The erythropoietin (Epo) gene is regulated by hypoxia-inducible cis-acting elements in the promoter and in a 3 enhancer, both of which contain consensus hexanucleotide hormone receptor response elements which are important for function. A group of 11 orphan nuclear receptors, transcribed and translated in vitro, were screened by the electrophoretic mobility shift assay. Of these, hepatic nuclear factor 4 (HNF-4), TR2-11, ROR␣1, and EAR3/COUP-TF1 bound specifically to the response elements in the Epo promoter and enhancer and, except for ROR␣1, formed DNA-protein complexes that had mobilities similar to those observed in nuclear extracts of the Epo-producing cell line Hep3B. Moreover, both anti-HNF-4 and anti-COUP antibodies were able to supershift complexes in Hep3B nuclear extracts. Like Epo, HNF-4 is expressed in kidney, liver, and

High-throughput metabolite profiling provides the opportunity to reveal metabolic mechanisms and identify biomarkers. Psoriasis is an immune-mediated chronic inflammatory disease. However, the role of metabolism in psoriasis pathogenesis remains unclear. Methods: Plasma samples of individuals (45 psoriasis and 45 sex‐, age-, and BMI-matched healthy controls) were collected. Non-targeted metabolomics and amino acid- or carnitine-targeted metabolomics were conducted, then, plasma samples of mice induced by imiquimod (IMQ) were subjected to the amino acid- and carnitine-targeted metabolomic profiling. Flow cytometry was used to study the effect of L-carnitine (LC(C0)) on IMQ-induced psoriatic inflammation. Results: Through the non-targeted metabolomics approach, we detected significantly altered amino acids and carnitines in psoriasis patients. Amino acid-targeted metabolomic profiling identified 37 amino acids altered in psoriasis, of these 23 were markedly upregulated, including essential amino acids (EAAs), and branched-chain amino acids (BCAAs), whereas glutamine, cysteine, and asparagine were significantly down-regulated. Carnitine-targeted metabolomic profiling identified 40 significantly altered carnitines, 14 of which included palmitoylcarnitine (C16) and were markedly downregulated in psoriasis, whereas hexanoylcarnitine (C6) and 3-OH-octadecenoylcarnitine (C18:1-OH) were significantly upregulated. Interestingly, glutamine, asparagine, and C16 levels were negatively correlated with the PASI score. Moreover, a higher abundance of LC(C0) was associated with markedly reduced IMQ-induced epidermal thickening and infiltration of Th17 cells in skin lesions, indicating LC(C0) supplementation as a potential therapy for psoriasis treatment. Conclusion: Our results suggested the metabolism of amino acids and carnitines are significantly altered in psoriasis, especially the metabolism of EAAs, BCAAs, and LC(C0), which may play key roles in the pathogenesis of psoriasis.

Aim. The study was to investigate the metabolic profile of urine metabolites and to elucidate their clinical significance in patients with colorectal cancer. Methods. Colorectal cancers from early stage and advanced stage were used in this study. Urine samples of colorectal cancer patients and healthy adults were collected and subjected to capillary electrophoresis mass spectrometry based on moving reaction boundary analysis. The metabolic data were analyzed by SPSS 17.0 to find urinary biomarkers for colorectal cancer. Results. The results indicated that the urine metabolic profiling of colorectal cancer patients had significant changes compared with the normal controls, and there were also differences between early stage and advanced colorectal cancer patients. Compared with the control group, the levels of isoleucine, valine, arginine, lactate acid and leucine increased (P < 0.05), but those of histidine, methionine, serine, aspartic acid, citric acid, succinate, and malic acid decreased in urine samples from colorectal cancer (P < 0.05). Furthermore, the levels of isoleucine and valine were lower in urine of patients with advanced colorectal cancer than those in early stage colorectal cancer (P < 0.05). Conclusion. The technique of capillary electrophoresis mass spectrometry based on MRB could reveal the significant metabolic alterations during progression of colorectal cancer, and the method is feasible and may be useful for the early diagnosis of colorectal cancer.

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We have examined the methylation profiles of the asparagine synthetase (ASY) promoter in a number of human leukemic cell lines in relation to their asparagine (ASN) requirements in vitro. Cells in which the promoter is highly methylated are auxotrophs and express ASY at very low levels. Electromobility shift assays (EMSA) of nuclear extracts with oligomers from the promoting region show, in addition to recognized transcription factor binding, a novel methyl binding protein specific for a 12 base consensus sequence, which includes a single methylated CpG. This sequence overlaps that of the amino-acid response unit of the ASY promoter, which is activated byATF4 and C/EBP. Competition by the methyl binding protein could account for the observed failure of the methylated promoter to bind these transcription factors and consequently, although other mechanisms can also be operative, for the specific repression of the gene. The ASY methyl binding protein (ASMB) is present in leukemic lymphoid and myeloid cells irrespective of their methylation status, and in normal lymphocytes after phytohemagglutinin stimulation. It has been purified by affinity chromatography and has a molecular size of 40 kDa in 10% SDS-polyacrylamide gels.