Kijeong Kim

Nucleotide change of codon 182 in the surface gene of hepatitis B virus genotype C leading to truncated surface protein is associated with progression of liver diseases

BACKGROUND & AIMS Hepatitis B virus (HBV) genotype C infection is associated with progression of hepatocellular carcinoma (HCC). Specific mutations of the HBV surface (S) gene have been reported to contribute to the development of HCC. In this study, novel nucleotide changes (sW182*) that result in a premature stop at codon 182 in the S gene of genotype C are investigated with regards to the development of HCC. METHODS A multi-probe real time PCR that enables rapid and reliable detection of sW182* was developed and applied to 292 DNA samples from Korean patients with diverse chronic liver diseases. RESULTS sW182* was detected in a total of 73 patients out of the 275 with positive amplification (26.5%). Its prevalence was significantly higher in patients with progressive forms of the disease (HCC and liver cirrhosis) than in patients with less severe forms of the disease (chronic hepatitis and carrier) [31.8% (56/176 patients) vs. 17.2% (17/99 patients); p=0.010]. In addition, an in vitro study using cell lines stable expressing the S protein with sW182* also strongly supported its relationship with HCC. CONCLUSIONS In the present study, we demonstrate that the sW182* of HBV could provide an important contribution to the progression of liver diseases, through molecular epidemiologic and in vitro studies.

Paired Bacillus anthracis Dps (Mini-ferritin) Have Different Reactivities with Peroxide

Dps (DNA protection during starvation) proteins, mini-ferritins in the ferritin superfamily, catalyze Fe2+/H2O2/O2 reactions and make minerals inside protein nanocages to minimize radical oxygen-chemistry (metal/osmotic/temperature/nutrient/oxidant) and sometimes to confer virulence. Paired Dps proteins in Bacillus, rare in other bacteria, have 60% sequence identity. To explore functional differences in paired Bacilli Dps protein, we measured ferroxidase activity and DNA protection (hydroxyl radical) for Dps protein dodecamers from Bacillus anthracis (Ba) since crystal structures and iron mineralization (iron-stain) were known. The self-assembled (200 kDa) Ba Dps1 (Dlp-1) and Ba Dps2 (Dlp-2) proteins had similar Fe2+/O2 kinetics, with space for minerals of 500 iron atoms/protein, and protected DNA. The reactions with Fe2+ were novel in several ways: 1) Ba Dps2 reactions (Fe2+/H2O2) proceeded via an A650 nm intermediate, with similar rates to maxi-ferritins (Fe2+/O2), indicating a new Dps protein reaction pathway, 2) Ba Dps2 reactions (Fe2+/O2 versus Fe2+/O2 + H2O2) differed 3-fold contrasting with Escherichia coli Dps reactions, with 100-fold differences, and 3) Ba Dps1, inert in Fe2+/H2O2 catalysis, inhibited protein-independent Fe2+/H2O2 reactions. Sequence similarities between Ba Dps1 and Bacillus subtilis DpsA (Dps1), which is regulated by general stress factor (SigmaB) and Fur, and between Ba Dps2 and B. subtilis MrgA, which is regulated by H2O2 (PerR), suggest the function of Ba Dps1 is iron sequestration and the function of Ba Dps2 is H2O2 destruction, important in host/pathogen interactions. Destruction of H2O2 by Ba Dps2 proceeds via an unknown mechanism with an intermediate similar spectrally (A650 nm) and kinetically to the maxi-ferritin diferric peroxo complex.

Reference genes for quantitative analysis on Clonorchis sinensis gene expression by real-time PCR

The accuracies of relative gene expressions as determined by quantitative real-time polymerase chain reaction are largely dependent on the variabilities of the reference genes used. Validation of the stabilities of reference genes under experimental conditions is an essential initial step for comparative studies on the expression levels of target genes in experimental groups. Using three total RNA samples extracted independently from Clonorchis sinensis metacercariae and adults, we determined the gene expression stabilities of eight reference gene candidates and the relative transcript levels of three target genes using the geNorm program. The reference genes found to be stably expressed in metacercariae and adults were phosphoglycerate kinase, β-actin, and calcyphosine; reference genes found to be stably expressed under γ-irradiated and non-irradiated conditions were succinate dehydrogenase, small nuclear ribonucleoprotein, and β-actin; and those stably expressed regardless of bile treatment were small nuclear ribonucleoprotein, phosphoglycerate kinase, and succinate dehydrogenase. According to our data, the expression levels of target genes are dependent on normalization factors, such as the CT values of single reference genes and the geometric mean of the CT values of three reference genes. When comparing C. sinensis gene expressions, we propose to employ the geometric mean of the CT values of more than three reference genes validated in the same experimental setting.

Combined effect of tumour necrosis factor‐α and interleukin‐13 polymorphisms on bronchial hyperresponsiveness in Korean children with asthma

Background TNF‐α and IL‐13, two pivotal pro‐inflammatory cytokines, are increased in asthmatic airways and may be linked to asthma susceptibility and/or bronchial hyperresponsiveness (BHR).

Development of a real‐time PCR‐based method for rapid differential identification of Mycobacterium species

Aims:  To develop a real‐time PCR method for rapid differential identification of many clinically important mycobacteria to the species level.

Development and Application of Multiprobe Real-Time PCR Method Targeting the hsp65 Gene for Differentiation of Mycobacterium Species from Isolates and Sputum Specimens

ABSTRACT We developed a multiprobe real-time PCR assay targeting hsp65 (HMPRT-PCR) to detect and identify mycobacterial isolates and isolates directly from sputum specimens. Primers and probes for HMPRT-PCR were designed on the basis of the hsp65 gene sequence, enabling the recognition of seven pathogenic mycobacteria, including Mycobacterium tuberculosis, M. avium, M. intracellulare, M. kansasii, M. abscessus, M. massiliense, and M. fortuitum. This technique was applied to 24 reference and 133 clinical isolates and differentiated between all strains with 100% sensitivity and specificity. Furthermore, this method was applied to sputum specimens from 117 consecutive smear-positive patients with smear results of from a trace to 3+. These results were then compared to those obtained using the rpoB PCR-restriction analysis method with samples from cultures of the same sputum specimens. The HMPRT-PCR method correctly identified the mycobacteria in 89 samples (76.0%, 89/117), and moreover, the sensitivity level was increased to 94.3% (50/53) for sputa with an acid-fast bacillus score equal to or greater than 2+. Our data suggest that this novel HMPRT-PCR method could be a promising approach for detecting pathogenic mycobacterial species from sputum samples and culture isolates routinely in a clinical setting.

Genetic variation of prevalent G1P[8] human rotaviruses in South Korea

The human rotavirus G1P[8] strain is one of the most common rotaviruses worldwide, including Korea. Six Korean G1P[8] human rotaviruses, isolated using cell culture techniques, were characterized on the basis of sequence differences in VP7, VP4, VP6, and NSP4 genes to elucidate the evolutionary relationships in the community. All strains had a long RNA electropherotype, supported by VP6 gene analysis, clearly associated with subgroup II specificity. The phylogenetic analysis of VP7 gene sequences showed that they all clustered into lineage I, as reported for G1 strains in Japan, China, Vietnam, and Thailand. In addition, phylogenetic analysis of the VP4 gene showed that they belong to two distinct lineages, P[8]‐II and P[8]‐III. With respect to the NSP4 gene, all strains belonged to genotype B. An understanding of the ecology and molecular evolution of rotaviruses circulating in the country is very important for the development of vaccines and vaccination strategies. This study provides new information concerning the genetic variability of the rotavirus strain G1P[8] occurring most commonly as a vaccine candidate. J. Med. Virol. 82: 886–896, 2010.

A Western Eurasian Male Is Found in 2000-Year-Old Elite Xiongnu Cemetery in Northeast Mongolia

We analyzed mitochondrial DNA (mtDNA), Y-chromosome single nucleotide polymorphisms (Y-SNP), and autosomal short tandem repeats (STR) of three skeletons found in a 2,000-year-old Xiongnu elite cemetery in Duurlig Nars of Northeast Mongolia. This study is one of the first reports of the detailed genetic analysis of ancient human remains using the three types of genetic markers. The DNA analyses revealed that one subject was an ancient male skeleton with maternal U2e1 and paternal R1a1 haplogroups. This is the first genetic evidence that a male of distinctive Indo-European lineages (R1a1) was present in the Xiongnu of Mongolia. This might indicate an Indo-European migration into Northeast Asia 2,000 years ago. Other specimens are a female with mtDNA haplogroup D4 and a male with Y-SNP haplogroup C3 and mtDNA haplogroup D4. Those haplogroups are common in Northeast Asia. There was no close kinship among them. The genetic evidence of U2e1 and R1a1 may help to clarify the migration patterns of Indo-Europeans and ancient East-West contacts of the Xiongnu Empire. Artifacts in the tombs suggested that the Xiongnu had a system of the social stratification. The West Eurasian male might show the racial tolerance of the Xiongnu Empire and some insight into the Xiongnu society.

Shikonin inhibits adipogenesis by modulation of the WNT/β-catenin pathway

AIM Our previous study showed for the first time that shikonin, a natural compound isolated from Lithospermun erythrorhizon Sieb. Et Zucc, inhibits adipogenesis and fat accumulation. This study was conducted to investigate the molecular mechanism of the anti-adipogenic effects of shikonin. MAIN METHODS Gene knockdown experiments using small interfering RNA (siRNA) transfection were conducted to elucidate the crucial role of β-catenin in the anti-adipogenic effects of shikonin. KEY FINDINGS Shikonin prevented the down-regulation of β-catenin and increased the level of its transcriptional product, cyclin D1, during adipogenesis of 3T3-L1 cells, preadipocytes originally derived from mouse embryo. β-catenin was a crucial mediator of the anti-adipogenic effects of shikonin, as determined by siRNA-mediated knockdown. Shikonin-induced reductions of the major transcription factors of adipogenesis including peroxisome proliferator-activated receptor γ and CCAAT/enhancer binding protein α, and lipid metabolizing enzymes including fatty acid binding protein 4 and lipoprotein lipase, as well as intracellular fat accumulation, were all significantly recovered by siRNA-mediated knockdown of β-catenin. Among the genes located in the WNT/β-catenin pathway, the levels of WNT10B and DVL2 were significantly up-regulated, whereas the level of AXIN was down-regulated by shikonin treatment. SIGNIFICANCE This study clearly shows that shikonin inhibits adipogenesis by the modulation of WNT/β-catenin pathway in vitro, and also suggests that WNT/β-catenin pathway can be used as a therapeutic target for obesity and related diseases using a natural compound like shikonin, even though the in vivo effects of shikonin and its clinical significance remain to be elucidated.

Mycobacterium paraterrae sp. nov. recovered from a clinical specimen: novel chromogenic slow growing mycobacteria related to Mycobacterium terrae complex

A previously unidentified, slowly growing scotochromogenic Mycobacterium was isolated from a Korean patient with symptomatic pulmonary infection. Phenotypically, this strain was generally similar to Mycobacterium terrae complex strains, however it uniquely produced orange pigmentation. Unique mycolic acid profiles and phylogenetic analyses based on three alternative chronometer molecules, 16S rRNA gene, hsp65 and rpoB, confirmed the taxonomic status of this strain as a novel species. These results support that this strain represents a novel Mycobacterium species. The name Mycobacterium paraterrae sp. nov. is proposed. The type strain is 05‐2522 (= DSM 45127 = KCTC 19556).