nc886 (=vtRNA2-1, pre-miR-886, or CBL3) is a newly identified non-coding RNA (ncRNA) that represses the activity of protein kinase R (PKR). nc886 is transcribed by RNA polymerase III (Pol III) and is intriguingly the first case of a Pol III gene whose expression is silenced by CpG DNA hypermethylation in several types of cancer. PKR is a sensor protein that recognizes evading viruses and induces apoptosis to eliminate infected cells. Like viral infection, nc886 silencing activates PKR and induces apoptosis. Thus, the significance of the nc886:PKR pathway in cancer is to sense and eliminate pre-malignant cells, which is analogous to PKR's role in cellular innate immunity. Beyond this tumor sensing role, nc886 plays a putative tumor suppressor role as supported by experimental evidence. Collectively, nc886 provides a novel example how epigenetic silencing of a ncRNA contributes to tumorigenesis by controlling the activity of its protein ligand.
Apoptosis ; Carcinogenesis ; DNA ; Epigenomics ; Immunity, Innate ; Protein Kinases ; RNA Polymerase III ; RNA, Untranslated*

OBJECTIVE: The individual cascades of the insulin-like growth factor-1 (IGF-1) signaling pathway and the molecular mechanism of aging have not been fully clarified. In the current study, we explored the effect of DNA polymerase delta 1 (POLD1) on the IGF-1 signaling pathway in cell aging. METHODS: First, we analyzed the relationship between IGF-1 and POLD1 expression in aging. To investigate the effect of IGF-1 on POLD1 expression and aging, the 2BS cells were incubated with young-age or old-age human serum, IGF-1 protein, or linsitinib. Next, the effect of IGF-1 on aging was examined in the 2BS cells with increased or decreased POLD1 expression to clarify the molecular mechanism. RESULTS: In this study, we found that IGF-1 expression increased and POLD1 expression decreased with aging in human serum and hippocampal tissues of SAMP8 mice, and a negative relationship between IGF-1 and POLD1 expression was observed. Furthermore, the cells cultured with old-age human serum or IGF-1 showed lower POLD1 expression and more pronounced senescence characteristics, and the effect could be reversed by treatment with linsitinib or overexpression of POLD1, while the effect of linsitinib on cell aging could be reversed with the knockdown of POLD1. CONCLUSION Taken collectively, our findings demonstrate that IGF-1 promotes aging by binding to IGF-1R and inhibiting the expression of POLD1. These findings offer a new target for anti-aging strategies.
Humans ; Animals ; Mice ; Insulin-Like Growth Factor I/pharmacology* ; Cellular Senescence ; Aging ; Hippocampus ; DNA Polymerase III

OBJECTIVETo investigate the antithrombin (AT) activity (AT: A) and AT antigen (AT: Ag) level in a Chinese family with type I antithrombin (AT) deficiency, and to explore the molecular mechanism of AT deficiency.

METHODSImmuno-nephelometry and chromogenic assay were used to detect the plasma level of AT: A and AT: Ag, respectively. Genomic DNA was isolated from the peripheral blood, and all the seven exons and exon-intron boundaries of AT gene were amplified by PCR and direct sequencing.

RESULTSThe plasma levels of AT: A and AT: Ag of the proband were 45% and 97 mg/L, respectively, which led to a type I AT deficiency. A heterozygous T to A mutation was found at nucleotide 9833 in exon 5 resulting in a Tyr363Stop nonsense mutation. The sequencing results from the pedigree indicated that four other members also had this mutation.

CONCLUSIONThis heterozygous nonsense mutation of T9833A in exon 5 resulting in venous thrombosis is a novel genetic defect of hereditary AT deficiency, which has not been described before.

Antithrombin III Deficiency ; genetics ; Antithrombins ; genetics ; Blood Coagulation Tests ; Female ; Humans ; Male ; Mutation ; Pedigree ; Polymerase Chain Reaction ; Sequence Analysis, DNA

BACKGROUND: Coronary artery spasm is an important mechanism in producing myocardial ischemia. But the exact mechanism of the spasm is not well known. We investigated the mutation of endothelial nitric oxide synthase (eNOS) that produce nitric oxide and relationship between eNOS mutation and coronary artery spasm. MATERIALS AND METHODS: Blood were drawn from the patients with angiographically proven coronary artery spasm and normal controls. DNA were extracted and polymerase chain reaction and restriction analysis with Nae I were performed to find T-786-- Cholesterol ; Chungcheongnam-do ; Coronary Vessels* ; DNA ; Female ; Humans ; Incidence* ; Korea ; Male ; Myocardial Ischemia ; Nitric Oxide ; Nitric Oxide Synthase Type III ; Polymerase Chain Reaction ; Prevalence ; Smoke ; Smoking ; Spasm* ; Triglycerides

BACKGROUND: Coronary artery spasm is an important mechanism in producing myocardial ischemia. But the exact mechanism of the spasm is not well known. We investigated the mutation of endothelial nitric oxide synthase (eNOS) that produce nitric oxide and relationship between eNOS mutation and coronary artery spasm. MATERIALS AND METHODS: Blood were drawn from the patients with angiographically proven coronary artery spasm and normal controls. DNA were extracted and polymerase chain reaction and restriction analysis with Nae I were performed to find T-786-- Cholesterol ; Chungcheongnam-do ; Coronary Vessels* ; DNA ; Female ; Humans ; Incidence* ; Korea ; Male ; Myocardial Ischemia ; Nitric Oxide ; Nitric Oxide Synthase Type III ; Polymerase Chain Reaction ; Prevalence ; Smoke ; Smoking ; Spasm* ; Triglycerides

Genome duplication in E. coli is carried out by DNA polymerase III, an enzyme complex consisting of ten subunits. Investigations of the biochemical and structural properties of DNA polymerase III require the expression and purification of subunits including α, ge, θ, γ, δ', δ, and β separately followed by in vitro reconstitution of the pol III core and clamp loader. Here we propose a new method for expressing and purifying DNA polymerase III components by utilizing a protein co-expression strategy. Our results show that the subunits of the pol III core and those of the clamp loader can be coexpressed and purified based on inherent interactions between the subunits. The resulting pol III core, clamp loader and sliding clamp can be reconstituted effectively to perform DNA polymerization. Our strategy considerably simplifies the expression and purification of DNA polymerase III and provides a feasible and convenient method for exploring other multi-subunit systems.
Cloning, Molecular ; DNA Polymerase III ; chemistry ; genetics ; metabolism ; DNA Replication ; DNA, Bacterial ; biosynthesis ; genetics ; Escherichia coli ; enzymology ; genetics ; Plasmids ; metabolism ; Polymerization ; Protein Engineering ; methods ; Protein Subunits ; chemistry ; genetics ; metabolism ; Recombinant Proteins ; chemistry ; genetics ; metabolism

OBJECTIVETo analyze sequences of the housekeeping genes including recA, dnaE, and mdh in different serogroups or different biotypes of Vibrio cholerae (VC) strains isolated from China.

METHODS AND RESULTSrecA, dnaE, and mdh genes of Vibrio cholerae were obtained by PCR, sequenced and analyzed. Forty-four variable bases were identified in the 500 bases of recA gene of 18 VC strains, and the mutation of only 3 variable bases could result in changes of 2 amino acids. In the 600 bases of dnaE genes of 18 strains, 32 variable bases were found and only 1 contributed to an amino acid change. In the 367 bases of mdh genes of 18 strains, only 1 variable base was identified whose mutation involved an amino acid convertion. Toxic EI Tor biotype (EVC) strains and toxic O139 serogroup strains were closely related. Non-toxic strains of different serogroups or types were lowly related. Non-toxic and toxic strains of different serogroups or types were lowly related.

CONCLUSIONToxic EVC and toxic O139 serogroup strains isolated from different areas of China may evolve from a common original strain, and toxic O139 VC strains may come from toxic EVC strains.

Bacterial Proteins ; genetics ; Base Sequence ; China ; DNA Polymerase III ; genetics ; Gene Expression Profiling ; Humans ; Molecular Sequence Data ; Phylogeny ; Rec A Recombinases ; genetics ; Sequence Analysis, DNA ; Sequence Homology, Nucleic Acid ; Vibrio cholerae ; classification ; genetics ; isolation & purification

BACKGROUND: The safety of plasma derivatives has been reinforced since 1980s by variable pathogen inactivation or elimination techniques. Nucleic acid amplification test (NAT) for the source plasma has also been implemented worldwide. Recently nanofiltration has been used in some country for ensuring safety of plasma derivatives to eliminate non-enveloped viruses such as parvovirus B19 (B19V) and hepatitis A virus (HAV). We evaluated the efficacy of nanofiltration for the elimination of B19V and HAV. METHODS: To verify the efficacy of nanofiltration, we adopted a 20 nm Viresolve NFP (Millipore, USA) in the scaling down (1:1,370) model of the antithrombin III production. As virus stock solutions, we used B19V reactive plasma and porcine parvovirus (PPV) and HAV obtained from cell culture. And 50% tissue culture infectious dose was consumed as infectious dose. The methods used to evaluate the virus-elimination efficacy were reverse-transcriptase polymerase chain reaction for B19V and the cytopathic effect calculation after filtration for PPV and HAV. RESULTS: B19V was not detected by RT-PCR in the filtered antithrombin III solutions with initial viral load of 6.42x10(5) IU/mL and 1.42x10(5) IU/mL before filtration. The virus-elimination efficacy of nanofiltration for PPV and HAV were > or =10(3.32) and > or =10(3.31), respectively. CONCLUSIONS: Nanofiltration would be an effective method for the elimination of B19V and HAV. It may be used as a substitute for NAT screening of these viruses in source plasma to ensure safety of plasma derivatives in Korea.
Antithrombin III/isolation & purification ; DNA, Viral/analysis ; Filtration/*methods ; Hepatitis A virus/genetics/*isolation & purification ; Humans ; Nanotechnology/*methods ; Parvovirus B19, Human/genetics/*isolation & purification ; RNA, Viral/analysis ; Reverse Transcriptase Polymerase Chain Reaction

Mucopolysaccharidosis (MPS) IIIB is a lysosomal storage disorder (LSD) caused by abnormalities of the enzyme alpha-N-acetylglucosaminidase (NAGLU) that is required for degradation of heparan sulfate. The patient in this study was a 4-yr-old boy. He presented with normal height and weight, pectus carinatum, and multiple persistent Mongolian spots on his back. He had mild dysmorphic features with prominent speech developmental delays and, to a lesser extent, motor developmental delays. The cetylpyridinium chloride precipitation test revealed excessive mucopolysacchariduria (657.2 mg glycosaminoglycan/g creatinine; reference range, <175 mg glycosaminoglycan/g creatinine). Thin layer chromatography showed urinary heparan sulfate excretion. NAGLU enzyme activity was significantly decreased in leukocytes (not detected; reference range, 0.9-1.51 nmol/hr/mg protein) as well as in plasma (0.14 nmol/hr/mg protein; reference range, 22.3-60.9 nmol/hr/mg protein). PCR and direct sequencing analysis of the NAGLU gene showed that the patient was a compound heterozygote for 2 mutations: c.200T>C (p.L67P) and c.1444C>T (p.R482W). The c.200T>C mutation was a novel finding. This is the first report of a Korean patient with MPS IIIB who was confirmed by molecular genetic analyses and biochemical investigation.
Acetylglucosaminidase/blood/*genetics ; Alleles ; Asian Continental Ancestry Group/*genetics ; Child, Preschool ; Chromatography, Thin Layer ; Heterozygote ; Humans ; Leukocytes/metabolism ; Male ; Mucopolysaccharidosis III/diagnosis/*genetics ; Mutation ; Polymerase Chain Reaction ; Republic of Korea ; Sequence Analysis, DNA

OBJECTIVETo evaluate the effects and possible mechanisms of rutaecarpine on angiotensin II (Ang II)-induced proliferation in cultured rat vascular smooth muscle cells (VSMCs).

METHODSVSMCs were isolated from Male Sprague-Dawley rat aorta, and cultured by enzymic dispersion method. Experiments were performed with cells from passages 3-8. The cultured VSMCs were randomly divided into control, model (Ang II 0.1 μmol/L), and rutaecarpine (0.3-3.0 μmol/L) groups. VMSC proliferation was induced by Ang II, and was evaluated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and cell counting. To examine the mechanisms involved in anti-proliferative effects of rutaecarpine, nitric oxide (NO) levels and NO synthetase (NOS) activity were determined. Expressions of VSMC proliferation-related genes including endothelial nitric oxide synthase (eNOS), and c-myc hypertension related gene-1 (HRG-1) were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSRutaecarpine (0.3-3.0 μmol/L) inhibited Ang II-induced VSMC proliferation and the best effects were achieved at 3.0 μmol/L. The Ang II-induced decreases in cellular NO contents and NOS activities were antagonized by rutaecarpine (P <0.05). Ang II administration suppressed the expressions of eNOS and HRG-1, while increased c-myc expression (P <0.05). All these effects were attenuated by 3.0 μmol/L rutaecarpine (P <0.05).

CONCLUSIONRutaecarpine is effective against Ang II-induced rat VSMC proliferation, and this effect is due, at least in part, to NO production and the modulation of VMSC proliferation-related gene expressions.

Angiotensin II ; pharmacology ; Animals ; Base Sequence ; Cell Proliferation ; drug effects ; Cells, Cultured ; DNA Primers ; Hemeproteins ; metabolism ; Indole Alkaloids ; pharmacology ; Male ; Muscle, Smooth, Vascular ; cytology ; drug effects ; Nitric Oxide Synthase Type III ; metabolism ; Proto-Oncogene Proteins c-myc ; metabolism ; Quinazolines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction