Ability Of Retinoic Acid Research Articles

Protein kinase A activation by retinoic acid in the nuclei of HL60 cells

BackgroundActivation of protein kinase A (PKA) occurs during retinoic acid (RA)-induced granulocytic differentiation of human promyelocytic leukemia HL60 cells. It is known that the RIIα regulatory subunit of PKA, is modified by RA (retinoylated) in the early stages of differentiation. We have investigated the effects of RA on PKA during cell differentiation in order to understand the potential significance of this process in the retinoylation of RIIα subunits. MethodsImmunoblotting, immunoprecipitation, confocal microscopy, PCR, and PKA activity assays were employed for characterizing the effects of RA on PKA. ResultsWe found that RA induces intracellular mobility of RIIα and the activation of PKA in HL60 cells. Increases in RIIα levels were observed in RA-treated HL60 cells. RA treatment altered intracellular localization of the PKA subunits, RIIα and Cα, and increased their protein levels in the nuclei as detected by both immunoblotting and immunostaining analyses. Coincident with the increase in nuclear Cα, RA-treated HL60 cells showed increases in both the protein phosphorylation activity of PKA and the levels of phosphorylated proteins in nuclear fractions as compared to control cells. In addition, RIIα protein was stabilized in RA-treated HL60 cells as compared to control cells. ConclusionsThese results suggest that RA stabilizes RIIα protein and activates PKA in the nucleus, with a resultant increase in the phosphorylation of nuclear proteins. General significanceOur evidence suggests that retinoylation of PKA might contribute to its stabilization and activation and that this could potentially participate in RA's ability to induce granulocytic differentiation of HL60 cells.

Abstract 422: Metabolic Regulation by miR-33 in Macrophages Controls Immune Effector Responses

Cellular metabolism is increasingly recognized to control immune cell fate and functions. MicroRNA-33 (miR-33) is a central regulator of cellular lipid metabolism that represses genes involved in cholesterol efflux and HDL biogenesis (Abca1, Abcg1) and fatty acid oxidation (Cpt1a, Crot, Ampk). Here we show that by altering the balance of aerobic glycolysis and mitochondrial oxidative phosphorylation, miR-33 inhibition instructs macrophage polarization to an M2 phenotype and shapes innate and adaptive immune responses. Targeted deletion of miR-33 in macrophages increases oxidative phosphorylation, enhances spare respiratory capacity, and induces the expression of genes that define M2 macrophage polarization (Arg1, Fizz1, Cd206, Ym1). Furthermore, inhibition of miR-33 in Abca1-/- macrophages showed that these changes are independent of effects on cholesterol efflux, but instead require miR-33 targeting of the energy sensor AMP-activated protein kinase (AMPK). Notably, inhibition of miR-33 markedly increased macrophage expression of the retinoic acid-producing enzyme Aldh1a2 and retinal dehydrogenase activity both in vitro and in vivo. Consistent with the ability of retinoic acid to foster inducible regulatory T cells, these macrophages had an enhanced capacity to induce FoxP3 expression in naïve CD4+ T cells. Finally, treatment of western diet-fed Ldlr-/- mice with miR-33 inhibitors for 8 weeks (conditions that do not alter HDL cholesterol levels) reduced atherosclerosis progression by 40%, and promoted the accumulation of M2 macrophages and FoxP3+ T regulatory cells in plaques. Collectively, these results identify a novel role for miR-33 in the regulation of macrophage inflammation and show that antagonism of miR-33 is atheroprotective, in part, by reducing plaque inflammation by promoting M2 macrophage polarization and regulatory T cell induction.

Retinoic acid restores adult hippocampal neurogenesis and reverses spatial memory deficit in vitamin A deprived rats.

A dysfunction of retinoid hippocampal signaling pathway has been involved in the appearance of affective and cognitive disorders. However, the underlying neurobiological mechanisms remain unknown. Hippocampal granule neurons are generated throughout life and are involved in emotion and memory. Here, we investigated the effects of vitamin A deficiency (VAD) on neurogenesis and memory and the ability of retinoic acid (RA) treatment to prevent VAD-induced impairments. Adult retinoid-deficient rats were generated by a vitamin A-free diet from weaning in order to allow a normal development. The effects of VAD and/or RA administration were examined on hippocampal neurogenesis, retinoid target genes such as neurotrophin receptors and spatial reference memory measured in the water maze. Long-term VAD decreased neurogenesis and led to memory deficits. More importantly, these effects were reversed by 4 weeks of RA treatment. These beneficial effects may be in part related to an up-regulation of retinoid-mediated molecular events, such as the expression of the neurotrophin receptor TrkA. We have demonstrated for the first time that the effect of vitamin A deficient diet on the level of hippoccampal neurogenesis is reversible and that RA treatment is important for the maintenance of the hippocampal plasticity and function.

Retinoic acid dampens LPS-induced NF-κB activity: results from human monoblasts and in vivo imaging of NF-κB reporter mice

Bacterial lipopolysaccharide (LPS) is a major inducer of systemic inflammatory reactions and oxidative stress in response to microbial infections and may cause sepsis. In the present study, we demonstrate that retinoic acid inhibits LPS-induced activation in transgenic reporter mice and human monoblasts through inhibition of nuclear factor κB (NF-κB). By using noninvasive molecular imaging of NF-κB luciferase reporter mice, we showed that administration of retinoic acid repressed LPS-induced whole-body luminescence, demonstrating in vivo the dynamics of retinoic acid's ability to repress physiologic response to LPS. Retinoic acid also inhibited LPS-induced NF-κB activity in the human myeloblastic cell line U937. Retinoic-acid-receptor-selective agonists mimicked — while specific antagonists inhibited — the effects of retinoic acid, suggesting the involvement of nuclear retinoic acid receptors. Retinoic acid also repressed LPS-induced transcription of NF-κB target genes such as IL-6, MCP-1 and COX-2. The effect of retinoic acid was dependent on new protein synthesis, was obstructed by a deacetylase inhibitor and was partly eliminated by a signal transducer and activator of transcription-1 (STAT1)/methyltransferase inhibitor, indicating that retinoic acid induces a new protein, possibly STAT1, that is involved in inhibiting NF-κB. This provides more evidence for retinoic acid's anti-inflammatory potential, which may have clinical implications in terms of fighting microbial infections.

Acute promyelocytic leukemia: New issues on pathogenesis and treatment response

Pathogenesis of acute promyelocytic leukemia appears to be one of the best understood among human malignancies. The ability of retinoic acid (RA) and arsenic trioxide to directly target the oncogenic promyelocytic leukemia-retinoic receptor A (PML-RARA) fusion protein also made this disease the first model for oncogene-targeted therapies. A set of recent data has significantly increased the complexity of our view of acute promyelocytic leukemia pathogenesis, as well as of therapeutic response. This review summarizes and discusses these findings, which yield novels questions and models.

Modulation of glutathione transferase P1-1 activity by retinoic acid in neuroblastoma cells.

The ability of retinoic acid to modulate glutathione S-transferase P1-1 (GSTP1-1) activity has important implications both for cancer prevention and for anticancer therapy. We investigated GSTP1-1 expression and activity in the human neuroblastoma cell line SK-N-BE(2) (genotype A*/B*) under basal conditions and during 48-h incubation with 0.1 microM all-trans-retinoic acid. The steady-state levels of glutathione transferase P1-1 mRNA and protein during 48-h incubation with all-trans-retinoic acid did not increase substantially, but we detected a significant reduction of GSTP1-1 specific activity. This reduction in enzymatic activity could not be ascribed to a differential action of retinoic acid on the gene variants A* and B*; indeed, the two GSTP1-1 isoforms have different affinities toward 1-chloro-2,4-dinitrobenzene (CDNB), while we found a substantial invariance of the K(m) (CDNB) in the cytosol during retinoid treatment. A modulatory effect of retinoic acid on other enzymes involved in glutathione transferase P1-1 metabolism, such as the retinoic acid-induced tissue trans-glutaminase, might be hypothesized, as well as a direct inactivation of GSTP1-1 by the oxidative stress that characterizes the early phases of apoptosis.

Effect of trans-retinoic acid and folic acid on apoptosis in human gastric cancer cell lines MKN-45 and MKN-28.

Induction of apoptosis has been implicated as an anticarcinogenic mechanism of both folic acid and retinoic acid. The ability of retinoic acid or folic acid to induce gastric cancer cell apoptosis was investigated in the human gastric cancer cell lines MKN-45 and MKN-28, and DNA fragmentation was studied in situ by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and DNA agarose gel electrophoresis. The rates of apoptosis in both the poorly differentiated MKN-45 and the well differentiated MKN-28 cell line were less than 5% after treatment with either retinoic or folic acid. Apoptosis may be induced by the administration of retinoic acid or folic acid, and the apoptosis indices of MKN-45 and MKN-28 cells were related to the doses of these drugs. The induction of gastric cancer cell apoptosis may play a role in the anticarcinogenic effect of retinoic acid and folic acid, both of which are potential agents for the treatment of human gastric cancer.

Induction of trk receptors by retinoic acid in a human embryonal carcinoma cell line.

Retinoic acid induced the differentiation of human embryonal carcinoma cells (NT2/D1) into several morphologically distinct cell types, including those resembling terminally differentiated postmitotic CNS neurones. The mechanism by which retinoic acid influences the process of neuronal differentiation in the CNS, however, remains unknown. In the present study, we have examined the ability of retinoic acid to induce the expression of the receptors that mediate the actions of the neurotrophins, using reverse transcription-polymerase chain reaction and Northern blot analysis. Our study demonstrated that the expression of mRNAs for three human trk receptors was significantly induced after treatment with retinoic acid. These findings suggest that the actions of retinoic acid on neuronal differentiation in the human CNS may potentially be mediated by the neurotrophins.

Potentiation of retinoic acid-induced U-937 differentiation into respiratory burst-competent cells by nitric oxide donors

All- trans retinoic acid (tretinoin) is a known inducer of differentiation of the human monoblastic cell line, U-937. We now report that the ability of retinoic acid (RA) to induce differentiation of U-937 cells into cells possessing respiratory burst activity is enhanced by the known nitric oxide-donating drugs glyceryl trinitrate, molsidomine and CAS 936, and by tetranitromethane in combination with cysteine. RA alone was a strong inducer of U-937 differentiation as indicated by the following responses to 12- O-tetradecanoylphorbol-13-acetate (TPA) stimulation: (1) increase in the percentage of cells staining with nitroblue tetrazolium (NBT); (2) increase in the total amount of formazan (the product of NBT reduction by O 2 −) as determined spectrophotometrically; (3) increase in hexose monophosphate shunt (HMPS) activity as assessed by [ 14C]CO 2 released from d-[1- 14C]glucose. RA was also able to increase mRNA levels for two respiratory burst-related genes and for glucose-6-phosphate dehydrogenase (G6PD), an HMPS enzyme. Other indications of differentiation were reduced cell proliferation, increased adherence and altered nuclear morphology. The observed increase in formazan production and HMPS activity and the reduction of cell proliferation due to RA were augmented by co-treatment with either glyceryl trinitrate, molsidomine, CAS 936 or tetranitromethane plus cysteine. Glyceryl trinitrate alone increased HMPS activity and G6PD mRNA levels and also reduced cell proliferation. Glyceryl trinitrate, molsidomine and CAS 936 are presumed to release nitric oxide and increase intracellular cGMP levels by stimulation of soluble guanylate cyclase. The mechanism of action of tetranitromethane is less certain, although it may also generate reactive nitrogen intermediates. These data suggest that a NO·/ cGMP pathway may augment a retinoic acid-mediated pathway to enhance maturation of U-937 cells with respect to the respiratory burst. Glyceryl trinitrate may act additionally by another pathway.

Cell-type specific interactions between retinoic acid and thyroid hormone in the regulation of expression of the gene encoding ornithine aminotransferase.

The purposes of this study were to determine whether expression of the gene encoding ornithine aminotransferase (OAT) in the rat liver and kidney is regulated by retinoic acid (RA) and to characterize further the role of thyroid hormone in regulating the expression of this gene. The level of OAT messenger RNA (mRNA) was reduced 70% in the liver of animals fed a vitamin A-deficient diet relative to that in animals fed a vitamin A-sufficient diet. RA, administered at a dose of 20 micrograms/rat to A-deficient rats for 1 or 3 days, restored OAT mRNA to near the level observed in animals fed the A-sufficient diet. Retinol was also effective in this regard. T3, when injected alone at a dose of 10 micrograms/100 g BW, had no effect on the level of OAT mRNA in the liver. However, when injected concurrently with RA, T3 blocked the ability of RA to induce OAT mRNA in the liver of rats fed the vitamin A-deficient diet. Animals made both vitamin A deficient and hypothyroid responded to RA in a manner similar to vitamin A-deficient animals. The vitamin A-deficient, hypothyroid rats responded somewhat differently to T3, however. T3 was unable to block the induction of OAT mRNA in the liver of vitamin A-deficient, hypothyroid rats when injected concurrently with RA for 1 day, but did block the induction of OAT mRNA by RA when these two hormones were injected concurrently for 3 days. These data indicate that RA and T3 exert opposing effects on the level of OAT mRNA in the liver. The effects of RA and T3 on OAT mRNA were markedly different in the kidney. Neither vitamin A deficiency nor RA had any apparent affect on the level of OAT mRNA in the kidney. T3, however, increased the level of OAT mRNA in the kidney of vitamin A-deficient rats. In the kidney of vitamin A-deficient, hypothyroid rats, T3 was unable to increase OAT mRNA when injected for 1 day, but did increase this mRNA when injected for 3 days. Together, these data indicate cell-type specific effects of both RA and T3 on the OAT gene.

Retinoic acid decreases attachment of JAR choriocarcinoma spheroids to a human endometrial cell monolayer in vitro

The ability of retinoic acid (RA) to modulate attachment of JAR choriocarcinoma multicellular spheroids to monolayers of a human uterine epithelial cell line (RL95-2) was examined using a centrifugal force-based adhesion assay. Exposure of choriocarcinoma spheroids to RA (10(-7) to 10(-5) M) over a 3-day culture period resulted in a dose-dependent decrease of attachment. Significant decreases in attachment were detected after 30 min (75 per cent versus 25 per cent) and 1 h (92 per cent versus 26 per cent) of confrontation-culture between choriocarcinoma and uterine cells for control versus 10(-5) M RA; by 5 h 100 per cent spheroid attachment was detected in all treatment groups. RA had no effect on cell proliferation in JAR spheroids, but 10(-5) M RA treatment induced a fivefold increase in secretion of human chorionic gonadotrophin (hCG), a known marker of conversion of cytotrophoblast to syncytiotrophoblast-like cells. These findings demonstrate that RA modulates cellular attachment and differentiation in choriocarcinoma spheroids in vitro.

Multiparametric Evaluation of Retinoic Acid-Induced Terminal Differentiation of Blastoid Cells from Acute Non-Lymphocytic Leukemia Patients in Vitro

Recent findings that retinoic acid (RA) induces terminal granulocytic differentiation of the human promyelocytic leukemia cell line HL-60 in vitro and blast cell maturation in patients suffering from acute non-lymphocytic leukemia (ANLL) prompted an investigation on the ability of this agent to induce terminal maturation in blast cells from ANLL patients in vitro. We tested the ability of RA at 3 x 10(-6) M, 3 x 10(-7) M and 3 x 10(8-) M concentrations to induce differentiation in blastoid cells from 16 patients with ANLL using cytochemical and cytologic parameters, in addition to cytofluorometric methods. Leukemic cells in primary culture from all the patients underwent cytochemical and biochemical changes after treatment with RA. However, the extent of differentiation-positive cell clones (D+ clones) varied from patient to patient. Morphologic maturation was observed in a significant number of bone marrow samples. Leukocyte alkaline phosphatase and NBT reduction ability of cells, which are biochemical markers of granulocytic differentiation, were also significantly increased with a simultaneous decrease in DNA and RNA synthesis (which was estimated using a Phywe ICP-11 impulse flow cytometer).

Actions and interactions of estradiol and retinoic acid in mouse anterior prostate gland.

Experiments were designed to define the ability of retinoic acid to block the estrogen-induced metaplasia in the mouse anterior prostate gland (coagulating gland), and to elucidate some of the biochemical correlates of the actions and interactions of these two compounds. In castrated mice, the estrogen-induced metaplasia of epithelial cells consisted of multi-layered, nonpolarized cells, which accumulated to fill the lumen of the acini. Retinoic acid had no discernable effect on epithelial morphology of castrates, but significantly reduced the estrogen-induced metaplasia. Likewise, the estrogen-induced increases in the prostatic wet weight, ratio of ribonucleic to deoxy ribonucleic acids (RNA/DNA) and glycosyltransferases, as well as the decreases in 21,000 and 28,000 Mr soluble proteins were prevented by retinoic acid; the retinoid had no effect on these various parameters when administered alone to castrates. In contrast, the cyclic 3',5'-adenosine monophosphate (cAMP)-dependent phosphorylations at 16,000, 18,000, and 25,000 Mr and the activities of the type II cAMP-dependent kinase were uniformly reduced by both estradiol and retinoic acid. Tests of the action of the anti-estrogen LY-156758 on estrogen and retinoid effects showed that for those parameters on which retinoic acid was anti-estrogenic, LY-156758 was also anti-estrogenic. However, the qualitatively similar effects of retinoic acid and estradiol, which were confined to selected aspects of protein phosphorylation, were not antagonized by LY-156758. It is concluded that in the mouse anterior prostate, retinoic acid has both anti-estrogenic and estrogenic actions, and the latter may occur independently of the estrogen receptor.

Effects of retinoic acid treatment on release of arachidonic acid by chondrogenic cells in response to ionophore A23187

Chondrogenic differentiation in mouse limb bud mesenchymal cells cultured at high density was suppressed by supplementation of the medium with retinoic acid in a dose-dependent fashion. Cells prelabeled with ( 3H) arachidonic acid were treated with 0.3 ug/ml retinoic acid. Treatment with retinoic acid increased the ( 3H) fatty acid in the triglyceride fraction. Furthermore, treatment with retinoic acid enhanced the release of ( 3H) fatty acid upon stimulation of these cells with the divalent ionophore A23187. These data permit the suggestion that there may be a correlation between altered lipid metabolism and retinoic acid's ability to disrupt chondrogenic differentiation.

Retinoic acid promotes phorbol ester-initiated macrophage differentiation in HL-60 leukemia cells without disappearance of protein kinase C

The ability of retinoic acid (RA) to promote 12- O-tetradecanoyl-phorbol-13-acetate (TPA)-initiated macrophage differentiation was examined in human promyelocytic leukemia cell line HL-60. One-hour exposure to 10 nM TPA and subsequent exposure for 48 h to 1 μM RA following removal of TPA rapidly induced the macrophage phenotype in 65% of the cells. This effect was comparable to continuous exposure for 48 h to TPA alone, but contrasted with the absence of macrophage-like cells after RA treatment alone or the induction of 10% of the cell population to a macrophage phenotype after 1-h exposure to TPA. The effect of TPA + RA was accompanied by increased cell adherence and increased nonspecific esterase activity but not by a change in the reduction of nitroblue tetrazolium. Protein kinase C (PK-C) activity was increased 35–40% in cells treated for 1 h with TPA alone or after subsequent exposure to RA. Cells treated for 48 h with RA exhibited a 2-fold increase in PK-C activity while cells exposed to TPA for 48 h lost all PK-C activity. The changes in PK-C activity in TPA + RA-treated cells were accompanied by Ca 2+/phospholipid(PL)-dependent phosphorylation in vitro of pp38 which is characteristic of treatment with RA alone, as well as the Ca 2+/PL-independent phosphorylation in vitro of pp82 and pp130 (vinculin) which is prevalent in cells treated continuously with TPA alone and is absent in RA-treated cells. These results indicate that the macrophage phenotype induced by TPA + RA is similar to that produced by continuous exposure to TPA alone with respect to their in vitro phosphoprotein patterns, cytochemical markers, cell adherence and morphology, but that the disappearance of PK-C is not an obligatory characteristic of these cells.

Regulation of acetylcholinesterase activity by retinoic acid in a human neuroblastoma cell line

The ability of retinoic acid (RA) to modulate acetylcholinesterase (AChE) activity in a human neuroblastoma cell line (LN-N-5) was examined. The specific activity of AChE was significantly increased 3 days after exposure of LA-N-5 to RA and reached its maximum values after 9 or more days of culturing. Dose-response experiments demonstrated that large increases of AChE occurred at RA concentrations between 10 −7 and 10 −6 M with maximum AChE values detected at 10 −6–10 −5 M. Increased AChE activity paralleled neurite outgrowth in LA-N-5 cultures. These findings demonstrate that RA can regulate specific AChE activity in human neuroblastoma cells in a manner consistent with neuronal maturation.

Increase in cell-surface N-acetylglucosaminide β(1→4)galactosyltransferase activity with retinoic acid-induced differentiation of F 9 embryonal carcinoma cells

Exposure of F 9 cells to all- trans-retinoic acid over a period of 6 days resulted in 4-fold induction of cell surface N-acetylglucosaminide β(1→4)galactosyltransferase (GT) activity. The retinoic acid-induced GT activity was further enhanced by treatment of the cells with 8-bromo cyclic AMP. The ability of retinoic acid alone, or retinoic acid in combination with 8-bromo cyclic AMP, to induce GT activity was inhibited by both actinomycin D and cycloheximide. These findings indicate that the induction of galactosyltransferase activity noted with differentiation of F 9 cells involves de novo synthesis of new enzyme protein.

Specific high-affinity binding and biologic action of retinoic acid in human neuroblastoma cell lines.

Neuroblastoma cells are a good model for neuronal development because of their ability to extend neurites in response to various stimuli, including retinoic acid. In the present experiments, we have examined five human neuroblastoma cell lines (LA-N-1, IMR-32, LA-N-5, SK-N-MC, and CHP-100) for the presence of cellular retinoic acid binding protein (CRABP), a receptor-like protein implicated in the molecular functioning of vitamin A. CRABP is identified and quantitated by sucrose gradient centrifugation, selective inhibition by the mercurial reagent p-chloromercuribenzene sulfonic acid (PCMBS), and saturation analysis. All five lines contain significant levels of cytosolic CRABP (2.5-7.5 pmol/mg of protein), which display typical properties of specific high affinity retinoic acid binding, a sedimentation coefficient of 2 S, and inhibition by PCMBS. Three of the lines (LA-N-1, IMR-32, and LA-N-5) are strongly growth inhibited by 1 microM retinoic acid in monolayer culture, whereas two (LA-N-1 and LA-N-5) undergo marked differentiation to a stellate, fusiform morphology with characteristic neurite outgrowths. The SK-N-MC and CHP-100 lines are relatively resistant to the antiproliferative effects of retinoic acid under these conditions. Nevertheless, all five lines are effectively inhibited by retinoic acid in their ability to form anchorage-independent colonies in soft agar. Thus, although CRABP is not necessarily correlated with growth inhibition in monolayer culture, it is associated with retinoic acid's ability to inhibit neuroblastoma colony formation in soft agar. More experiments will be required to determine if this effect on growth in soft agar reflects the putative ability of retinoic acid to convert tumorigenic neuroblastoma cell lines into the normal differentiated phenotype.

Retinoids and inhibition of ornithine decarboxylase activity.

The tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) causes a dose-dependent induction (up to 300-fold) of ornithine decarboxylase activity in the epidermis of intact mice within 5 hours after the application to the skin of from 1 to 10 nmoles of the ester in 0.2 ml of acetone. Enzyme activity regresses with a half-life of 17 minutes, the shortest known for any enzyme. The induction is inhibited in a dose-dependent manner by a single application of from 0.034 to 3.4 nmoles of retinoic acid within an hour before to an hour after phorbol ester treatment. Of over thirty natural and synthetic retinoids that were tested for this property, all-trans-retinoic acid was most effective. The ability of retinoic acid and its congeners to inhibit tumor promotion by the phorbol ester correlated with the effect of dose, structure, and time of treatment on the induction of ornithine decarboxylase. It appears that a permanently increased level of the enzyme and its product, putrescine, may be one of the essential components of the mechanism of tumor promotion.

Lack of enhancement of experimental photocarcinogenesis by topical retinoic acid.

A controversy exists regarding the ability of retinoic acid to enhance photocarcinogenesis. Divergent results have been obtained with albino hairless mice. We examined this issue with the lightly pigmented variety. We followed two designs: 1. ultraviolet light and topical retinoic acid were given concomitantly while the retinoic acid was continued for many weeks after stopping irradiation; 2. tumors were first induced by ultraviolet light and then treated topically with retinoic acid. In both studies, retinoic acid did not enhance photocarcinogenesis with regard to latent period, tumor yield or tumor progression. It appears that different treatment schedules and different varieties of mice can produce widely disparate results.