Effects of fibrates on human organic anion-transporting polypeptide 1B1-, multidrug resistance protein 2- and P-glycoprotein-mediated transport.

The effects of different fibric acid derivatives (bezafibrate, clofibrate, clofibric acid, fenofibrate, fenofibric acid and gemfibrozil) on human organic anion transporting-polypeptide 1B1 (OATP2, OATP-C, SLC21A6), multidrug resistance protein 2 (MRP2/ABCC2) and MDR1-type P-glycoprotein (P-gp/ABCB1) were examined in vitro. Cyclosporin A (a known inhibitor of OATP1B1 and P-gp), MK-571 (a known inhibitor of MRP2) and cimetidine (an organic cation) were also tested. Bezafibrate, fenofibrate, fenofibric acid and gemfibrozil showed concentration-dependent inhibition of estradiol 17-beta-D-glucuronide uptake by OATP1B1-stably transfected HEK cells, whereas clofibrate and clofibric acid did not show any significant effects up to 100 microM. Inhibition kinetics of gemfibrozil, which exhibited the most significant inhibition on OATP1B1, was shown to be competitive with a Ki = 12.5 microM. None of the fibrates showed any significant inhibition of MRP2-mediated transport, which was evaluated by measuring the uptake of ethacrynic acid glutathione into MRP2-expressing Sf9 membrane vesicles. Only fenofibrate showed moderate P-gp inhibition as assessed by measuring cellular accumulation of vinblastine in a P-gp overexpressing cell-line. Cyclosporin A significantly inhibited OATP1B1 and P-gp, whereas only moderate inhibition was observed on MRP2. The rank order of inhibitory potency of MK-571 was determined as OATP1B1 (IC50: 0.3 microM) > MRP2 (4 microM) > P-gp (25 microM). Cimetidine did not show any effects on these transporters. In conclusion, neither MRP2- nor P-gp-mediated transport is inhibited significantly by the fibrates tested. Considering the plasma protein binding and IC50 values for OATP1B1, only gemfibrozil appeared to have a potential to cause drug-drug interactions by inhibiting OATP1B1 at clinically relevant concentrations.

Gene electrotransfer results in a high-level transduction of rat skeletal muscle and corrects anemia of renal failure.

We have investigated the efficacy of a gene transfer strategy based on plasmid DNA electroinjection for the correction of anemia associated with renal failure. An expression plasmid encoding the rat erythropoietin (EPO) cDNA under the control of the CMV promoter as constructed and utilized for this work. Electroinjection of pCMV/rEPO in different rat muscles yielded sustained and long-term EPO production and secretion. The muscle-produced EPO corrected the anemia in five of six nephrectomized rats, used as a model of renal failure. The efficiency of muscle transduction was comparable in rats and mice injected with equivalent amounts of DNA per kilogram of body weight. These results demonstrate that gene electrotransfer can be applied to produce therapeutically significant levels of erythropoietin in chronic renal failure.

Leptin receptor-mediated regulation of cholinergic neurotransmitter phenotype in cells of central nervous system origin.

Leptin is an adipocyte-secreted hormone that regulates body weight and exerts effects on hematopoiesis, reproduction, and immunity. The leptin receptor (OBR) shares sequence similarity and signaling capabilities with receptors for cytokines of the ciliary neurotrophic factor (CNTF) family. Our previous finding that CNTF and leptin exert similar anti-obesity effects and activate common neuronal signaling pathways, prompted us to investigate whether leptin may share with CNTF the ability to regulate the expression of specific neuronal genes. To this end, we established a cell line, derived from the murine septal cholinergic neuronal cell line SN-56, which stably expresses OBR. In this cell line, termed SN-56/OBR, leptin induces STAT transcription factor activation and STAT-dependent reporter gene expression in a manner similar to that of CNTF. Furthermore, in SN-56/OBR cells both CNTF and leptin produce changes in neurotransmitter and neuropeptide phenotype characteristic of cholinergic neurons, such as an increase in choline acetyltransferase and vasoactive intestinal polypeptide, and a decrease in neuropeptide Y expression. SN-56/OBR cells thus constitute an interesting new model system to investigate leptin action in cells of central nervous system origin. Possible physiological implications of OBR's intrinsic ability to regulate cholinergic phenotypic markers are discussed.

Agonistic and antagonistic variants of ciliary neurotrophic factor (CNTF) reveal functional differences between membrane-bound and soluble CNTF alpha-receptor.

Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR) and the signal-transducing beta-subunits gp130 and leukemia inhibitory factor receptor-beta (LIFR). CNTFR can function in either membrane-bound or soluble forms. The membrane-bound form mediates the neuronal actions of CNTF, whereas the soluble form serves to confer cytokine responsiveness to non-neuronal cells expressing gp130 and LIFR. The objective of this work was to analyze whether the two receptor isoforms differ in their ability to interact functionally with CNTF and related proteins. Two new types of CNTF variants, characterized by weakened interactions with either CNTFR or both LIFR and gp130, were developed, and the biological activities of these and other mutants were determined in non-neuronal versus neuronal cells, as well as in non-neuronal cells transfected with an expression vector for CNTFR. Membrane anchoring of CNTFR was found to render the CNTF receptor complex relatively insensitive to changes in agonist affinity for either alpha- or beta-receptor subunits and to promote a more efficient interaction with a gp130-depleting antagonistic variant of CNTF. As a result of this phenomenon, which can be rationalized in terms of the multivalent nature of CNTF receptor interaction, CNTF variants display striking changes in receptor selectivity.

Ciliary neurotrophic factor corrects obesity and diabetes associated with leptin deficiency and resistance.

Receptor subunits for the neurocytokine ciliary neurotrophic factor (CNTF) share sequence similarity with the receptor for leptin, an adipocyte-derived cytokine involved in body weight homeostasis. We report here that CNTF and leptin activate a similar pattern of STAT factors in neuronal cells, and that mRNAs for CNTF receptor subunits, similarly to the mRNA of leptin receptor, are localized in mouse hypothalamic nuclei involved in the regulation of energy balance. Systemic administration of CNTF or leptin led to rapid induction of the tis-11 primary response gene in the arcuate nucleus, suggesting that both cytokines can signal to hypothalamic satiety centers. Consistent with this idea, CNTF treatment of ob/ob mice, which lack functional leptin, was found to reduce the adiposity, hyperphagia, and hyperinsulinemia associated with leptin deficiency. Unlike leptin, CNTF also reduced obesity-related phenotypes in db/db mice, which lack functional leptin receptor, and in mice with diet-induced obesity, which are partially resistant to the actions of leptin. The identification of a cytokine-mediated anti-obesity mechanism that acts independently of the leptin system may help to develop strategies for the treatment of obesity associated with leptin resistance.

The receptor super-antagonist Sant7.

Interleukin-6 (IL-6) plays a central role in the pathogenesis of multiple myeloma, acting both as a growth and a survival factor for myeloma cells. IL-6 has been recently shown to possess three topologically distinct receptor binding sites: site 1 for binding to the subunit specific chain IL-6R alpha and sites 2 and 3 for the interaction with two separate subunits of the signalling chain gp130. We have generated a set of IL-6 receptor antagonists carrying substitutions that abolish interaction with gp130 at either site 2 alone (site 2 antagonist) or at both sites 2 and 3 (site 2+3 antagonist). In addition, substitutions were introduced at site 1 that increased affinity for IL-6R alpha. When tested as growth inhibitors on a representative set of IL-6-dependent human myeloma cell lines (XG-1, XG-2, XG-4 and XG-6), although site 2 antagonists were effective on 3 out of 4 of the cell lines, only the site 2+3 antagonist Sant7 showed full antagonism on the entire spectrum of cells tested. Moreover, IL-6 receptor antagonists were also pro-apoptotic factors for myeloma cells. Their capacity to induce cell death was directly related to the impairment of binding to gp130 and to their ability to fully block intracellular signalling. In fact, the most potent inducer of apoptosis was again Sant7, which also counteracted the protective autocrine effect excercised by the endogenously produced IL-6. On the basis of these results we propose the super-antagonist Sant7 as a possible candidate for the immunotherapy of multiple myeloma.

Interleukin 6 receptor superantagonists are potent inducers of human multiple myeloma cell death.

Interleukin-6 (IL-6) plays a central role in the pathogenesis of multiple myeloma, acting as both a growth and a survival factor for myeloma cells. A series of IL-6 receptor antagonists that are IL-6 variants has been recently obtained, the affinity of which for the ligand-specific receptor chain IL-6R alpha has been maintained or even increased, but the signaling of which is impaired by not being able to bind and/or dimerize the signaling chain gp130. Although IL-6 antagonists have been shown to inhibit the growth of IL-6-dependent myeloma, no information has been gathered on their ability to induce myeloma cell death. We show here that IL-6 receptor antagonists are pro-apoptotic factors for the IL-6-dependent human myeloma cell line XG-1. Their capacity to induce cell death is in direct relation to their affinity for IL-6R alpha, degree of gp130 binding impairment, and efficiency to inhibit intracellular signaling events. Interestingly, the most potent pro-apoptotic molecule, Sant7, counteracts the protective autocrine effect exercised by the limited amounts of IL-6 produced by XG-1 cells and is thus able to induce cell death at higher rate than just IL-6 deprivation. These findings are particularly relevant for the therapy of multiple myeloma.

Rational design of a receptor super-antagonist of human interleukin-6.

Interleukin-6 (IL-6) is a differentiation and growth factor for a variety of cell types and its excessive production plays a major role in the pathogenesis of multiple myeloma and post-menopausal osteoporosis. IL-6, a four-helix bundle cytokine, is believed to interact sequentially with two transmembrane receptors, the low-affinity IL-6 receptor (IL-6R alpha) and the signal transducer gp130, via distinct binding sites. In this paper we show that combined mutations in the predicted A and C helices, previously suggested to establish contacts with gp130, give rise to variants with no bioactivity but unimpaired binding to IL-6R alpha. These mutants behave as full and selective IL-6 receptor antagonists on a variety of human cell lines. Furthermore, a bifacial mutant was generated (called IL-6 super-antagonist) in which the antagonist mutations were combined with amino acid substitutions in the predicted D helix that increase binding for IL-6R alpha. The IL-6 super-antagonist has no bioactivity, but improved first receptor occupancy and, therefore, fully inhibits the wild-type cytokine at low dosage. The demonstration of functionally independent receptor binding sites on IL-6 suggests that it could be possible to design super-antagonists of other helical cytokines which drive the assembly of structurally related multisubunit receptor complexes.

Cloning and developmental expression of LFB3/HNF1 beta transcription factor in Xenopus laevis.

We have cloned the Xenopus laevis homologue of the LFB3/HNF1 beta transcription factor. RNase protection and in situ hybridisation experiments show that XLFB3 transcription starts in the gastrulating endoderm at stage 10.5 (mid-gastrula). At later stages, XLFB3 transcripts within the endoderm are restricted to mid- and hindgut and to their derivative organs and tissues. XLFB3 is also expressed in the neuroectoderm and in the pronephros anlage. XLFB3 is not expressed in the rostral part of all three germ layers, with coincident anterior borders that are shifted anteriorly by treatment of developing embryos with retinoic acid. XLFB3 is a useful marker of early endoderm differentiation and its expression pattern along the antero-posterior axis, as well as the response to retinoic acid treatment, suggests a role in early morphogenesis.

Ribosomal RNA genes of Phaseolus coccineus. I.

rDNA fragments, including the whole intergenic spacer (IGS) region of P. coccineus, were cloned into dephosphorylated pUC 13 plasmid. Four clones of different insert size were analysed. Restriction patterns and physical maps of these length variants (pPH1, pPH2, pPH5, pPH6) were performed through complete Eco RI cleavage and partial digestion with Hpa II, Hae III, Sau 3AI, Sma I. Evidence was obtained that the length heterogeneity of the four genes was mainly due to a differing number of about 170 bp sub-repeating elements in the IGS. Indeed, there are 16 of these in pPH1, about 34 in pPH2, 10 in pPH5 and about 60 in pPH6. The sequence analysis of pPH6 sub-repeats revealed that there are two types of sub-repeats: short ones (S) of 162 bp and long ones (L) of 176 bp. The homology between S and L is high (93.8%). S and L elements are present in at least three of the four genes investigated, as shown by a restriction pattern obtained with Hae III digestion to completion. The relative frequency of S and L types, however, differs among the four genes. The possible functional meaning of the sub-repeat structure is discussed on the basis of the homology between the S and L sequences on the one hand and on the other the ribosomal sequences of: i) Xenopus promoter(s); ii) wheat block A sub-repeats; iii) presumptive promoter(s) of wheat.

A centromeric satellite DNA in the European plethodontid salamanders (Amphibia, Urodela).

A highly repeated satellite DNA (Hy500) located in the centromeric heterochromatin of the European plethodontid salamander Speleomantes (formerly Hydromantes) was studied. The Hy500 family represents about 1% of the Speleomantes supramontis genome and has a major repeating unit of about 500 base pairs, which may have evolved from the progressive amplification of shorter sequences. This centromeric satellite is conserved in all the Speleomantes species, which nevertheless show distinct patterns of chromosomal distribution, which are of relevance as to their phylogenetic relationships.

Synergistic trans-activation of the human C-reactive protein promoter by transcription factor HNF-1 binding at two distinct sites.

The promoter region of the human C-reactive protein (CRP) gene comprises two distinct regions (APREs, for Acute Phase Responsive Elements) each one containing information necessary and sufficient for liver specific and IL-6 inducible expression in human hepatoma Hep3B cells. In this paper we show that both APREs contain a low affinity binding site for the liver specific transcription factor HNF-1/LF-B1. The two sites are separated by approximately 80 bp. Mutations in either of the two sites abolish inducible expression. The same effect is specifically obtained in cotransfection competition experiments when the human albumin HNF-1 site is used as competitor. However, HNF-1 is not the intranuclear mediator of IL-6 because synthetic promoters formed by multimerized copies of different HNF-1 binding sites are not transcriptionally activated by this cytokine. An expression vector encoding full length HNF-1 is capable of trans-activating transcription from the wild-type CRP promoter but not from mutants which have lost the ability to bind HNF-1. Moreover, the level of trans-activation observed with the natural promoter containing both HNF-1 binding sites is far greater than the level of mutated variants containing only one of the two sites. This result strongly suggests that two HNF-1 molecules bound simultaneously to sites distant from each other can act synergistically to activate gene expression.