucleic acid (mRNA) encoding luteinizing hormone releasing hormone (LHRH) level in the hypothalamus of ovariectomized estradiol-primed prepubertal rats.

In order to investigate the mechanism underlying ovarian steroid action on gene expression of hypothalamic luteinizing hormone releasing hormone (LHRH), changes in LHRH mRNA level were determined by RNA-blot hybridization assay. Twenty-eight-day-old female rats were ovariectomized (OVX) and implanted with Silastic capsule containing either 17 beta-estradiol (E) or vehicle (V). Two days later (day 30), OVX + E-primed rats were given s.c. progesterone (P, 1 mg) 6 h prior to decapitation. Four experimental groups were studied: (1) intact, (2) OVX + V, (3) OVX + E, and (4) OVX + E + P-treated rats. Poly(A) RNA fractions from hypothalami (40-50/group) were isolated, blotted onto nitrocellulose paper and hybridized with 32P-end-labeled LHRH oligonucleotides (29 mer) which are complementary to rat LHRH mRNA. The hypothalamic LHRH mRNA signal markedly attenuated 2 days following ovariectomy. E replacement to OVX rats slightly increased LHRH mRNA level, which is lower than that of the intact group. However, a single injection of P to OVX + E-treated rats notably augmented the LHRH mRNA level over that observed in the intact group. In addition, LHRH content and release in vitro were examined to correlate with changes in LHRH gene expression. Ovariectomy and the replacement of E and/or P resulted in a similar fashion of changes in LHRH release and content as compared to alteration of LHRH mRNA level. This study clearly demonstrates that P increases LHRH mRNA level in the hypothalamus of OVX + E-primed immature rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Gene for selenium-dependent glutathione peroxidase maps to human chromosomes 3, 21 and X.

A human glutathione peroxidase cDNA has been used as a probe to hybridize to DNAs isolated from human - rodent somatic cell hybrids that have segregated human chromosomes. A 609 bp probe which contains the entire coding region hybridizes to human chromosomes 3, 21 and Xp. Fragments of the cDNA coding sequence and of the 3' untranslated region were also used as probes. These fragments hybridized to each of the three chromosomes with the same efficiency, suggesting similarity between the loci, whereas an intronic probe detected only the gene on chromosome 3. The general organization of each gene was determined from the hybridization data. The data suggest that the locus on chromosome 3 is a functional gene containing a single intron and a pattern of restriction sites identical to those found in the cDNA coding sequence. The data also suggest that the sequences on chromosomes X and 21 have equal conservation of the 3' untranslated and coding sequences but do not contain introns, providing evidence that the latter two sequences are processed pseudogenes. A simple two allele polymorphism in PvuII digests was detected at the locus on chromosome 21.

Animals↗