RESUMO
Neuronal calcium sensor-1 or Frequenin is a calcium sensor widely expressed in the nervous system, with roles in neurotransmission, neurite outgrowth, synaptic plasticity, learning, and motivated behaviours. Neuronal calcium sensor-1 has been implicated in neuropsychiatric disorders including autism spectrum disorder, schizophrenia, and bipolar disorder. However, the role of neuronal calcium sensor-1 in behavioural phenotypes and brain changes relevant to autism spectrum disorder have not been evaluated. We show that neuronal calcium sensor-1 deletion in the mouse leads to a mild deficit in social approach and impaired displaced object recognition without affecting social interactions, behavioural flexibility, spatial reference memory, anxiety-like behaviour, or sensorimotor gating. Morphologically, neuronal calcium sensor-1 deletion leads to increased dendritic arbour complexity in the frontal cortex. At the level of hippocampal synaptic plasticity, neuronal calcium sensor-1 deletion leads to a reduction in long-term potentiation in the dentate gyrus, but not area Cornu Ammonis 1. Metabotropic glutamate receptor-induced long-term depression was unaffected in both dentate and Cornu Ammonis 1. These studies identify roles for neuronal calcium sensor-1 in specific subregions of the brain including a phenotype relevant to neuropsychiatric disorders.
Assuntos
Comportamento de Escolha/fisiologia , Cognição/fisiologia , Potenciação de Longa Duração/genética , Proteínas Sensoras de Cálcio Neuronal/genética , Plasticidade Neuronal/genética , Neuropeptídeos/genética , Reconhecimento Psicológico/fisiologia , Animais , Ansiedade/genética , Região CA1 Hipocampal/fisiologia , Giro Denteado/fisiopatologia , Lobo Frontal/patologia , Camundongos , Camundongos Knockout , Receptores de Glutamato Metabotrópico , Filtro Sensorial/genética , Comportamento Social , Interação Social , Memória Espacial/fisiologiaRESUMO
Cognitive dysfunction is a core feature of dementia and a prominent feature in psychiatric disease. As non-redundant regulators of intracellular cAMP gradients, phosphodiesterases (PDE) mediate fundamental aspects of brain function relevant to learning, memory, and higher cognitive functions. Phosphodiesterase-4B (PDE4B) is an important phosphodiesterase in the hippocampal formation, is a major Disrupted in Schizophrenia 1 (DISC1) binding partner and is itself a risk gene for psychiatric illness. To define the effects of specific inhibition of the PDE4B subtype, we generated mice with a catalytic domain mutant form of PDE4B (Y358C) that has decreased ability to hydrolyze cAMP. Structural modeling predictions of decreased function and impaired binding with DISC1 were confirmed in cell assays. Phenotypic characterization of the PDE4B(Y358C) mice revealed facilitated phosphorylation of CREB, decreased binding to DISC1, and upregulation of DISC1 and ß-Arrestin in hippocampus and amygdala. In behavioral assays, PDE4B(Y358C) mice displayed decreased anxiety and increased exploration, as well as cognitive enhancement across several tests of learning and memory, consistent with synaptic changes including enhanced long-term potentiation and impaired depotentiation ex vivo. PDE4B(Y358C) mice also demonstrated enhanced neurogenesis. Contextual fear memory, though intact at 24 h, was decreased at 7 days in PDE4B(Y358C) mice, an effect replicated pharmacologically with a non-selective PDE4 inhibitor, implicating cAMP signaling by PDE4B in a very late phase of consolidation. No effect of the PDE4B(Y358C) mutation was observed in the prepulse inhibition and forced swim tests. Our data establish specific inhibition of PDE4B as a promising therapeutic approach for disorders of cognition and anxiety, and a putative target for pathological fear memory.
Assuntos
Tonsila do Cerebelo/fisiologia , Ansiedade/enzimologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/fisiologia , Medo/fisiologia , Hipocampo/fisiologia , Memória/fisiologia , Tonsila do Cerebelo/citologia , Tonsila do Cerebelo/enzimologia , Animais , Arrestinas/metabolismo , Condicionamento Clássico/fisiologia , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Espinhas Dendríticas/enzimologia , Comportamento Exploratório/fisiologia , Feminino , Hipocampo/citologia , Hipocampo/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Neurogênese , Plasticidade Neuronal , Neurônios/citologia , Neurônios/fisiologia , Fosforilação , Transdução de Sinais , beta-ArrestinasRESUMO
Understanding the mechanisms of memory formation is fundamental to establishing optimal educational practices and restoring cognitive function in brain disease. Here, we show for the first time in a non-primate species, that spatial learning receives a special bonus from self-directed exploration. In contrast, when exploration is escape-oriented, or when the full repertoire of exploratory behaviors is reduced, no learning bonus occurs. These findings permitted the first molecular and cellular examinations into the coupling of exploration to learning. We found elevated expression of neuronal calcium sensor 1 (Ncs1) and dopamine type-2 receptors upon self-directed exploration, in concert with increased neuronal activity in the hippocampal dentate gyrus and area CA3, as well as the nucleus accumbens. We probed further into the learning bonus by developing a point mutant mouse (Ncs1(P144S/P144S)) harboring a destabilized NCS-1 protein, and found this line lacked the equivalent self-directed exploration learning bonus. Acute knock-down of Ncs1 in the hippocampus also decoupled exploration from efficient learning. These results are potentially relevant for augmenting learning and memory in health and disease, and provide the basis for further molecular and circuit analyses in this direction.
Assuntos
Comportamento Exploratório , Aprendizagem , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Neuropeptídeos/metabolismo , Animais , Região CA3 Hipocampal/metabolismo , Giro Denteado/metabolismo , Meio Ambiente , Técnicas de Silenciamento de Genes , Masculino , Camundongos Endogâmicos C57BL , Núcleo Accumbens/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptores de Dopamina D2/metabolismo , Memória EspacialRESUMO
While rat ultrasonic vocalizations (USVs) are known to vary with anticipation of an aversive vs. positive stimulus, little is known about USVs in adult mice in relation to behaviors. We recorded the calls of adult C57BL/6J male mice under different environmental conditions by exposing mice to both novel and familiar environments that varied in stress intensity through the addition of bright light or shallow water. In general, mouse USVs were significantly more frequent and of longer duration in novel environments. Particularly, mice in dimly-lit novel environments performed more USVs while exhibiting unsupported rearing and walking behavior, and these calls were mostly at high frequency. In contrast, mice exhibited more low frequency USVs when engaging in supported rearing behavior in novel environments. These findings are consistent with data from rats suggesting that low-frequency calls are made under aversive conditions and high-frequency calls occur in non-stressful conditions. Our findings increase understanding of acoustic signals associated with exploratory behaviors relevant to cognitive and motivational aspects of behavior.
RESUMO
Several lines of evidence have suggested that some naturally occurring mutations of hepatitis B virus (HBV) play a critical role in hepatocellular carcinoma (HCC). Here, we describe a novel HCC-related pre-S2 mutation, F141L. To prove the relationship between the F141L mutation and HCC, molecular epidemiology studies using MboII PCR restriction analysis (PRA) were performed, and the molecular mechanism was investigated through construction of a stable hepatocyte cell line expressing the large surface HB protein (LHB) with the F141L mutation (F141L-LHB). Application of MboII PRA to samples from 241 Korean patients with chronic liver diseases of different clinical stages confirmed that F141L mutants were significantly related to HCC, even in comparison to liver cirrhosis (HCC, 26.3% of patients, or 26/99; liver cirrhosis, 3.8% of patients, or 2/52; P = 0.001). By studying stable cell lines, we found that F141L-LHBs could induce cell cycle progression by downregulating the p53 and p21 pathways and upregulating CDK4 and cyclin A. Furthermore, we found that in a colony-forming assay, the colony-forming rates in cell lines expressing F141L-LHBs were about twice as high as those of the wild type. In conclusion, our results suggest that F141L-LHBs may contribute importantly to the pathogenesis of HCC by inducing cell proliferation and transformation. So, the F141L mutation examined in this study could serve as a diagnostic marker for the prognosis of HCC.
Assuntos
Carcinoma Hepatocelular/virologia , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Neoplasias Hepáticas/virologia , Mutação , Precursores de Proteínas/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Viral , Genótipo , Vírus da Hepatite B/classificação , Hepatite B Crônica/patologia , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Neoplasias Hepáticas/patologia , RiscoRESUMO
Deletions and insertions in the hepatitis B virus (HBV) X region have been associated with severe forms of liver disease, including hepatocellular carcinoma (HCC). However, the molecular epidemiologic features of this virus have been described rarely. Deletions and insertions in the X region were determined by direct sequencing in a Korean cohort of 267 patients with different clinical statuses. Deletions and insertions were observed in two sets of six patients each (2.2%, 6/267). The prevalence of deletions or insertions was significantly higher in patients with severe liver disease, HCC, or cirrhosis of the liver (7.2%, 10/132) compared to patients who were carriers or had chronic hepatitis (1.5%, 2/135) (P = 0.017). All deletions in six strains were concentrated at the C terminal end of HBx, encompassing the 113th to 154th codons. A total of four novel types of insertions (PKLL, GM, FFN, and tt) were observed in six patients. Of particular interest, all six strains with insertions were accompanied by double mutations in the basal core promoter (BCP). In conclusion, these results suggest that deletions or insertions in the X region may contribute to disease progression in Korean patients with genotype C infection.
Assuntos
Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Mutagênese Insercional , Deleção de Sequência , Transativadores/genética , Adulto , Idoso , Povo Asiático , Carcinoma Hepatocelular/virologia , Portador Sadio/virologia , DNA Viral/química , DNA Viral/genética , Feminino , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/patologia , Humanos , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , República da Coreia , Análise de Sequência de DNA , Índice de Gravidade de Doença , Proteínas Virais Reguladoras e AcessóriasRESUMO
Although Korea is one of the endemic areas for hepatitis B virus infection (HBV), the prevalence of deletions in HBV preS region occurring naturally have not been determined. In the present study, the prevalence of preS deletions was determined in terms of clinical state and HBeAg serostatus in 120 patients with different clinical features [59 HBeAg positive, 61 HBeAg negative; 38 asymptomatic carriers, 21 patients with chronic hepatitis, 21 patients with liver cirrhosis, 40 patients with hepatocellular carcinoma (HCC)]. A total of 37 strains (30.8%) harbored deletions in the preS region. Overall, the frequencies of preS deletions tended to increase gradually according to the degree of the clinical severity of liver disease. The prevalence of preS1 deletions in HCC patients tended to be higher than in patients with liver cirrhosis (32.5% vs. 19%). The prevalence of preS2 deletions in HBeAg negative patients was significantly higher than in HBeAg positive patients (23% vs. 6.8%). The type of deletion encountered most frequently was one disrupting the preS1 start codon [14/37 strains (37.8%)], which showed a very high prevalence in HCC patients (9/40, 22.5%; HCC vs. asymptomatic carriers, P=0.048). These results suggest that there might be the discrepancy between preS1 and preS2 mutations in the mechanism of enhancing the progression of chronic liver disease, especially the development of HCC and to maintain tolerance during the stage of immune tolerance. Specific deletion of the type disrupting preS1 start codon may play important roles in hepatocarcinogenesis, at least in Korean patients with chronic HBV infection.
Assuntos
Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Precursores de Proteínas/genética , Deleção de Sequência , Adulto , Sequência de Aminoácidos , Feminino , Genótipo , Antígenos de Superfície da Hepatite B/química , Vírus da Hepatite B/classificação , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Precursores de Proteínas/químicaRESUMO
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)).
Assuntos
Pneumopatias/microbiologia , Infecções por Mycobacterium/microbiologia , Mycobacterium/classificação , Mycobacterium/crescimento & desenvolvimento , Idoso , Proteínas de Bactérias/genética , Sequência de Bases , Chaperonina 60 , Chaperoninas/genética , DNA Bacteriano/análise , RNA Polimerases Dirigidas por DNA/genética , Humanos , Masculino , Dados de Sequência Molecular , Mycobacterium/genética , Mycobacterium/isolamento & purificação , Ácidos Micólicos/análise , Micobactérias não Tuberculosas/classificação , Micobactérias não Tuberculosas/genética , Fenótipo , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Escarro/microbiologiaRESUMO
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.
Assuntos
Carcinoma Hepatocelular/virologia , Genoma Viral , Vírus da Hepatite B/genética , Neoplasias Hepáticas/virologia , Mutação , Sequência de Bases , Antígenos E da Hepatite B/genética , Vírus da Hepatite B/classificação , Humanos , Regiões Promotoras GenéticasRESUMO
Mycobacterium xenopi is a nontuberculous mycobacterium (NTM) that rarely causes pulmonary disease in Asia. Here we describe the first case of M. xenopi pulmonary disease in Korea. A 66-year-old man was admitted to our hospital with a 2-month history of productive cough and hemoptysis. His past medical history included pulmonary tuberculosis 44 years earlier, leading to a right upper lobectomy. Chest X-ray upon admission revealed cavitary consolidation involving the entire right lung. Numerous acid-fast bacilli were seen in his initial sputum, and M. xenopi was subsequently identified in more than five sputum cultures, using molecular methods. Despite treatment with clarithromycin, rifampicin, ethambutol, and streptomycin, the infiltrative shadow revealed on chest X-ray increased in size. The patient's condition worsened, and a right completion pneumonectomy was performed. The patient consequently died of respiratory failure on postoperative day 47, secondary to the development of a late bronchopleural fistula. This case serves as a reminder to clinicians that the incidence of NTM infection is increasing in Korea and that unusual NTM are capable of causing disease in non-immunocompromised patients.
Assuntos
Pneumopatias/diagnóstico , Pneumopatias/microbiologia , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Mycobacterium xenopi/isolamento & purificação , Idoso , Proteínas de Bactérias/genética , Proteínas de Choque Térmico/genética , Humanos , Coreia (Geográfico) , Pneumopatias/diagnóstico por imagem , Masculino , Infecções por Mycobacterium não Tuberculosas/diagnóstico por imagem , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium xenopi/classificação , Mycobacterium xenopi/genética , Filogenia , Radiografia , Análise de Sequência de DNARESUMO
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.
Assuntos
Carcinoma Hepatocelular/virologia , Elementos Facilitadores Genéticos/genética , Genoma Viral , Antígenos E da Hepatite B/sangue , Vírus da Hepatite B/classificação , Neoplasias Hepáticas/virologia , Transativadores/genética , Evolução Molecular , Deleção de Genes , Duplicação Gênica , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Vírus da Hepatite B/patogenicidade , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Recombinação Genética , Análise de Sequência de DNA , Proteínas Virais Reguladoras e AcessóriasRESUMO
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)).
Assuntos
Pneumopatias/microbiologia , Infecções por Mycobacterium/microbiologia , Mycobacterium/classificação , Sequência de Bases , DNA Bacteriano/genética , DNA Ribossômico/genética , Feminino , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mycobacterium/genética , RNA Ribossômico 16S/genéticaRESUMO
In a previous study, we found that the phylogenetic analysis of partial rpoB sequences can be used effectively to phylogenetically differentiate Streptomyces spp. [B.J. Kim, C.J. Kim, J. Chun, Y.H. Koh, S.H. Lee, J.W. Hyun, C.Y. Cha, Y.H. Kook, Phylogenetic analysis of the genera Streptomyces and Kitasatospora based on partial RNA polymerase beta-subunit gene (rpoB) sequences, Int. J. Syst. Evol. Microbiol. 54 (2004) 593-598]. In the present study, we analyzed the partial rpoB gene sequences of 19 reference Streptomyces strains associated with potato scab. Furthermore, to empirically confirm the usefulness of rpoB gene analysis for the phylogenetic differentiation of Streptomyces spp., we applied the proposed system to 27 potato scab isolates obtained from the Korean provinces of Jeju-do and Kangwon-do. Phylogenetic relationships among these isolates using the devised rpoB gene-based methods were generally similar to those reported for 16S rRNA gene-based analysis. Isolates from potato scab lesion in Korea were also clearly differentiated into their phylogenetic groups by this method. In addition, the deduced RpoB amino acid sequences were also found to be useful for differentiating these strains. Our data demonstrate that the rpoB gene-based method can be used as a means of complementing other genetic methods such as 16S rRNA gene analysis or DNA-DNA hybridization to phylogenetically differentiate potato scab related Streptomyces spp.
Assuntos
Doenças das Plantas/microbiologia , RNA Ribossômico 16S/análise , Solanum tuberosum/microbiologia , Streptomyces/classificação , DNA Ribossômico/análise , RNA Polimerases Dirigidas por DNA/genética , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Streptomyces/genéticaRESUMO
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.
Assuntos
Proteínas de Bactérias/genética , Chaperoninas/genética , Impressões Digitais de DNA , DNA Bacteriano/genética , Mycobacterium/isolamento & purificação , Polimorfismo de Fragmento de Restrição , Escarro/microbiologia , Técnicas Bacteriológicas , Chaperonina 60 , DNA Bacteriano/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Humanos , Mycobacterium/genética , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
DNA Viral/genética , Genoma Viral , Vírus da Hepatite B/classificação , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Polimorfismo Genético , Adulto , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Humanos , Coreia (Geográfico) , Masculino , Dados de Sequência Molecular , Filogenia , Mutação Puntual , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Deleção de Sequência , Homologia de SequênciaRESUMO
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.
Assuntos
Vírus da Hepatite B/classificação , Vírus da Hepatite B/genética , Hepatite B Crônica/epidemiologia , Epidemiologia Molecular , Adulto , Idoso , Feminino , Genótipo , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/virologia , Humanos , Coreia (Geográfico)/epidemiologia , Masculino , Pessoa de Meia-Idade , Filogenia , Análise de Sequência de DNA , SorotipagemRESUMO
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.