BACKGROUND: Alcohol is known to affect two epigenetic phenomena, DNA methylation and DNA hydroxymethylation, and iron is a cofactor of ten-eleven translocation (TET) enzymes that catalyze the conversion from methylcytosine to hydroxymethylcytosine. In the present study we aimed to determine the effects of alcohol on DNA hydroxymethylation and further effects of iron on alcohol associated epigenetic changes. METHODS: Twenty-four male Sprague-Dawley rats were fed either Lieber-DeCarli alcohol diet (36% calories from ethanol) or Lieber-DeCarli control diet along with or without iron supplementation (0.6% carbonyl iron) for 8 weeks. Hepatic non-heme iron concentrations were measured by colorimetric assays. Protein levels of hepatic ferritin and transferrin receptor were determined by Western blotting. Methylcytosine, hydroxymethylcytosine and unmodified cytosine in DNA were simultaneously measured by liquid chromatography/mass spectrometry method. RESULTS: Iron supplementation significantly increased hepatic non-heme iron contents (P < 0.05) but alcohol alone did not. However, both alcohol and iron significantly increased hepatic ferritin levels and decreased hepatic transferrin receptor levels (P < 0.05). Alcohol reduced hepatic DNA hydroxymethylation (0.21% ± 0.04% vs. 0.33% ± 0.04%, P = 0.01) compared to control, while iron supplementation to alcohol diet did not change DNA hydroxymethylation. There was no significant difference in methylcytosine levels, while unmodified cytosine levels were significantly increased in alcohol-fed groups compared to control (95.61% ± 0.08% vs. 95.26% ± 0.12%, P = 0.03), suggesting that alcohol further increases the conversion from hydroxymethylcytosine to unmodified cytosine. CONCLUSIONS: Chronic alcohol consumption alters global DNA hydroxymethylation in the liver but iron supplementation reverses the epigenetic effect of alcohol.
Alcohol Drinking* ; Alcohols ; Animals ; Blotting, Western ; Cytosine ; Diet ; DNA Methylation ; DNA* ; Epigenomics ; Ferritins ; Humans ; Iron* ; Liver ; Male ; Methods ; Rats* ; Rats, Sprague-Dawley ; Receptors, Transferrin ; Spectrum Analysis

OBJECTIVETo establish and evaluate a protein microarray method for combined measurement of serum ferritin (SF) and soluble transferrin receptor (sTfR).

METHODSMicroarrayer was used to print both anti-SF antibodies I and anti-sTfR antibodies I on each protein microarray. Anti-SF antibodies II and anti-sTfR antibodies II were used as detection antibodies and goat antibodies coupled to Cy3 were used as antibodies III. The detection conditions of the quantitative analysis method for simultaneous measurement of SF and sTfR with protein microarray were optimized and evaluated. The protein microarray was compared with commercially available traditional tests with 26 serum samples.

RESULTSBy comparison experiment, mouse monoclonal antibodies were chosen as the probes and contact printing was chosen as the printing method. The concentrations of SF and sTfR probes were 0.5 mg/mL and 0.5 mg/mL respectively, while those of SF and sTfR detection antibodies were 5 μg/mL and 0.36 μg/mL respectively. Intra- and inter-assay variability was between 3.26% and 18.38% for all tests. The regression coefficients comparing protein microarray with traditional test assays were better than 0.81 for SF and sTfR.

CONCLUSIONThe present study has established a protein microarray method for combined measurement of SF and sTfR.

Animals ; Antibodies, Monoclonal ; analysis ; Ferritins ; blood ; Mice ; Protein Array Analysis ; methods ; Rabbits ; Receptors, Transferrin ; blood

OBJECTIVETo study the clinical utility of measuring reticulocyte hemoglobin content (CHr) in the diagnosis of iron deficiency anemia (IDA) in children.

METHODSOne hundred children with IDA at ages of 1 to 6 years and 50 healthy children were enrolled. Red blood cell parameters, CHr, hemoglobin (Hb), red blood count (RBC) and mean corpusular volume (MCV), were determined using the Blood Cell Analyzer. Serum ferritin (SF) levels were determined using radioimmunoassay double antibody techique. Soluble serum transferrin (sTfR) levels were determined using ELISA.

RESULTSThe values of Hb (100 ± 6 g/L vs 126 ± 8 g/L) and CHr (18 ± 5 pg vs 31 ± 3 pg) in the IDA group were significantly lower than normal controls (P<0.01). SF levels (11 ± 4 μg/L) in the IDA group were also lower than normal controls (59 ± 36 μg/L) (P<0.01). In contrast, the values of sTfR in the IDA group were significantly higher than normal controls (4.8 ± 2.1 mg/L vs 1.4 ± 0.6 mg/L; P<0.01). In both groups, there was a positive correlation between the values of CHr and Hb [r=0.540 (control group), r=0.734 (IDA group); P<0.01]. In the IDA group, CHr was positively correlated with SF(r=0.464; P<0.01) and negatively correlated with sTfR(r=-0.450; P<0.01). When the cut-off value of CHr was 27.8 pg, the sensitivity and specificity for the diagnosis of IDA were 88.0% and 90.0%, respectively and the area under the ROC curve was 0.948.

CONCLUSIONSCHr can be used as an index for the diagnosis of IDA in children.

Anemia, Iron-Deficiency ; blood ; diagnosis ; Child ; Child, Preschool ; Female ; Hemoglobins ; analysis ; Humans ; Infant ; Male ; ROC Curve ; Receptors, Transferrin ; blood ; Reticulocytes ; chemistry

The study investigated the effects of red clover extract (RCE) on mouse T macrophages and lymphocytes in vitro. The cell toxic effect of RCE was estimated by MTT assay. Multiple-fluorescence staining plus flow cytometry were used to detect the effect of RCE on CD69/CD25/CD71 expression of mouse T lymphocytes stimulated by Con A; CFDA-SE staining plus flow cytometry were used to analyze the effect of RCE on proliferation of T lymphocytes activated by Con A; The effect of RCE on nitric oxide (NO) secretion of mouse macrophages stimulated by lipopolysaccharide (LPS) for 24 h was assayed by Griess reagent system. We found that RCE had potent anti-inflammatory effects on mice. RCE had little cell toxic effect on mouse lymphocytes and macrophages. RCE strongly inhibited the excessive production of inflammatory mediators (NO, CD69, CD25, CD71), in a dose-dependent manner, like cyclosporine A injection. RCE could inhibit proliferation of CD3+ T lymphocytes. These data suggested that RCE might exhibit anti-inflammatory effect by inhibiting the activation and proliferation of mouse lymphocytes and the NO secretion of mouse macrophages.
Animals ; Anti-Inflammatory Agents ; isolation & purification ; pharmacology ; Antigens, CD ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; metabolism ; CD3 Complex ; analysis ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Interleukin-2 Receptor alpha Subunit ; metabolism ; Lectins, C-Type ; Lymphocyte Activation ; drug effects ; Macrophages ; cytology ; secretion ; Male ; Mice ; Mice, Inbred BALB C ; Nitric Oxide ; secretion ; Plants, Medicinal ; chemistry ; Receptors, Transferrin ; metabolism ; T-Lymphocytes ; cytology ; drug effects ; metabolism ; Trifolium ; chemistry

OBJECTIVETo investigate the systemic changes of iron metabolism following manganese exposure.

METHODSNinety-seven welders and 91 workers with no history of exposure to manganese were recruited from the same factory in Beijing serving as the exposure group and the control group respectively. The welding rods used were type J422. The concentration of the manganese in the air of the work place was determined respectively with the national standard method. The serum iron and manganese, ferritin, transferrin and transferrin receptors were measured with the graphite furnace atomic absorption spectrophotometry and ELISA in both groups.

RESULTSThe permissible concentration-STEL of ambient Mn in welders' breathing zone ranged from 0.53 mg/m(3) to 2.19 mg/m(3), while the permissible concentration-TWA of ambient Mn was between 0.29 mg/m(3) and 0.92 mg/m(3) in the breathing zone of the workplace. Serum Mn and Fe concentrations in welders were about 1.40 times (P < 0.0l) and 1.2 times (P < 0.01), respectively, higher than those of control subjects. At the same time, the transferrin concentrations in serum were significantly higher (about 1.2 times, P < 0.05) in welders than in controls. In contrast, transferrin receptors were significantly lower (about 1.2 times) in exposed subjects than controls (P = 0.001). There was no difference in serum ferritin between the two groups (P = 0.112). Although there was no significant trend, the serum ferritin level was increased by 18% in comparison with that of the control. The abnormal percentage of serum Fe and Serum Mn in welders were 55.67% and 67.01% respectively, higher than those of control subjects. In addition, the correlations between all indicators and the duration of employment were not observed.

CONCLUSIONThe long term exposure to the manganese can induce the disorder of the iron metabolism, which is found in the expression of increase of the serum iron and transferrin as well as the decrease of transferrin receptors.

Female ; Ferritins ; blood ; Humans ; Iron ; metabolism ; Iron Metabolism Disorders ; chemically induced ; Male ; Manganese ; adverse effects ; Occupational Exposure ; adverse effects ; Receptors, Transferrin ; blood ; Transferrin ; analysis ; Welding

OBJECTIVETo investigate the feasibility of (99m)Tc-transferrin ((99m)Tc-Tf) as an transferrin receptor imaging agent in nude mice bearing human hepatoma SMMC-7721 tumor.

METHODSBiodistribution of (99m)Tc-Tf in Balb/c mice was assayed, and transferrin receptor imaging in Balb/c nu/nu nude mice bearing human hepatocellular carcinoma SMMC-7721 xenografts was carried out, with or without inhibition of transferrin receptor with unlabeled transferrin. The receptor radioligand binding assay with (125)I-transferrin as ligand was established, and the number of receptors per cell and the K(d) were worked out.

RESULTS(99m)Tc-Tf was mainly distributed in the liver, blood and kidneys. At 18 h post injection, the ratios of tumor/blood, tumor/liver and tumor/muscle were 24.2, 1.7 and 35.5, respectively. The ratio of tumor uptake at 18 h without unlabeled transferrin pretreatment to that with pretreatment was 16.3. The number of receptors per cell was 2.4 x 10(5) and the K(d) was found to be 4.46 nmol/L.

CONCLUSIONAll those results indicate that (99m)Tc-Tf is specifically localized in tumor lesions of nude mice. (99m)Tc-Tf may be useful in detection of hepatocellular carcinoma.

Animals ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Female ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Organotechnetium Compounds ; pharmacokinetics ; Random Allocation ; Receptors, Transferrin ; analysis ; Tissue Distribution ; Transferrin ; pharmacokinetics

In order to investigate the potential of human ERMAP gene in erythroid cell differentiation, K562 cells were induced to erythroid lineage by Ara-C and to macrophage lineage by TPA, human ERMAP mRNA was detected by fluorescent quantitative PCR. The results showed that human ERMAP mRNA increased while K562 cells were induced to erythroid lineage after treatment with Ara-C at 2.5 x 10(-6) mmol/L/L and 1.0 x 10(-6) mmol/L/L. Human ERMAP mRNA not changed while K562 cells were induced to macrophage lineage after treatment with TPA at 2.0 x 10(-6) mmol/L/L and 1.0 x 10(-6) mmol/L/L. It is concluded that human ERMAP gene plays an important role in differentiation and proliferation of erythroid cells.
Antigens, CD ; analysis ; Antigens, Differentiation, Myelomonocytic ; analysis ; Blood Group Antigens ; genetics ; Butyrophilins ; Cell Differentiation ; drug effects ; genetics ; Cytarabine ; pharmacology ; Erythrocytes ; cytology ; metabolism ; ultrastructure ; Flow Cytometry ; Gene Expression ; drug effects ; Humans ; K562 Cells ; Macrophages ; cytology ; metabolism ; ultrastructure ; Microscopy, Electron ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Transferrin ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sialic Acid Binding Ig-like Lectin 3 ; Tetradecanoylphorbol Acetate ; pharmacology ; Time Factors

Extracellular Signal-Regulated MAP Kinases ; metabolism ; Glomerular Mesangium ; immunology ; Glycosylation ; Humans ; Immunoglobulin A ; metabolism ; Phosphorylation ; Receptors, Fc ; analysis ; genetics ; Receptors, Transferrin ; analysis ; genetics

OBJECTIVETo study trend of dynamic change in level of serum transferrin receptor (sTfR) in the process of iron supplementation to provide evidence for sTfR in evaluating the efficacy of iron supplementation.

METHODSTotally, 942 child-bearing-age women aged 18 to 45 years were selected from Longfang City, Hebei Province and Shunyi County, Beijing. Biochemical indicators of iron metabolism were measured for all of them, including serum levels of ferritin (SF) and zinc protoporphyrin (ZPP), and hemoglobin (Hb). According to the current criteria for assessing iron status, women were screened for iron deficiency erythropoiesis (IDE) or iron deficiency anemia (IDA). Seventy-two women agreed to participate in the study, and 59 of them finished whole dynamic observations with signed informed consent. Four capsules of ferrous L-threonate (containing 7 mg of iron element per capsule) were administered for women with IDE every other day and for women with IDA every day, respectively, for 12 weeks. Serum biochemical indicators and level of sTfR were measured in 0 wk, 3 wk, 6 wk, 9 wk and 12 wk, respectively, during the process of iron supplementation, and their dynamic changes were observed.

RESULTSLevel of sTfR in women with IDE and IDA was (26.62 +/- 10.57) nmol/L and (41.25 +/- 21.96) nmol/L, respectively, significantly higher than normal level. During the process of iron supplementation, level of sTfR changed as the following characteristics. In women with IDE, level of sTfR kept stable within the first 3 weeks of iron supplementation, then dropped gradually and progressively, reached to normal, with (17.86 +/- 5.57) nmol/L, in the 12 wk after iron supplementation. In women with IDA, level of sTfR dropped quickly within the first 3 wk of iron supplementation, then dropped slowly until the 9th wk and kept stable, and reached to normal level in the 12 wk, with (19.54 +/- 5.94) nmol/L and a ratio of sTfR/SF of 12.23 +/- 4.34. Ratio of sTfR/SF changed as level of sTfR during the process of iron supplementation. Level of sTfR correlated reversely with levels of Hb and SF and positively with level of ZPP.

CONCLUSIONSerum level of sTfR in child-bearing age women gradually decreased to normal with the restoration of their normal iron status during the process of iron supplementation and could be used as a specific indicator for assessing efficacy of iron supplementation.

Adolescent ; Adult ; Anemia, Iron-Deficiency ; blood ; drug therapy ; Biomarkers ; blood ; Dietary Supplements ; Female ; Hemoglobins ; analysis ; Humans ; Iron ; administration & dosage ; therapeutic use ; Middle Aged ; Protoporphyrins ; blood ; Receptors, Transferrin ; blood

OBJECTIVETo explore the isolation, purification and expansion of human umbilical cord blood mesenchymal stem cells (MSCs) into neuron-like cells in vitro.

METHODSHuman cord blood samples were obtained sterilely with 20 U/ml preservative-free heparin. MSCs were isolated by lymphocyte separation medium (density 1.077 g/ml), and purified and expanded with Mesencult trade mark medium. The surface antigen expression of MSCs was detected by flow cytometry. The passage 2, 5 and 8 of the expanded MSCs were induced to differentiate to neuron-like cells. Specific markers and structures were detected by immunohistochemistry and histochemistry methods.

RESULTSThe number of MSCs increased two- to three-fold with each expanded passage. 6.6 x 10(5) primary MSCs were expanded ten passages to reach a number of 9.9 x 10(8), and was increased about 1.5 x 10(3)-fold. Flow cytometry showed that MSCs did not express antigens CD(34), CD(11a) and CD(11b), but expressed strongly CD(29) and weakly CD(71), which was identical to human bone marrow-derived MSCs. 70% cells exhibited typical neuron-like phenotype after induction. Immunohistochemistry staining showed that all of the induced different-passage MSCs expressed neurofilament (NF) and neuron-specific enolase (NSE). Special Nissl body was found by histochemistry.

CONCLUSIONMSCs in human umbilical cord blood can expand in vitro and differentiate into non-mesenchymal cells.

Antigens, CD ; analysis ; Antigens, Differentiation, B-Lymphocyte ; analysis ; Cell Count ; Cell Differentiation ; Cell Division ; Fetal Blood ; cytology ; Flow Cytometry ; Humans ; Immunohistochemistry ; Integrin beta1 ; analysis ; Mesoderm ; chemistry ; cytology ; Neurofilament Proteins ; analysis ; Neurons ; cytology ; Phosphopyruvate Hydratase ; analysis ; Receptors, Transferrin ; Stem Cells ; chemistry ; cytology