ng-Yong Cha

Molecular Epidemiology of Hepatitis B Virus (HBV) Genotypes and Serotypes in Patients with Chronic HBV Infection in Korea

Objectives: Although hepatitis B virus (HBV) is endemic to Korea, no large-scale survey of HBV genotypes and serotypes based on sequence analysis has been performed. Methods: In the present study, we genotyped and serotyped HBV strains from 209 patients in two Korean regions, Seoul (107 patients) and Jeju (102 patients), an island off the southeastern Korean coast. Analyses were conducted using the direct sequencing method targeting the partial surface (S) gene (541 bp). Results: Phylogenetic analysis showed that all HBV strains from the 209 patients belonged to genotype C2 (100%). Of the 209 patients, 193 (92.3%), 12 (5.7%) and 1 (0.5%) were found to have the adr, adw and ayr serotypes, respectively. The other three strains (1.5%) showed unique serotype and were not typeable by sequence analysis. No HBV strains characteristic of Jeju island were observed. Conclusions: The extraordinary predominance of genotype C2 in chronic Korean patients, which is known to be associated with more severe liver disease than genotype B, suggests that the clinical manifestations of Korean HBV chronic patients are likely to differ from those found in other Asian countries, especially in Japan and Taiwan, where genotypes B and C coexist.

Mycobacterium senuense sp. nov., a slowly growing, non-chromogenic species closely related to the Mycobacterium terrae complex.

A previously undescribed, slowly growing, non-chromogenic mycobacterium, isolated from a Korean patient with a symptomatic pulmonary infection, is described as representing a novel species. Its 16S rRNA gene sequence was unique and phylogenetic analysis based on 16S rRNA gene sequences showed that this organism belonged to the Mycobacterium terrae subclade. Phenotypically, the strain was generally similar to M. terrae and Mycobacterium nonchromogenicum, but its growth rate was slower than those of other M. terrae complex strains. A unique mycolic acid profile and phylogenetic analysis based on two different alternative chronometer molecules, hsp65 and rpoB, confirm the taxonomic status of this strain as a representative of a novel species. The name Mycobacterium senuense sp. nov. is proposed, with the type strain 05-832(T) (=DSM 44999(T) =KCTC 19147(T)).

Human Cytomegalovirus IE1-72 Protein Interacts with p53 and Inhibits p53-Dependent Transactivation by a Mechanism Different from That of IE2-86 Protein

ABSTRACT Infection of host cells with human cytomegalovirus (HCMV) induces cell cycle dysregulation. Two HCMV immediate-early (IE) proteins, IE1-72 and IE2-86, are promiscuous transactivators that have been implicated in the dysregulatory events. Cellular p53 protein is accumulated to high levels in HCMV-infected cells, but the indicative marker of p53 transcriptional activity, p21, is markedly decreased. Both IE1-72 and IE2-86 were able to transactivate the p53 promoter and interact with p53 protein in DNA-transfected or HCMV-infected cells. HCMV UL84, a multiregulatory protein expressed in early periods of HCMV infection, also interacted with p53. HCMV IE1-72 prevented or disrupted p53 binding to p53-specific DNA sequences, while IE2-86 and/or UL84 enhanced p53 binding and induced supershift of this DNA-protein complex. Both HCMV IE1-72 and IE2-86 were able to inhibit p53-dependent transcriptional activation in plasmid-transfected cells. IE1-72, rather than IE2-86, was found to be responsible for p21 downregulation in HCMV-infected HEL cells. DNA transfection analysis using IE1-72 mutants revealed that exon 2/3 and the zinc finger region of IE1-72 are essential for IE1-72s effect on the repression of p53-dependent transcriptional activation. These data suggest that HCMV IE1-72 and/or IE2-86 transactivates the p53 promoter and induces p53 accumulation, but HCMV IE1-72 represses the p53 transactivation activity by a unique binding hindrance mechanism different from that of IE2-86. Thus, various modes of viral IE proteins and p53 interactions might result in multiple outcomes, such as stimulation of cellular DNA synthesis, cell cycle progression and cell cycle arrest, and prevention of program cell death.

Clinical usefulness of human cytomegalovirus antigenemia assay after kidney transplantation.

Background. Human (H) cytomegalovirus (CMV) infections are a major cause of morbidity and mortality among kidney transplants. We performed a prospective study to evaluate the clinical usefulness of HCMV antigenemia assay for preemptive treatment after kidney transplantation. Methods. A total of 100 patients were followed up by HCMV antigenemia assay at posttransplantation weeks 1, 3, 5, 7, 9, 13, 17, and 21. Asymptomatic patients with positive antigenemia were observed without specific antiviral therapy. Results. Most patients had been given cyclosporine A- and prednisolone-based immunosuppressive therapy (99.0%) and were HCMV seropositive before transplantation (99.0%). A positive antigenemia assay was detected in 41 patients among 97 eligible patients. Symptomatic CMV diseases were observed in 10 of 41 patients. HCMV infections were related to history of acute rejection and use of antithymocyte globulin. HCMV-related symptoms and signs were clearly correlated with the level of antigenemia. All patients who had an HCMV antigenemia titer of higher than 50 per 400,000 leukocytes developed HCMV-related symptoms and signs during the follow-up period. This criterion showed the highest positive predictive value and specificity in the development of symptomatic HCMV infection. Conclusions. Data suggest that HCMV antigenemia titer can be used as a useful guide to preemptive treatment of HCMV infection after kidney transplantation in HCMV-positive donor and recipient.

The DNA hybridization assay using single-walled carbon nanotubes as ultrasensitive, long-term optical labels.

Single walled carbon nanotubes (SWNTs) exhibit strong Raman signals as well as fluorescence emissions in the near infrared region. Such signals do not blink or photobleach under prolonged excitation, which is an advantage in optical nano-biomarker applications. In this paper, we present single-stranded DNA conjugated SWNT probes to locate a particular sequence of DNA within a complex genome. Chromosomal DNAs of human fibroblasts and Escherichia coli are used as a target and a control, respectively. Southern blotting, which uses photostable Raman signals of nanotubes instead of fluorescent dyes, demonstrates excellent sensitivity and specificity of the probes. The results show that SWNTs may be used as generic nano-biomarkers for the precise detection of specific kinds of genes.

Antiviral effects of 28-deacetylsendanin on herpes simplex virus-1 replication

The compound purified from the fruit of Melia azedarach exerted an antiviral effect on herpes simplex virus-1 (HSV-1) in Vero cells. It was identified as 28-deacetylsendanin (28-DAS). The 50% inhibitory concentration (IC50) of 28-DAS was 1.46 microg/ml without cytotoxicity at 400 microg/ml on Vero cells. Electron microscopy showed that low electron-dense cores of newly synthesized nucleocapsids remained in swollen nuclei and no extracellular virus particles were observed at 15 h p.i. Consistent with this result, it was confirmed by a plaque assay that few infectious progeny viruses were released from the 28-DAS-treated virus-infected cells at 24 h p.i. Intracellular viruses in 28-DAS-treated virus-infected cells were 23% of untreated and infected cells. The synthesis of thymidine kinase (TK) was reduced by 28-DAS at early stage. In conclusion, 28-DAS inhibited the replication of HSV-1, reduced the synthesis of HSV-1 TK, and led to the formation of defective nucleocapsids.

Differentiation of Mycobacterial Species by hsp65 Duplex PCR Followed by Duplex-PCR-Based Restriction Analysis and Direct Sequencing

ABSTRACT Here we describe a novel duplex PCR method which can differentiate Mycobacterium tuberculosis and nontuberculosis mycobacteria (NTM) strains by amplifying hsp65 DNAs of different sizes (195 and 515 bp, respectively). The devised technique was applied to 54 reference and 170 clinical isolates and differentiated all strains into their respective groups with 100% sensitivity and specificity. Furthermore, a duplex PCR-restriction analysis (duplex PRA) and a direct sequencing protocol were developed to differentiate NTM strains at the species and subspecies levels based on previously reported hsp65 DNA sequences (H. Kim et al., Int. J. Syst. Evol. Microbiol. 55:1649-1656, 2005) and then applied to 105 NTM clinical isolates. All NTM isolates were clearly differentiated at the species and subspecies levels by subsequent procedures (PRA or direct sequencing) targeting 515-bp NTM duplex PCR amplicons. Our results suggest that novel duplex PCR-based methods are sensitive and specific for identifying mycobacterial culture isolates at the species level.

Characterization of two hepatitis B virus populations in a single Korean hepatocellular carcinoma patient with an HBeAg-negative serostatus: a novel X-Gene-deleted strain with inverted duplication sequences of upstream enhancer site II.

Objectives: The aim of the study was to elucidate mutation patterns related to hepatocarcinogenesis in a Korean hepatocellular carcinoma (HCC) patient. Methods: We analyzed full genome sequences of 6 hepatitis B virus (HBV) clones from an HCC patient. Results: This patient harbored 2 HBV populations with genomes of different lengths (3,221 and 2,212 bp). In addition, we found 2 characteristic features not described so far in the full-genome sequence of deleted strains. First, 3 large deletion events (847, 144 and 48 bp) and a premature termination of the 182th codon of the surface antigen could lead to truncated or possibly nonfunctional forms of all HBV proteins. Second, these showed a novel mutation type not reported to date, which is a complex of an inverted duplication of 36-bp sequences containing an upstream enhancer site II (UEII), a remote insertion, and a large deletion event of the X region by homologous recombination. Conclusion: The fact that UEII is a binding site of liver-specific nuclear factor, which is expressed only in highly differentiated liver cells such as cancerous HepG2, strongly suggests a relationship between this novel mutation and hepatocarcinogenesis in this patient.

Human Cytomegalovirus (HCMV) IE1 Plays Role in Resistance to Apoptosis with Etoposide in Cancer Cell Line by Cdk2 Accumulation

Human cytomegalovirus (HCMV) has many strategies to survive the attack of the host. HCMV infection of host cells induces cellular activation and disturbance of the cell cycle. It is possible that HCMV modulates the behavior of certain cancer cells that are susceptible to HCMV infection. This study was performed to identify the possible mechanism of resistance to apoptotic stimuli in some cancer cell lines by HCMV infection. HCMV‐infected cancer cells showed resistance to apoptosis induced by the topoisomerase II inhibitor etoposide. UMG1–2, which constitutively expresses HCMV immediate‐early protein‐1 (IE1), had resistance to apoptosis induced by etoposide as compared with the parental cell line U373MG. Measurement of caspases activity with fluorogenic substrates in etoposide‐treated U373MG and UMG1–2 cells and the direct activation of caspase‐3 with peptides containing arginine‐glycine‐aspartate in U373MG and UMG1–2 cells revealed that the inhibition level of apoptosis by HCMV IE1 would be upstream of caspase‐3 in the caspase cascade pathway. Cellular expression of Cdk2 was increased in UMG1–2 after etoposide treatment while the expression of E2F‐1 in UMG1–2 was decreased as compared with that in U373MG. The Cdk2 inhibitor, roscovitine, decreased the resistance to apoptosis on etoposide‐treated UMG1–2. These results suggest that aberrant HCMV infection confers resistance to anticancer drugs on some cancer cells and protects cells from apoptosis, possibly due to the deregulation of cyclin‐dependent kinase by HCMV immediate‐early protein.

Inhibition of p53 transcriptional activity by human cytomegalovirus UL44

Human cytomegalovirus (HCMV) stimulates cellular synthesis of DNA and proteins and induces transition of the cell cycle from G1 to S and G2/M phase, in spite of increased amounts of p53 in the infected cells. The immediate early protein IE2–86 kDa (IE86) tethers a transcriptional repression domain to p53; however, its repression of p53 function is not enough to abrogate the G1 checkpoint function of p53. Other HCMV proteins that suppress the activity of p53 were investigated in this study. Of the HCMV proteins that bind to p53 when assessed by immunoprecipitation and immunoblot analysis, HCMV UL44 was chosen as a candidate protein. It was found that reporter gene containing p53 consensus sequence was activated by transfection with wild type p53, but when plasmids of p53 with IE86 or UL44 were co‐transfected, p53 transcriptional activity was decreased to 3–7% of the p53 control in a dose‐dependent manner. When the deletion mutant of UL44 was co‐transected with p53, the carboxyl one‐third portion of UL44 had little effect on inhibition of p53 transcriptional activity. The amount of mRNA p21 was measured in H1299 by real time PCR after transfection of the combination of p53 and UL44 vectors and it was found that p21 transcription by p53 was inhibited dose‐dependently by UL44. Increased G0/G1 and decreased S phases in p53 wild type‐transfected H1299 cells were recovered to the level of p53 mutant type‐transfected ones by the additional transfection of UL44 in a dose‐dependent manner. In conclusion, the transcriptional activity of p53 is suppressed by UL44 as well as by IE86.