Predisposition of genetic disease by modestly decreased expression of GCH1 mutant allele

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.

Increased lysine N-methylation of a 23-kDa protein during hepatic regeneration

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 C18 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.

A Family with A Missense Mutation in the SCN5A Gene

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 electrocardiogram.

Detection and Typing of HSV-1, HSV-2, CMV and EBV by Quadruplex PCR

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.

Cysteine carboxyl O-methylation of human placental 23 kDa protein

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.

Protein methylation in cellular proliferation and differentiation: Non-histone nuclear methyl acceptor protein(s) during 3'-methyl-4-dimethylaminoazobenzeneinduced hepatocarcinogenesis

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 (1)Gly-Val-Pro-Leu-(5)X-Arg-Leu-Phe-Asp-(10)His-Ala-Met-Leu-Gln-(15)Ala -His-Arg-Ala-His-(20)Glu, having 94.7% sequence homology with human chorionic somatomammotropin precursor A and B.

General Anesthetics and Norepinephrine and Epinephrine in Plasma / 대한마취과학회지

This was done to measure the plasma catecholamine concentration esp. norepinephrine and epinephrine in the volunteers and in the operated patents after administration of thiopental sodium, after administration of pancuronium and after the inhalation of halothane. Also studied was the interrelation between blood pressure and pulse rate and catecholamine. Ten healthy subjects were studied under general anesthesia. When compared with the results from patients in the operating room. 1) In the operating room, plasma norepinephrine levels increased 55+/-7.2. pg/ml; The increase int plasma epinephrine was not statistically significant: The increases of the systolic and diastolic pressure were significant : The increase in heart rate was not significant. 2) After the administration of thiopental sodium, plasma concentration of norepinphrine was not significantly increased: the change of plasma eninephrine was not significantly decreased: the increase in systolic and diastolic pressure was less marked: the increase in pulse rate was significant. 3) After the administration of pancuronium, plasma norepinephrine levels were not increased significanty: the decrease in plasma epinephrine was not significant: the increase of systolic pressure was not significant: the diastolic pressure and pulse rate were significant. 4) After the inhalation of halothane, plasma norepinephrine and epinephrine concentration increased significant and the change of the pulse rate was significantly increased.

A Study of Changes ia Norepinephrine and Epiaephrine during Endotracheal Intubation / 대한마취과학회지

This work was done to measure the plasma concentration of catecholamines esp. norepinephrine and epineparine after eadotracheal intubation and so study the interrelation between the blood pressure and pulse rate and catecholamines. All seven patients studied were normotensive men with general anesthesia including eadotracheal intubation, systolic blood pressure, diastolic blood pressure and pulse rate were increased to 36+/-4.72 torr, 31+/-5.71 torr, and 34+/-6.23min. respectively. Norepinephrine and epinsphrine were also increased to 165+/-28.55pg/ml and 88+/-27.97pg/ml respectively. Those results show that the rise of blood pressure and pulse rate after endotracheal intubation may result from increased catecholamines.