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 G(1) to S and G(2) /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 G(1) 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.

Role of T lymphocytes in liver abscess formation by Bacteroides fragilis in mice.

The underlying mechanisms of liver abscess formation have not been fully elucidated with regard to the interaction between bacterial virulence factors and the immune response. The objective of this study was to determine the role of the host T cells in liver abscess formation caused by Bacteroides fragilis. We developed a liver abscess mouse model with inoculation of B. fragilis through the hepatic portal vein and examined the role of T cells by studying T cell-deficient mice, as well as conducting adoptive T cell transfer experiments. No microabscess was formed in the αß T cell receptor-positive (αßTCR(+)) T cell-depleted mice, in contrast to the results for the control mice. In addition, the αßTCR knockout (KO) mice showed significantly lower numbers of microabscesses, and the abscesses were smaller in size than those in the wild-type mice. Adoptive transfer of T cells purified from the wild-type mice into the αßTCR KO mice resulted in liver abscess formation in those mice. These findings suggest that T cells play an essential role in liver abscess formation caused by B. fragilis in mice.

Radiation exposure and cancer incidence in a cohort of nuclear power industry workers in the Republic of Korea, 1992-2005.

This study examines for the first time cancer incidence between radiation and non-radiation workers in nuclear power facilities in the Republic of Korea. Radiation workers were defined as persons who were issued with a dosimeter at nuclear power facilities, until 2005. All analyses were conducted on male workers only (in total 16,236 individuals) because of the sparseness of females. Statistical analyses were carried out using the standardized incidence ratio (SIR), to compare the cancer risks of radiation and non-radiation workers with those of the general population, and the chi(2) trend test was used to investigate any increase in cancer rates with dose. Poisson regression was also used to estimate the rate ratio (RR) and the excess relative risk (ERR) after considering the confounding effect due to smoking. During 1992-2005, 99 cancer cases in 63,503 person-years were observed among 8,429 radiation workers, while 104 cancer cases were observed in 48,301 person-years among 7,807 non-radiation workers. When compared with the site- and age-specific cancer rates for the male population of Korea, the SIR for all cancers combined was 1.07 [95% confidence interval (CI) 0.87-1.30] for radiation workers, and 0.88 (95% CI 0.72-1.06) for non-radiation workers, respectively. The RR for radiation workers compared with non-radiation workers was 1.18 (95% CI 0.89-1.58) for all cancers combined. The SIRs for thyroid cancer were noticeably high for both radiation and non-radiation workers, possibly due to the screening effect, but analysis of the RR showed that there was no statistically significant difference in thyroid cancer incidence rates between the two groups. For lung cancer, radiation workers showed a higher incidence rate as compared to non-radiation workers, with the RR being 3.48 (95% CI 1.19-11.48). A chi(2) trend test showed that there was no evidence for an increase in cancer rate with increasing cumulative dose for all cancers combined (p = 0.5108). The ERR per Sievert was estimated to be 1.69 (95% CI -2.07 to 8.21) for all cancers combined assuming a 10 years lag time. Consequently, a significant excess of cancer incidence among radiation workers in the nuclear power industry in Korea was not observed. Further follow-up and an expansion of the cohort are needed to overcome the lack of statistical power in the study.

Human cytomegalovirus IE1-72 protein interacts with p53 and inhibits p53-dependent transactivation by a mechanism different from that of IE2-86 protein.

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 IE1-72's 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.

Differentiation of mycobacteria in sputa by duplex polymerase chain reaction for mycobacterial hsp65 gene.

Early differentiation of mycobacteria in sputa is crucial. This study was set to evaluate the usefulness of a newly developed duplex polymerase chain reaction (PCR) for hsp65 gene-based method in differentiating mycobacteria in sputum with a positive acid-fast bacilli (AFB) smear before culturing. One hundred forty-seven sputa with positive AFB smear were included for the analysis. Mycobacterial species were identified using a newly developed duplex PCR for hsp65 gene followed by a nested PCR-direct sequencing and the conventional colony-based method. Final decision of mycobacterial species were made based on 1) results of species identification based on mycobacterial colonies or 2) results of species identification of other sputa from the same patients and clinical findings. The duplex PCR-based method correctly identified 83.2% sputa from tuberculosis patients and 82.2% sputa from nontuberculous mycobacteria patients, whereas the colony-based method correctly identified 86.1% and 77.8%, respectively. Sensitivity and specificity of the colony-based method for Mycobacterium tuberculosis were 86.1% and 100%, respectively, whereas those of the duplex PCR-based method were 83.2% and 95.6%, respectively. The duplex PCR-based method, to differentiate mycobacterial species in sputa, produced comparable results as those of the colony-based identification method.

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)).

Comparison of full genome sequences between two hepatitis B virus strains with or without preC mutation (A1896) from a single Korean hepatocellular carcinoma patient.

This report describes the full-length sequences of 2 HBV clones from a hepatocellular carcinoma (HCC) patient, one with preC mutation (1896A) and the other without preC mutation. The high level of discrepancy in mutation frequency between these 2 strains was observed in the Core (C) region among 4 ORFs. These data support previous results that Korean HBV strains, belonging to genotype C2, are prone to mutations. It is possible that the mutations (BCP and preC mutations) associated with the HBeAg defective production might contribute to the diversity of mutations related to HBV persistence, playing an important role in hepatocarcinogenesis in this patient.

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.

Mycobacterium seoulense sp. nov., a slowly growing scotochromogenic species.

A previously undescribed, slowly growing, scotochromogenic mycobacterium was isolated from a patient with symptomatic pulmonary infection during hsp65 sequence-based identification of Korean clinical isolates. Phenetic characteristics of this strain were generally similar to those of Mycobacterium nebraskense and Mycobacterium scrofulaceum. However, some phenetic characteristics differentiated it from these two species. Its 16S rRNA gene sequences were unique and phylogenetic analysis based on 16S rRNA gene sequences placed the organism in the slowly growing Mycobacterium group close to M. nebraskense and M. scrofulaceum. Its unique mycolic acid profiles and the results of phylogenetic analysis based on two independent alternative chronometer molecules, hsp65 and rpoB, confirmed the taxonomic status of this strain as representing a novel species. These data support the conclusion that this strain represents a novel mycobacterial species, for which the name Mycobacterium seoulense sp. nov. is proposed. The type strain is strain 03-19(T) (=DSM 44998(T)=KCTC 19146(T)).

Direct application of AvaII PCR restriction fragment length polymorphism analysis (AvaII PRA) targeting 644 bp heat shock protein 65 (hsp65) gene to sputum samples.

To evaluate the usefulness of the AvaII PRA method targeting 644-bp hsp65 gene for the direct detection of pathogenic mycobacteria from clinical specimens, we applied this method to 40 sputum samples and compared the results to those obtained by IS 6110 PCR. Although this method showed a sensitivity slightly lower than IS 6110 PCR (97.5% vs. 100%), it detected infections of M. avium complex (MAC) in two patients, which was not possible by IS 6110 PCR. We conclude that AvaII PRA is a highly effective method for directly detecting pathogenic mycobacteria in primary clinical specimens.

Analysis of hepatitis B virus quasispecies distribution in a Korean chronic patient based on the full genome sequences.

Although Korea is a hepatitis B virus (HBV) endemic area, relatively few full-length genome sequences are available. In particular, no comparative analysis has been performed on the full-genome sequences of different HBV quasispecies from a single Korean patient. This report describes the full-length sequences of five HBV clones (two clones with shorter PCR amplicons and three clones with longer amplicons). Large deletions, that is, 685-bp, 487-bp, and 144-bp, that might interfere with the production of normal proteins were observed in four of five clones. Double mutations in the basal core promoter (BCP) region (T1762/A1764) were detected in two clones but no precore mutations (A1896) were detected in any of the five clones. These data support previous results that genotype C, in particular Korean clones of this genotype, is prone to mutations. Two independent mechanisms, namely, the deletions of long lengths and amino acid substitutions followed by BCP double mutations might contribute to the diversity of HBV quasispecies. Considering the importance of HBV quasispecies as HBV variant sources, the distribution of HBV quasispecies in mutation prone genotype C prevalent areas like Korea, should be monitored to improve the management of chronic HBV infections and to control HBV variants that arise due to the administration of vaccine or antiviral therapy.

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.

Differentiation of Mycobacterial species by hsp65 duplex PCR followed by duplex-PCR-based restriction analysis and direct sequencing.

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.

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.

Mechanism of natural rifampin resistance of Streptomyces spp.

In a previous phylogenetic study of the genus Streptomyces using the rpoB gene, N531, which stands for an aspargine residue in position 531 of RpoB instead of serine (S531), known to be associated with natural rifampin resistance in several organisms, was also observed in the RpoB of several Streptomyces species. To determine whether N531 is associated with the rifampin resistance of Streptomyces strains, we analyzed the rifampin minimum inhibitory concentrations (MICs) of 11 strains of the N531 RpoB type (putative rifampin resistant strains) and of 12 strains of the S531 RpoB type. (putative rifampin susceptible strains). In general, the N531 RpoB types showed higher MIC levels (16-128 microg/ml) than the S531 RpoB types (0-8 microg/ml). To determine the isolation frequencies of N531 RpoB types versus rifampin concentration, we applied screening methods involving different rifampin concentrations (0, 20 and 100 microg/ml) to Korean soils. Higher isolation frequencies of the N531 RpoB types were observed at the higher rifampin concentrations. In addition, during the course of this study we developed an allele specific PCR method to detect rifampin resistant Streptomyces strains. Our results strongly suggested that N531 might be involved in a major mechanism of natural rifampin resistance in strains of the genus Streptomyces.

PCR restriction fragment length polymorphism analysis (PRA)-algorithm targeting 644 bp Heat Shock Protein 65 (hsp65) gene for differentiation of Mycobacterium spp.

A method based on PCR-restriction fragment length polymorphism analysis (PRA) using a novel region of the hsp65 gene was developed for the rapid and exact identification of mycobacteria to the species level. A 644 bp region of hsp65 in 62 mycobacteria reference strains, and 4 related bacterial strains were amplified, and the amplified DNAs were subsequently digested with restriction enzymes, namely, AvaII, HphI, and HpaII. Most of the mycobacteria species were easily differentiated at the species level by the developed method. In particular, the method enabled the separation of M. avium, M. intracellulare and M. tuberculosis to the species level by AvaII digestion alone. An algorithm was constructed based on the results and a blind test was successfully performed on 251 clinical isolates, which had been characterized by conventional biochemical testing. Our results suggest that this novel PRA offers a simple, rapid, and accurate method for the identification of mycobacteria culture isolates at the species level.

Differentiation of Mycobacterium species by analysis of the heat-shock protein 65 gene (hsp65).

The nucleotide sequences (604 bp) of partial heat-shock protein genes (hsp65) from 161 Mycobacterium strains containing 56 reference Mycobacterium species and 105 clinical isolates were determined and compared. hsp65 sequence analysis showed a higher degree of divergence between Mycobacterium species than did 16S rRNA gene analysis. Generally, the topology of the phylogenetic tree based on the hsp65 DNA sequences was similar to that of the 16S rRNA gene, thus revealing natural relationships among Mycobacterium species. When a direct sequencing protocol targeting 422 bp sequences was applied to 70 non-tuberculous mycobacterium (NTM) clinical isolates, all NTMs were clearly identified. In addition, an XhoI PCR restriction fragment length polymorphism analysis method for the differentiation of Mycobacterium tuberculosis complex from NTM strains was developed during this study. The results obtained suggest that 604 bp hsp65 sequences are useful for the phylogenetic analysis and species identification of mycobacteria.

Molecular taxonomy of a soil actinomycete isolate, KCCM10454 showing neuroprotective activity by 16S rRNA and rpoB gene analysis.

Epilepsy constitutes a significant public health problem, and even the newest drugs and neurosurgical techniques have proven unable to cure the disease. In order to select a group of isolates which could generate an active compound with neuroprotective or antiepileptic properties, we isolated 517 actinomycete strains from soil samples taken from Jeju Island, in South Korea. We then screened these strains for possible anti-apoptotic effects against serum deprivation-induced hippocampal cell death, using the 3-(4, 5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) assay as an in vitro test. The excitotoxic glutamate analog, kainic acid (KA), was used to induce seizures in experimental mice in our in vivo tests. As a result of this testing, we located one strain which exhibited profound neuroprotective activity. This strain was identified as a Streptomyces species, and exhibited the rifampin-resistant genotype, Asn(AAC)442, according to the results of 16S rRNA and rpoB gene analyses.

Prevalence of naturally occurring surface antigen variants of hepatitis B virus in Korean patients infected chronically.

Although Korea is one of the endemic areas of hepatitis B virus (HBV) infection, the prevalence of naturally occurring variants in the major hydrophilic region (MHR) of the surface (S) gene of HBV has not been determined. In the present study, the prevalence of these variants was examined in terms of the clinical state, and HBeAg serostatus in a large series of Korean patients with chronic HBV infection by direct sequencing analysis of part of the S gene containing the MHR of HBV isolated from 101 chronic HBV patients (51 HBeAg-positive and 50 HBeAg-negative): 37 were asymptomatic carriers, 21 had chronic hepatitis, 20 had liver cirrhosis, and 23 had hepatocellular carcinoma (HCC). Forty-seven MHR variants (46.5%) of the 101 patients were detected, involving a total of 59 amino acid substitutions at 12 positions inside and 14 position outside the 'a' determinant, and 33 'a' determinant variants (32.7%). A total of 17 novel variants and 14 novel mutation patterns were detected. The prevalence of MHR variants in HBeAg-negative patients tended to be higher than in HBeAg-positive patients (54.0% vs.39.2%) and the prevalence of MHR variants in HCC and liver cirrhosis tended to be higher than in asymptomatic carriers (65.2% vs. 40.5% and 50.0% vs. 40.5%, respectively). In conclusion, three important findings were found in the present study. First, an unexpectedly high prevalence of naturally occurring MHR variants was found in Korean chronic patients. Second, several novel variants associated with mutations outside the 'a' determinant were detected. Finally, a higher prevalence of MHR variants was associated with HBeAg-negative serostatus and severe liver disease, particularly HCC.

Decreased serum level of antibody against human cytomegalovirus in patients with Behçet's disease.

Behçet's disease (BD) is a connective tissue disorder characterized by recurrent orogenital ulcer, uveitis, and skin lesions. Recurrent aphthous ulcer is associated with human cytomegalovirus (HCMV). To investigate the possible role of HCMV in BD, we measured the titers of IgG, IgM, and IgA anti-HCMV antibodies in 73 Korean patients with BD, 50 with scleroderma, 70 with systemic lupus erythematosus, and 50 from healthy controls by indirect immunofluorescent staining. The titer of IgG anti-HCMV antibody was significantly lower in patients with BD than in controls (geometric mean 3115.4 vs 9687.6, P = 0.0001 by Wilcoxon's rank sum test), as was the titer of IgA anti-HCMV antibody (geometric mean 1.9 vs 15.7, P = 0.0001, Wilcoxon's rank sum test). In conclusion, we found significantly lower antibody responses to HCMV in patients with BD.