Preparation of a pure autoantigen by way of recombinant DNA technology has an important value in an accurate diagnosis or prognosis of an autoimmune disease. BCOADC-E2 subunit, a mitochondrial protein, has been known to be the autoantigen of primary biliary cirrhosis (PBC), a chronic autoimmune liver disease, as well as idiopathic dilated cardiomypathy (IDCM), a chronic autoimmune heart disease. Recombinant form of this molecule had been expressed in E. coli but with low yield and severe degradation. Furthermore, sera from IDCM patients failed to recognized BCOADC-E2 molecule produced in prokaryotic expression system. In this study, a recombinant bovine BCOADC-E2 fusion protein has been expressed in insect cells using baculovirus expression system and analyzed anti-BCOADC-E2 reactivity in sera from patients with PBC or with IDCM. Optimal production of the recombinant fusion protein has been achieved at 20 multiplicity of infection (MOI), and the protein was affinity-purified using metal-binding resins. The affinity-purified BCOADC-E2 protein was successfully recognized by sera from PBC patients, but not by sera from IDCM patients suggesting that the different auto-immune response against BCOADC-E2 is needed to be elucidated in terms of epitope recognition.
Acetyltransferases/metabolism ; Acetyltransferases/immunology ; Acetyltransferases/genetics* ; Animal ; Baculoviridae/genetics ; Cardiomyopathy, Congestive/immunology ; Cattle ; Human ; Immune Sera ; Insects/cytology* ; Ketone Oxidoreductases/metabolism ; Ketone Oxidoreductases/immunology* ; Ketone Oxidoreductases/genetics* ; Liver Cirrhosis, Biliary/immunology ; Multienzyme Complexes/metabolism ; Multienzyme Complexes/immunology* ; Multienzyme Complexes/genetics* ; Protein Engineering/methods ; Recombinant Proteins/isolation & purification ; Recombinant Proteins/immunology ; Recombinant Proteins/genetics*

OBJECTIVETo evaluate the roles of various cytokines, histone acetyltransferase (HAT) and histone deacetylase (HDAC) in the pathogenesis of bronchial asthma.

METHODSBALB/C mice were randomly assigned to control, untreated asthma, hormone treatment and TSA treatment groups. Bronchial asthma was induced by intraperitoneal injections and atomization inhalation of ovalbumin (OVA) in the asthma, hormone treatment and trichostatin (TSA) treatment groups. The mice in the hormone treatment and TSA treatment groups were administered with dexamethasone 1.0 mg/kg and TSA 1.0 mg/kg respectively by an intraperitoneal injection 30 minutes before challenge of asthma. At 24 hours after the last challenge, IL-4, IL-8 and IFN- levels in serum were measured using ELISA, and activities of HAT and HDAC in peripheral blood mononuclear cells (PBMC) were determined by the enzyme linked immunofluorescence assay.

RESULTSThe serum levels of IL-4 and IL-8 in the untreated asthma group were higher than in the control, hormone treatment and TSA treatment groups (P<0.05). There was no difference in the serum levels of IL-4 and IL-8 among the control, hormone treatment and TSA treatment groups (P>0.05). The activity of HDAC in the untreated asthma group was lower than in the control, hormone treatment and TSA treatment groups (P<0.05). Hormone treatment significantly decreased the activity of HAT compared with the untreated asthma group (P<0.05). There was no difference in the activities of HAT and HDAC among the control, hormone treatment and TSA treatment groups (P>0.05).

CONCLUSIONSThe decreased activity of HDAC leads to an increased secretion of inflammatory factors and thus induces asthma.

Animals ; Asthma ; enzymology ; etiology ; immunology ; Cytokines ; blood ; Histone Acetyltransferases ; physiology ; Histone Deacetylases ; physiology ; Male ; Mice ; Mice, Inbred BALB C ; Th1 Cells ; immunology ; Th2 Cells ; immunology

Yeast Elongation protein 3 (yElp3), the catalytic subunit of the multi-subunit histone acetyltransferase elongator complex, is involved in histone acetylation and transcription, exocytosis and tRNA modification. To study the complex function of yElp3 in yeast, we amplified the yElp3 gene fragment encoding 73aa in the N-terminal from plasmid pYES2-yElp3, and then cloned it into pMXB10 to construct the recombinant plasmid pMXB10-yElp3-219. We expressed the fusion protein in E. coli BL21 (DE3), then purified it by chin affinity column, and finally obtained the soluble purified protein (8.0 kD), which was used to immune the rabbits for acquiring antiserum. ELISA and Western blotting indicated that the polyclonal antibody was of high titration and specificity. Chromatin immunoprecipitation (ChIP) assay with this antibody suggested that yhElp3 exerted the transcriptional regulatory function directly through its presence on the SSA3 gene; this might be the reason that it can rescue the delay activation of SSA3 in elp3delta cells.
Amino Acid Sequence ; Antibodies ; analysis ; immunology ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Fungal ; Histone Acetyltransferases ; biosynthesis ; genetics ; immunology ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Saccharomyces cerevisiae Proteins ; biosynthesis ; genetics ; immunology

Human arrest defective 1(hARD1) is an acetyltransferase catalyzing the N-terminal acetylation of proteins after translation. The high expression of hARD1 could be an indicator of the breast cancer. In current study, we produced an anti-hARD lp monoclonal antibody that could specifically recognize ARD1 in breast cancer tissues by using the immunohistochemical assay. The full-length His-tag hARD1 protein (1-235 aa) was over-expressed in Escherichia coli, and purified recombinant protein was injected into Balb/c mice to perform immunization procedure. Eight stable positive monoclonal cell lines were isolated. ELISA results demonstrated that all light chains of antibodies were kappa, and the heavy chains displayed three subtypes IgG1, IgG2a and IgG2b, respectively. A monoclonal antibody, which could specifically recognize hARD1 protein in breast cancer tissues, was identified by screening different cancer tissues using antibody-specificity method. Further, the specificity of the antibody was confirmed by Western blotting analysis. Our study would facilitate breast cancer diagnosis by using this ARD1 monoclonal antibody in clinic. Also, this antibody could be used as an important tool for further investigating the role of ARD1 in tumorigenesis.
Acetyltransferases ; genetics ; immunology ; Animals ; Antibodies, Monoclonal ; biosynthesis ; genetics ; immunology ; Biomarkers, Tumor ; biosynthesis ; immunology ; Breast Neoplasms ; immunology ; metabolism ; pathology ; Escherichia coli ; genetics ; metabolism ; Female ; Humans ; Immunization ; Mice ; Mice, Inbred BALB C ; N-Terminal Acetyltransferase A ; N-Terminal Acetyltransferase E ; Recombinant Proteins ; biosynthesis ; genetics ; immunology

Mucopolysaccharidosis (MPS) III has 4 enzymatically distinct forms (A, B, C, and D), and MPS IIIC, also known as Sanfilippo C syndrome, is an autosomal recessive lysosomal storage disease caused by a deficiency of heparan acetyl-CoA:alpha-glucosaminide N-acetyltransferase (HGSNAT). Here, we report a case of MPS IIIC that was confirmed by molecular genetic analysis. The patient was a 2-yr-old girl presenting with skeletal deformity, hepatomegaly, and delayed motor development. Urinary excretion of glycosaminoglycan (GAG) was markedly elevated (984.4 mg GAG/g creatinine) compared with the age-specific reference range (<175 mg GAG/g creatinine), and a strong band of heparan sulfate was recognized on performing thin layer chromatography. HGSNAT enzyme activity in leukocytes was 0.7 nmol/17 hr/mg protein, which was significantly lower than the reference range (8.6-32 nmol/17 hr/mg protein). PCR and direct sequencing of the HGSNAT gene showed 2 mutations: c.234+1G>A (IVS2+1G>A) and c.1150C>T (p.Arg384*). To the best of our knowledge, this is the first case of MPS IIIC to be confirmed by clinical, biochemical, and molecular genetic findings in Korea.
Acetyltransferases/*genetics ; Asian Continental Ancestry Group/*genetics ; Base Sequence ; Child, Preschool ; Chromatography, Thin Layer ; Female ; Glycosaminoglycans/urine ; Heparitin Sulfate/chemistry/metabolism ; Humans ; Leukocytes/immunology/metabolism ; Mucopolysaccharidosis III/*diagnosis/genetics/radiography ; Mutation ; Republic of Korea ; Sequence Analysis, DNA

Human arrest defective 1 (hARD1) is an acetyltransferase; its physiological significance remains unclear. To explore the relationship between ARD1 protein and tumors, we detected the hARD1 protein in tumor tissues in vivo. We cloned hARD1 gene from Hela cell and construct recombinant plasmid pET28b-hARD1. The recombinant plasmid was transformed into E. coli BL21 (DE3)plysS. hARD1 protein was expressed by inducing with IPTG(1 mmol/L) and purified up to 95% through Ni2+ chelation affinity chromatography. We used the purified hARD1 protein as antigen immunized the Balb/c mice and obtained the hARD1 specific polyclonal antiserum. Through immunohistochemical analysis of different tumor tissues in vivo, we found that hARD1 expressed at high frequency in breast cancer, prostate cancer and lung cancer, especially, hARD1 expression frequency in breast cancer was up to 70%, which is higher than in the other tumors. These results indicate that the high expression level of hARD1 could be an indicator of the breast cancer. This new finding would be a foundation to further explore the relationship between breast tumor and hARD1.
Acetyltransferases ; analysis ; genetics ; immunology ; Amino Acid Sequence ; Animals ; Antibodies ; blood ; immunology ; Base Sequence ; Biomarkers, Tumor ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Female ; Humans ; Immune Sera ; biosynthesis ; Immunization ; Immunohistochemistry ; Lung Neoplasms ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; N-Terminal Acetyltransferase A ; N-Terminal Acetyltransferase E ; Prostatic Neoplasms ; metabolism ; pathology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology