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Biomedical subjects

S L Lee

Publications and source records attributed to S L Lee.

At least 109 records · Page 6Linked to original sources
PubMed1996

Luteinizing hormone deficiency and female infertility in mice lacking the transcription factor NGFI-A (Egr-1).

The immediate-early transcription factor NGFI-A (also called Egr-1, zif/268, or Krox-24) is thought to couple extracellular signals to changes in gene expression. Although activins and inhibins regulate follicle-stimulating hormone (FSH) synthesis, no factor has been identified that exclusively regulates luteinizing hormone (LH) synthesis. An analysis of NGFI-A-deficient mice derived from embryonic stem cells demonstrated female infertility that was secondary to LH-beta deficiency. Ovariectomy led to increased amounts of FSH-beta but not LH-beta messenger RNA, which suggested a pituitary defect. A conserved, canonical NGFI-A site in the LH-beta promoter was required for synergistic activation by NGFI-A and steroidogenic factor-1 (SF-1). NGFI-A apparently influences female reproductive capacity through its regulation of LH-beta transcription.

Animals↗
PubMed1996

5-Oxo-eicosanoids and hematopoietic cytokines cooperate in stimulating neutrophil function and the mitogen-activated protein kinase pathway.

The newly defined eicosatetraenoates (ETEs), 5-oxoETE and 5-oxo-15(OH)-ETE, share structural motifs, synthetic origins, and bioactions with leukotriene B4 (LTB4). All three eicosanoids stimulate Ca2+ transients and chemotaxis in human neutrophils (PMN). However, unlike LTB4, 5-oxoETE and 5-oxo-15(OH)-ETE alone cause little degranulation and no superoxide anion production. However, we show herein that, in PMN pretreated with granulocyte-macrophage or granulocyte colony-stimulating factor (GM-CSF or G-CSF), the oxoETEs become potent activators of the last responses. The oxoETEs also induce translocation of secretory vesicles from the cytosol to the plasmalemma, an effect not requiring cytokine priming. To study the mechanism of PMN activation in response to the eicosanoids, we examined the activation of mitogen-activated protein kinase (MAPK) and cytosolic phospholipase A2 (cPLA2). PMN expressed three proteins (40, 42, and 44 kDa) that reacted with anti-MAPK antibodies. The oxoETEs, LTB4, GM-CSF, and G-CSF all stimulated PMN to activate the MAPKs and cPLA2, as defined by shifts in these proteins' electrophoretic mobility and tyrosine phosphorylation of the MAPKs. However, the speed and duration of the MAPK response varied markedly depending on the stimulus. 5-OxoETE caused a very rapid and transient activation of MAPK. In contrast, the response to the cytokines was rather slow and persistent. PMN pretreated with GM-CSF demonstrated a dramatic increase in the extent of MAPK tyrosine phosphorylation and electrophoretic mobility shift in response to 5-oxoETE. Similarly, 5-oxoETE induced PMN to release some preincorporated [14C]arachidonic acid, while GM-CSF greatly enhanced the extent of this release. Thus, the synergism exhibited by these agents is prominent at the level of MAPK stimulation and phospholipid deacylation. Pertussis toxin, but not Ca2+ depletion, inhibited MAPK responses to 5-oxoETE and LTB4, indicating that responses to both agents are coupled through G proteins but not dependent upon Ca2+ transients. 15-OxoETE and 15(OH)-ETE were inactive while 5-oxo-15(OH)-ETE and 5(OH)-ETE had 3- and 10-fold less potency than 5-oxoETE, indicating a rather strict structural specificity for the 5-keto group. LY 255283, a LTB4 antagonist, blocked the responses to LTB4 but not to 5-oxoETE. Therefore, the oxoETEs do not appear to operate through the LTB4 receptor. In summary, the oxoETEs are potent activators of PMN that share some but not all activities with LTB4. The response to the oxoETEs is greatly enhanced by pretreatment with cytokines, indicating that combinations of these mediators may be very important in the pathogenesis of inflammation.

Arachidonic Acid↗
PubMed1996

Pathways for degradation of the catalytic subunit of cAMP-dependent protein kinase differ in wild-type and kinase-negative S49 mouse lymphoma cells.

The catalytic subunit of cAMP-dependent protein kinase radiolabeled with [35S]methionine in wild-type S49 mouse lymphoma cells was degraded with half-lives of approximately 9.2 h in unstimulated cells and approximately 4.5 h in cells stimulated with a membrane-permeable cAMP analog. Turnover in kinase-negative mutant cells was about three times faster than in stimulated wild-type cells and appeared to involve a unique 47-kDa intermediate. Levels of catalytic subunit protein revealed by Western immunoblotting were consistent with the measured differences in turnover, but whereas the protein was mostly soluble in wild-type cell extracts, it was almost entirely insoluble in the mutant cell extracts. A substantial fraction of the catalytic subunit labeled in a 5-min pulse was soluble in kinase-negative cell extracts, but most of this material was rendered insoluble by incubating the cells for an additional 30 min before extraction. Degradation of the catalytic subunit in kinase-negative, but not in wild-type, cells was inhibited strongly by two specific peptide aldehyde inhibitors of the proteasomal chymotrypsin-like activity. An inhibitor of the proteasomal protease that prefers branched-chain amino acids had less of an effect on catalytic subunit degradation in the mutant cells.

Animals↗
PubMed1996

5-Oxo-eicosatetraenoate is a broadly active, eosinophil-selective stimulus for human granulocytes.

5-Oxo-eicosatetraenoate (5-oxoETE) is gaining recognition as a chemotactic factor for eosinophilic (Eo) as well as neutrophilic (Neu) polymorphonuclear leukocytes. We found that the eicosanoid was far stronger than C5a, platelet-activating factor (PAF), leukotriene B4 (LTB4), or FMLP in stimulating Eo chemotaxis. Moreover, it had weak intrinsic degranulating effects on otherwise unstimulated Eo, produced prominent degranulation responses in Eo primed by granulocyte-macrophage CSF, and enhanced the Eo-degranulating potencies of PAF, C5a, LTB4, and FMLP by up to 10,000-fold. Low picomolar levels of 5-oxoETE also induced Eo to activate mitogen-activated protein kinases (MAPKs), as defined by shifts in the electrophoretic mobility and tyrosine phosphorylation of two immunodetectable proteins, p44 and p42. 5-OxoETE was > or = 100-fold weaker or unable to stimulate any of these responses in Neu. Finally, 5-oxo-15-hydroxy-ETE and 5-hydroxy-ETE activated both cell types, but were weaker than 5-oxoETE and had Eo/Neu potency ratios approaching unity. 5-OxoETE, thus, is uniquely potent and selective in promoting Eo not only to migrate, but also to release granule enzymes and activate MAPKs. By triggering MAPK activation, the eicosanoid may also influence the production of anaphylactoid lipids (e.g., PAF), arachidonic acid metabolites, and cytokines. 5-OxoETE therefore possesses a biologic profile well suited for mediating Eo-dominated allergic reactions in vivo.

Arachidonic Acids↗
PubMed1996

Methods for studying synthesis, turnover, and phosphorylation of catalytic subunit of cAMP-dependent protein kinase in mammalian cells.

Because of the low abundance of the two major isoforms (C alpha and C beta) of catalytic (C) subunit of cAMP-dependent protein kinase, it has been difficult to monitor their expression and virtually impossible to quantify their synthesis, phosphorylation, and turnover in intact mammalian cells. We now describe sensitive and quantitative immunochemical methods using a goat antibody raised against the recombinant C alpha isoform of murine C subunit that enable studies of the expression and metabolism of C subunit in cultured cells. The antibody reacts well with C alpha and C beta isoforms of murine C subunit and with C subunits from rat, hamster, and human cell lines, so it should have widespread utility. Immunoreactivity with bovine heart C subunit was substantially weaker. For quantitation of C subunit radioactivity in extracts of cells labeled metabolically with [35S]methionine, we developed a two-cycle immunoadsorption protocol that reduces nonspecific adsorption to negligible levels. A tritium-labeled, truncated C subunit marker protein is added to extracts as an internal marker to monitor C subunit recoveries in different samples. For analysis of expression of C subunit isoforms in different cells or tissues, we describe a nonradioactive Western immunoblot procedure that can quantitate C subunit in amounts as low as 12 pg. Using extraction conditions that either stabilize or destabilize the phosphate on Thr-197, we show how the relative expression and phosphorylation of C alpha and C beta isoforms can be estimated from SDS-gel patterns resulting from either immunoblot or immunoadsorption procedures.

Animals↗
PubMed1996

Does increased spending on alcoholism treatment lead to lower cirrhosis death rates?

The purpose of this study was to see if recent changes in the funding of alcoholism programmes in the United States were related to changes in liver cirrhosis death rates. Data on per-capita spending, per-capita alcohol consumption and cirrhosis death rates were gathered from various sources for the years 1979 and 1989 for the 50 states and the District of Columbia. Regression analysis showed that greater increases in spending on alcoholism across US states were associated with greater declines in cirrhosis mortality rates. Since alcohol-related deaths cause large productivity losses and treatment is relatively cheap this creates large savings for society.

Alcoholism↗
PubMed1996

Unimpaired macrophage differentiation and activation in mice lacking the zinc finger transplantation factor NGFI-A (EGR1).

The zinc finger protein NGFI-A (also called EGR1, Krox24, or zif268) is a candidate regulator of myeloid cell differentiation. Evidence supporting this hypothesis is twofold. First, NGFI-A antisense oligonucleotides prevent macrophage differentiation in HL-60 and U937 myeloid leukemia cell lines and in normal bone marrow cells. Second, enforced expression of NGFI-A blocks granulocytic differentiation and promotes macrophage differentiation in HL-60 cells and in the hematopoietic progenitor cell line 32D. We sought to determine the effect of NGFI-A deficiency on macrophage differentiation and function in vivo by examining native bone marrow cells from mice homozygous for a disrupted allele of NGFI-A derived from gene-targeted ES cells. Macrophages were observed in peripheral blood and several tissues, indicating that NGFI-A was not required for the formation of a variety of macrophage compartments. No differences in myeloid cell differentiation were observed between wild-type and NGFI-A-/- bone marrow cells cultured in the presence of macrophage, granulocyte-macrophage, or granulocyte colony-stimulating factor (M-CSF, GM-CSF, or G-CSF). Activation of NGFI-A-/- macrophages was comparable to that of wild-type macrophages as determined by nitric oxide production and increased cell surface expression of class II major histocompatibility complex molecules. Moreover, NGFI-A-/- mice showed no increased mortality or bacteria] burden when challenged with Listeria monocytogenes. Together, these results indicate that NGFI-A is not required for macrophage differentiation or activation.

Animals↗
PubMed1996

Nitroprusside inhibits serotonin-induced mitogenesis and tyrosine phosphorylation of smooth muscle cells.

Serotonin (5-hydroxytryptamine, 5-HT) produces hyperplasia and hypertrophy of bovine pulmonary artery smooth muscle cells (SMC) in culture. The growth responses are associated with early elevations of c-myc and actin gene expressions and are blocked by agents that elevate cellular adenosine 3',5'-cyclic monophosphate or inhibit 5-HT transport or tyrosine phosphorylation. A rapid enhancement of tyrosine phosphorylation of a 120-kDa protein (p120) is associated with the 5-HT-induced mitogenesis. In the present studies, sodium nitroprusside (SNP, 10-100 micromol/l), a NO-generating agent, dose dependently inhibited 5-HT-induced thymidine incorporation by SMC. Inhibition of the 5-HT stimulatory effect was also observed with isosorbide dinitrate and nitroglutathione, which are also NO donors. Incubation of cells with 8-bromoguanosine 3',5'-cyclic monophosphate (1 micromol/l) mimicked the antimitogenic action of SNP. The antiproliferative effect of SNP was inhibited by hemoglobin (50 micromol/l) and potentiated by superoxide dismutase (200 U/ml), supporting the role of NO in the process. Enhancement of tyrosine phosphorylation of p120 by 5-HT was prevented by preincubation with SNP or exogenously added guanosine 3',5'-cyclic monophosphate. The data indicate that 5-HT acts as a mitogen for SMC through a signal transduction pathway involving tyrosine phosphorylation. SNP likely prevents the 5-HT-induced mitogenesis of SMC through elevation of intracellular guanosine 3',5'-cyclic monophosphate and inhibition of tyrosine phosphorylation of p120.

Actins↗
PubMed1996

Dehydration natriuresis in male rats is mediated by oxytocin.

In a previous study in rats we demonstrated the existence of osmoregulatory natriuretic mechanisms distinct from the natriuretic mechanisms that are dependent on volume stimulation. At the same time, we found that oxytocin (OT) receptors were important mediators of natriuresis induced by hypernatremia but not of that induced by isotonic volume expansion. In the present study, the role of OT in dehydration natriuresis was examined in conscious rats. Dehydration for 24 h caused hypernatremia (from 142.1 +/- 0.4 to 147.7 +/- 0.7 mmol/l) and natriuresis accompanied by an approximately 30% spontaneous reduction of food intake. In conjunction with renal retention of water caused by an increase in circulating vasopressin, the natriuresis and probably the reduction of food intake can help to counteract the rise in body fluid osmolality. This natriuresis could not be fully explained by the reduction in plasma aldosterone. Plasma OT concentration had increased from 15.5 +/- 1.2 to 23.8 +/- 2.0 pg/ml at the end of 24 h of dehydration. Intravenous infusion of a selective OT-receptor antagonist [Mpa1,D-Tyr(Et)2, Thr4, Orn8]-OT using osmotic minipumps prevented dehydration natriuresis. It is concluded that in a dehydration-induced hypernatremic state OT is released, inducing natriuresis and facilitating sodium homeostasis. This mechanism is activated by Na osmoreceptors, but is not primarily dependent on the volume status.

Aldosterone↗
PubMed1995

31P nuclear magnetic resonance spectroscopic evidence that canine nucleus pulposus and anulus fibrosus cells differ in principal organophosphorus biomolecules.

STUDY DESIGN: 31P Nuclear magnetic resonance spectroscopy was used to compare amounts and types of principal organophosphorus biomolecules in canine intervertebral disc tissues ex vivo. OBJECTIVE: The goal of this study was to compare principal organophosphorus biomolecules in nucleus pulposus, anulus fibrosus, and in isolated nucleus pulposus and anulus fibrosus cells. SUMMARY OF BACKGROUND DATA: There is no published information on principal organophosphorus biomolecules in disc tissues for any animal. METHODS: Canine nucleus pulposus and anulus fibrosus were shown by hematoxylin-eosin staining to be healthy tissues characteristic of adult dogs. Viable cells liberated from these disc tissues by sequential protease digestion were directly visualized by light microscopy of wet mounts. 31P nuclear magnetic resonance spectra were recorded at room temperature for 12 hours according to conventional published methods. RESULTS: No resonances were detected for intact nucleus pulposus and anulus fibrosus tissue. 31P nuclear magnetic resonance spectra of viable chondrocyte-like cells from anulus fibrosus featured two distinctive resonance peaks consistent with phosphomonoesters and phosphodiesters. After undigested anulus fibrosus was removed, no resonances were detected. 31P nuclear magnetic resonance spectra of viable chondrocyte-like cells from nucleus pulposus before and after removal of undigested tissue featured only one resonance peak consistent with phosphodiesters. CONCLUSIONS: A phosphomonoester-containing biomolecule is present in healthy canine anulus fibrosus tissue but not in nucleus pulposus tissue, nor in nucleus pulposus cells or anulus fibrosus cells. A phosphodiester-containing biomolecule is present principally in nucleus pulposus cells. This study demonstrated that canine chondrocyte-like cells from nucleus pulposus and anulus fibrosus are phenotypically distinctive in principal organophosphorus biomolecules.

Animals↗
PubMed1995

Unimpaired thymic and peripheral T cell death in mice lacking the nuclear receptor NGFI-B (Nur77).

T cell hybridomas require the immediate-early gene NGFI-B (nur77) for T cell receptor (TCR)-mediated apoptosis, a model for negative selection of self-reactive T cells. TCR-mediated death was examined in mice bearing an NGFI-B loss-of-function mutation, either by administration of antibodies to CD3 (anti-CD3) or in two well-characterized transgenic models expressing self-reactive TCRs. Both the extent and the rate of thymocyte death were unimpaired. Anti-CD3-induced death was normal in CD4+ peripheral T cells, in which death is mediated predominantly by the Fas signaling pathway. Thus, no unique requirement for NGFI-B is observed for thymic or peripheral T cell death.

Animals↗
PubMed1995

Growth and differentiation proceeds normally in cells deficient in the immediate early gene NGFI-A.

NGFI-A (also known as EGR-1, zif/268, and Krox-24) is a zinc finger transcription factor induced in many cell types by a variety of growth and differentiation stimuli. To determine if NGFI-A plays a requisite role in these processes, we used homologous recombination to mutate both alleles of NGFI-A in embryonic stem (ES) cells and examined its effect on growth and differentiation. We find that ES cells lacking NGFI-A exhibit similar growth rates and serum-induced gene expression profiles compared to wild-type parental cells. They are capable of differentiating into neurons, cardiac myocytes, chondrocytes, and squamous epithelium. Chimeric mice were generated from targeted ES cells, and their progeny were crossed to produce homozygous mutant mice. Growth and histological analyses of mice lacking NGFI-A confirm the finding in ES cells that NGFI-A is not required for many of the processes associated with its expression and suggest that the function of NGFI-A is either more subtle in vivo or masked by redundant expression provided by other gene family members such as NGFI-C, Krox-20, or EGR3.

Animals↗
PubMed1995

Kinetic and crystallographic studies of thrombin with Ac-(D)Phe-Pro-boroArg-OH and its lysine, amidine, homolysine, and ornithine analogs.

The X-ray crystallographic structure of Ac-(D)Phe-Pro-boroArg-OH [DuP714, Ki = 0.04 nM; Kettner, C., Mersinger, L., & Knabb, R. (1990) J. Biol. Chem. 265, 18289] complexed with human alpha-thrombin shows the boron atom covalently bonded to the side-chain oxygen of the active site serine, Ser195. The boron adopts a nearly tetrahedral geometry, and the boronic acid forms a set of interactions with the protein that mimic the tetrahedral transition state of serine proteases. Contributions of the arginine side chain to inhibitor affinity were examined by synthesis of the ornithine, lysine, homolysine, and amidine analogs of DuP714. The basic groups interact with backbone carbonyl groups, water molecules, and an aspartic acid side chain (Asp189) located in the thrombin S1 specificity pocket. The variation in inhibition constant by 3 orders of magnitude appears to reflect differences in the energetics of interactions made with thrombin and differences in ligand flexibility in solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Amidines↗
PubMed1995

Status epilepticus in 37 Chinese children: aetiology and outcome.

OBJECTIVE: The aetiology and clinical features of status epilepticus (SE) are described, with the aim of defining any relationship between risk factors and clinical outcome. METHODOLOGY: A retrospective review was performed of 37 Chinese children admitted to Queen Mary Hospital, Hong Kong, from 1989 to 1993 with the diagnosis of SE. RESULTS: Eighty-six per cent had onset before 5 years of age; 60% were due to an acute central nervous system (CNS) insult, 11% were idiopathic, 13% had a pre-existing CNS insult, 5% were febrile and 11% were due to progressive encephalopathy. An abnormal neurological status was present in 24% before the episode of SE, and a history of seizures before the onset of SE was present in 35% of patients. Fifty-four per cent of the episodes of SE were generalized. The mortality rate was 11% during the period of follow up but no deaths were attributed to SE. Neurological sequelae were observed in 27% of patients and recurrent SE occurred in 12%. CONCLUSIONS: In those patients with normal neurological status before an episode of SE and without acute CNS insult or progressive encephalopathy, the outcome was favourable.

Age of Onset↗
PubMed1995

Adrenocortical function and regulation of the steroid 21-hydroxylase gene in NGFI-B-deficient mice.

The immediate-early gene NGFI-B encodes an orphan nuclear receptor that binds DNA as a monomer and activates transcription through a canonical response element (NBRE). NGFI-B is expressed under basal conditions and in response to external stimuli in many mammalian tissues. In particular, NGFI-B expression is dramatically elevated in the adrenal cortex in response to stress and in Y1 adrenocortical cells in response to adrenocorticotropin. NGFI-B activates transcription through an NBRE of the gene encoding 21-hydroxylase (P450c21) in Y1 cells. Steroidogenic factor 1 (SF-1), a homolog of NGFI-B, also activates the P450c21 promoter. To examine the influence of these factors on P450c21 expression in vivo and the function of the hypothalamic-pituitary-adrenocortical axis as a whole, we generated NGFI-B (-/-) mice. These mice thrive and reproduce normally and maintain normal basal adrenocorticotropin, corticosterone, and P450c21 mRNA levels. In response to increases in adrenocorticotropin, NGFI-B (-/-) and wild-type mice demonstrated equivalent increases in serum corticosterone levels. Furthermore, and in contrast to in vitro results, no increases in P450c21 mRNA levels were observed in response to increases in adrenocorticotropin in NGFI-B (-/-) or wild-type mice. While SF-1 mRNA levels were not increased with increased steroidogenic demand, adrenal expression of Nurr1, a close homolog of NGFI-B, was induced to a greater extent by lipopolysaccharide in NGFI-B (-/-) mice than in wild-type mice. Finally, when the administration of dexamethasone for suppression was stopped, P450c21 mRNA and serum corticosterone levels recovered at the same rate in wild-type and NGFI-B (-/-) mice. Thus, while NGFI-B appears poised to affect the structure and function of the adrenal gland, the gland functions normally in its absence, suggesting that other factors, including Nurr1 and SF-1, are sufficient to drive P450c21 expression in mice and maintain normal steroidogenesis.

Adrenal Cortex↗
PubMed1995

Specificity of antinuclear antibodies in primary biliary cirrhosis.

OBJECTIVE: To study the specific autoantibodies against nuclear antigens in patients with primary biliary cirrhosis (PBC). METHODS: Sera from 21 patients with PBC were tested for antinuclear antibody (ANA) by indirect immunofluorescence on human epithelial (HEp)-2 cells, and for antibodies to various nuclear antigens by enzyme linked immunosorbent assay (ELISA) using different purified proteins or recombinant proteins as antigens. RESULTS: ANA detected in 10 of 21 patients (48%) with PBC included five anti-centromere antibody (ACA), two speckled, two homogeneous and one nuclear dot staining. ACA were present in 24% of PBC patients. By ELISA, anti-histone antibodies were detected in 81% of PBC patients, anti-ssDNA antibodies in 71% and anti-dsDNA in 10%, anti-topoisomerase-1 antibodies in 24%, anti-Sm/RNP antibodies in 24%, anti-La-48(SS-A) antibodies in 21%, and anti-Ro-60(SS-A) and anti-Ro-52(SS-A) antibodies in 30% and 25%, respectively. CONCLUSIONS: The high frequencies of various antibodies directed against intracellular proteins and nucleic acids in patients with PBC suggests that PBC is a multisystem autoimmune disease which is similar to other systemic autoimmune diseases.

Antibodies, Antinuclear↗
PubMed1995

Natriuretic role of endogenous oxytocin in male rats infused with hypertonic NaCl.

The involvement of endogenous oxytocin (OT) in the renal natriuretic response to an intravenous load of hypertonic and isotonic saline was investigated in anesthetized male rats with use of a selective OT-receptor antagonist [Mpa1,D-Tyr(Et)2,Thr4,Orn8]-OT (OT-ant). NaCl was infused (0.05 mmol.min-1.kg body wt-1 iv for 120 min) as a hypernatremic (HNa) solution or as an isotonic solution producing volume expansion (VE). HNa markedly increased sodium excretion from 0.25 +/- 0.06 to 10.04 +/- 0.62 mumol/min, whereas VE yielded only a moderate increase from 0.25 +/- 0.04 to 1.64 +/- 0.15 mumol/min. The increase in potassium excretion and urine flow rate during HNa was similar to that during VE. There were no significant changes in mean arterial pressure in either case. Administration of OT-ant delayed the natriuresis induced by HNa and reduced it by 60% but did not affect that induced by VE. Plasma OT concentration was significantly increased from 10.2 +/- 1.7 to 18.8 +/- 1.8 pg/ml at the end of HNa stimulation. In conclusion, the natriuresis in response to the intravenous sodium load was much more pronounced when NaCl was administered as a hypertonic solution than as an infusion inducing isotonic VE. Direct evidence was produced to assert that endogenous OT and its receptors are highly involved in the natriuresis elicited by systemic hypernatremic stimulation.

Animals↗