Biomedical subjects

R Kaempfer

Publications and source records attributed to R Kaempfer.

At least 19 recordsLinked to original sources

PubMed1992

Binding of ATP and messenger RNA by the beta-subunit of eukaryotic initiation factor 2.

In addition to forming a ternary complex with Met-tRNA(f) and GTP, eukaryotic initiation factor 2 (eIF-2) recognizes a specific site in mRNA molecules. Both binding activities are regulated by ATP, which itself binds tightly and specifically to eIF-2. Denaturation of eIF-2 with urea leads to complete loss of Met-tRNA(f) binding activity, while mRNA binding activity is stable. Hence, distinct conformational features in eIF-2 are required for ternary complex formation and for binding of mRNA. Chromatography of eIF-2 over ATP-agarose, in denaturing conditions that induce polypeptide subunit dissociation, results in selective retention of the beta-subunit of eIF-2. Isolated beta-subunit is capable of binding mRNA as well as ATP. Cibacron blue 3G-A binds tightly to eIF-2 and inhibits the binding of mRNA. This inhibition is relieved upon addition of ATP, showing that Cibacron blue 3G-A competes with ATP for eIF-2. eIF-2 beta subunit, active in binding of mRNA, is recovered upon chromatography of eIF-2 in denaturing conditions over matrix-bound Cibacron blue 3G-A. These results show that the ability of eIF-2 to bind mRNA and its ability to bind ATP are both lodged within remarkably stable domains of its beta-subunit. During initiation of protein synthesis, the eIF-2 beta subunit may thus interact with three ligands important for translational control: Met-tRNA(f), mRNA and ATP.

Adenosine Triphosphate↗

PubMed1992

Is cord blood erythropoietin a marker of intrapartum hypoxia?

A sensitive assay was used to compare the biological activity of cord serum erythropoietin in two groups of infants born with or without labor-induced hypoxia. The mean cord serum erythropoietin activity in 161 infants delivered after vaginal labor was 116 +/- 36 mU/mL, and was indistinguishable from that observed in 23 infants delivered by preplanned, elective cesarean section, 114 +/- 12 mU/mL (P = .75). The bioassay measured effective erythropoietin activity, including the contribution of potentiators in serum. These results indicate that duration and intensity of labor are insufficient to cause a significant increase in effective erythropoietin activity.

Cesarean Section↗

PubMed1991

Aberrant regulation of interleukin-2 but not of interferon-gamma gene expression in Down syndrome (trisomy 21).

The regulated expression of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) genes was analyzed in peripheral blood mononuclear cells derived from 29 noninstitutionalized Down syndrome individuals and compared to that of 32 normal donors. Culture conditions were chosen that measure the transient, phytohemagglutinin-induced expression of IL-2 and IFN-gamma mRNA, as well as the intactness of post-transcriptional and suppressor T cell-dependent mechanisms that control this expression. The latter was achieved by analyzing, respectively, the superinduction of IL-2 and IFN-gamma mRNA occurring upon culture with cycloheximide or after low-dose gamma-irradiation. A convenient, sensitive, and quantitative assay for specific mRNA was devised, suitable for measuring mRNA levels expressed in cells from 1 ml of peripheral blood. Analysis of individuals with Down syndrome revealed a pronounced decrease in inducibility of the IL-2 gene. By contrast, induction of IFN-gamma mRNA was as vigorous as that observed for normal donors. In cells from trisomic subjects, superinduction of IFN-gamma mRNA by cycloheximide was at least as extensive as for normal donors, while in the case of IL-2 mRNA, it was weaker. These abnormal patterns of IL-2 gene expression were seen irrespective of age. Our findings demonstrate a selective impairment of IL-2 gene expression in Down syndrome, rather than a general deficiency in helper T cells.

Adult↗

PubMed1991

Regulation of human interleukin-2 and interferon-gamma gene expression by suppressor T lymphocytes.

Concomitant with induction of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) gene expression in human tonsil cells, mitogenic stimulation induces a transient activation of cells able to effectively suppress expression of these genes. Induction of IL-2 and IFN-gamma genes largely precedes appearance of suppressor cell activity, allowing expression of both genes to occur before strong down-regulation is exerted by activated suppressor cells. Suppressive activity induced in one cell population can inhibit IL-2 and IFN-gamma gene expression in another population from the same donor. The distinct nature of suppressor cells is supported by the absence of down-regulation of IL-2 gene expression in a helper cell line, MLA-144; yet, in these cells, negative control can be expressed when active suppressor cells are introduced. Our findings support the concept that actual levels of IL-2 and IFN-gamma gene activity are regulated to a large extent by the differential kinetics of activation of suppressor cells on one hand and of cells expressing the IL-2 and IFN-gamma genes on the other.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1990

Expression of the human IL-2 receptor on lymphocytes involves rapid turnover of its p55 alpha-subunit (Tac).

Upon mitogenic stimulation, both mRNA encoding the p55 alpha-subunit (Tac) of the human IL-2R alpha and IL-2R alpha protein are induced and expressed in tonsil lymphocyte populations over several days. Using a quantitative dot-blot immunoassay for the IL-2R alpha subunit, a rapid disappearance of this polypeptide from cells is demonstrated in the presence of the translation inhibitor, cycloheximide. The half-life of IL-2R alpha subunit protein is 2 to 3 h. This decline in cell-associated IL-2R alpha subunit is matched by a rapid decline in IL-2R alpha on the cell surface and is not accompanied by any increase in soluble IL-2R alpha protein. Long term expression of the IL-2R on the cell surface is thus the result of continual synthesis and rapid breakdown of IL-2R alpha chains in the cell. Steady state expression of the IL-2R after an immune stimulus hence depends upon continuous expression of the IL-2R alpha subunit gene. Rapid turnover of the unstable alpha-subunit on the cell surface provides a novel mechanism for sensitive control of functional IL-2R expression.

Blotting, Northern↗

PubMed1990

Distinct epitopes in eukaryotic initiation factor 2 for binding of mRNA and for ternary complex formation with methionyl-tRNA(f) and GTP.

Eukaryotic initiation factor 2 (eIF-2) forms a ternary complex with methionyl-tRNA(fMet) and GTP on one hand, and it binds to a specific site in mRNA molecules on the other. Antibodies directed against eIF-2 were used to analyze these dual binding activities. A monoclonal antibody directed against the beta-subunit of eIF-2, 5A4, is able to inhibit ternary complex formation as well as binding of mRNA, showing that this subunit is essential for both binding activities of eIF-2. However, a polyclonal antibody, PR1, is able to distinguish between these activities in the eIF-2 molecule. In the presence of PR1, binding of mRNA by eIF-2 is inhibited completely, yet ternary complex formation with methionyl-tRNA(fMet) and GTP is stimulated more than 5-fold. Apparently, specific antibodies to eIF-2 can induce a conformational change in inactive factor molecules that permits them to form ternary complexes. These results show that distinct epitopes in eIF-2 are involved in binding of mRNA and in ternary complex formation with methionyl-tRNA(fMet) and GTP.

Animals↗

PubMed1990

Binding of ATP to eukaryotic initiation factor 2. Differential modulation of mRNA-binding activity and GTP-dependent binding of methionyl-tRNAMetf.

Eukaryotic initiation factor 2 (eIF-2) is shown to bind ATP with high affinity. Binding of ATP to eIF-2 induces loss of the ability to form a ternary complex with Met-tRNAf and GTP, while still allowing, and even stimulating, the binding of mRNA. Ternary complex formation between eIF-2, GTP, and Met-tRNAf is inhibited effectively by ATP, but not by CTP or UTP. Hydrolysis of ATP is not required for inhibition, for adenyl-5'-yl imidodiphosphate (AMP-PNP), a nonhydrolyzable analogue of ATP, is as active an inhibitor; adenosine 5'-O-(thiotriphosphate) (ATP gamma S) inhibits far more weakly. Ternary complex formation is inhibited effectively by ATP, dATP, or ADP, but not by AMP and adenosine. Hence, the gamma-phosphate of ATP and its 3'-OH group are not required for inhibition, but the beta-phosphate is indispensible. Specific complex formation between ATP and eIF-2 is shown 1) by effective retention of Met-tRNAf- and mRNA-binding activities on ATP-agarose and by the ability of free ATP, but not GTP, CTP, or UTP, to effect elution of eIF-2 from this substrate; 2) by eIF-2-dependent retention of [alpha-32P]ATP or dATP on nitrocellulose filters and its inhibition by excess ATP, but not by GTP, CTP, or UTP. Upon elution from ATP-agarose by high salt concentrations, eIF-2 recovers its ability to form a ternary complex with Met-tRNAf and GTP. ATP-induced inhibition of ternary complex formation is relieved by excess Met-tRNAf, but not by excess GTP or guanyl-5'-yl imidodiphosphate (GMP-PNP). Thus, ATP does not act by inhibiting binding of GTP to eIF-2. Instead, ATP causes Met-tRNAf in ternary complex to dissociate from eIF-2. Conversely, affinity of eIF-2 for ATP is high in the absence of GTP and Met-tRNAf (Kd less than or equal to 10(-12) M), but decreases greatly in conditions of ternary complex formation. These results support the concept that eIF-2 assumes distinct conformations for ternary complex formation and for binding of mRNA, and that these are affected differently by ATP. Interaction of ATP with an eIF-2 molecule in ternary complex with Met-tRNAf and GTP promotes displacement of Met-tRNAf from eIF-2, inducing a state favorable for binding of mRNA. ATP may thus regulate the dual binding activities of eIF-2 during initiation of translation.

Adenosine Diphosphate↗

PubMed1990

Relief of cytotoxicity and enhancement of interferon inducer activity of double-stranded RNA by eukaryotic initiation factor 2.

Double-stranded RNA (dsRNA) is a powerful interferon inducer but also possesses toxic properties. dsRNA was shown previously to inhibit translation by causing the inactivation of eukaryotic initiation factor 2 (eIF-2) and to bind tightly to this protein. The cytotoxicity of dsRNA was analyzed simultaneously with the induction of interferon in murine fibroblast cultures. Incubation of dsRNA with eIF-2 leads to a significant reduction in toxicity, concomitant with a marked stimulation of interferon induction. The enhancement of dsRNA-dependent interferon induction by eIF-2 is sensitive to a monoclonal antibody directed against the beta-subunit of eIF-2. When injected in combination with dsRNA, eIF-2 potentiates the dsRNA-dependent survival of mice infected with a lethal dose of Mengo virus.

Animals↗

PubMed1990

The potential to express or suppress human interleukin-2 and interferon-gamma genes is not restricted to distinct cell subsets.

Cell surface markers CD4, CD8, Leu8 and Leu15 (CD11) were used to separate human lymphoid cell subsets with monoclonal antibody-coated immunomagnetic beads. We show that each of these subsets is able to suppress the induction of IL-2 and IFN-gamma genes effectively. This is manifested by a pronounced superinduction of IL-2 and IFN-gamma mRNA, as well as IFN-gamma protein, in cell populations depleted of one of these subsets. Co-culture of cell subsets with total cell populations or depleted ones, on the other hand, leads to severe inhibition of expression of these genes. In these experiments, cells in suppressor subsets exhibit little, if any, expression of IL-2 and IFN-gamma genes. By contrast, depending on donor and lymphoid tissue examined (tonsils or peripheral blood mononuclear cells), CD4, CD8, Leu8, and Leu15 cell subsets are also able to express IL-2 or IFN-gamma genes to high levels. Moreover, in Leu8+ cells that do not express the IFN-gamma gene, extensive expression of both mRNA and protein can be elicited by inhibiting the activation of suppressor cells with gamma-irradiation before induction. These results support the concept that the potential to express or suppress human IL-2 and IFN-gamma genes is not restricted to distinct cell subsets. Suppression or expression can be elicited in cells carrying a given surface marker, depending on the state of the immune system in a lymphoid tissue.

Antigens, CD↗

PubMed1990

A constitutive antibody in normal human serum directed against rabbit bone marrow cells: lack in parturients, neonates, and hematologic disorders.

Normal human serum effectively inhibits a bioassay for erythropoietin based on DNA synthesis by rabbit erythroid precursors. This heat-sensitive inhibitory activity is readily lost on dilution of serum, revealing the presence of erythropoietin-potentiating activity. Inhibitory activity is caused by a rapid cytotoxic effect on rabbit bone marrow cells; mouse cells are less sensitive. Cytotoxic activity is removed from serum by adsorption to protein A, is not expressed at 4 degrees C, and is neutralized by anti-C3c complement antibody. Cytotoxicity is inhibited by EGTA; the effect of EGTA is reversed by addition of Ca2+ ions. These findings show that cytotoxicity is exerted through an antibody via the classical pathway of complement-dependent cell lysis. Although serum from healthy, adult human donors consistently contains cytotoxic activity, no such activity is observed in most serum samples from neonates, parturients, and patients with severe anemia. Patients with polycythemia or chronic renal failure occasionally lack cytotoxic activity in their serum. Serum samples lacking cytotoxic activity were found to be deficient in the antibody component in 34 out of 35 cases examined. These results show that an antibody directed against rabbit cells is constitutively present in normal human serum but is absent in a number of pathologic situations as well as being absent in neonates and parturients.

Anemia↗

PubMed1989

Superinduction of the human gene encoding low density lipoprotein receptor.

Expression of human LDL receptor mRNA and protein is induced in human glioblastoma-astrocytoma cells upon transfer into lipoprotein-deficient medium, a mode of induction likely to be physiological. The presence of cycloheximide (CHX) leads to up to 7.5-fold superinduction of LDL receptor mRNA within 4 hr and, upon removal of this inhibitor, to superinduction of LDL receptor protein as well. The extent of superinduction of LDL receptor mRNA reaches over 40-fold beyond the level expressed in medium containing regular serum. Despite its extensive superinduction, LDL receptor mRNA decays rapidly in the presence of CHX. Stabilization of LDL receptor mRNA is thus unlikely to account for the observed superinduction. These results show that normally the LDL receptor gene is expressed to only a small fraction of its full potential.

Cell Line↗

PubMed1987

Superinduction of the human gene encoding immune interferon.

Mitogen-induced interferon-gamma (IFN-gamma) gene expression was analyzed in human tonsil cells by titration of IFN-gamma activity and by quantitation of IFN-gamma mRNA. Expression of the IFN-gamma gene can be superinduced extensively by two distinct methods: exposure to various inhibitors of translation, or to low doses of gamma-irradiation. gamma-Irradiated cells produce, after exposure to cycloheximide, up to 12-fold greater amounts of IFN-gamma activity. Within as little as 4 h after the addition of translation inhibitors, IFN-gamma mRNA levels rise 3- to 5-fold. Superinduction acts to increase the size of the wave of IFN-gamma mRNA. Primary transcription of the IFN-gamma gene does not increase in cells superinduced by cycloheximide, nor can superinduction be explained by stabilization of IFN-gamma mRNA sequences. These findings show that, during normal induction, a labile protein acts post-transcriptionally to repress the accumulation of mature IFN-gamma mRNA sequences. The superinductive effects of cycloheximide and gamma-irradiation on levels of IFN-gamma are additive, suggesting that they affect different aspects of IFN-gamma gene expression. Superinduction by gamma-irradiation also has a post-transcriptional basis and is consistent with the possibility that expression of the IFN-gamma gene is normally controlled by the action of suppressor T cells. Even though the genes for human IFN-gamma and for interleukin-2 are both superinducible, a striking difference in the regulation of expression of these lymphokine genes is observed. Superinduction of IFN-gamma mRNA is not due to superinduction of interleukin-2.

Cells, Cultured↗

PubMed1985

DNA synthesis by erythroid precursors in a completely defined medium: a rapid, sensitive, and convenient bioassay for erythropoietin.

We detail a novel, sensitive, and reproducible in vitro bioassay for erythropoietin that can be performed conveniently in any laboratory and is well suited for analysis of large numbers of samples. The assay measures DNA synthesis by a cohort of highly erythropoietin-responsive red cell precursors appearing in bone marrow of anemic rabbits after treatment with a single dose of actinomycin D. The assay is conducted in a completely defined culture medium that totally dispenses not only with serum but also with serum-replacing factors. Under well-defined conditions, incorporation of [3H]thymidine by the cells depends specifically on erythropoietin. A stimulation index of up to 40-fold is obtained at 50 mU/ml of the hormone. The assay is linear in the range 0-50 mU/ml and not saturated before 1 U/ml of erythropoietin. Sample volumes of 1-30 microliter suffice for assay. Assay cells can be frozen in aliquots that retain their viability and ability to respond to erythropoietin over extended periods. Using microtiter-plate techniques, cells from one rabbit suffice for over 5000 triplicate erythropoietin determinations. Concentrations of 0.1-0.2 mU/well of erythropoietin can be detected. Erythropoietin values determined in sera from a variety of patients correlate extremely well with values obtained by the colony formation method. The ability to follow erythropoietin-dependent DNA synthesis and multiple cell divisions by a cohort of erythroid precursors in completely defined culture conditions may find application in controlled studies of red cell development.

Anemia↗

PubMed1979