The association of uterine cervical microbiota with an increased risk for cervical intraepithelial neoplasia in Korea.

Recent studies have suggested potential roles of the microbiome in cervicovaginal diseases. However, there has been no report on the cervical microbiome in cervical intraepithelial neoplasia (CIN). We aimed to identify the cervical microbiota of Korean women and assess the association between the cervical microbiota and CIN, and to determine the combined effect of the microbiota and human papillomavirus (HPV) on the risk of CIN. The cervical microbiota of 70 women with CIN and 50 control women was analysed using pyrosequencing based on the 16S rRNA gene. The associations between specific microbial patterns or abundance of specific microbiota and CIN risk were assessed using multivariate logistic regression, and the relative excess risk due to interaction (RERI) and the synergy index (S) were calculated. The phyla Firmicutes, Actinobacteria, Bacteroidetes, Proteobacteria, Tenericutes, Fusobacteria and TM7 were predominant in the microbiota and four distinct community types were observed in all women. A high score of the pattern characterized by predominance of Atopobium vaginae, Gardnerella vaginalis and Lactobacillus iners with a minority of Lactobacillus crispatus had a higher CIN risk (OR 5.80, 95% CI 1.73-19.4) and abundance of A. vaginae had a higher CIN risk (OR 6.63, 95% CI 1.61-27.2). The synergistic effect of a high score of this microbial pattern and oncogenic HPV was observed (OR 34.1, 95% CI 4.95-284.5; RERI/S, 15.9/1.93). A predominance of A. vaginae, G. vaginalis and L. iners with a concomitant paucity of L. crispatus in the cervical microbiota was associated with CIN risk, suggesting that bacterial dysbiosis and its combination with oncogenic HPV may be a risk factor for cervical neoplasia.

Cancer cells promote survival through depletion of the von Hippel-Lindau tumor suppressor by protein crosslinking.

Nuclear factor-κB (NF-κB) and insulin-like growth factor-1 (IGF-1)-mediated signaling is associated with different tumors including renal cell carcinoma. NF-κB- and IGF-1-mediated signaling is found to be inhibited in the presence of wild-type von Hippel-Lindau (VHL) tumor suppresser gene. Therefore, negative regulator of VHL may be a good target for regulating NF-κB and IGF-1R. In this study, we found that VHL, a tumor suppressor protein that downregulates the NF-κB activity and the stability of IGF-1R was depleted by TGase 2 through polymerization via crosslinking and proteasomal degradation in kidney, breast and ovary cancer cell lines. We also found that TGase 2 knockdown promotes hypoxia-inducible factor 1α (HIF-1α) degradation, and thereby decrease HIF-1α transcriptional activity. Importantly, VHL expression was decreased in vivo in TGase-2-transgenic mice, and this was associated with increased NF-κB activity and the levels of expression of IGF-1R, HIF-1α and erythropoietin in kidney tissue. These results suggest a novel mechanism of regulation of the VHL tumor suppressor by TGase 2 that appears to be independent of the known cancer regulatory mechanisms.

Frequent homozygous deletion of the LKB1/STK11 gene in non-small cell lung cancer.

LKB1/STK11 is a tumor suppressor and a negative regulator of mammalian target of rapamycin signaling. It is inactivated in 30% of lung cancer cell lines but only 5-15% of primary lung adenocarcinomas. There is evidence that homozygous deletion (HD) of chromosome 19p at the LKB locus contributes to the inactivation of the gene in primary human lung cancers. Here, we used several complementary genetic approaches to assess the LKB1 locus in primary non-small cell lung cancers (NSCLCs). We first analyzed 124 NSCLC cases for allelic imbalance using eight microsatellite markers on chromosome 19p, which revealed an overall rate of 65% (80 of 124) loss of heterozygosity (LOH). We next used chromogenic in situ hybridization (CISH) to directly examine the chromosomal status of the LKB1 locus. In all, 65 of 124 LOH tested samples were available for CISH and 58 of those (89%) showed either loss of one copy of chromosome 19p (LOH, 40 of 65 cases, 62%) or both copies (HD 18 of 65 cases, 28%). The occurrence of HD was significantly more frequent in Caucasian (35%) than in African-American patients (6%) (P=0.04). A total of 62 of 124 samples with LOH at one or both markers immediately flanking the LKB1 gene were further analyzed by directly sequencing the complete coding region, which identified 7 of 62 (11%) tumors with somatic mutations in the gene. Jointly, our data identified total inactivation of the LKB1 gene by either HD or LOH with somatic mutation in 39% of tested samples, whereas loss of chromosome 19p region by HD or LOH at the LKB1 region occured in 90% of NSCLC.

A novel tumor-associated mucin of gastrointestinal carcinoma.

PURPOSE: To identify a new tumor-associated antigen, a monoclonal antibody, SC142, was produced by immunizing mice with a stomach cancer cell line. The tumor specificity of mAb SC142 was studied by immunohistochemical staining, and the biochemical characteristics of this new gastrointestinal tumor-associated antigen were also studied. METHODS: The expression of SC142-reactive antigen was investigated in various cancers by immunohistochemical staining. The SC142-reactive antigen was characterized by immunoblotting, sodium metaperiodate treatment assay, O-glycanase digestion assay, and lectin binding assay. RESULTS: The SC142-reactive antigen was highly expressed in 78% of gastric cancers (29/37) and 87% of colon cancers (27/31). No normal colon or stomach tissues remote from the tumor were positive for the antigen. The antibody also reacted with other tumors of epithelial origin such as lung squamous cell cancer (2/4), breast ductal cancer (2/20), bladder transitional cell carcinoma (4/6), and uterine cancer (3/16). Western blot analysis of the antigen revealed glycoprotein(s) which migrated as a smear ranging from the origin of the gel to about the 80 kDa region. The reactivity of this antigen with SC142 was reduced by sodium metaperiodate treatment or O-glycanase digestion, but not by N-glycanase, suggesting that the epitope is an O-glycan. In lectin-binding assay, this antigen reacted only with wheat germ agglutinin but not with Ricinus communis agglutinin, Datura stramonium agglutinin, and Sambucus nigra agglutinin. CONCLUSIONS: Our findings indicate that the antigen defined by SC142 is a tumor-associated antigen that could differentiate the gastrointestinal cancer cells from the normal cells. Therefore, SC142 may become a valuable tool for the immunohistochemical diagnosis and tumor immunoscintigraphy of the gastrointestinal cancer patients.

A novel nonsense mutation of the GTP cyclohydrolase I gene in a family with dopa-responsive dystonia.

We report a new nonsense mutation in the GTP cyclohydrolase I (GCH1) gene in a family with dopa-responsive dystonia. Two sisters and three children of the sisters are affected. The exons of the GCH1 gene were amplified by PCR and sequenced. The substitution of thymine for cytosine at nucleotide position 142 causing a nonsense mutation (Q48X) in exon 1 was identified in all of the five affected patients. There were three asymptomatic carriers of the mutation in the family.

Characterization and large-scale expression of the recombinant cysteine proteinase from adult Clonorchis sinensis.

Cysteine proteinases play important roles in the pathogenesis of several parasitic infections and have been proposed as targets for the structure-based approach of drug design. As the first step toward applying this strategy to design inhibitors as antiparasitic agents for Clonorchis sinensis, we overexpressed and characterized the 24-kDa cysteine proteinase from adult worms. First, the partial cysteine proteinase gene from C. sinensis was cloned by performing reverse transcription polymerase chain reaction (RT-PCR) with degenerate oligonucleotide primers derived from conserved cysteine proteinase sequences. The 5' and the 3' regions of the cysteine proteinase gene were amplified using the PCR protocol for the rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The cDNA has an open reading frame of 981 bp, and the deduced amino acid sequence shares similarity with the cathepsin L-like cysteine proteinases from Schistosoma mansoni, Paragonimus westermani metacercaria, Fasciola hepatica, and human cathepsin L by 52%, 47%, 34%, and 29%, respectively. The cysteine proteinase was then overexpressed in the yeast Pichia pastoris as an active enzyme on a large-scale basis (19.7 mg/L). The active recombinant enzyme was purified from culture media using a Ni2+-NTA-agarose affinity column and gel filtration chromatography. This 24-kDa recombinant protein exhibited a substrate preference for Z-Phe-Arg-AMC (benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amino-4-methyl-coumarin) compared with Z-Arg-Arg-AMC, and the activity was inhibited by E-64 (L-trans-epoxysuccinylleucylamido(4-quanidino)butane).

Mutation analysis of Korean patients with citrullinemia.

Citrullinemia is an autosomal recessive disease due to the mutations in the argininosuccinate synthetase (ASS) gene. Mutation analysis was performed on three Korean patients with citrullinemia. All of the three patients had the splicing mutation previously reported as IVS6-2A>G mutation. Two had Gly324Ser mutation and the other patient had a 67-bp insertion mutation in exon 15. The IVS6-2A>G mutation was reported to be found frequently in Japanese patients with citrullinemia, but Caucasian patients showed the extreme mutational heterogeneity. Although a limited number of Korean patients were studied, the IVS6-2A>G mutation appears to be one of the most frequent mutant alleles in Korean patients with citrullinemia. The Gly324Ser mutation identified in two patients also suggests the possible high frequency of this mutation in Korean patients as well.

The first successful prenatal diagnosis on a Korean family with citrullinemia.

DNA prenatal diagnosis was successfully performed on a family with citrullinemia. The father carried the G324S mutation and the mother carried the IVS6-2A > G mutation in the argininosuccinate synthase gene. They had a previous child with citrullinemia who died in the week after birth owing to complicated hyperammonemia. The lost child turned out to be a compound heterozygote. DNA was extracted from the cultured amniotic cells after amniocentesis done at 18-week gestation. For the detection of the G324S mutation, the PCR and restriction fragment length polymorphism method was used, and for the IVS6-2A > G mutation, allele-specific PCR was performed. The fetus was found to carry G324S but not IVS6-2A > G, suggesting a heterozygote carrier. Pregnancy was continued and a healthy boy was born. Plasma amino acid analysis performed on the third day after birth was normal and the serial ammonia level was in the normal range. A molecular study on his genomic DNA after birth also agreed with the previous fetal DNA analysis. He is now 2-months old with normal growth and development.

Cloning of a cysteine proteinase gene from Acanthamoeba culbertsoni.

Free living amoeba, including pathogenic Acanthamoeba culbertsoni, are widely distributed in soil and fresh water. It has been found that cysteine proteinases are more active in pathogenic strains of amoeba whereas serine proteinases are found in both pathogenic and nonpathogenic strains. Cysteine proteinases thus play important roles in the pathogenesis of several parasitic infections and have been proposed as targets for the structure-based strategy of drug design. As the first step toward applying this strategy to design inhibitors as antiparasitic agents for A. culbertsoni, we isolated and sequenced the full length clone of a cysteine proteinase gene from A. culbertsoni by performing reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotide primers derived from conserved cysteine proteinase sequences. The 5' and the 3' regions of the cysteine proteinase gene were amplified using the PCR protocol for the rapid amplification of cDNA ends (RACE). It has an open reading frame of 1359 bp. The deduced amino acid sequence has the sequence homology with the cysteine proteinase genes of Paragonimus westermani metacercaria, Schistosoma mansoni, human cathepsin L and Fasciola hepatica, each by 45.3%, 45.9%, 57.9% and 50.8% respectively. Sequence analysis and alignment showed significant similarity to other eukaryotic cysteine proteinases, including the conservation of the cysteine, histidine, and asparagine residues that form the catalytic triad. A 1.5 kbp mRNA was detected on Northern blot analysis using full-length cysteine proteinase cDNA as a probe. The A. culbertsoni cysteine proteinase gene (AcCP2) was found to contain Ex3Rx3Wx2N at the proregion and also a proline/threonine-rich C-terminal extension. Therefore, it has cathepsin L-like characteristics. Phylogenetic analysis based on the amino acid sequences of cysteine proteinase indicated that AcCP2 was closely related with papaya, while it was remotely related with those of Schistosoma.

Cysteine carboxyl O-methylation of human placental 23 kDa protein.

C-Terminal carboxyl methylation of a human placental 23 kDa protein catalyzed by membrane-associated methyltransferase has been investigated. The 23 kDa protein substrate methylated was partially purified by DEAE-Sephacel, hydroxyapatite and Sephadex G-100 gel filtration chromatographies. The substrate protein was eluted on Sephadex G-100 gel filtration chromatography as a protein of about 29 kDa. In the absence of Mg2+, the methylation was stimulated by guanine nucleotides (GTP, GDP and GTPgammaS), but in the presence of Mg2+, only GTPgammaS stimulated the methylation which was similar to the effect on the G25K/rhoGDI complex. AFC, an inhibitor of C-terminal carboxyl methylation, inhibited the methylation of human placental 23 kDa protein. These results suggests that the substrate is a small G protein different from the G25K and is methylated on C-terminal isoprenylated cysteine residue. This was also confirmed by vapor phase analysis. The methylated substrate protein was redistributed to membrane after in vitro methylation, suggesting that the methylation of this protein is important for the redistribution of the 23 kDa small G protein for its putative role in intracellular signaling.

Paclitaxel reduces anti-dsDNA antibody titer and BUN, prolonging survival in murine lupus.

We evaluated the effect of paclitaxel on the severity of autoimmunity in the murine model of systemic lupus erythematosus (SLE), NZB x NZW F1 mice. Fifteen 20 week old (NZB x NZW) F1 female mice were given a dose of 10 mg/kg paclitaxel by the intraperitoneal route on three alternate days followed by 7.5 mg/kg on three additional alternate days. This pattern of treatment was repeated every 4 weeks for a period of 28 weeks. 20 control mice were injected intraperitoneally with an equal volume of the vehicle used. Serum anti-double stranded DNA (dsDNA) antibody titers and the blood urea nitrogen (BUN) were significantly diminished in the paclitaxel treated group compared to the vehicle treated group. While the onset of proteinuria appeared to be delayed in the experimental group, the difference was not significant. Survival rate improved significantly in paclitaxel treated group (p = 0.04 by log-rank test). These results suggest that paclitaxel is beneficial in the suppression of autoimmunity in this strain of mice by reducing the anti-dsDNA antibody titer and the BUN, prolonging survival.

Molecular and genetic characterization of two anti-DNA antibodies derived from patients with systemic lupus erythematosus.

Anti-double stranded(ds) DNA antibody is one of markers of systemic lupus erythematosus (SLE). Two human monoclonal anti-DNA antibody-producing cell lines were established from two SLE patients. One cell line secreted IgG isotype antibody (KSUG) and the other secreted IgM isotype antibody (KSUN). The light chains of the two immunoglobulins were lambda chains. The nucleotide sequences for the immunoglobulin variable region genes of the two antibodies were determined and compared to germline sequences. The heavy and lambda light chains of KSUG were VH3 family and V lambda IIIb, respectively. The heavy and lambda light chains of KSUN were VH4 family and V lambda IX, respectively. Antibody KSUG, IgG isotype, showed somatic mutations, whereas KSUN, IgM isotype, used the germline gene without mutation. These findings reconfirm the current paradigms that IgM anti-DNA antibodies are produced by utilizing germline genes whereas IgG anti-DNA antibodies are produced by somatic mutations.

Cloning of a cysteine proteinase cDNA of adult Paragonimus westermani by polymerase chain reaction.

Cysteine proteinase cDNA fragment from adult mammalian trematode, Paragonimus westermani was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using degenerate oligonucleotide primers derived from conserved cysteine proteinase sequences. The 5' and the 3' regions of the cysteine proteinase gene were amplified using the PCR protocol for the rapid amplification of cDNA ends (RACE). It has an open reading frame of 804 bp. The deduced amino acid sequence consists of 268 amino acids. Sequence analysis and alignment showed significant sequence similarity to other eukaryotic cysteine proteinases and conservation of the cysteine, histidine, and asparagine residues that form the catalytic triad. The cysteine proteinase cDNA fragment was also subcloned in the expression vector pET and expressed as a C-terminal His-tag fusion protein in Escherichia coli.

Developmental human sexuality: the introduction of a new curriculum in child psychiatry training.

The training objectives, content and course evaluation results of a new curriculum offering, Developmental Human Sexuality, for child and adult psychiatric residents are described and discussed.