Mitochondrial Protein Nfu1 Influences Homeostasis of Essential Metals in the Human Fungal Pathogen Cryptococcus neoformans

Mitochondrial protein Nfu1 plays an important role in the assembly of mitochondrial Fe-S clusters and intracellular iron homeostasis in the model yeast Saccharomyces cerevisiae. In this study, we identified the Nfu1 ortholog in the human fungal pathogen Cryptococcus neoformans. Our data showed that C. neoformans Nfu1 localized in the mitochondria and influenced homeostasis of essential metals such as iron, copper and manganese. Marked growth defects were observed in the mutant lacking NFU1, which suggests a critical role of Nfu1 in Fe-S cluster biosynthesis and intracellular metal homeostasis in C. neoformans.

Use of hydrophilic extra-viral domain of canine distemper virus H protein for enzyme-linked immunosorbent assay development

Simple methods for measuring the levels of serum antibody against canine distemper virus (CDV) would assist in the effective vaccination of dogs. To develop an enzyme-linked immunosorbent assay (ELISA) specific for CDV, we expressed hydrophilic extra-viral domain (HEVD) protein of the A75/17-CDV H gene in a pET 28a plasmid-based Escherichia (E.) coli vector system. Expression was confirmed by dot and Western blotting. We proposed that detection of E. coli-expressed H protein might be conformation-dependent because intensities of the reactions observed with these two methods varied. The H gene HEVD protein was further purified and used as an antigen for an ELISA. Samples from dogs with undetectable to high anti-CDV antibody titers were analyzed using this HEVD-specific ELISA and a commercial CDV antibody detection kit (ImmunoComb). Levels of HEVD antigenicity measured with the assays and immunochromatography correlated. These data indicated that the HEDV protein may be used as antigen to develop techniques for detecting antibodies against CDV.

Microsatellite instability in endometrioid type endometrial adenocarcinoma is associated with poor prognostic indicators.

Microsatellite instability (MSI) has been reported in 25% to 45% of sporadic endometrial carcinoma. The clinicopathologic and molecular characteristics of MSI-high phenotype in colorectal and gastric carcinomas have been widely investigated; however, the clinicopathologic impact of MSI on endometrial carcinomas remained unclear. This study was performed to determine the clinicopathologic and molecular significance of MSI in endometrial carcinomas. We analyzed the MSI status using National Cancer Institute-recommended 5 microsatellite markers, and the immunohistochemical profiles of various regulatory proteins of cell cycle and apoptosis using tissue microarray in 100 endometrial carcinomas. The results were compared between MSI-high and MSI(-) groups as for the traditional clinicopathologic prognostic parameters and the immunoreactivities of various regulatory proteins. We especially focused on the endometrioid type adenocarcinoma to exclude the bias from nonendometrioid type adenocarcinomas with more aggressiveness and a close association with MSI(-) phenotype. The incidence of MSI-high phenotype was significantly higher in endometrioid type than in nonendometrioid serous type (20% vs. 0%, P<0.001). It showed orderly increase in the frequencies of MSI-high phenotype in higher histologic grade (13% vs. 21% vs. 50% in histologic grade I, II, and III, P=0.039). The MSI-high phenotype was related with the presence of lymphovascular invasion (P=0.008), deep myometrial invasion (P=0.040), and the higher clinical stages (P=0.018) independent of tumor grade. We also found a correlation between MSI-high phenotype and higher cyclin A and skp2 immunoreactivity (P=0.03 and 0.05, respectively), known to be the poor prognostic molecular indicators. According to these results, the MSI may represent the poor prognostic impact on the endometrioid type endometrial adenocarcinoma.

A Feasibility Study of Adenosine Triphosphate-based Chemotherapy Response Assay (ATP-CRA) as a Chemosensitivity Test for Lung Cancer / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: A chemosensitivity test can reflect the differences in responses of individual cancer patients to chemotherapeutic agents. The adenosine triphosphate-based chemotherapy response assay (ATP-CRA)is an accurate method, which does not require a large amount of tissue specimen. So far, no studies have evaluated the utility of the ATP-CRA in Korea. Therefore, we investigated the clinical usefulness of the ATP-CRA in 53 patients with lung cancer. MATERIALS AND METHODS: Tumor tissues were obtained from bronchoscopic biopsies or surgical resections. The validity of ATP-CRA was assessed focusing on the success rate, experimental error level (intraassay mean coefficient of variation [CV]) and reproducibility. RESULTS: The overall success rate of ATP-CRA was 90.6% (48/53). Normal cells were effectively eliminated from the tumor tissues with the use of ficoll gradient centrifugation and immunomagnetic separation, which was confirmed using loss of heterozygosity analysis of the 3p deletion. The mean CV of ATP assays was 10.5+/-4.6%. The reproducibility of ATP assays was 94+/-3.8%. The results of the ATP assays were reported to physicians within 7 days of specimen collection. More than 6 anticancer drugs were tested on the tumor specimens obtained from bronchoscopic biopsies. CONCLUSION: The ATP-CRA is a stable, accurate and potentially practical chemosensitivity test in patients with lung cancer.

Genital human papillomavirus genotyping by HPV oligonucleotide microarray in Korean commercial sex workers.

Because of the diversity in human papillomavirus (HPV) distribution, according to the population and region, detailed investigations of HPV genotypes are important in designing more effective HPV vaccines for any given country. HPV DNA oligonucleotide microarray was used to investigate the distribution of HPV genotypes among commercial sex workers. The prevalence of HPV in Korean commercial sex workers was 47%, with HPV-16 and HPV-51 as the dominant genotypes. HPV subtypes in 148 commercial sex workers comprised 70 with one genotype, 42 with two genotypes, 17 with three genotypes, and 19 with four or more genotypes. HPV-40, the most dominant low-risk genotype, was not detected in single-infection commercial sex workers. All women with multiple infections of low-risk genotypes had the HPV-40 genotype. This molecular epidemiological study of genital HPV will be useful for the development of a favorable strategy to prevent the spread of this potentially serious infection.

Multiple HPV infection in cervical cancer screened by HPVDNAChip.

This study determined the distribution of high-risk HPV type infection in cervical cancer using newly developed oligonucleotide chips (HPVDNAChips). The study subjects included 80 cases of cervical neoplasia and 746 controls with a normal Pap smear. For HPV genotyping, the commercially available HPVDNAChips was used. The risk of cervical cancer was increased in women with a family history of cervical cancer (adjusted OR=2.3, 95% CI: 0.92-6.17) and in smokers (adjusted OR=3.2, 95% CI: 1.45-7.06). There was also a trend of increased risk with the number of full term pregnancies (P(for trend)<0.001). There were only 7.2% (54 of 746) infected high-risk HPV types in the control, whereas 54.5% (six of 11) and 76.5% (52 of 68) were infected in the CIN and cervical cancer, respectively. Multiple HPV infection was observed in 0.5% (three of 592) of the control group but in 9.1% (seven of 77) of cases. Multivariate analysis revealed that subjects infected with multiple HPV types had a 31.8-fold (95% CI: 7.50-134.75) higher risk of cervical cancer, while the single HPV type had a 19.9-fold increased risk (95% CI: 10.90-36.18) (P(for trend)<0.001). These results show that the detection and typing of HPV infection by HPVDNAChip can be a useful in clinical applications because it provides information on multiple infections and the types of HPV in addition to HPV infection status.

Detection and typing of HPV genotypes in various cervical lesions by HPV oligonucleotide microarray.

OBJECTIVES: This study was conducted to evaluate a clinical efficacy of human papillomavirus (HPV) oligonucleotide microarray (Biomedlab Co., Seoul, South Korea) for the detection of HPVs in various cervical lesions. RESULTS: HPV DNAs from 234 patients were analysed by two methods. Among those, 212 patients were classified into 5 groups according to the histologic diagnosis: chronic cervicitis, cervical intraepithelial neoplasia (CIN) I, CIN II, CIN III, and invasive cervical carcinoma. PCR-RFLP could detect 7 types of high-risk HPVs (HPV 16/18/31/33/35/52/58) and HPV microarray could detect 15 types of high-risk HPVs (HPV 16/18/31/33/35/39/45/51/52/56/58/59/66/68/69) and 7 types of low-risk HPVs (HPV 6/11/34/40/42/43/44).HPV genotyping by HPV oligonucleotide microarray revealed that HPV16 was the most frequent type (30.2%) in all specimens tested and was significantly more frequent in CIN III and invasive carcinomas than other lesions. METHODS: HPV DNAs were detected in 158 and 174 of the 234 cervical samples by PCR-RFLP and HPV microarray, respectively. The correlation between the two methods was good in detecting HPVs in general (kappa index = 0.69) and HPVs 31 and 52 (kappa index = 0.70 and 0.70, respectively) and excellent in detecting HPVs 16, 18, 33, 35, and 58 (kappa index = 0.90, 0.88, 0.92, 0.77, and 0.84, respectively). Double HPV infection was detected in 10 cases and one triple infection was detected. By combining cytology and HPV testing, the sensitivity was improved to 87.5, 95.5, 96.1, and 97.2% in CIN I, CIN II, CIN III, and carcinoma, respectively. CONCLUSIONS: This results suggest that HPV oligonucleotide microarray is a highly comparable method to the previously used PCR-RFLP method for the detection of HPVs in cervical specimens. Genetic informations for HPV infection in cervical specimens may offer new strategies in manipulating the patients harboring cervical intraepithelial neoplasia and cervical carcinoma.

HPV oligonucleotide microarray-based detection of HPV genotypes in cervical neoplastic lesions.

BACKGROUND: In this study we examined the use of a new-human papillomavirus (HPV) detection method, the HPV oligonucleotide microarray system (Biomedlab Co., Korea), which we compared with the well-established HPV DNA detection system (Hybrid Capture II; HC-II, Digene Co.). This new method prompted us to develop a new HPV genotyping technique, using the oligonucleotide microarray, to detect the generic and type-specific sequence of HPV types. In particular, we undertook the evaluation of the clinical efficacy of the HPV oligonucleotide microarray for detecting HPV in cervical neoplastic lesions. METHODS: One hundred forty patients were involved and classified into three groups according to their histopathologic diagnoses: Group I (nonspecific chronic cervicitis; n = 61), Group II (low-grade squamous intraepithelial lesion (SIL); koilocytosis, and mild dysplasia; n = 39), and Group III (high-grade SIL; moderate, severe dysplasia and in situ carcinoma; n = 40). Cytological diagnoses were based on the Bethesda System and cervical samples were analyzed by the two methods. The HPV oligonucleotide microarray detected 15 types of high-risk HPV (HPV-16/-18/-31/-33/-35/-39/-45/-51/-52/-56/-58/-59/-66/-68/-69) and 7 types of low-risk HPV (HPV-6/-11/-34/-40/-42/-43/-44). RESULTS: In 105 of the 140 cervical samples (75%), HPV DNAs were examined using the HC-II method. HPV detection rates using the HPV microarray agreed with those of HC-II. One HC-II-positive, but HPV microarray-negative, case occurred in the low-grade SIL (Group II) and was later confirmed negative for HPV. The other HPV microarray-positive but HC-II-negative case was found to be HPV-18 by PCR. Low-risk types of HPV were detected in 3 of 39 low-grade SIL cases (Group II) using the HPV microarray. HPV-16 was the most frequent type (32.1%) in all specimens tested, and was significantly more frequent in low-grade or high-grade intraepithelial lesions (Groups II or III) than in normal controls (Group I) (P < 0.05). HPV-58 was the second most common type (17.5%) in Group III. The HPV microarray was found to have advantages in terms of identifying the HPV genotypes and cases of multiple HPV infection. Double HPV infections were detected in 12 cases and triple HPV infections in 7 cases. Two cases were positive for four types of HPV (HPV-16/18/33/35, HPV-16/18/58/68). The sensitivity of HPV testing (HC-II; 94.9%, HPV microarray; 93.7%) for identifying patients with squamous intraepithelial lesion was significantly better than the sensitivity of cytology (77.1%, P < 0.05). On using multiple logistic regression analysis to estimate the relative risk of SIL versus HPV type, HPV-16-positive cases were found to have a 7.5-fold risk of SIL (95% CI = 3.28-16.51; P < 0.01). HPV-33 and HPV-58 were found to be significantly related to high-grade SILs (P < 0.01). CONCLUSIONS: Our results suggest that the HPV oligonucleotide microarray is highly comparable to HC-II for detecting HPV in cervical specimens. The HPV oligonucleotide microarray provides useful information on viral genotype and multiple HPV infections in HPV-related cervical lesions. Genetic information on HPV in cervical specimens might be a particular benefit of the new procedure in the management of cervical neoplastic lesions

Correlation of cervical carcinoma and precancerous lesions with human papillomavirus (HPV) genotypes detected with the HPV DNA chip microarray method.

BACKGROUND: Human papillomavirus (HPV) infection is considered to play an important role in the development of cervical carcinoma, and it is known that certain HPV types, such as HPV-16 and HPV-18, are highly associated with cervical carcinoma. However, the pathologic behavior of other HPV types remains unclear. Recently, a new HPV detection technique, the HPV DNA chip, was introduced. The HPV DNA chip harbors 22 HPV probes and has the advantage of being able to detect 22 HPV types simultaneously. To evaluate the quality of the HPV DNA chip method and to identify HPV types related to cervical carcinoma and precancerous lesions, the authors performed HPV typing in cervical specimens from 1983 patients and compared their cytologic and histologic diagnoses. METHODS: The HPV DNA chip was used for HPV typing. Among 1983 patients who were tested for HPV types, cervical smear cytology was performed in 1650 patients, and 677 of those patients underwent cervical biopsy. RESULTS: Among the 1650 smears that were examined cytologically, 92.7% (114 of 123 smears) of low-grade squamous intraepithelial lesions (LSILs), 98.1% (106 of 108 smears) of high-grade squamous intraepithelial lesions (HSILs), and 96.3% (51 of 53 smears) of carcinomas were HPV positive, compared with only 35.1% of smears with normal cytology that were HPV positive. HPV-16 was the most prevalent type (chi-square test; P < 0.01) in LSILs (28.5%), in HSILs (51.9%), and in carcinomas (62.5%) followed by HPV-58 and a group of low-risk types (HPV-6, HPV-11, HPV-34, HPV-40, HPV-42, HPV-43,and HPV-44) in LSILs. HPV-58 (15.7%), HPV-18 (6.7%), and HPV-52 (4.6%) were the next most prevalent types after HPV-16 in HSILs. HPV-18 (11.4%) and HPV-58 (11.4%) were the second most common types in carcinomas. HPV-58 had the highest positive predictive value (54.9%) for the detection of histologically confirmed HSIL or carcinoma, whereas HPV 16 had the highest negative predictive value (80.6%). The sensitivity (96.0%) of the HPV test using the DNA chip method for detecting HSIL or carcinoma was superior compared with the sensitivity of cytologic diagnosis (83.6%). CONCLUSIONS: The HPV DNA chip provides a very sensitive method for detecting 22 HPV genotypes with reasonable sensitivity (96.0%) and reasonable negative predictive value (96.9%), and it overcomes the low sensitivity of cytologic screening for the detection of HSIL or carcinoma. HPV-58, HPV-52, and HPV-56, as well as HPV-16 and HPV-18, were associated highly with HSIL and carcinoma in the current large series. In addition, multiple HPV infection was associated less frequently with cervical carcinoma and with precancerous lesions compared with normal cytology.

Genotyping of 22 human papillomavirus types by DNA chip in Korean women: comparison with cytologic diagnosis.

OBJECTIVE: More sensitive and reliable methods than individual testing (such as polymerase chain reaction, restriction fragment length polymorphism, and Southern blot) should be developed as screening tools for the detection of latent human papillomavirus. Today, the new Bethesda system recommends human papillomavirus testing as an adjuvant to the conventional Papanicolaou smear for more comprehensive identification of women at certain risk of cervical neoplasia. We performed human papillomavirus genotyping with the newly designed human papillomavirus DNA chip, which is based on polymerase chain reaction for high-throughput screening power, and compared the results with the results of a Papanicolaou smear according to the new Bethesda system. STUDY DESIGN: Polymerase chain reaction amplifications of the human papillomavirus L1 region from biologic samples were hybridized to silanized glass slides by a microarrayer, which comprised 22 specific oligonucleotide probes to their genotypes, consisting of 15 high-risk and 7 low-risk types. Two cervical cancer cell lines and 20 plasmids that contained each type of the human papillomavirus whole genome were used for the evaluation of this method; in all cases, the cancer cell lines and plasmids showed clear positive signals on their corresponding positions. A comparative study that used 685 cervicovaginal swabs was performed by human papillomavirus DNA chip microarray together with Papanicolaou diagnosis. RESULTS: Human papillomavirus was identified as positive in 31.9% of the 414 control samples and in 78.6% of the 271 neoplastic lesions. The major prevailing human papillomavirus genotypes were human papillomavirus types 16, 58, and 18, in descending order of incidence (average overall, 78.8%). Almost all of the remaining cases were comprised of human papillomavirus types 39, 52, 56, and 51. The frequency of multiple infection of human papillomavirus was highest in low-grade squamous intraepithelial lesion but was lowest in squamous cell carcinoma. All cases that exhibited infection of single human papillomavirus type 58 were squamous cell carcinoma. CONCLUSION: Human papillomavirus types 16, 18, and 58 were confirmed to be major causative factors for cervical carcinogenesis. Low-grade squamous intraepithelial lesion is a heterogeneous entity that is composed of different human papillomavirus subtypes and prevails in younger women (<40 years old). The human papillomavirus chip has potential use as a high-throughput screening test.