Peroxisome proliferator-activated receptors beta/delta: emerging roles for a previously neglected third family member.

PURPOSE OF REVIEW: Peroxisome proliferator-activated receptors alpha, beta/delta and gamma are members of the nuclear receptor superfamily. They mediate the effects of fatty acids and their derivatives at the transcriptional level, and are considered to be lipid sensors that participate in the regulation of energy homeostasis. Compared with the alpha and gamma peroxisome proliferator-activated receptor isotypes, peroxisome proliferator-activated receptor beta functions have long remained an enigma. In this review, we focus on emerging knowledge about peroxisome proliferator-activated receptor beta activation and roles. RECENT FINDINGS: We review recent data that suggest key roles in basic cell functions, such as proliferation, differentiation and survival, and in embryonic development and lipid metabolism in peripheral tissues. SUMMARY: The newly unveiled roles of peroxisome proliferator-activated receptor beta in important basic cell functions certainly justify a further exploration of its potential as a therapeutic target in pathologies such as metabolic syndrome X or skin diseases.

Identification of an expanded population of activated CD4(+) CD25(+) T cells expressing CD45RO and IL-7R in kidney transplant recipients

Recent studies have shown that CD4+ CD25+ T cells belong to two functionally different T lymphocytes, i.e. regulatory T cells (Treg) or activated T cells (Tact), which can be distinguished based on the expression of CD45RO and IL-7R: Treg (FoxP3+) are CD45RO+ IL-7R- , whereas Tact (FoxP3- ) are CD45RO+ IL- 7R+. In order to determine if a CD4+ CD25+ CD45RO+ IL-7R+ activated T cell population might be identified in kidney transplant recipients, we studied 27 healthy subjects (HS) and 23 kidney recipients, of whom 17 had stable graft function under standard immunosuppression (IS), 5 had biopsy-proven chronic humoral rejection (CHR), and one was a stable "tolerant" patient who had discontinued IS for more than 2 years. Phenotypical analysis by flow cytometry and functional assays by MLR were performed. Overall, the Tact population was found to be significantly increased in 87% of the transplant recipients (mean: 18.8±10.1% of CD4+ CD25+ T cells) compared to HS (mean: 4.5±2.0%; P<0.0001). In the 5 patients with CHR, this Tact population was highly expanded (31.3±9.3%; P<0.0001), whereas it was comparable to HS in the "tolerant" recipient (4.7%). Intermediate levels (16.0±6.9%; P<0.0001) were found in the 17 stable recipients. In CHR, the proliferative capacity of the Tact population was found to be 5-fold higher when stimulated by irradiated donor PBMC as compared to a stimulation by irradiated 3rd party PBMC. After kidney transplantation, an expanded circulating CD4+ CD25+ T cell population characterized by the expression of CD45RO and IL-7R was found in most recipients, particularly in those with CHR. In a patient with long-term operational tolerance, this Tact population was similar to HS. Measuring circulating Tact may become a useful monitoring tool after transplantation.

Transport and metabolism of [214-C]abscisic acid in maize root

The tips of intact maize (cv. LG 11) roots, maintained vertically, were pretreated with a droplet of buffer solution or a bead of anion exchange resin, both containing [214-C]abscisic acid (ABA). A significant basipetal ABA movement was observed and two metabolites of ABA (possibly phaseic acid and dihydrophaseic acid) were found. ABA pretreatment enhanced the gravireaction of 10 mm apical root segments kept both in the dark and in the light. The possibility that ABA could be one of the endogenous growth inhibitors produced or released by the cap cells is discussed.

Production of low phytic acid rice by hairpin RNA- and artificial microRNA-mediated silencing of OsMIK in seeds

To produce agronomically competitive rice with nutritionally superior, environmentally safe phytic acid (PA) levels, hairpin RNA (hpRNA)- and artificial microRNA (amiRNA)-mediated gene silencing approaches were explored to reduce both myo-inositol kinase gene (OsMIK) expression and PA accumulation in rice seeds. hpRNA and amiRNA sequences targeted to OsMIK (hpMIK and amiMIK), under the control of a rice Ole18 promoter, were transformed into the rice cultivar Nippon-bare. Fourteen and 21 independent transgenic events were identified containing the hpMIK and amiMIK constructs, respectively, from which five stable homozygous transgenic lines of each were developed together with their null siblings. Southern blotting demonstrated transgene integration into the genome and quantitative real-time PCR showed that gene silencing was restricted to seeds. OsMIK transcripts were significantly reduced in both transgenic amiMIK and hpMIK seeds, which had PA levels reduced by 14.9-50.2 and 38.1-50.7 %, respectively, compared with their respective null siblings. There were no systematic significant differences in agronomic traits between the transgenic lines and their non-transgenic siblings, and no correlation between seed PA contents and decreased rates of seed germination and seedling emergence. The results of the present study suggest that Ole 18-driven OsMIK silencing via hpRNA and amiRNA could be an effective way to develop agronomically competitive low phytic acid rice.

Five new CYLD mutations in skin appendage tumors and evidence that aspartic acid 681 in CYLD is essential for deubiquitinase activity.

Brooke-Spiegler syndrome, familial cylindromatosis, and familial trichoepithelioma are autosomal-dominant genetic predispositions for benign tumors of skin appendages caused by mutations in the CYLD gene localized on chromosome 16q12-q13. The encoded protein functions as ubiquitin-specific protease (UBP), which negatively regulates NF-kappaB and c-Jun N-terminal kinase (JNK) signaling. We investigated five families affected with these skin neoplasms and identified four premature stop codons and the novel missense mutation D681G in a family in which 11 of 12 investigated tumors were trichoepitheliomas. CYLD protein harboring this missense mutation had a significant reduced ability to inhibit TNF receptor-associated factor (TRAF)2- and TRAF6-mediated NF-kappaB activation, tumor necrosis factor-alpha (TNFalpha)-induced JNK signaling, and to deubiquitinate TRAF2. CYLD-D681G was coimmunoprecipitated by TRAF2, but was unable to cleave K63-linked polyubiquitin chains. Aspartic acid 681 is highly conserved in CYLD homologues and other members of the UBP family, but does not belong to the Cys and His boxes providing the CYLD catalytic triad (Cys601, His871, and Asp889). As reported previously, the homologous residue D295 of HAUSP/USP-7 forms a hydrogen bond with the C-terminal end of ubiquitin and is important for the enzymatic activity. These results underline that D681 in CYLD is required for cleavage of K63-linked polyubiquitin chains.

Isoelectric focusing of alpha-1 acid glycoprotein (orosomucoid) in immobilized pH-gradients with 8M urea: detection of its desialylated variants using an alkaline phosphatase-linked secondary antibody system.

A method allowing a clear separation of the different variants of desialylated alpha 1-acid glycoprotein (orosomucoid) has been developed using isoelectric focusing in immobilized pH gradients, supplemented with 8 M urea and 2% v/v 2-mercaptoethanol. Immunoblotting with two antibody-steps afforded high sensitivity and permitted the detection of about 700 pg of alpha 1-acid glycoprotein in a 20 microL plasma sample diluted 1:28 672. A one year old bloodstrain, kept at room temperature, could easily be phenotyped.

Genetic analysis of phenoxyalkanoic acid degradation in Sphingomonas herbicidovorans MH.

Phenoxyalkanoic acid degradation is well studied in Beta- and Gammaproteobacteria, but the genetic background has not been elucidated so far in Alphaproteobacteria. We report the isolation of several genes involved in dichlor- and mecoprop degradation from the alphaproteobacterium Sphingomonas herbicidovorans MH and propose that the degradation proceeds analogously to that previously reported for 2,4-dichlorophenoxyacetic acid (2,4-D). Two genes for alpha-ketoglutarate-dependent dioxygenases, sdpA(MH) and rdpA(MH), were found, both of which were adjacent to sequences with potential insertion elements. Furthermore, a gene for a dichlorophenol hydroxylase (tfdB), a putative regulatory gene (cadR), two genes for dichlorocatechol 1,2-dioxygenases (dccA(I/II)), two for dienelactone hydrolases (dccD(I/II)), part of a gene for maleylacetate reductase (dccE), and one gene for a potential phenoxyalkanoic acid permease were isolated. In contrast to other 2,4-D degraders, the sdp, rdp, and dcc genes were scattered over the genome and their expression was not tightly regulated. No coherent pattern was derived on the possible origin of the sdp, rdp, and dcc pathway genes. rdpA(MH) was 99% identical to rdpA(MC1), an (R)-dichlorprop/alpha-ketoglutarate dioxygenase from Delftia acidovorans MC1, which is evidence for a recent gene exchange between Alpha- and Betaproteobacteria. Conversely, DccA(I) and DccA(II) did not group within the known chlorocatechol 1,2-dioxygenases, but formed a separate branch in clustering analysis. This suggests a different reservoir and reduced transfer for the genes of the modified ortho-cleavage pathway in Alphaproteobacteria compared with the ones in Beta- and Gammaproteobacteria.

Ampakine CX546 bolsters energetic response of astrocytes: a novel target for cognitive-enhancing drugs acting as alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor modulators.

Glutamate was previously shown to enhance aerobic glycolysis i.e. increase glucose utilization and lactate production with no change in oxygen levels, in mouse cortical astrocytes by a mechanism involving glutamate uptake. It is reported here that a similar response is produced in both hippocampal and cerebellar astrocytes. Application of the cognitive-enhancing drug CX546 promoted further enhancement of glucose utilization by astrocytes from each brain area following glutamate exposure. alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors represent the purported molecular target of cognitive-enhancing drugs such as CX546, and the presence of AMPA receptor subunits GluR1-4 was evidenced in astrocytes from all three regions by immunocytochemistry. AMPA itself did not stimulate aerobic glycolysis, but in the presence of CX546, a strong enhancement of glucose utilization and lactate production was obtained in cortical, hippocampal and cerebellar astrocytes. The effect of CX546 was concentration-dependent, with an EC(50) of 93.2 microm in cortical astrocytes. AMPA-induced glucose utilization in the presence of CX546 was prevented by the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the negative modulator GYKI 52466. In addition, the metabolic effect of CX546 in the presence of AMPA was mimicked by the AMPA receptor modulator cyclothiazide. Our data suggest that astrocyte energetics represents a novel target for cognitive-enhancing drugs acting as AMPA receptor modulators.

RXR: from partnership to leadership in metabolic regulations.

Vitamin A signaling occurs through nuclear receptors recognizing diverse forms of retinoic acid (RA). The retinoic acid receptors (RARs) bind all-trans RA and its 9-cis isomer (9-cis RA). They convey most of the activity of RA, particularly during embryogenesis. The second subset of receptors, the rexinoid receptors (RXRs), binds 9-cis RA only. However, RXRs are obligatory DNA-binding partners for a number of nuclear receptors, broadening the spectrum of their biological activity to the corresponding nuclear receptor-signaling pathways. The present chapter more particularly focuses on RXR-containing transcriptional complexes for which RXR is not only a structural component necessary for DNA binding but also acts as a ligand-activated partner. After positioning RXR among the nuclear receptor superfamily in the first part, we will give an overview of three major signaling pathways involved in metabolism, which are sensitive to RXR activation: LXR:RXR, FXR:RXR, and PPAR:RXR. The third and last part is focused on RXR signaling and its potential role in metabolic regulation. Indeed, while the nature of the endogenous ligand for RXR is still in question, as we will discuss herein, a better understanding of RXR activities is necessary to envisage the potential therapeutic applications of synthetic RXR ligands.

Folic acid: one of life's essentials

This leaflet explains what folic acid is and how it can help to protect the unborn child against conditions such as spina bifida.

Discovery of amino acid variants in the human glucose-dependent insulinotropic polypeptide (GIP) receptor: the impact on the pancreatic beta cell responses and functional expression studies in Chinese hamster fibroblast cells.

The two incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), are insulinotropic factors released from the small intestine to the blood stream in response to oral glucose ingestion. The insulinotropic effect of GLP-1 is maintained in patients with Type II (non-insulin-dependent) diabetes mellitus, whereas, for unknown reasons, the effect of GIP is diminished or lacking. We defined the exon-intron boundaries of the human GIP receptor, made a mutational analysis of the gene and identified two amino acid substitutions, A207 V and E354Q. In an association study of 227 Caucasian Type II diabetic patients and 224 matched glucose tolerant control subjects, the allelic frequency of the A207 V polymorphism was 1.1% in Type II diabetic patients and 0.7% in control subjects (p = 0.48), whereas the allelic frequency of the codon 354 polymorphism was 24.9% in Type II diabetic patients versus 23.2% in control subjects. Interestingly, the glucose tolerant subjects (6% of the population) who were homozygous for the codon 354 variant had on average a 14% decrease in fasting serum C-peptide concentration (p = 0.01) and an 11% decrease in the same variable 30 min after an oral glucose load (p = 0.03) compared with subjects with the wild-type receptor. Investigation of the function of the two GIP receptor variants in Chinese hamster fibroblasts showed, however, that the GIP-induced cAMP formation and the binding of GIP to cells expressing the variant receptors were not different from the findings in cells expressing the wildtype GIP receptor. In conclusion, amino acid variants in the GIP receptor are not associated with random Type II diabetes in patients of Danish Caucasian origin or with altered GIP binding and GIP-induced cAMP production when stably transfected in Chinese hamster fibroblasts. The finding of an association between homozygosity for the codon 354 variant and reduced fasting and post oral glucose tolerance test (OGTT) serum C-peptide concentrations, however, calls for further investigations and could suggest that GIP even in the fasting state regulates the beta-cell secretory response.

Carbon isotopic ratio analysis by gas chromatography/combustion/isotope ratio mass spectrometry for the detection of gamma-hydroxybutyric acid (GHB) administration to humans.

Since GHB (gamma-hydroxybutyric acid) is naturally produced in the human body, clinical and forensic toxicologists must be able to discriminate between endogenous levels and a concentration resulting from exposure. To suggest an alternative to the use of interpretative concentration cut-offs, the detection of exogenous GHB in urine specimens was investigated by means of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). GHB was isolated from urinary matrix by successive purification on Oasis MCX and Bond Elute SAX solid-phase extraction (SPE) cartridges prior to high-performance liquid chromatography (HPLC) fractioning using an Atlantis dC18 column eluted with a mixture of formic acid and methanol. Subsequent intramolecular esterification of GHB leading to the formation of gamma-butyrolactone (GBL) was carried out to avoid introduction of additional carbon atoms for carbon isotopic ratio analysis. A precision of 0.3 per thousand was determined using this IRMS method for samples at GHB concentrations of 10 mg/L. The (13)C/(12)C ratios of GHB in samples of subjects exposed to the drug ranged from -32.1 to -42.1 per thousand, whereas the results obtained for samples containing GHB of endogenous origin at concentration levels less than 10 mg/L were in the range -23.5 to -27.0 per thousand. Therefore, these preliminary results show that a possible discrimination between endogenous and exogenous GHB can be made using carbon isotopic ratio analyses.