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The methylation of a 23-kDa nuclear protein increased after partial hepatectomy and methylation returned to basal levels after the initial stage of regeneration. The methylating enzyme was partially purified from rat liver by ammonium sulfate precipitation, DEAE-anion exchange chromatography and Butyl-Sepharose chromatography. The 23-kDa protein was purified from a nuclear fraction of liver tissue with SP-Sepharose. When the 23-kDa protein was methylated with the partially purified methyltransferase and analyzed on C(18) high performance liquid chromatography (HPLC), the methylated acceptor amino acid was monomethyl lysine (MML). Previously, only arginine N-methylation of specific substrate proteins has been reported during liver regeneration. However, in this report, we found that lysine N-methylation increased during early hepatic regeneration, suggesting that lysine N-methylation of the 23-kDa nuclear protein may play a functional role in hepatic regeneration. The methyltransferase did not methylate other proteins such as histones, hnRNPA1, or cytochrome C, suggesting the enzyme is a 23-kDa nuclear protein- specific lysine N-methyltransferase.

Purpose: To identify a new tumor-associated antigen, a monoclonal antibody, SC142, was produced by immunizing mice with a stomach cancer cell line. The tumor specificity of mAb SC142 was studied by immunohistochemical staining, and the biochemical characteristics of this new gastrointestinal tumor-associated antigen were also studied. Methods: The expression of SC142-reactive antigen was investigated in various cancers by immunohistochemical staining. The SC142-reactive antigen was characterized by immunoblotting, sodium metaperiodate treatment assay, O-glycanase digestion assay, and lectin binding assay. Results: The SC142-reactive antigen was highly expressed in 78% of gastric cancers (29/37) and 87% of colon cancers (27/31). No normal colon or stomach tissues remote from the tumor were positive for the antigen. The antibody also reacted with other tumors of epithelial origin such as lung squamous cell cancer (2/4), breast ductal cancer (2/20), bladder transitional cell carcinoma (4/6), and uterine cancer (3/16). Western blot analysis of the antigen revealed glycoprotein(s) which migrated as a smear ranging from the origin of the gel to about the 80 kDa region. The reactivity of this antigen with SC142 was reduced by sodium metaperiodate treatment or O-glycanase digestion, but not by N-glycanase, suggesting that the epitope is an O-glycan. In lectin-binding assay, this antigen reacted only with wheat germ agglutinin but not with Ricinus communis agglutinin, Datura stramonium agglutinin, and Sambucus nigra agglutinin. Conclusions: Our findings indicate that the antigen defined by SC142 is a tumor-associated antigen that could differentiate the gastrointestinal cancer cells from the normal cells. Therefore, SC142 may become a valuable tool for the immunohistochemical diagnosis and tumor immunoscintigraphy of the gastrointestinal cancer patients.

An accelerating effect of methyl-deficient diet (MDD) on hepatocarcinogenesis and methylation pattern of nuclear protein(s) by S-adenosylmethionine: protein arginine N-methyltransferase (protein methylase I, PM-I) have been studied with 3'-methyl-4-dimethyl- aminoazobenzene(MeDAB)-treated rats. The MDD+MeDAB-fed group produced typical cancer cells in the liver almost two weeks earlier than the control synthetic diet (CSD)+MeDAB-fed group. Protein methylase I (PM-I) activity in the livers of MDD alone fed rats began to increase at around 2 weeks after MDD-feeding, reaching a peak at 4 weeks and declining thereafter. When nuclei isolated either from normal livers or from cholangiocarcinoma cells were incubated with PM-I preparation from normal liver, 16 and 23-kDa nuclear proteins were the major methylated proteins, regardless of the source of the nuclei. However, when the above mentioned nuclei were incubated with PM-I preparations either from MDD alone fed livers or MDD+ MeDAB-induced cholangiocarcinoma cells, the methylation of 23-kDa protein was not detected. The result suggests that there is a hitherto-unknown PM-I specific to 23 kDa nuclear protein which was lost during methyl deficient diet feeding and hepatocarcinogenesis. The N-terminal 20 amino acids sequence of the 23-kDa protein was found to be ¹Gly-Val-Pro-Leu-?X-Arg-Leu-Phe-Asp-¹?His-Ala-Met-Leu-Gln-¹?Ala-His-Arg-Ala-His-²?Glu, having 94.7% sequence homology with human chorionic somatomammotropin precursor A and B.

Allele frequencies for the 10 STR loci, D6S1043, D9S925, D7S821, D4S2368, D21S2055, GATA193A07, D12S391, D10S2326, D15S822 and D18S51 were obtained from a sample of 217-310 unrelated Koreans. In this study, 2 out of the 10 loci did not meet Hardy-Weinberg expectation. The combined probability of identity for 10 loci tested was 4.93 x 10(-14).

Park, H., Hong, K-M., Sakanari, J. A., Choi, J-H., Park, S-K., Kim, K-Y., Hwang, H-A., Paik, M-K., Yun, K-J., Shin, C-H., Lee, J-B., Ryu, J-S., Min, D-Y. 2001. Paragonimus westermani: Cloning of a cathepsin F-like cysteine proteinase from the adult worm. Experimental Parasitology98, 223 –227.

We report a new nonsense mutation in the GTP cyclohydrolase I (GCH1) gene in a family with dopa-responsive dystonia. Two sisters and three children of the sisters are affected. The exons of the GCH1 gene were amplified by PCR and sequenced. The substitution of thymine for cytosine at nucleotide position 142 causing a nonsense mutation (Q48X) in exon 1 was identified in all of the five affected patients. There were three asymptomatic carriers of the mutation in the family.

DNA prenatal diagnosis was successfully performed on a family with citrullinemia. The father carried the G324S mutation and the mother carried the IVS6-2A > G mutation in the argininosuccinate synthase gene. They had a previous child with citrullinemia who died in the week after birth owing to complicated hyperammonemia. The lost child turned out to be a compound heterozygote. DNA was extracted from the cultured amniotic cells after amniocentesis done at 18-week gestation. For the detection of the G324S mutation, the PCR and restriction fragment length polymorphism method was used, and for the IVS6-2A > G mutation, allele-specific PCR was performed. The fetus was found to carry G324S but not IVS6-2A > G, suggesting a heterozygote carrier. Pregnancy was continued and a healthy boy was born. Plasma amino acid analysis performed on the third day after birth was normal and the serial ammonia level was in the normal range. A molecular study on his genomic DNA after birth also agreed with the previous fetal DNA analysis. He is now 2-months old with normal growth and development.

C-Terminal carboxyl methylation of a human placental 23 kDa protein catalyzed by membrane-associated methyltransferase has been investigated. The 23 kDa protein substrate methylated was partially purified by DEAE-Sephacel, hydroxyapatite and Sephadex G-100 gel filtration chromatographies. The substrate protein was eluted on Sephadex G-100 gel filtration chromatography as a protein of about 29 kDa. In the absence of Mg2+, the methylation was stimulated by guanine nucleotides (GTP, GDP and GTPgammaS), but in the presence of Mg2+, only GTPgammaS stimulated the methylation which was similar to the effect on the G25K/rhoGDI complex. AFC, an inhibitor of C-terminal carboxyl methylation, inhibited the methylation of human placental 23 kDa protein. These results suggests that the substrate is a small G protein different from the G25K and is methylated on C-terminal isoprenylated cysteine residue. This was also confirmed by vapor phase analysis. The methylated substrate protein was redistributed to membrane after in vitro methylation, suggesting that the methylation of this protein is important for the redistribution of the 23 kDa small G protein for its putative role in intracellular signaling.

To clarify the correlation of the proteinase activity with pathogenicity of Clonorrhis sinensis, the proteinase activity either in excretory-secretory products (ESP) or in crude extracts of adult C. sinensis was examined. Substrate gel electrophoresis of the ESP and crude extracts revealed four distinct enzyme bands, which were differently inhibited by the specific proteinase inhibitors. The proteinase of the ESP with molecular mass of 24 kDa, was purified 23-fold with 14.5% yield by spectra gel ACA 44 gel filtration. It exhibited optimal pH at 7.5 in sodium phosphate (0.1 M). Its activity was inhibited specifically by N-ethylmaleimide (NEMI and antipain whereas potentiated 1.9 folds in the presence of 5 mM dithiothreitol (DTT). Cytotoxicity of the proteinase increased in a dose- dependent manner up to 120 ㎍/ml while reduced by NEM and antipain, indicating that cysteine proteinase was responsible for the cytotoxicity. This result shows that the 24 kDa cysteine proteinase is deeply correlated with the pathogenicity of C. sinensis infection. Key words: ChDnorchis sinenis, cysteine proteinase, cytotoxicity

1. 1. Protein methylase I (S-adenosylmethionine[:]protein-arginine N-methyltransferase; EC 2.1.1.23) which methylates protein-bound arginine residues has been purified from human term placenta 400-fold with an approximate yield of 6%. 2. 2. When histone was used as in vitro substrate, the methylation products were found to be NG-mono-, NG, NG-di- and NG, N'G-dimethylarginine. The enzyme was found to be sensitive toward Cu2+ with Ki, value of 8 x lO-5M. The Km value for S-adenosyl-L-methionine was 5 x lO-6M. 3. 3. When this partially purified protein methylase I was incubated with isolated human placental nuclei and S-adenosyl-L-Imethyl-'Hlmethionine, the major endogenous [methyl-3H]-labeled proteins were protein species of 23,38,45 and 68 kDa, the 23 kDa species being the most predominant. 4. 4. The endogenous enzyme activity during the pregnancy increased significantly, reaching more than 4 times the initial activity at the end of term.

Recently it was shown that single nucleotide polymorphisms (SNPs) can explain individual variation because of the small changes of the gene expression level and that the 50% decreased expression of an allele might even lead to predisposition to cancer. In this study, we found that a decreased expression of an allele might cause predisposition to genetic disease. Dopa responsive dystonia (DRD) is a dominant disease caused by mutations in GCH1 gene. The sequence analysis of the GCH1 in a patient with typical DRD symptoms revealed two novel missense mutations instead of a single dominant mutation. Family members with either of the mutations did not have any symptoms of DRD. The expression level of a R198W mutant allele decreased to about 50%, suggesting that modestly decreased expression caused by an SNP should lead to predisposition of a genetic disease in susceptible individuals.

The development of a multiplex polymerase chain reaction (PCR) method for rapid and accurate detection and typing of herpes simplex virus type 1 (HSV-1), and type-2 (HSV-2), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) is very important for clinical diagnosis to allow the deliver of therapy as early as possible. Large scale amplifications by multiplex PCR of viral DNA can lower the cost and time for viral diagnosis. In this study, therefore sensitive quadruplex PCR was achieved by optimizing parameters such as primers, and 1.5 mM magnesium and 200 uM dNTPs concentrations. The concentrations of HSV-1, HSV-2, CMV and EBV primers were 0.5, 0.3, 0.25 and 0.25 pmoles, respectively. Optimal annealing temperature was 54 degrees C. Employing these conditions, we could detect 10 copies of reconstructed template plasmid DNA, which were cloned to vectors containing target sequences of viral DNA. PCR products of 271 bp for HSV-1, 231 bp for HSV-2, 368 bp for CMV, and 326 bp for EBV were separated on 5.0% polyacrylamide gel electrophoresis and confirmed by direct sequencing. The present study showed that the quadruplex PCR assay described herein has potential application in clinical diagnosis, when rapid, accurate detection and typing of viruses HSV-1, HSV-2, CMV or EBV are necessary.

Brugada syndrome, an autosomal dominantly inherited form of ventricular fibrillation, is characterized by ST- segment elevation in leads V1-3 and right bundle-branch block on surface electrocardiogram. It is caused by mutations in the cardiac sodium channel gene, SCN5A, and to the best of our knowledge, there has been no report of this mutation in Korea. Three members of a family were heterozygous for a G to T substitution at the nucleotide position 5851 in exon 28 of the SCN5A gene. This nucleotide alteration makes a missense mutation, leading to a valine to leucine substitution (V1951L), in the carboxy terminal region of the sodium channel a subunit. We report here a missense mutation in a Korean family with Brugada-type electrocar-diogram. (Korean Circulation J 2003;33(2):150-154)

1. 1. Protein methylase II was purified from human placenta approx. 8700-fold with a yield of 14%. 2. 2. Unlike protein methylase II from other sources, the activity of human placenta enzyme was completely inhibited by 2 mM Cu2+. Other divalent ions were without effect. 3. 3. Human chorionic gonadotropin (HCG), immunoglobulin A and calf thymus histones served as good in vitro substrates for the enzyme, particularly HCG. 4. 4. The Km for S-adenosyl- l -methionine and Ki for S-adenosyl- l -homocysteine were 2.08 × 10−6 and 5.8 × 10−7 M, respectively. 5. 5. The protein methylase II activity in human placenta changed with gestational age, the activity at 1st and 2nd trimester being approximately twice that of term placenta.