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배경 및 목적 :갑상선자극인자-방출호르몬(Thyro-tropin-releasing hormone, TRH)의 분비 및 유전자 발현은 당질코르티코이드에 의하여 촉진되거나 억제되는데, 이러한 반응은 조직에 따라 다르다. 조직 특이성의 기전을 규명하기 위해서는 우선 TRH 유전자의 당질코르티코이드 반응요소(glucocorticoid response element, CRE)의 위치와 특성을 규명되어야 한다. 최근 본 연구자등은 TRH 유전자의 촉진자 위 -200 bp와 -220 bp 사이에 GRE가 존재하는데, -200 bp와 -205 bp 사이에 GRE 공감서열의 half site인 TGTTCT가 있고 그 양 옆으로 TPA 반응요소(TPA response element, TRE)와 유사한 서열 TACTCA가 연결되어 있는 특이한 구조를 가지고 있음을 보고하였다. 본 연구는 이 CRE/TRE 복합서열이 당질코르티코이드와 TPA에 반응을 하고, 전사인자인 당질코르티코이드 수용체(glucocoiticoid receptor, GR)와 c-Jun이 결합을 하는지 여부를 조사하기 위하여 수행되었다. 방법 : 배양된 HeLa 세포에 CRE/TRE 복합서열을 포함하고 있는 TRH 유전자의 촉진자를 luciferase 유전자 앞에 연결시킨 플라즈미드를 Fugene 6를 이용하여 주입한 후, 덱사메타손(DEX)과 TPA(12-O-tetradecanoyl phobol-13-acetate)로 자극하고 세포를 파괴시켜luciferase 활성을 측정하였다. GRE와 TRE의 염기서열 일부를 다른 뉴클레오타이드로 치환한 여러 가지 돌연변이를 제작하여 동일한 방법으로 실험하여 야생형의 전사활성 정도와 비교하였다. 그리고, GR과 c-Jun을 GRE/TRE 복합서열과 결합시킨 후 겔지연분석을 시행하였다. 연구결과 : DEX와 TPA는 야생형의 전사활성을 각각 2.5배, 4배 증가시켰고, 동시에 두 가지로 자극하였을 때는 10배 증가시켰다. GRE의 두 가지 돌연변이들은 DEX에 전혀 반응을 보이지 않았으며, TPA에 대한 반응도 야생형의 50% 정도로 저하되었다. TRE의 3'쪽 돌연변이와 5'쪽 돌연변이의 DEX에 대한 반응과 TPA에 대한 반응은 각각 야생형의 20%, 25%이었으나, 양쪽 돌연변이의 반응은 야생형과 유사하였다. 겔지연분석상 GR과 CRE/TRE 복합서열에 의한 합체는 한 개의 띠로 보였으며, GRE 돌연변이는 야생형에 비하여 GR과의 결합 친화도가 현저히 낮았다. c-Jun에 의한 복합체는 두 개의 띠로 보였는데, 복합서열의 결합 친화도는 TRE 공감서열에 비하여 현저히 낮았다. 3'과 5' TRE 돌연변이들도 야생형에 비하여 c-Jun과의 결합 친화도가 현저히 낮았으나, 양쪽 돌연변이의 친화도는 야생형의 친화도와 유사하였다. GR과 c-Jun을 동시에 결합시켰을 경우와 두 단백이 결합된 분자량이 큰 복합체는 관찰되지 않았으며, GR에 의한 복합체는 c-Jun에 의한 복합체보다 분자량이 현저히 작아 단일체로 결합되었을 것을 암시하였다. 결론 : 쥐 TRH 유전자의 전사조절 부위에 존재하는 GRE/TRE 복합서열은 GR과 c-Jun이 결합하여 상호작용을 할 수 있는 장소를 제공하여 줌으로써 HeLa 세포에서는 당질코르티코이드와 TPA가 전사 활성을 상승적으로 촉진할 수 있다. 향후 당질코르티코이드에 의하여 TRH 유전자의 전사가 억제되는 세포에서는 이 복합서열에서 GR과 다른 전사인자들이 어떻게 상호작용하여 전사를 억제하는지 연구가 필요할 것으로 사료된다. We previously demonstrated that the promoter of rat TRH gene has GRE half site (TGTTCT) between-210 bp and-205 bp flanking with similar sequences of TPA response element (TRE). TAGTCA, at a distance of several base pairs from the GRE half site. It prompt us to hypothesize that this composite GRE/TRE sequence can provide a site for interaction between glucocorticoid receptor (GR) and c-Jun. Thus, we investigated whether the composite sequence mediates transcriptional regulation induced by dexamethasone (DEX) and 12-O-tetradecanoyl phobol-13-acetate (TPA), and it binds GR and c-Jun. The Luciferase expressing plasmids that contain a part of rat TRH promoter including the composite sequence or its mutants were transfected into HeLa cells by Fugene 6. After the cells were incubated overnight with DEX and TPA, the luciferase activity was measured in a chemiluminometer. A gel retardation assay was performed after binding of the labeled composite sequence or its mutants with GR and c-Jun. DEX increased the transcriptional activity of the plasmid containing the wild type GRE by 2.5 folds, and TPA increased the transcriptional activity by 4 folds. The simultaneous stimulation with DEX and TPA synergistically increased the transcriptional activity by 10 folds. Two mutants whose GRE half site was altered showed no response to DEX. and suppressed the TPA-induced or both agents-induced transcriptional activity by 50%. Two mutants whose TRE-like site was altered suppressed the DEX-induced transcriptional activity by 20%, TPA-induced transcriptional activity by 25%. and both agents-induced transcriptional activity by 50%. Gel retardation assay showed that the composite sequence formed a complex with GR and its mutants bound to GR with a remarkably less affinity, c-Jun also bound to the composite sequence to form two complexes with a less affinity compared to the AP-1 consensus sequence. The mutants of the TRE-like sequence bound to c-Jun with a significantly lower affinity compared to that of the wild type. Simulateous binding of the composite sequence with GR and c-Jun did not form any larger complex. The complex of GR and the composite sequence was much smaller than that formed by c-Jun, suggesting that GR binds to the composite sequence as a monomer. These result suggest that the composite sequence of GRE half site and TRE-like site on the promoter of rat TRH gene provides binding sites for GR and c-Jun, which mediate the interaction between two signal transduction pathways.
In molecular diagnostics, the methodological development and application of detecting and quantifying nucleic acid has been made over the past decades. Nucleic acid analysis based polymerase chain reaction(PCR) has been dominated among various techniques, such as PCR, sequencing, microarray and others. Detection and quantification of disease-related genes gives us a important information that may be used to predict disease progression, signs of infection and the efficacy of therapeutic agent. traditional PCR is based on exponential amplification and quantification of nucleic acids may be determined by comparing the number of amplification cycles and amount of PCR product to those of a reference specimen. However, many factors can complicate the calculation of quantification, resulting in uncertainties and inaccuracies. Digital PCR is considered to have overcome the critical problems of insufficient quantification in traditional PCR. The difference between dPCR and traditional PCR lies on the measuring method for nucleic acids amounts. Absolute quantification through clonal amplification is a key factor in digital PCR. It has many possible applications which include the detection and quantification of low-level pathogens, viral load, rare allele, copy number variation, and relative gene expression. Along with the more advanced development in technology of digital PCR, the clinical applications of digital PCR will be greatly expanded. Therefore, the present review describes the principle, technological types and clinical applications of digital PCR as useful molecular diagnostic tool.
Corticotropin-releasing hormone(CRH) is a major regulator of the hypothalamic-pituitaryadrenal axis(HPA). In inflammatory stress, the cytokines TNF-, IL-6 stimulate the production of CRH, a 41 amino acids neuropeptide, in the hypothalamus. The release of CRH leads to pituitary production of adrenocorticotropic hormone, followed by glucocorticoid secretion by the adrenal cortex. Glucocorticoids suppress namy components of the inflammatory process. Recently, CRH and CRH receptor were reported to be located in the periphery such as Immune system and chronic inflammatory sites as rheumatoid arthritis (RA). Cyclooxygenase consisted of two isoforms, COX-1 and COX-2, converts arachdonate to prostaglandins(PGs) which are important mediators of inflammation. insymoviocyte in RA, it was described that COX-2 mRNA was markedly increased by inflammatory agents, PMA or IL-1 and COX-1 transcripts were not modulated. We examined the modulation of COX by immune CRH in cultured normal and rheumatoid synoviocytes. Our results were shown that COX-1 mRNA expression decreased with the each stimulation of PMA and IL-1 in normal synoviocyte. In RA synoviocyte, PMA and IL-1 were increased mRNA expression of COX-1. In simultaneous treatment with CRH, PMA group was decreased, but IL-1 group was increased mRNA expression of COX-1. COX-2 mRNA expression was slightly increased by the treatment with PMA and highly increased by IL-1. After CRH treatment, PMA and IL-1 addictively increased COX-2 mRNA expression. We think that these results are contributed to the influence of increased cANP by CRH on the promoter of COX-2 in normal and synoviocytes. Because many cytokines, neuropeptides, and signal transduction pathways are involved in chronic inflammation, the exact role of CRH on inflammation is not fully elucidated. To achieve this goal, further experiments are needed.
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Human papillomavirus(HPV) has been known as one of the important pathogenic agent in uterine cervical carcinoma. The molecular works such as PCR enable the detection of large number of HPV genotypes obtained from viginal swab. Many of the PCR-based methods for HPV detection involve an amplification step followed by any of a number of methods for distinguishing different HPV types. In this study, we adopted the DNA chip technology enabling a HPV type-specific differentiation both low-risk group(type-6, 11, 34, 40, 42, 43, and 44) and high-risk group(type-16, 18, 31, 33, 35, 39, 45, 51,52,54, 56, and 58). MY09/MY11 and GP5+/GP6+primers covered LI region are used in nested PCR to improve PCR amplification. HPV type-specific probes for DNA chip were modified with NH2-C6, followed by spotting on silylated slides, washing slides and hybridization with each PCR products. Of 163 DNA samples chosen randomly, 42 samples were negative, 8 ones for low-risk group of HPV and 96 ones for high-risk group of HPV. Especially, co-infections with various HPVs were shown in 17 samples. A recent study found that multiple HPV is a factor in persistent HPV infection, resulting in the development of cervical dysplasia. This result emphasized the necessity to detect multiple HPV infection. The application of DNA-chip to determinate specific HPV typing will be a stronger candidate than any other PCR-based methods. Furthermore, the sequencing data of the positive PCR products were shown no discrepancy with DNA chip results. This means that DNA chip is very useful tool for both HPV detection and typing.
Over the past decades, the development and application of molecular diagnostics has been made in all fields of laboratory medicine as in vitro diagnostics. Although conventional methods are mainly used, there is an increasing trend towards molecular diagnostics. These techniques are superior to conventional ones in rapid detection, higher sensitivity and specificity. Polymerase chain reaction(PCR)-based systems has dominated in various nucleic acid-based techniques, such as PCR, sequencing, DNA microarray, lab-on-a-chip(LOC) and etc. PCR-based systems can detect the etiologic agents through the amplification of disease-related gene directly from clinical samples and have been undergone various modification for more rapid, accurate and applicable to diagnostic tests. Sequencing analysis ensures exact identification and better characterization of the etiologic agents. Some like array-based systems offer the multiparameter results where many pathogen-related markers and gene mutations can be analysed simultaneously. The development of LOC chip should allow point-of-care testing for the revolutionary improvement of healthcare. Along with the development of proteomic or metabolomic-based tests, More advanced diagnostic tools will expand the applications of molecular diagnostic testing. The user-friendly automation about both the high throughput and the use of minimal quantity of sample makes these technologies more widely available.
Papillomaviruses infect a wide variety of animals, including humans. The human papillomavirus (HPV), in particular, is one of the most common causes of sexually transmitted disease. More than 200 types of HPV have been identified by DNA sequence data, and 85 HPV genotypes have been well characterized to date. HPV can infect the basal epithelial cells of the skin or inner tissue linings, and are, accordingly, categorized as either cutaneous or mucosal type. HPV is associated with a panoply of clinical conditions, ranging from innocuous lesions to cervical cancer. In the early 1980s, studies first reported a link between cervical cancer and genital HPV infection. Genital HPV infections are now recognized to be a major risk factor in at least 95% of cervical cancers. 30 different HPV genotypes have been identified as causative of sexually transmitted diseases, most of which induce lesions in the cervix, vagina, vulva, penis, and anus, as the result of sexual contact. There is also direct evidence demonstrating that at least four of these genotypes are prerequisite factors in cervical cancer. The main aim of this review was to evaluate the current literature regarding the pathovirology, diagnostics, vaccines, therapy, risk groups, and further therapeutic directions for HPV infections. In addition, we reviewed the current status of HPV infections in South Korean women, as evidenced by our data.
Antimicrobial resistance has increased remarkably over the decades and is a challenge that has been associated with high mortality and medical costs. Antibiotic resistant pathogens that can be difficult to treat are currently responsible for approximately 90% of infection related deaths. Multidrug resistance has become a main concern about how to identify and treat infection of pathogenic organism. For the persistent outbreak of new types of virulence and resistant bacteria, diverse patterns on multidrug resistance is becoming more common and make it more difficult to treat or may be untreatable with conventional antimicrobial therapies. But we are living in the era of a shortage of effective prevention, suitable therapies and a few new antibiotics. There are urgent demands on the development of novel treatment strategies and an effective alternative antibiotics. Understanding molecular mechanisms of anitbiotic resistance is necessary to look for ways to develop new substantial approaches to manage and prevent occcurrence of antibiotic resistance. These mechanisms allow pathgenic bacteria to survive or to grow well in the administration of antibiotics. Various genetic and phenotypic mechanisms that include antibiotic degradation, target modification and change of permeability through the bacterial membrane have been described. Technologies for Antibiotic susceptibility testing(AST) are constantly evolved to enhance rapid turn around time with the ultimate goal of targeted antibiotic therapy and increasing recovery rates. It would be continued to perform Clinical applications of advanced AST applying the new technologies to identify antimicrobial resistance pathogens more accurately. The latest advances of diagnostic technologies on antimicrobial resistance and their applications in clinical microbiology laboratories are discussed in this review.
Conventional culture-based identification systems are well standardized and widely adopted to detect and identify infectious pathogens in the microbiological diagnostics. These traditional methods as gold standard have the advantages of high sensitivity, high specificity and low cost, whereas they shows the disadvantages on intensive labor, long test time and non-growing some microorganisms that not grow in artificial medium. A fast and reliable identification of pathogens is a keyfacts in clinical diagnostics. In order to meet these problems, the new technological methods has been applied on the identification of clinically important pathogens over the last decades. Although microbiological analysis is too complexed to automate because of the various reasons, automation of culture system is becoming a reality in recent years in microbiological laboratories. Revolutionary nucleic acid analysis such as polymerase chain reaction(PCR), hybridization, sequencing and others has been increasingly used and considered to compensate or replace traditional culture-based methods in the future. Another technique is the matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF/MS). These new methods do not need bacterial culture to confirm the pathogens and have fast, easy and high-throughput features. Along with the more advanced technologies to be able to identify pathogens accurately, the clinical applications of them in microbial laboratories will be sustainedly performed. The recent trend of microbial diagnostic technologies and their clinical applications in microbiological laboratories would be discussed in this review.
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