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Since 15-deoxy-delta(12,14)-prostaglandin J(2) (15dPGJ(2)) has been identified as an endogenous ligand of PPARgamma thus inducing adipogenesis, it has been reported to play active parts in numerous cellular regulatory mechanisms. As 15dPGJ(2) has been shown to covalently bind several peptides and proteins, we investigated whether it also covalently binds PPARgamma. We first observed that after incubation of 15dPGJ(2) with recombinant PPARgamma, the quantity of free 15dPGJ(2) measured was always lower than the initial amount. We then measured the ability of the labeled agonist rosiglitazone to displace the complex PPARgamma(2)/15dPGJ(2) obtained after pre-incubation. We observed that the binding of rosiglitazone was dependent on the initial concentration of 15dPGJ(2). Finally using MALDI-TOF mass spectrometry analysis, after trypsinolysis of an incubate of the PPARgamma(2) ligand binding domain (GST-LBD) with 15dPGJ2, we found a fragment (m/z = 1314.699) corresponding to the addition of 15dPGJ(2) (m/z = 316.203) to the GST-LBD peptide (m/z = 998.481). All these observations demonstrate the existence of a covalent binding of 15dPGJ(2) to PPARgamma, which opens up new perspectives to study the molecular basis for selective activities of PPARs.

Melanin-concentrating hormone (MCH) is a cyclic peptide, mainly involved in the regulation of skin pigmentation in teleosts and feeding behavior in mammals. The human keratinocyte SVK14 cell line has been previously shown to express binding sites for the MCH analog [125I]-[Phe13,3-iodo-Tyr19]MCH. We report here that: (1) this binding site similarly recognized [125I]-[3-iodo-Tyr13]MCH; (2) its pharmacological profile clearly differed from those observed at the two human MCH receptor subtypes, MCH1-R and MCH2-R; (3) MCH did not induce any effect on second messenger systems (including cAMP, calcium, and MAP kinase signaling pathways), and (4) no mRNAs corresponding to the MCH receptors were found. In conclusion, the binding site characterized in the SVK14 cell line is distinct from the MCH1 and MCH2 receptors and deserves therefore further investigation.

We report the characterization by genomics-based approach of the human H4-receptor gene structure. The H4-receptor gene have been mapped by radiation hybrid experiments (Gene Bridge 4) on chromosome 18q11.2, between the AFMBB11WH5 and CHLC.GATA85D10 markers. The H4-receptor gene spans more than 21 kbp and contains three exons separated by two large introns (>7 kbp). RT-PCR analysis showed that the H4-receptor gene encoded a 3.7 kb mRNA which did not seem to be alternatively spliced within its coding region. The H4-receptor transcripts were found to be highly expressed in peripheral tissues implicated in inflammatory responses such as leukocytes, spleen, lung, and liver. In addition, low expression level of the H4-receptor mRNA was also detected in several human brain regions. Analysis of the 5'-flanking region of the H4-receptor gene did not reveal the existence of canonical TATA or CAAT-box. However, several putative regulatory elements mediating TNFalpha or IL-6-stimulated transcriptional activation were detected. The uteroglobin promoter binding factor, known to mediate anti-inflammatory response of uteroglobin, in the lung, was also found in this region. Thus, the description of the H4-receptor gene promoter region will facilitate the elucidation of its transcriptional control by factors secreted during inflammatory responses.

Transcriptional mechanisms controlling human UCP3 gene expression in skeletal muscle remain poorly understood. Experiments based on plasmid electrotransfer into tibialis anterior muscle of C57/BL6 male mice were set up in order to functionally analyze the hUCP3 gene promoter. These transfection experiments showed that a 6300 bp region upstream of the transcription initiation site was sufficient to mediate maximal promoter activity. Further analyses with a series of 5(')-deleted constructs demonstrated that the hUCP3 gene minimal promoter was located between nucleotides -284 and -40. Furthermore, an essential region was identified between nucleotides -284 and -224. The analysis of this region revealed a putative response element for PPAR located between nucleotides -281 and -269. Finally, mutations of potential cis-acting elements within the hUCP3 minimal promoter showed the presence of two TATA boxes (-198/-194 and -45/-41) required for constitutive UCP3 gene expression. To our knowledge, this is the first time that molecular characterization of the UCP3 promoter has been achieved using an in vivo experimental model.

Uncoupling protein 3 (UCP3), a member of the UCP family, mainly expressed in skeletal muscle could be responsible for thermogenesis in humans. Since little is known about its regulation, we studied the 5' flanking region of the human UCP3 (hUCP3) gene, which potentially contains the promoter sequences. We report the hUCP3 transcription initiation on a G located 764 nucleotides upstream the A contained in the first translated codon. Therefore, hUCP3 first exon has 669 bases of untranslated sequence. We also report the cloning and sequencing of seven kilobases from the gene 5' end and analyze the features of the potential proximal promoter. The MyoD family binding motif, called E-box, is the most abundant on this region. Other muscle-specific motives present in the potential proximal promoter include a MEF2 site as well as binding sequences for ubiquitous factors such as GC box and two CAAT boxes. Additionally, three putative peroxisome proliferator and one thyroid hormone response elements (PPRE and TRE, respectively) are found, which suggest a potential role for the peroxisome proliferator-activated receptor (PPAR) and thyroid hormone in human UCP3 gene expression. The description of the promoter region of the UCP3 gene will facilitate the elucidation of its transcriptional control.

Rat liver microsomes contain 3α-hydroxysteroid dehydrogenase (HSD) (EC 1.1.1.50) and dihydrodiol dehydrogenase (DHD) (EC 1.3.1.20) activities. The two enzyme activities were solubilized by 10% Triton X-100 or 0.4% sodium deoxycholate. Unlike the cytosolic enzyme ( Penning & Talalay (1983) Proc. Natl. Acad. Sci. U.S.A. , 80 , 4505 ), the microsomal HSD and DHD activities were not inhibited by indomethacin. Chromatography of the microsomal Triton X-100 extract on Affigel Blue and then on Phenyl-Sepharose gave an HSD preparation containing no detectable (less than 3 – 5%) DHD activity, whereas chromatography of the deoxycholate extract on Phenyl-Sepharose provided a DHD preparation that lacked measurable HSD activity. These results are in sharp contrast to the cytosolic enzyme where both HSD and DHD activities could be copurified to homogeneity (Penning et al . (1984) Biochem. J. 222 , 601).

A major peak of tyrosine protein kinase activity was partially purified from a Triton X100 extract of HL-60. This preparation submitted to high pressure gel filtration was eluted at a volume corresponding to a mass of 35/40 kD. This activity was insensitive to EGF and insulin. Autoradiographs of the preparations incubated with [gamma P32]-ATP and separated by electrophoresis do not give any evidence that autophosphorylation occurs for that particular tyrosine protein kinase. Furthermore, we failed to immunoprecipitate the enzyme with a specific antiphosphotyrosine antibody and anti v-src antibody. All the data presented herein suggest that this enzyme has not been previously purified.