Characterization of the human germ cell nuclear factor gene

The vertex potential (N2, P2) of the laser-evoked potential (LEP) is preceded by a small negativity (N1). The role of the secondary somatosensory cortex (SII) in generation of the N1 is established for the upper but not for the lower limb. We therefore investigated the N1 after painful radiant heat stimulation of hand and foot dorsum in 22 subjects. LEPs were recorded from the scalp with midline and temporal electrodes. After hand stimulation N1 was maximal in the contralateral temporal lead (mean peak latency 156 +/- 23 ms). After foot stimulation N1 was maximal in the same lead (200 +/- 22 ms). In the ipsilateral temporal lead, N1 appeared significantly smaller and later. N2 and P2 were maximal in midline electrodes for both stimulus sites. The latency shift between hand and foot stimulation was identical for all three components. These results suggest a contribution of temporo-parietal cortex (e.g. SII) to the N1 generation for stimulation of upper and lower limb.     

An orphan nuclear receptor lacking a zinc-finger DNA-binding domain: interaction with several nuclear receptors

BACKGROUND: Affections of the lacrimal drainage system are often seen by ophthalmologists. Inspection, palpation, diagnostic rinsing and sounding can distinguish anatomical stops before or after tear sac. For final diagnostics however more apparative examinations are necessary. The ultrasonic examination with contrast media is a simple method for diagnostics of affections of the lacrimal drainage system besides the dacryocystography, the scintigraphy and other ones. PATIENTS AND METHODS: On 12 patients with a dysfunctional lacrimal drainage system and 12 normal controls the ultrasonic examination with instillation of contrast media: silicon oil, glycerine, dispersions of almond oil and viscoelastic substances was performed. All examinations were performed with the 13 B-scan. RESULTS: The lacrimal drainage way was detectable from the canaliculus to the middle nasolacrimal duc CONCLUSION: An additional advantage of the ultrasonic examination with contrast-media is the neglect of radiation, the simple and often repeatable examination method, especially the enhancement of the contrast of different valves and stenoses and mucinous deposits in stationary anatomical variations and dynamic defects.     

Dimeric binding of the mouse germ cell nuclear factor

The mouse germ cell nuclear factor (GCNF), a member of the nuclear receptor superfamily, is highly expressed during spermatogenesis, oogenesis, and during neuronal embryonic differentiation. The in vitro translated receptor binds autonomously to the direct repeat of the sequence 5'-AGGTCA-3'. To gain insights into the determinants necessary for DNA binding, I have generated truncated GCNF molecules by introducing carboxy-terminal deletions into expression constructs. An electrophoretic mobility-shift assay with these polypeptides shows that amino acids in addition to the core DNA-binding domain are important for specific binding. To address the question of whether the protein binds as monomer, homodimer, or heterodimer, I used different approaches. Analysis of the full-length protein was possible with GCNF polypeptides that contain epitopes of six consecutive histidines. Using a monoclonal antibody directed against these epitopes, I demonstrate that two GCNF molecules bind to a direct repeat. Dimerization between wild-type and truncated GCNF is shown by an electrophoretic mobility-shift analysis with a mixture of the proteins. In addition, I show that there is no in vitro interaction of GCNF with the retinoid X receptor, a promiscous partner of many nuclear receptors. The data suggest that GCNF may excert its in vivo function independently of other nuclear receptors.     

Evolution of the nuclear receptor superfamily: early diversification from an ancestral orphan receptor

From a database containing the published nuclear hormone receptor (NR) sequences I constructed an alignment of the C, D and E domains of these molecules. Using this alignment, I have performed tree reconstruction using both distance matrix and parsimony analysis. The robustness of each branch was estimated using bootstrap resampling methods. The trees constructed by these two methods gave congruent topologies. From these analyses I defined six NR subfamilies: (i) a large one clustering thyroid hormone receptors (TRs), retinoic acid receptors (RARs), peroxisome proliferator-activated receptors (PPARs), vitamin D receptors (VDRs) and ecdysone receptors (EcRs) as well as numerous orphan receptors such as RORs or Rev-erbs; (ii) one containing retinoid X receptors (RXRs) together with COUP, HNF4, tailless, TR2 and TR4 orphan receptors; (iii) one containing steroid receptors; (iv) one containing the NGFIB orphan receptors; (v) one containing FTZ-F1 orphan receptors; and finally (vi) one containing to date only one gene, the GCNF1 orphan receptor. The relationships between the six subfamilies are not known except for subfamilies I and IV which appear to be related. Interestingly, most of the liganded receptors appear to be derived when compared with orphan receptors. This suggests that the ligand-binding ability of NRs has been gained by orphan receptors during the course of evolution to give rise to the presently known receptors. The distribution into six subfamilies correlates with the known abilities of the various NRs to bind to DNA as homo- or heterodimers. For example, receptors heterodimerizing efficiently with RXR belong to the first or the fourth subfamilies. I suggest that the ability to heterodimerize evolved once, just before the separation of subfamilies I and IV and that the first NR was able to bind to DNA as a homodimer. From the study of NR sequences existing in vertebrates, arthropods and nematodes, I define two major steps of NR diversification: one that took place very early, probably during the multicellularization event leading to all the metazoan phyla, and a second occurring later on, corresponding to the advent of vertebrates. Finally, I show that in vertebrate species the various groups of NRs accumulated mutations at very different rates.     

The nuclear orphan receptor CAR-retinoid X receptor heterodimer activates the phenobarbital-responsive enhancer module of the CYP2B gene

PBREM, the phenobarbital-responsive enhancer module of the cytochrome P-450 Cyp2b10 gene, contains two potential nuclear receptor binding sites, NR1 and NR2. Consistent with the finding that anti-retinoid X receptor (RXR) could supershift the NR1-nuclear protein complex, DNA affinity chromatography with NR1 oligonucleotides enriched the nuclear orphan receptor RXR from the hepatic nuclear extracts of phenobarbital-treated mice. In addition to RXR, the nuclear orphan receptor CAR was present in the same enriched fraction. In the phenobarbital-treated mice, the binding of both CAR and RXR was rapidly increased before the induction of CYP2B10 mRNA. In vitro-translated CAR bound to NR1, but only in the presence of similarly prepared RXR. PBREM was synergistically activated by transfection of CAR and RXR in HepG2 and HEK293 cells when the NR1 site was functional. A CAR-RXR heterodimer has thus been characterized as a trans-acting factor for the phenobarbital-inducible Cyp2b10 gene.     

Cross-talk between orphan nuclear hormone receptor RZRalpha and peroxisome proliferator-activated receptor alpha in regulation of the peroxisomal hydratase-dehydrogenase gene

The genes encoding the peroxisomal beta-oxidation enzymes enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD) and fatty acyl-CoA oxidase (AOx) are coordinately regulated by peroxisome proliferator-activated receptor alpha (PPARalpha)/9-cis-retinoic acid receptor (RXRalpha) heterodimers that transactivate these genes in a ligand-dependent manner via upstream peroxisome proliferator response elements (PPRE). Here we demonstrate that the monomeric orphan nuclear hormone receptor, RZRalpha, modulates PPARalpha/RXRalpha-dependent transactivation in a response-element dependent manner. Electrophoretic mobility shift analysis showed that RZRalpha bound specifically as a monomer to the HD-PPRE but not the AOx-PPRE. Determinants in the HD-PPRE for binding of RZRalpha were distinct from those required for interaction with PPARalpha/RXRalpha heterodimers. In transient transfections, RZRalpha stimulated ligand-mediated transactivation by PPARalpha from an HD-PPRE luciferase reporter in the absence of exogenously added RXRalpha, but did not affect PPARalpha-dependent transactivation of an AOx-PPRE reporter gene. These data illustrate cross-talk between the RZRalpha and PPARalpha signaling pathways at the level of the HD-PPRE in the regulation of the HD gene and characterize additional factors governing the regulation of peroxisomal beta-oxidation.     

Induction of mitogen-inducible nuclear orphan receptor by interleukin 1 in human synovial and gingival fibroblasts

High levels of interleukin 1 (IL-1) found in inflammatory diseases such as rheumatoid arthritis and periodontitis act on the local fibroblasts, resulting in an altered phenotype characterized by hyperplasia and the production of inflammatory mediators and destructive enzymes. The goal of this study was to identify genes induced as an early response to IL-1 in synovial and gingival fibroblasts which might play a regulatory role in the cascade of events leading to their activation. Using the technique of mRNA differential display, we have identified the mitogen-inducible nuclear orphan receptor (MINOR) as a gene up-regulated by IL-1 in human synovial and gingival fibroblasts. The rapid induction of both mRNA and DNA binding activity suggests that MINOR may play an important early role in regulating the response of fibroblasts to inflammation.     

An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway

PURPOSE: To determine the effects of 28 d of creatine supplementation during training on body composition, strength, sprint performance, and hematological profiles. METHODS: In a double-blind and randomized manner, 25 NCAA division IA football players were matched-paired and assigned to supplement their diet for 28 d during resistance/agility training (8 h x wk[-1]) with a Phosphagen HP (Experimental and Applied Sciences, Golden, CO) placebo (P) containing 99 g x d(-1) of glucose, 3 g x d(-1) of taurine, 1.1 g x d(-1) of disodium phosphate, and 1.2 g x d(-1) of potassium phosphate (P) or Phosphagen HP containing the P with 15.75 g x d(-1) of HPCE pure creatine monohydrate (HP). Before and after supplementation, fasting blood samples were obtained; total body weight, total body water, and body composition were determined; subjects performed a maximal repetition test on the isotonic bench press, squat, and power clean; and subjects performed a cycle ergometer sprint test (12 x 6-s sprints with 30-s rest recovery). RESULTS: Hematological parameters remained within normal clinical limits for active individuals with no side effects reported. Total body weight significantly increased (P < 0.05) in the HP group (P 0.85 +/- 2.2; HP 2.42 +/- 1.4 kg) while no differences were observed in the percentage of total body water. DEXA scanned body mass (P 0.77 +/- 1.8; HP 2.22 +/- 1.5 kg) and fat/bone-free mass (P 1.33 +/- 1.1; HP 2.43 +/- 1.4 kg) were significantly increased in the HP group. Gains in bench press lifting volume (P -5 +/- 134; HP 225 +/- 246 kg), the sum of bench press, squat, and power clean lifting volume (P 1,105 +/- 429; HP 1,558 +/- 645 kg), and total work performed during the first five 6-s sprints was significantly greater in the HP group. CONCLUSION: The addition of creatine to the glucose/taurine/electrolyte supplement promoted greater gains in fat/bone-free mass, isotonic lifting volume, and sprint performance during intense resistance/agility training.     

The role of T cell receptor dimerization for T cell antagonism and T cell specificity

Endemic nephropathy is a chronic renal disease with a high prevalence in a geographically limited area of Croatia. It has also been recorded in some parts of Bosnia, Serbia, Bulgaria and Romania. Despite numerous studies conducted to date, the etiology of this disease has not been clarified. Pathological studies of the kidney in the early stage of endemic nephropathy have shown renal tubules to be the primary sites of the pathologic process with an interstitial tissue reaction, whereas glomerular alterations are of a secondary character. Tubulointerstitial lesions can thus account for the symptoms of the disease, i.e. tubular proteinuria and reduced urine concentration capacity and urine acidification. Also, an increased incidence of malignant tumours of the urinary tract was found in the same geographic area.     

A novel nuclear receptor heterodimerization pathway mediated by orphan receptors TR2 and TR4

A unique heterodimerization pathway involving orphan receptors TR2 and TR4 is demonstrated. TR2 and TR4 preferentially form heterodimers in solution as well as on DNA elements containing a direct repeat-5 (DR5). The in vitro interaction between TR2 and TR4 is demonstrated by the yeast and the mammalian two-hybrid interaction assays, the pull-down assay, and the gel mobility shift assay. The in vivo interaction is demonstrated by following the intracellular localization of fusion receptors tagged with a green fluorescent protein. The dimerization is mediated by the ligand binding domains, and the three leucine residues on helix 10 of TR2 are critical for this interaction. In addition, coexpression of these two receptors exerts a much stronger repressive activity on a DR5-containing reporter than expressing either receptor alone. In the developing testis, TR2 and TR4 are coexpressed in the same testicular cell populations and exhibit a parallel pattern of expression along development. The preferential heterodimerization between TR2 and TR4 and their coexistence in specific germ cell populations suggest a physiological role of TR2/TR4 heterodimers in germ cell development.     

Characterization of a cell cycle-dependent nuclear autoantigen

Analysis of reactivity to nuclear antigens in autoimmune sera revealed a serum that produced a previously undescribed cell cycle-dependent immunofluorescence staining pattern. By indirect immunofluorescence using HEp-2 cells as substrate, the serum generated a speckled and nucleolar pleomorphic staining pattern. This characteristic immunofluorescence pattern was detected in different cell lines from various species indicating that the antigen was highly conserved. This serum immunoprecipitated a 85 kDa protein using an extract from [35S]-labeled HeLa cells. Indirect immunofluorescence of proliferating mouse 3T3 cells displayed the characteristic pleomorphic staining which was not observed in serum-starved cells. Resting human and mouse peripheral blood lymphocytes were negative in immunofluorescence while mitogen-stimulated lymphocytes were positive. Germinal centers of mice two weeks after immunization with 2-phenyl-oxazolone showed speckled immunofluorescence staining in the dark zones whereas unimmunized mice were completely negative. Cell synchronization experiments showed a characteristic sequence of locations of the antigen during the cell cycle. In G1, cells were completely negative. In late G1, G1/S and S phase, speckles were visible. In early G2, speckles were visible, and later in G2, the nucleoli were positive. During mitosis chromosomes were stained. Further characterization of this antibody specificity and cloning of cDNA are in progress.