Stimulation of HLA-A,B,C by IFN-alpha. The derivation of Molt 4 variants and the differential expression of HLA-A,B,C subsets.

Spontaneous mutants with altered HLA-A,B,C response to interferon-alpha (IFN-alpha) were isolated from the human thymus leukemia cell line Molt 4. Using fluorescein isothiocyanate (FITC)-conjugated W6/32 (a monoclonal antibody to HLA-A,B,C) and the fluorescence-activated cell sorter, the cells with highest and lowest fluorescence after 24-48 h of IFN-alpha treatment were selected and expanded. After several cycles of selection, mutant clones with low (greater than 10% of wild-type) and high (three times better) response were obtained. A similar protocol was employed to derive high responder mutants with the monoclonal antibody YT76, which recognises a subset of HLA strongly induced by IFN-alpha. Stable clones were derived for which YT-HLA induction was 7-fold that of Molt 4 cells and for which HLA induction occurred at 100-fold lower concentrations of IFN-alpha. The high response phenotype of the mutants was not accompanied by a significant increase in the constitutive level of expression of HLA-A,B,C (in the absence of IFN). The increase in the level of HLA-A,B,C expression after IFN-alpha treatment is mostly accounted for by the increase in the expression of a subset of HLA molecules, detected by the monoclonal antibody YT76 including HLA-B molecules.     

The phorbol ester TPA stimulates the expression of functional beta-adrenoceptors in human T lymphoblasts Molt 3.

In crude membranes from human T lymphoblasts Molt 3 cultured under standard conditions, the adenylate cyclase system was stimulated by GTP, its beta gamma-imido analogue (p[NH]ppG,) NaF and forskolin, but not by isoprenaline, prostaglandin E1 and vasoactive intestinal peptide. TPA (tumour-promoting agent phorbol ester) added at low concentration (3.2 nM) to the culture medium induced a marked increase in functional beta 2-adrenoceptors. Competition curves of [125I]cyanopindolol with the antagonist ICI 118.551 and four beta-adrenergic agonists indicated that the emergence of functional beta 2-adrenoceptors corresponded to one class of binding sites, shifting from a high-affinity state for agonists to a low-affinity state in the presence of p[NH]ppG. This expression of beta 2-adrenoceptors after a 4 h lag period depended on newly formed mRNA and protein synthesis as judged by the inhibitory effects of actinomycin D and cycloheximide. Further effects of TPA included alterations of the stimulatory G-protein Gs and/or the catalytic unit of adenylate cyclase.     

Modulation of tubulin mRNA levels by interferon in human lymphoblastoid cells.

Blot hybridization with labeled tubulin cDNA showed that treatment of Ramos cells, a human cell line of lymphoblastoid origin, with either alpha or beta interferon (IFN) induced a marked increase in the amount of tubulin mRNA sequences. The level of tubulin mRNA sequences increased rapidly after exposure of cells to IFN-alpha and reached a maximum after 1 h of treatment, which was four times the control level. Treatment with IFN-beta induced a maximal increase after 4 h; the amount of tubulin mRNA sequences was seven times higher than the control level. The mRNA extracted from IFN-treated and nontreated cells was translated in vitro in a reticulocyte lysate cell-free system containing [35S]methionine. Electrophoretic analysis of the labeled cell-free products showed an increase in the amount of translatable tubulin mRNA that parallels the time course of induction of tubulin mRNA sequences. Two-dimensional gel electrophoresis of the labeled protein products directed by mRNA indicates that IFN caused a more pronounced increase in the level of alpha-tubulin than beta-tubulin mRNA. Treatment with colchicine, which disrupts the cell microtubules, caused a marked decrease in the tubulin mRNA content. Concomitant treatment of the cells with colchicine and IFN abolished the interferon-dependent induction of tubulin mRNA.     

Opioid and non-opioid binding of beta-endorphin to bovine adrenal medullary membranes

Binding of human beta-endorphin (beta h-EP) to bovine adrenal medullary membranes was characterized using [125I]Tyr27-beta h-EP [( 125I]beta h-EP) as a primary ligand. The specific binding of [125I]beta h-EP was time-dependent, saturable and stereospecific. Analysis of a saturation isotherm revealed two apparent classes of specific binding sites with dissociation constants of 2.4 and 34 nM. The extent of maximum inhibition of specific [125I]beta h-EP binding by either levorphanol, morphine, naloxone, dynorphin A (1-13) or D-Ala2-D-Leu5-enkephalin was similar to each other and remained partial (60-70%). Levorphanol eliminated the high affinity component but showed no effect on the low affinity component of [125I]beta h-EP binding. beta h-EP(1-31) displaced completely the [125I]beta h-EP binding. However, beta h-EP(1-23) only partially (approximately 80%) inhibited the [125I]beta h-EP binding. beta h-EP(6-31) showed inhibitory activity on [125I]beta h-EP binding. These results suggest that [125I]beta h-EP binding to bovine adrenal medullary membranes consists of a high affinity opioid-sensitive component and a low affinity non-opioid component. The non-opioid component of [125I]beta h-EP binding may be related to COOH-terminal of the beta h-EP molecule.     

Cellular metabolism of 2',3'-dideoxycytidine, a compound active against human immunodeficiency virus in vitro

The nucleoside analog 2',3'-dideoxycytidine (ddCyd) has been shown to inhibit the infectivity and cytopathic effect of human immunodeficiency virus on human OKT4+ lymphocytes in vitro. Metabolism of ddCyd by human T-lymphoblastic cells (Molt 4) negative for human immunodeficiency virus and OKT4 was examined. Molt 4 cells accumulated ddCyd and its phosphorylated derivatives into acid-soluble and acid-insoluble material in a dose-dependent manner. For each concentration tested, 2',3'-dideoxycytidine triphosphate represented 40% of the total acid-soluble pool of ddCyd metabolites. Uptake of 5 microM ddCyd was linear for 4 h after addition of drug. Efflux of ddCyd metabolites from cells followed a biphasic course with an initial retention half-life of 2.6 h for 2',3'-dideoxycytidine triphosphate. DNA, but not RNA, of cells incubated with [3H]ddCyd became radiolabeled. Nuclease and phosphatase treatment of DNA followed by reverse-phase high pressure liquid chromatography showed that the nucleoside was incorporated into DNA in its original form. ddCyd was not susceptible to deamination by human Cyd-dCyd deaminase. It was a poor substrate for human cytoplasmic and mitochondrial dCyd kinases, with Km values of 180 +/- 30 and 120 +/- 20 microM, respectively. DNA polymerases alpha, beta, and gamma varied in their sensitivity to inhibition by ddCTP with Ki values of 110 +/- 40, 2.6 +/- 0.3, and 0.016 +/- 0.008 microM, respectively; however, inhibition was competitive with dCTP in each case.     

A slow component of intramembranous charge movement during sarcoplasmic reticulum calcium release in frog cut muscle fibers

Cut muscle fibers from Rana temporaria were mounted in a double Vaseline-gap chamber and equilibrated with an end-pool solution that contained 20 mM EGTA and 1.76 mM Ca (sarcomere length, 3.3-3.8 microns; temperature, 14-16 degrees C). Sarcoplasmic reticulum (SR) Ca release, delta[CaT], was estimated from changes in myoplasmic pH (Pape, P.C., D.- S. Jong, and W.K. Chandler. 1995. J. Gen. Physiol. 106:259-336). The maximal value of delta[CaT] obtained during a depleting depolarization was assumed to equal the SR Ca content before stimulation, [CaSR]R (expressed as myoplasmic concentration). After a depolarization to -55 to -40 mV in fibers with [CaSR]R = 1,000-3,000 microM, currents from intramembranous charge movement, Icm, showed an early I beta component. This was followed by an I gamma hump, which decayed within 50 ms to a small current that was maintained for as long as 500 ms. This slow current was probably a component of Icm because the amount of OFF charge, measured after depolarizations of different durations, increased according to the amount of ON charge. Icm was also measured after the SR had been depleted of most of its Ca, either by a depleting conditioning depolarization or by Ca removal from the end pools followed by a series of depleting depolarizations. The early I beta component was essentially unchanged by Ca depletion, the I gamma hump was increased (for [CaSR]R > 200 microM), the slow component was eliminated, and the total amount of OFF charge was essentially unchanged. These results suggest that the slow component of ON Icm is not movement of a new species of charge but is probably movement of Q gamma that is slowed by SR Ca release or some associated event such as the accompanying increase in myoplasmic free [Ca] that is expected to occur near the Ca release sites. The peak value of the apparent rate constant associated with this current, 2-4%/ms at pulse potentials between -48 and -40 mV, is decreased by half when [CaSR]R approximately equal to 500-1,000 microM, which gives a peak rate of SR Ca release of approximately 5-10 microM/ms.     

Reversible, positive cooperative interaction of 11 beta-chloromethyl-[3H]estradiol-17 beta with the calf uterine estrogen receptor

The binding of 11 beta-chloromethyl-[3H]estradiol-17 beta [3H]CME2) with the calf uterine estrogen receptor was investigated. The equilibrium binding analysis indicated a positive cooperative interaction yielding curvilinear Scatchard plots and Hill coefficients of 1.4-1.5. This positive cooperative interaction of [3H]CME2 was indistinguishable from the typical cooperative interaction of [3H]estradiol with the receptor. The apparent relative association constant and the relative binding affinity of CME2 for the estrogen receptor measured by competitive binding assay were 146 and 184%, respectively. The dissociation kinetics of [3H]CME2 from the receptor was biphasic, composed of a fast dissociating component (15%, t1/2 = 4 min at 0 degrees C; 9%, t1/2 = 4 min at 28 degrees C) and a slow dissociating component (85%, t1/2 greater than 50 h at 0 degrees C; 91%, t1/2 greater than 50 h at 28 degrees C). The dissociation kinetics of [3H]estradiol was also biphasic: the t1/2 of the fast dissociating component was 4 min at 0 and 28 degrees C and approximately 200 min for the slow dissociating component at both temperatures. The fraction of the slow [3H]estradiol dissociating component increased from 56 to 92% upon warming. Ethanol extraction and trichloroacetic acid treatment proved that the binding of [3H]CME2 is fully reversible. The unusual dissociation kinetics and the binding mechanism of CME2 are discussed.     

1H-Imidazo[4,5-c]quinoline derivatives as novel potent TNF-alpha suppressors: synthesis and structure-activity relationship of 1-, 2-and 4-substituted 1H-imidazo[4,5-c]quinolines or 1H-imidazo[4,5-c]pyridines

Structural modification of imiquimod (1), which is known as an interferon-alpha (IFN-alpha) inducer, for the aim of finding a novel and small-molecule tumor necrosis factor-alpha (TNF-alpha) suppressor and structure-activity relationship (SAR) are described. Structural modification of a imiquimod analogue, 4-amino-1-[2-(1-benzyl-4-piperidyl)ethyl-1H-imidazo[4,5-c]quinoline (2), which had moderate TNF-alpha suppressing activity without IFN-alpha inducing activity, led to a finding of 4-chloro-2-phenyl-1-[2-(4-piperidyl)ethyl]-1H-imidazo[4,5-c]quinoline (10) with potent TNF-alpha suppressing activity. The relation between conformational direction of 2-(4-piperidyl)ethyl group at position 1 and TNF-alpha suppressing activity is also demonstrated by NMR.     

Synergistic antiviral and antiproliferative activities of Escherichia coli-derived human alpha, beta, and gamma interferons

The antiviral and antiproliferative effects of highly purified Escherichia coli-derived human interferons (IFNs) were examined in human melanoma cells (Hs294T). Antiproliferative activity was monitored by measuring inhibition of cell multiplication, and antiviral activity was determined by inhibition of herpes simplex virus type 1 replication. Treatment of cells with IFN-gamma in combination with IFN-alpha A or IFN-beta 1 resulted in potentiation of both antiproliferative and antiviral activities. In contrast, combination treatments composed of IFN-alpha A and IFN beta 1 yielded inconsistent results. Some combinations reflected additive responses, whereas others were antagonistic. To examine correlations between IFN-induced biological activities and interactions of the different IFNs with cell surface receptors, in vivo [35S]methionine-labeled IFN-alpha A was prepared. Binding studies indicated the presence of 2,980 +/- 170 receptors per cell, each with an apparent Kd of (8.4 +/- 1.3) X 10(-11) M. Results from competitive binding studies suggested that Hs294T cells possess at least two types of IFN receptors: one which binds IFN-alpha A and IFN-beta 1 and another to which IFN-gamma binds.     

Different induction patterns of mRNA for IFN-alpha and -beta in human mononuclear leukocytes after in vitro stimulation with herpes simplex virus-infected fibroblasts and Sendai virus

Human peripheral blood mononuclear leukocytes (PBL) were stimulated in vitro by HSV type 1-infected glutaraldehyde-fixed fibroblasts, or Sendai virus (SV). The PBL containing mRNA for IFN-alpha 2 or -beta 1 were clearly identified by RNA-RNA in situ hybridization by using 35S-labeled alpha 2- and beta 1-probes. Although the two inducers gave similar levels of IFN in the culture medium (about 20 U/10(4) PBL), the patterns of expression of mRNA at the cellular level differed. The HSV induced only IFN-alpha mRNA in the PBL, with a lag of 1 to 2 h, and with a peak frequency of about 10 labeled cells/10(4) PBL at 6 h. Grain counts were high, the majority of cells having more than 50 grains. They were morphologically medium to large lymphocytes. The HSV-infected glutaraldehyde-fixed fibroblasts therefore induce IFN-alpha 2 mRNA in infrequent but highly efficient PBL, each cell capable of producing as much as 2 antiviral units of IFN-alpha. In contrast, SV induced both IFN-alpha 2 and -beta 1 mRNA in PBL, and without clear lags. IFN-beta 1 mRNA-positive PBL peaked somewhat earlier (4 h) than cells containing IFN-alpha 2 mRNA (6 h), and their mean frequencies were approximately 80 and 60/10(4) PBL, respectively, in a panel of PBL from six blood donors. Grain counts were lower than with the HSV inducer, the majority of cells having less than 50 grains, and most labeled cells were morphologically monocytes. The frequency of labeled PBL rapidly decreased with increasing culture time with both the SV and HSV inducers, was low at 12 h and almost absent at 24 h.     

PHOSPHATIDYLINOSITOL AND OTHER LIPIDS IN A MAMMALIAN SYMPATHETIC GANGLION: EFFECTS OF NEURONAL ACTIVITY ON INCORPORATION OF LABELLED INOSITOL,PHOSPHATE, GLYCEROL AND ACETATE1

Abstract— Superior cervical ganglia from adult rats were incubated for 1–6 h in a physiological salt solution containing ³²P_i [2-³H]inositol, [U-¹⁴C]glycerol, or [U-¹⁴C]acetate. Control ganglia were at rest throughout incubation, while the preganglionic nerves of the experimental ganglia were stimulated at 5/s, starting after 1 h of incubation. Responses were monitored by recording the action potentials in a postganglionic nerve. Radioactivity of phospholipids was counted after separation of the lipids by paper chromatography. Specific activity of free inositol and the gamma-phosphate of ATP were measured, the latter by using the hexokinase reaction with [¹⁴C]glucose, isolating the product, and counting its content of ³²P and ¹⁴C. At rest, labelling of phosphatidylinositol (PI), phosphatidylcholine and phosphatidylethanolamine proceeded at constant rates for at least 8 h with all precursors which entered them, except that labelling with glycerol slowed after 2–4 h. During stimulation the rate of incorporation of ³²P into PI approximately doubled, as previously reported. The increased rate remained constant for 3 h and then reverted to approximately the resting rate, although the electrical response continued unabated for 16 h. This decrease in rate of ³²P-labelling of PI in the ganglion could not be accounted for by transport into the postganglionic nerves. In stimulated preparations, after 4 h of incubation the labelling of PI was increased above the resting level by 53 ± 5% (mean ±s.e.m. ) with [³H]inositol, 97 ± 6% with ³²P_i, 24 ± 6% with [¹⁴C]glycerol and ?3 ± 10% with [¹⁴C]acetate. The increase with glycerol was thus statistically significant, in contrast with the findings of others on brain, where an increase of this size has neither been demonstrated nor excluded. There were no accompanying effects of stimulation on the specific activities of the gamma-P of ATP or of the free inositol within the ganglion that were sufficient to explain the difference between the labelling of PI with P and that with inositol.     

Alteration of intramolecular disulfides in insulin receptor/kinase by insulin and dithiothreitol: insulin potentiates the apparent dithiothreitol-dependent subunit reduction of insulin receptor

Dithiothreitol (DTT) was observed to increase both beta-subunit autophosphorylation and exogenous substrate phosphorylation of the insulin receptor in the absence of insulin. The natural protein reducing agent thioredoxin was also observed to increase the insulin receptor beta-subunit autophosphorylation. The activation of the insulin receptor/kinase by both DTT and thioredoxin was found to be additive with that of insulin. Further, the increase in the insulin receptor beta-subunit autophosphorylation in the presence of DTT and insulin was demonstrated to be due to an increase in the initial rate of autophosphorylation without alteration in the extent of phosphorylation. Similarly, the increase in the exogenous substrate phosphorylation was due to an increase in the Vmax of phosphorylation without significant effect on the apparent Km of substrate binding. In the presence of relatively low concentrations of DTT, insulin was found to potentiate the apparent insulin receptor subunit reduction of the native alpha 2 beta 2 heterotetrameric complex into alpha beta heterodimers, when observed by silver staining of sodium dodecyl sulfate-polyacrylamide gels. N-[3H]Ethylmaleimide ([3H]NEM) labeling in the absence of DTT pretreatment demonstrated that only the beta subunit had accessible sulfhydryl group(s). However, treatment of insulin receptors with DTT increased the amount of [3H]NEM labeling in the beta subunit as well as exposing sites on the alpha subunit. Further, incubation of the insulin receptors with the combination of DTT and insulin also demonstrated the apparent insulin-potentiated subunit reduction without any increase in the total amount of [3H]NEM labeling.(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigation of the redox centres of periplasmic selenate reductase from Thauera selenatis by EPR spectroscopy

Periplasmic SER (selenate reductase) from Thauera selenatis is classified as a member of the Tat (twin-arginine translocase)-translocated (Type II) molybdoenzymes and comprises three subunits each containing redox cofactors. Variable-temperature X-band EPR spectra of the purified SER complex showed features attributable to centres [3Fe-4S]1+, [4Fe-4S]1+, Mo(V) and haem-b. EPR-monitored redox-potentiometric titration of the SerABC complex (SerA-SerB-SerC, a hetero-trimetric complex of alphabetagamma subunits) revealed that the [3Fe-4S] cluster (FS4, iron-sulfur cluster 4) titrated as n=1 Nernstian component with a midpoint redox potential (E(m)) of +118+/-10 mV for the [3Fe-4S]1+/0 couple. A [4Fe-4S]1+ cluster EPR signal developed over a range of potentials between 300 and -200 mV and was best fitted to two sequential Nernstian n=1 curves with midpoint redox potentials of +183+/-10 mV (FS1) and -51+/-10 mV (FS3) for the two [4Fe-4S]1+/2+ cluster couples. Upon further reduction, the observed signal intensity of the [4Fe-4S]1+ cluster decreases. This change in intensity can again be fitted to an n=1 Nernstian component with a midpoint potential (E(m)) of about -356 mV (FS2). It is considered likely that, at low redox potential (E(m) less than -300 mV), the remaining oxidized cluster is reduced (spin S=1/2) and strongly spin-couples to a neighbouring [4Fe-4S]1+ cluster rendering both centres EPR-silent. The involvement of both [3Fe-4S] and [4Fe-4S] clusters in electron transfer to the active site of the periplasmic SER was demonstrated by the re-oxidation of the clusters under anaerobic selenate turnover conditions. Attempts to detect a high-spin [4Fe-4S] cluster (FS0) in SerA at low temperature (5 K) and high power (100 mW) were unsuccessful. The Mo(V) EPR recorded at 60 K, in samples poised at pH 6.0, displays principal g values of g3 approximately 1.999, g2 approximately 1.996 and g1 approximately 1.965 (g(av) 1.9867). The dominant features at g2 and g3 are not split, but hyperfine splitting is observed in the g1 region of the spectrum and can be best simulated as arising from a single proton with a coupling constant of A1 (1H)=1.014 mT. The presence of the haem-b moiety in SerC was demonstrated by the detection of a signal at g approximately 3.33 and is consistent with haem co-ordinated by methionine and lysine axial ligands. The combined evidence from EPR analysis and sequence alignments supports the assignment of the periplasmic SER as a member of the Type II molybdoenzymes and provides the first spectro-potentiometric insight into an enzyme that catalyses a key reductive reaction in the biogeochemical selenium cycle.     

The beta 2-microglobulin dissociation rate is an accurate measure of the stability of MHC class I heterotrimers and depends on which peptide is bound.

Stable, recombinant, water-soluble complexes of HLA-A2 and HLA-B27 were reconstituted from 125I-labeled beta 2-microglobulin (beta 2m), a synthetic peptide, and HLA H chain fragments expressed as inclusion bodies in the Escherichia coli cytoplasm. Using this system, we were able to show: 1) the t1/2 of beta 2m dissociation from HLA complexes at 37 degrees C varied from approximately 40 h to less than 1 h, depending on the peptide employed for reconstitution. Peptide length and composition were found to be critical factors in determining the beta 2m dissociation rate. Endogenous peptides form complexes that are about as stable as those formed with typical antigenic peptides. 2) Peptide exchange reactions, in which an exogenous peptide replaces the peptide that is already bound by the class I molecule, proceed readily for complexes that have rapid beta 2m dissociation rates. Thus, difficulties in demonstrating peptide binding to complexes that contain endogenous peptides can be attributed to the stability of the endogenous peptide/class I molecule complex. 3) The peptide exchange reaction does not require concomitant beta 2m dissociation. 4) Distal parts of the class I molecule, which are not directly involved in peptide binding or beta 2m binding, have a major impact on the stability of class I molecules. Thus, these studies show that the dissociation rate of beta 2m is an excellent measure of how tightly a given peptide binds to class I MHC molecules, that the ability to bind peptide is tightly coupled to the binding of beta 2m and vice versa, and that regions of the molecule distal from the binding site influence the stability of peptide binding.     

Lewis X-containing glycans are specific and potent competitive inhibitors of the binding of ZP3 to complementary sites on capacitated, acrosome-intact mouse sperm

Mammalian fertilization requires a cascade of interactions between sperm and the egg's zona pellucida (ZP). O-linked glycans on mouse glycoprotein ZP3 have been implicated in mediating one step of the fertilization process, the firm adhesion of acrosome-intact sperm to the ZP. Experiments to identify structural requirements of a sperm-binding glycan have demonstrated that a Lewis X (Le(x))-containing glycan (Gal beta 4[Fuc alpha 3]GlcNAc-R) was a potent, competitive inhibitor of in vitro sperm-ZP binding (Johnston et al. J Biol Chem 1998; 273: 1888-1895). However, those experiments did not define the particular step in the fertilization pathway that was blocked. The experiments described herein test the hypothesis that Le(x)-containing glycans are specific, competitive inhibitors of the binding of Alexa Fluor 568 fluorochrome (Alexa(568))-labeled ZP3 to sperm and, thus, bind the same sperm surface sites as ZP3. Dose-response analyses demonstrated that these glycans are potent inhibitors (IC(50) approximately 180 nM), which at saturation, reduced Alexa(568)-ZP3 binding by approximately 70%. A Lewis A (Le(a))-capped glycan (Gal beta 3[Fuc alpha 4]GlcNAc) was also a potent inhibitor (IC(50) approximately 150-200 nM), but at saturation, it reduced Alexa(568)-ZP3 binding by only 30%. In contrast, nonfucosylated glycans with nonreducing GlcNAc beta 4 or Gal beta 4 residues did not compete; neither did sialyl-Le(x) (Neu5Ac alpha 3Gal beta 4[Fuc alpha 3]GlcNAc-Lewis X) nor sulfo-Le(x) (3'-O-SO(3)-Lewis X). However, at saturation, Gal alpha 3Gal beta 4GlcNAc beta 3Gal beta 4Glc reduced Alexa(568)-ZP3 binding by approximately 70% but with moderate apparent affinity (IC(50) approximately 3000 nM). Fluorescence microscopy revealed that Alexa(568)-labeled Le(x)-Lac-BSA, Le(a)-Lac-BSA, and ZP3 bound to the same sperm surface domains. However, Le(a)-Lac did not inhibit binding of Alexa(568)-Le(x)-Lac-BSA, and Le(x)-Lac did not inhibit binding of Alexa(568)-Le(a)-Lac-BSA. Finally, Le(x)-Lac and Le(a)-Lac had an additive inhibitory effect on Alexa(568)-ZP3 binding. Thus, Le(x) is a ligand for a major class of ZP3 binding sites on mouse sperm, whereas Le(a) binding defines a different but less-abundant class of sites.     

Characterization of [11C]vorozole binding in ovarian tissue in rats throughout estrous cycle in association with conversion of androgens to estrogens in vivo and in vitro

Estrogen levels vary in a cyclic fashion during the rat estrous cycle, reaching peak concentrations during proestrus. Previously, it was suggested that the preovulatory peak in estrogen production in rats in vivo is regulated by other control mechanisms than concentration of precursor and amount of aromatase enzyme, changing the specific activity of the enzyme. To explore this hypothesis, ovarian binding of [11C]vorozole in vivo and in vitro, representing the amount of active aromatase, and conversion activity of ovarian homogenate were assayed together with serum androstenedione (A4) and estradiol-17beta (E2) levels during the estrous cycle in rats. The reducing ovarian [11C]vorozole binding in vivo from proestrus +4 up to +8h might indicate that the ovarian aromatase is blocked, probably to prevent premature increase of E2 levels. Thereafter (between proestrus +9 and +13h), the binding dramatically increases (aromatase enzyme is unblocked), to enable increased E2 synthesis. In addition, during the latter period, serum E2 levels were strongly correlated with serum A4 levels after adjustment for amount of ovarian aromatase (P=0.03), but not with amount of aromatase adjusted for levels of A4 (P=0.13), which might indicate changes in specific activity of the aromatase enzyme. Significant correlation between Kd and serum E2 levels during the same period indicated that aromatase-precursor affinity might be involved in the regulation of the enzyme-specific activity. This conclusion is done assuming that [11C]vorozole binding mimics that of the substrate (A4). The [11C]vorozole in vivo technique keeps auto- and paracrine mechanisms intact, and might therefore yield additional information about biological processes compared with traditional in vitro techniques.     

HLA expression in hemopoietic development. Class I and II antigens are induced in the definitive erythroid lineage and differentially modulated by fetal liver cytokines.

We investigated the expression of HLA Ag on hemopoietic progenitors (burst-forming unit E, CFU-E, and CFU-granulocyte-macrophage) and precursors from human embryonic fetal liver (FL) and peripheral blood at 5 to 9 wk postconception. The expression on progenitors was evaluated by complement-mediated cytotoxicity followed by assay of residual progenitors in clonogenic culture; immunofluorescence and RIA were used for differentiated precursors. HLA Class I and II Ag are not expressed on the primitive erythroid lineage, i.e., on yolk sac-derived megaloblasts circulating in 5- to 6-wk peripheral blood. However, they are gradually induced on the definitive erythroid lineage in FL. Their expression on progenitors is first detected at 6 wk and shows a progressive increase through 9 wk, up to greater than or equal to 50% of adult values. A similar expression pattern is observed for FL erythroblasts. Incubation of 6-wk FL erythroid cells with IFN-alpha, -beta, or -gamma, or TNF-alpha induces a sharp rise of the expression of HLA class I, but not HLA class II Ag on both progenitors and precursors. In contrast, incubation with PHA-stimulated adult leukocyte-conditioned medium causes a marked increase of both HLA-ABC and HLA class II Ag expression. We hence investigated the effect of cytokines present in leukocyte-conditioned medium on the expression of HLA class II Ag: although IL-1 alpha, IL-2, and granulocyte-macrophage-CSF do not exert a significant action, IL-1 beta and IL-4 induce a marked increase of HLA class II, but not class I, Ag expression on 6-wk FL progenitors and precursors. Low amounts of IFN-gamma, TNF-alpha, and IL-1 beta were detected in the supernatants and extracts of 6-wk FL cells. The concentrations of these cytokines in both supernatants and extracts sharply increase in the 7- to 8-wk period, particularly for TNF-alpha and IL-1 beta, thus indicating a direct correlation with the rise of HLA Ag expression on FL erythroid cells in the same period. In conclusion, the expression of HLA class I and II Ag is not detected on primitive megaloblasts, but is gradually induced on definitive FL progenitors and precursors, possibly via production of specific cytokines in the FL microenvironment, i.e., IFN-gamma and TNF-alpha for class I Ag and IL-1 beta for class II Ag.