Interleukin-1 receptor antagonist gene (IL1RN) polymorphism possibly associated to severity of rheumatic carditis in a Brazilian cohort

Aims: To evaluate the IL1RN polymorphism as a possible marker for Rheumatic Fever (RF) susceptibility or disease severity. Methods: The genotypes of 84 RF patients (Jones criteria) and 84 normal race-matched controls were determined through the analysis of the number of 86-bp tandem repeats in the second intron of IL1RN. The DNA was extracted from peripheral-blood leukocytes and amplified with specific primers. Clinical manifestations of RF were obtained through a standardized questionnaire and an extensive chart review. Carditis was defined as new onset cardiac murmur that was perceived by a trained physician with corresponding valvae regurgitation or stenosis on echocardiogram. Carditis was classified as severe in the presence of congestive heart failure or upon the indication for cardiac surgery. The statistical association among the genotypes, RF and its clinical variations was determined. Results: The presence of allele I and the genotype A1/A1 were found less frequently among patients with severe carditis when compared to patients without this manifestation (OR = 0.11, p = 0.031; OR = 0.092, p = 0.017). Neither allele I nor allele 2 were associated with the presence of RF (p = 0.188 and p = 0.106), overall carditis (p = 0.578 and p = 0.767), polyarthritis (p = 0.343 and p = 0.313) and chorea (p = 0.654 and p = 0.633). Conclusion: In the Brazilian population, the polymorphism of the IL-1ra gene is a relevant factor for rheumatic heart disease severity. (C) 2009 Elsevier Ltd. All rights reserved.

Comparison of the effects of tramadol, codeine, and ketoprofen alone or in combination on postoperative pain and on concentrations of blood glucose, serum cortisol, and serum interleukin-6 in dogs undergoing maxillectomy or mandibulectomy

Objective-To compare analgesic effects of tramadol, codeine, and ketoprofen administered alone and in combination and their effects on concentrations of blood glucose, serum cortisol, and serum interleukin (IL)-6 in dogs undergoing maxillectomy or mandibulectomy. Animals-42 dogs with oral neoplasms. Procedures-30 minutes before the end of surgery, dogs received SC injections of tramadol (2 mg/kg), codeine (2 mg/kg), ketoprofen (2 mg/kg), tramadol + ketoprofen, or codeine + ketoprofen (at the aforementioned dosages). Physiologic variables, analgesia, and sedation were measured before (baseline) and 1, 2, 3, 4, 5, and 24 hours after surgery. Blood glucose, serum cortisol, and serum IL-6 concentrations were measured 1, 3, 5, and 24 hours after administration of analgesics. Results-All treatments provided adequate postoperative analgesia. Significant increases in mean +/- SD blood glucose concentrations were detected in dogs receiving tramadol (96 +/- 14 mg/dL), codeine (120 +/- 66 mg/dL and 96 +/- 21 mg/dL), ketoprofen (105 +/- 22 mg/dL), and codeine + ketoprofen (104 +/- 16 mg/dL) at 5, 1 and 3, 5, and 3 hours after analgesic administration, respectively, compared with preoperative (baseline) values. There were no significant changes in physiologic variables, serum IL-6 concentrations, or serum cortisol concentrations. Dogs administered codeine + ketoprofen had light but significant sedation at 4, 5, and 24 hours. Conclusions and Clinical Relevance-Opioids alone or in combination with an NSAID promoted analgesia without adverse effects during the 24-hour postoperative period in dogs undergoing maxillectomy or mandibulectomy for removal of oral neoplasms. (Am J Vet Res 2010;71:1019-1026)

Effects of estrogen receptor alpha and beta gene deletion on estrogenic induction of progesterone receptors in the locus coeruleus in female mice

Locus coeruleus (LC) is involved in the LHRH regulation by gonadal steroids. We investigated the expression of progesterone and estrogen receptors (PR; ER) in LC neurons of ER alpha (alpha ERKO) or ER beta (beta ERKO) knockout mice, and their wild-type (alpha WT and beta WT). Immunocytochemical studies showed that LC expresses PR and both ERs, although ER beta was more abundant. Estradiol benzoate (EB) decreased ER alpha-positive cells in WT and beta ERKO mice, and progesterone caused a further reduction, whereas none of the steroids influenced ER beta expression. ER beta deletion increased ER alpha while ER alpha deletion did not alter ER beta expression. In both WT mice, EB increased PR expression, which was diminished by progesterone. These steroid effects were also observed in alpha ERKO animals but to a lesser extent, suggesting that ER alpha is partially responsible for the estrogenic induction of PR in LC. Steroid effects on PR in beta ERKO mice were similar to those in the alpha ERKO but to a lesser extent, probably because PR expression was already high in the oil-treated group. This expression seems to be specific of LC neurons, since it was not observed in other areas studied, the preoptic area and ventromedial nucleus of hypothalamus. These findings show that LC in mice expresses alpha ER, beta ER, and PR, and that a balance between them may be critical for the physiological control of reproductive function.

GABA(A) receptor alpha-subunit proteins in human chronic alcoholics

Antibodies were raised against specific peptides from N-terminal regions of the alpha (1) and alpha (3) isoforms of the GABA(A) receptor, and used to assess the relative expression of these proteins in the superior frontal and primary motor cortices of 10 control, nine uncomplicated alcoholic and six cirrhotic alcoholic cases were matched for age and post-mortem delay. The regression of expression on post-mortem delay was not statistically significant for either isoform in either region. In both cortical areas, the regression of a, expression on age differed significantly between alcoholic cases, which showed a decrease, and normal controls, which did not. Age had no effect on alpha (3) expression. The alpha (1) and alpha (3) isoforms were found to be expressed differentially across cortical regions and showed a tendency to be expressed differentially across case groups. In cirrhotic alcoholics, alpha (1) expression was greater in superior frontal than in motor cortex, whereas this regional difference was not significant in controls or uncomplicated alcoholics. In uncomplicated alcoholics, alpha (3) expression was significantly lower in superior frontal than in motor cortex. Expression of alpha (1) was significantly different from that Of alpha (3) in the superior frontal cortex of alcoholics, but not in controls. In motor cortex, there were no significant differences in expression between the isoforms in any case group.

Peroxisome proliferator-activated receptor alpha in the human breast cancer cell lines MCF-7 and MDA-MB-231

Peroxisome proliferator-activated receptor (PPAR) alpha is a ligand-activated transcription factor that has been linked with rodent hepatocarcinogenesis. It has been suggested that PPARalpha mRNA expression levels are an important determinant of rodent hepatic tumorigenicity. Previous work in rat mammary gland epithelial cells showed significantly increased PPARalpha mRNA expression in carcinomas, suggesting the possible role of this isoform in rodent mammary gland carcinogenesis. In this study we sought to determine whether PPARalpha is expressed and dynamically regulated in human breast cancer MCF-7 and MDA-MB-231 cells. Having established the presence of PPARalpha in both cell types, we then examined the consequence of PPARa activation, by its ligands Wy-14,643 and clofibrate, on proliferation. With real-time reverse transcriptase-polymerase chain reaction, we showed that PPARalpha mRNA was dynamically regulated in MDA-MB-231 cells and that PPARalpha activation significantly increased proliferation of the cell line. In contrast, PPARalpha expression in MCF-7 cells did not change with proliferation during culture and was present at significantly lower levels than in MDA-MB-231 cells. However, PPARalpha ligand activation still significantly increased the proliferation of MCF-7 cells. The promotion of proliferation in breast cancer cell lines following PPARalpha activation was in stark contrast to the effects of PPARgamma-activating ligands that decrease proliferation in human breast cancer cells. our results established the presence of PPARalpha in human breast cancer cell lines and showed for the first time that activation of PPARalpha in human breast cancer cells promoted proliferation. Hence, this pathway may be significant in mammary gland tumorigenesis. (C) 2002 Wiley-Liss, Inc.

Characterization of the retinoid orphan-related receptor-alpha coactivator binding interface: A structural basis for ligand-independent transcription

The retinoid orphan-related receptor-alpha (RORalpha) is a member of the ROR subfamily of orphan receptors and acts as a constitutive activator of transcription in the absence of exogenous ligands. To understand the basis of this activity, we constructed a homology model of Rill using the closely related TRbeta as a template. Molecular modeling suggested that bulky hydrophobic side chains occupy the RORa ligand cavity leaving a small but distinct cavity that may be involved in receptor stabilization. This model was subject to docking simulation with a receptor-interacting peptide from the steroid receptor coactivator, GR-interacting protein-1, which delineated a coactivator binding surface consisting of the signature motif spanning helices 3-5 and helix 12 [activation function 2 (AF2)]. Probing this surface with scanning alanine mutagenesis showed structural and functional equivalence between homologous residues of RORalpha and TRbeta. This was surprising (given that Rill is a ligand-independent activator, whereas TRbeta has an absolute requirement for ligand) and prompted us to use molecular modeling to identify differences between Rill and TRbeta in the way that the All helix interacts with the rest of the receptor. Modeling highlighted a nonconserved amino acid in helix 11 of RORa (Phe491) and a short-length of 3.10 helix at the N terminus of AF2 which we suggest i) ensures that AF2 is locked permanently in the holoconformation described for other liganded receptors and thus 2) enables ligand-independent recruitment of coactivators. Consistent with this, mutation of RORa Phe491 to either methionine or alanine (methionine is the homologous residue in TRbeta), reduced and ablated transcriptional activation and recruitment of coactivators, respectively. Furthermore, we were able to reconstitute transcriptional activity for both a deletion mutant of Ill lacking All and Phe491 Met, by overexpression of a GAL-AF2 fusion protein, demonstrating ligand-independent recruitment of AF2 and a role for Phe491 in recruiting AF2.

Abnormal spontaneous interleukin 8 receptor expression: a brief report of two cases

Interleukin 8 (CXCL8) is an autocrine chemokine specific for the chemoattraction and activation of granulocytes, NKT cells and T lymphocytes. Patients with tuberculosis and latent Mycobacterium tuberculosis infection were assessed for the spontaneous expression of CXCR1 (CD128) and CXCR2 on lymphocytes and monocytes. Compared with ex vivo profiles, increased spontaneous CXCR2 expression and normal CXCR1 expression were found on lymphocytes in two out of 59 individuals. Monocytes showed normal ex vivo profiles for both receptors. After stimulation with purified protein derivative, the in vitro levels of CXCL8 were below the median levels of all patients with prior tuberculosis. Spontaneous CXCR2 modulation did not cause notable variation in the in vitro levels of CXCL8.

Structure-function relationships for the interleukin 2 receptor system

Receptors for interleukin 2 (IL-2) esit in at least three forms which differ in their subunit compositio, their affinity for ligand and their ability to mediate a cellular reponse. Type I receptors occur following cellular acitivation and consist of the 55,000 m. w. glycoprotein Tac. These receptors bind IL-2 with a low affinity, do not internalize ligand and have not been definitively associated with any response. Type II receptors, on the other hand, conssit of one or more glycoproteins of 70,000 m. w. which have been termed "beta ([beta]) chains." They bind IL-2 with an intermediate affinity and rapidly internalize the ligand. [Beta] proteins mediate many cellular IL-2-dependent reponses, including the short-term activation of natural killer cells and the induction of Tac protein expression. Type III receptors consist of a ternary complex of the Tac protein, the [beta] chain(s) and IL-2. They are characterized by a paricularly high affinity for ligand association. Type III receptors also internalize ligand and mediate IL-2-dependent responses at low factor concentrations. The identification of two independent IL-2-binding molecules, Tac and [beta], thus provides the elusive molecular explanation for the differences in IL-2 receptor affinity and suggests the potential for selective therapeutic manipulation of IL-2 reponses.

Promoter rearrangements cause species-specific hepatic regulation of the glyoxylate reductase/hydroxypyruvate reductase gene by the peroxisome proliferator-activated receptor alpha.

In liver, the glyoxylate cycle contributes to two metabolic functions, urea and glucose synthesis. One of the key enzymes in this pathway is glyoxylate reductase/hydroxypyruvate reductase (GRHPR) whose dysfunction in human causes primary hyperoxaluria type 2, a disease resulting in oxalate accumulation and formation of kidney stones. In this study, we provide evidence for a transcriptional regulation by the peroxisome proliferator-activated receptor alpha (PPARalpha) of the mouse GRHPR gene in liver. Mice fed with a PPARalpha ligand or in which PPARalpha activity is enhanced by fasting increase their GRHPR gene expression via a peroxisome proliferator response element located in the promoter region of the gene. Consistent with these observations, mice deficient in PPARalpha present higher plasma levels of oxalate in comparison with their wild type counterparts. As expected, the administration of a PPARalpha ligand (Wy-14,643) reduces the plasma oxalate levels. Surprisingly, this effect is also observed in null mice, suggesting a PPARalpha-independent action of the compound. Despite a high degree of similarity between the transcribed region of the human and mouse GRHPR gene, the human promoter has been dramatically reorganized, which has resulted in a loss of PPARalpha regulation. Overall, these data indicate a species-specific regulation by PPARalpha of GRHPR, a key gene of the glyoxylate cycle.

Multimeric complexes of the PML-retinoic acid receptor alpha fusion protein in acute promyelocytic leukemia cells and interference with retinoid and peroxisome-proliferator signaling pathways.

The t(15;17) chromosomal translocation, specific for acute promyelocytic leukemia (APL), fuses the PML gene to the retinoic acid receptor alpha (RAR alpha) gene, resulting in expression of a PML-RAR alpha hybrid protein. In this report, we analyzed the nature of PML-RAR alpha-containing complexes in nuclear protein extracts of t(15;17)-positive cells. We show that endogenous PML-RAR alpha can bind to DNA as a homodimer, in contrast to RAR alpha that requires the retinoid X receptor (RXR) dimerization partner. In addition, these cells contain oligomeric complexes of PML-RAR alpha and endogenous RXR. Treatment with retinoic acid results in a decrease of PML-RAR alpha protein levels and, as a consequence, of DNA binding by the different complexes. Using responsive elements from various hormone signaling pathways, we show that PML-RAR alpha homodimers have altered DNA-binding characteristics when compared to RAR alpha-RXR alpha heterodimers. In transfected Drosophila SL-3 cells that are devoid of endogenous retinoid receptors PML-RAR alpha inhibits transactivation by RAR alpha-RXR alpha heterodimers in a dominant fashion. In addition, we show that both normal retinoid receptors and the PML-RAR alpha hybrid bind and activate the peroxisome proliferator-activated receptor responsive element from the Acyl-CoA oxidase gene, indicating that retinoids and peroxisome proliferator receptors may share common target genes. These properties of PML-RAR alpha may contribute to the transformed phenotype of APL cells.

Differential roles of T cell receptor alpha and beta chains in ligand binding among H-2Kd-restricted cytolytic T lymphocyte clones specific for a photoreactive Plasmodium berghei circumsporozoite peptide derivative.

To study the interaction of T cell receptor with its ligand, a complex of a major histocompatibility complex molecule and a peptide, we derived H-2Kd-restricted cytolytic T lymphocyte clones from mice immunized with a Plasmodium berghei circumsporozoite peptide (PbCS) 252-260 (SYIPSAEKI) derivative containing photoreactive Nepsilon-[4-azidobenzoyl] lysine in place of Pro-255. This residue and Lys-259 were essential parts of the epitope recognized by these clones. Most of the clones expressed BV1S1A1 encoded beta chains along with specific complementary determining region (CDR) 3beta regions but diverse alpha chain sequences. Surprisingly, all T cell receptors were preferentially photoaffinity labeled on the alpha chain. For a representative T cell receptor, the photoaffinity labeled site was located in the Valpha C-strand. Computer modeling suggested the presence of a hydrophobic pocket, which is formed by parts of the Valpha/Jalpha C-, F-, and G-strands and adjacent CDR3alpha residues and structured to be able to avidly bind the photoreactive ligand side chain. We previously found that a T cell receptor specific for a PbCS peptide derivative containing this photoreactive side chain in position 259 similarly used a hydrophobic pocket located between the junctional CDR3 loops. We propose that this nonpolar domain in these locations allow T cell receptors to avidly and specifically bind epitopes containing non-peptidic side chains.

Malignant transformation of DMBA/TPA-induced papillomas and nevi in the skin of mice selectively lacking retinoid-X-receptor alpha in epidermal keratinocytes.

Retinoid-X-receptor alpha (RXRalpha), a member of the nuclear receptor (NR) superfamily, is a ligand-dependent transcriptional regulatory factor. It plays a crucial role in NR signalling through heterodimerization with some 15 NRs. We investigated the role of RXRalpha and its partners on mouse skin tumor formation and malignant progression upon topical DMBA/TPA treatment. In mutants selectively ablated for RXRalpha in keratinocytes, epidermal tumors increased in size and number, and frequently progressed to carcinomas. As keratinocyte-selective peroxisome proliferator-activated receptor gamma (PPARgamma) ablation had similar effects, RXRalpha/PPARgamma heterodimers most probably mediate epidermal tumor suppression. Keratinocyte-selective RXRalpha-null and vitamin-D-receptor null mice also exhibited more numerous dermal melanocytic growths (nevi) than control mice, but only nevi from RXRalpha mutant mice progressed to invasive human-melanoma-like tumors. Distinct RXRalpha-mediated molecular events appear therefore to be involved, in keratinocytes, in cell-autonomous suppression of epidermal tumorigenesis and malignant progression, and in non-cell-autonomous suppression of nevi formation and progression. Our study emphasizes the crucial role of keratinocytes in chemically induced epidermal and melanocytic tumorigenesis, and raises the possibility that they could play a similar role in UV-induced tumorigenesis, notably in nevi formation and progression to melanoma.

Proline- and acidic amino acid-rich basic leucine zipper proteins modulate peroxisome proliferator-activated receptor alpha (PPAR alpha) activity.

In mammals, many aspects of metabolism are under circadian control. At least in part, this regulation is achieved by core-clock or clock-controlled transcription factors whose abundance and/or activity oscillate during the day. The clock-controlled proline- and acidic amino acid-rich domain basic leucine zipper proteins D-site-binding protein, thyrotroph embryonic factor, and hepatic leukemia factor have previously been shown to participate in the circadian control of xenobiotic detoxification in liver and other peripheral organs. Here we present genetic and biochemical evidence that the three proline- and acidic amino acid-rich basic leucine zipper proteins also play a key role in circadian lipid metabolism by influencing the rhythmic expression and activity of the nuclear receptor peroxisome proliferator-activated receptor α (PPARα). Our results suggest that, in liver, D-site-binding protein, hepatic leukemia factor, and thyrotroph embryonic factor contribute to the circadian transcription of genes specifying acyl-CoA thioesterases, leading to a cyclic release of fatty acids from thioesters. In turn, the fatty acids act as ligands for PPARα, and the activated PPARα receptor then stimulates the transcription of genes encoding proteins involved in the uptake and/or metabolism of lipids, cholesterol, and glucose metabolism.

A-kinase-anchoring protein-Lbc anchors IκB kinase ^6; to support interleukin-6-mediated cardiomyocyte hypertrophy.

In response to stress, the heart undergoes a pathological remodeling process associated with hypertrophy and the reexpression of a fetal gene program that ultimately causes cardiac dysfunction and heart failure. In this study, we show that A-kinase-anchoring protein (AKAP)-Lbc and the inhibitor of NF-κB kinase subunit ^6; (IKK^6;) form a transduction complex in cardiomyocytes that controls the production of proinflammatory cytokines mediating cardiomyocyte hypertrophy. In particular, we can show that activation of IKK^6; within the AKAP-Lbc complex promotes NF-κB-dependent production of interleukin-6 (IL-6), which in turn enhances fetal gene expression and cardiomyocyte growth. These findings provide a new mechanistic hypothesis explaining how hypertrophic signals are coordinated and conveyed to interleukin-mediated transcriptional reprogramming events in cardiomyocytes.

Regulation of the peroxisome proliferator-activated receptor alpha gene by glucocorticoids.

This study demonstrates that the expression of the peroxisome proliferator-activated receptor alpha (PPAR alpha) is regulated by glucocorticoid hormones in hepatocytes. Hydrocortisone, dexamethasone, and triamcinolone stimulated PPAR alpha mRNA synthesis in a dose-dependent manner in primary rat hepatocyte cultures. This glucocorticoid stimulation was inhibited by RU 486, a specific glucocorticoid antagonist. Moreover, in contrast to glucocorticoid hormones, the mineralocorticoid aldosterone had only a weak effect, suggesting that the hormonal stimulation of PPAR alpha was mediated by the glucocorticoid receptor. The induction was not prevented by cycloheximide treatment of the hepatocytes, indicating that it was mediated by preexisting glucocorticoid receptor. Finally, the RNA synthesis inhibitor actinomycin D abolished the stimulatory effect of dexamethasone, and nuclear run-on analysis showed an increase of PPAR alpha transcripts after hormonal induction. Thus, the PPAR alpha gene is an early response gene of glucocorticoids that control its expression at the transcriptional level.