Identification of differentially-expressed genes by DNA methylation in cervical cancer.

To identify novel cervical cancer-related genes that are regulated by DNA methylation, integrated analyses of genome-wide DNA methylation and RNA expression profiles were performed using the normal and tumor regions of tissues from four patients; two with cervical cancer and two with pre-invasive cancer. The present study identified 19 novel cervical cancer-related genes showing differential RNA expression by DNA methylation. A number of the identified genes were novel cervical cancer-related genes and their differential expression was confirmed in a publicly available database. Among the candidate genes, the epigenetic regulation and expression of three genes, CAMK2N1, ALDH1A3 and PPP1R3C, was validated in HeLa cells treated with a demethylating reagent using methylation-specific polymerase chain reaction (PCR) and quantitative PCR, respectively. From these results, the expression of the CAMK2N1, ALDH1A3 and PPP1R3C genes are were shown to be suppressed in cervical cancers by DNA methylation. These genes may be involved in the progression or initiation of cervical cancer.

Expression and function of the testis-predominant protein LYAR in mice.

Mammalian spermatogenesis is a complex process involving an intrinsic genetic program of germ cell-specific and -predominant genes. In the present study, we analyzed the Ly-1 reactive clone (Lyar) gene in the mouse. Lyar, which is known to be expressed abundantly in the testis, encodes a nucleolar protein that contains a LYAR-type C2HC zinc finger motif and three nuclear localization signals. We herein confirmed that Lyar is expressed predominantly in the testis, and further showed that this expression is specific to germ cells. Protein analyses with an anti-LYAR antibody demonstrated that the LYAR protein is present in spermatocytes and spermatids, but not in sperm. To assess the functional role of LYAR in vivo, we used a genetrap mutagenesis approach to establish a LYAR-null mouse model. Lyar mutant mice were born live and developed normally. Male mutant mice lacking LYAR were fully fertile and showed intact spermatogenesis. Taken together, our results demonstrate that LYAR is strongly preferred in male germ cells, but has a dispensable role in spermatogenesis and fertility.

Identification of heat shock protein 5, calnexin and integral membrane protein 2B as Adam7-interacting membrane proteins in mouse sperm.

In mammals, sperm acquire their motility and ability to fertilize eggs in the epididymis. This maturation process involves the acquisition of particular proteins from the epididymis. One such secretory protein specifically expressed in the epididymis is Adam7 (a disintegrin and metalloprotease 7). Previous studies have shown that Adam7 that resides in an intracellular compartment of epididymal cells is transferred to sperm membranes, where its levels are dependent on the expression of Adam2 and Adam3, which have critical roles in fertilization. Here, using a proteomics approach based on mass spectrometry, we identified proteins that interact with Adam7 in sperm membranes. This analysis revealed that Adam7 forms complexes with calnexin (Canx), heat shock protein 5 (Hspa5), and integral membrane protein 2B (Itm2b). Canx and Hspa5 are molecular chaperones, and Itm2b is a type II integral membrane protein implicated in neurodegeneration. The interaction of Adam7 with these proteins was confirmed by immunoprecipitation-Western blot analysis. We found that Adam7 and Itm2b are located in detergent-resistant regions known to be highly correlated with membrane lipid rafts. We further found that the association of Adam7 with Itm2b is remarkably promoted during sperm capacitation owing to a conformational change of Adam7 that occurs in concert with the capacitation process. Thus, our results suggest that Adam7 functions in fertilization through the formation of a chaperone complex and enhanced association with Itm2b during capacitation in sperm.

Concurrent endometrial carcinoma following hysterectomy for atypical endometrial hyperplasia.

OBJECTIVE: To evaluate the prevalence of concurrent endometrial carcinoma in women diagnosed with atypical endometrial hyperplasia (AEH) by endometrial biopsy. STUDY DESIGN: We retrospectively analyzed the medical records of 126 patients who underwent hysterectomies for AEH diagnosed by endometrial biopsy from 1999 to 2008. AEH was initially diagnosed by dilatation and curettage (98 cases) or endometrial biopsy with a Z-sampler (24 cases). The remaining four cases were diagnosed by hysteroscopic polypectomy. The results of the endometrial biopsies were graded on an ordinal scale and were compared with pathologic features obtained at the hysterectomy. RESULTS: In patients preoperatively diagnosed with AEH by biopsy, hysterectomy specimens revealed a rate of simple or complex endometrial hyperplasia without atypia of 27% with AEH and normal proliferative phases found in 54.7 and 7.9% of specimens, respectively. The incidence of endometrial carcinoma was considerably high (13/126, 10.3%). Eleven of 13 cases were confined to the endometrium and the remaining two were located at the adenomyosis without myometrial invasion. All patients with endometrial carcinoma displayed coexisting atypical complex hyperplasia following hysterectomy. CONCLUSIONS: Biopsy specimens showing AEH, particularly atypical complex hyperplasia, are associated with a risk of coexisting endometrial carcinoma. When considering management strategies for women with a biopsy diagnosis of AEH, clinicians should take into account the considerable rate of concurrent endometrial cancer and the discrepancy with pathologic diagnosis. Treatment modalities may differ depending on population as the rates of concurrent endometrial cancer with AEH and myometrial invasion vary by geographical location.

The effects of polymorphisms in methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), and methionine synthase reductase (MTRR) on the risk of cervical intraepithelial neoplasia and cervical cancer in Korean women.

The purpose of the study was to investigate the association between cervical cancer risk and single-nucleotide polymorphisms (SNPs) in three one-carbon metabolism genes, methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), and methionine synthase reductase (MTRR) in Korean women. Twelve SNPs were identified in MTHFR, MTR, and MTRR in the 927 case-control samples, which included 165 cervical intraepithelial neoplasia 1 (CIN1), 167 cervical intraepithelial neoplasia 2 and 3 (CIN2/3), 155 cervical cancer patients, and 440 normal controls. The frequencies of the genotypes and haplotypes were assessed in the controls, CINs, and cervical cancers. Individual carriers of the variant allele C of MTHFR A1298C (rs1801131) had a 0.64-fold [95% confidence interval (CI): 0.42-0.98] decreased risk for CIN2/3 compared with common homozygotes. However, no significant association was found between most other variants and cervical cancer risk. The results also identified an increased CIN1 risk in carriers with at least one copy of haplotype 3 in the MTHFR gene (odds ratio, 1.88; 95% CI: 1.03-3.42). In conclusion, there was no significant association between most SNPs in MTHFR, MTR, or MTRR and the risk of CIN and cervical cancer in Korean women. In addition, there was no significant association of MTHFR haplotypes with risk of CIN2/3 and cervical cancer.

Functional polymorphism in manganese superoxide dismutase and antioxidant status: their interactions on the risk of cervical intraepithelial neoplasia and cervical cancer.

OBJECTIVE: Manganese superoxide dismutase (MnSOD), the primary antioxidant enzyme in mitochondria, plays a key role in protecting cells from oxidative stress. Furthermore, the MnSOD rs4880 polymorphism is associated with enzyme activity. The authors evaluated the interaction between MnSOD genotypes and cervical carcinogenesis risk and the modulating effects of serum antioxidant nutrient status (beta-carotene, lycopene, zeaxanthin/lutein, retinol, alpha-tocopherol and gamma-tocopherol). METHODS: Cases and controls for this study were recruited between June 2006 and July 2007 (263 controls, 84 cervical intraepithelial neoplasia (CIN), 94 CIN 2/3, and 99 cases of cervical cancer). The MnSOD polymorphism at rs4880T/C was examined using SNaPshot assays. Serum antioxidant vitamin concentrations were measured by reverse-phase gradient high-pressure liquid chromatography. Odds ratios (OR) and 95% confidence intervals (95%CI) were estimated after adjusting for age, menopause, parity, oral contraceptive use, smoking and alcohol consumption. RESULTS: No association was found between the MnSOD rs4880 polymorphism and cervical cancer. However, genotypes significantly modified the risk of cervical cancer in association with the serum statuses of micronutrients (P(interaction)<0.05 for beta-carotene, lycopene, zeaxanthin/lutein, alpha-tocopherol, and gamma-tocopherol). Decreased CIN1 risk in association with the MnSOD rs4880 variant genotype was also observed particularly for subjects with higher beta-carotene and gamma-tocopherol levels. Similar results were observed for lycopene and alpha-tocopherol in relation to the risk of CIN2/3. CONCLUSION: Our findings suggest that a higher antioxidant micronutrients status may decrease the risk of CIN and cervical cancer and modify the effect of the MnSOD polymorphism on disease risk.

Plasma carotenoids, retinol and tocopherol levels and the risk of ovarian cancer.

OBJECTIVE: We investigated the relation between plasma carotenoids, retinol and tocopherol levels and ovarian cancer risk in Korean women. DESIGN: Hospital-based case-control study. SETTING: Six tertiary medical institutes in Korea. POPULATION: Forty-five epithelial ovarian cancers and 135 age-matched controls. METHODS: Preoperative plasma concentrations of beta-carotene, lycopene, zeaxanthin plus lutein, retinol, alpha-tocopherol, and gamma-tocopherol were measured by reverse-phase, gradient high-pressure liquid chromatography. MAIN OUTCOME MEASURES: Odds ratios (OR) and 95% confidence intervals (95%CI) were estimated by tertiles to evaluate the effect of micronutrients on endometrial cancer risk after adjustment for body mass (BMI) index, menopause, parity, oral contraceptive use, smoking status, and alcohol consumption status. RESULTS: Women in the highest tertile for beta-carotene had 0.12-times the risk of ovarian cancer of in the lowest tertile (OR 0.12; 95%CI 0.04-0.36). Women with the highest tertiles of lycopene (OR 0.09; 95%CI 0.03-0.32), zeaxanthin/lutein (OR 0.21; 95%CI 0.09-0.52), retinol (OR 0.45; 95%CI 0.21-0.98), alpha-tocopherol (OR 0.23; 95%CI 0.10-0.53) and gamma-tocopherol (OR 0.28; 95%CI 0.11-0.70) had lower risk of ovarian cancer than women in the lowest tertiles. Results were consistent across strata of socio-epidemiologic factors. CONCLUSIONS: Micronutrients, specifically ss-carotene, lycopene, zeaxanthin, lutein, retinol, alpha-tocopherol, and gamma-tocopherol, may play a role in reducing the risk of ovarian cancer.

Preoperative levels of plasma micronutrients are related to endometrial cancer risk.

OBJECTIVE: To examine the relation between the plasma concentration of antioxidant micronutrients and endometrial cancer risk in Korean women. DESIGN: Hospital-based case-control study. SETTING: Seven tertiary medical institutes in Korea. POPULATION: Incidence of 28 endometrial cancer cases were identified and 140 age-matched controls selected for the same period. METHODS: Preoperative plasma concentrations of beta-carotene, lycopene, zeaxanthin plus lutein, retinol, alpha-tocopherol, and gamma-tocopherol were measured by reverse-phase, gradient high-pressure liquid chromatography. Conditional logistic regression was used to evaluate micronutrient effect after adjustment for body mass index (BMI), menopause, parity, oral contraceptive use, smoking status, and alcohol consumption status. MAIN OUTCOME MEASURES: Effect of micronutrients on endometrial cancer risk. RESULTS: The mean concentration of plasma beta-carotene (p=0.001), lycopene (p=0.008), zeaxanthin plus lutein (p=0.031), retinol (p=0.048), and gamma-tocopherol (p=0.046) were significantly lower in endometrial cancer patients than in controls. Plasma levels of beta-carotene (p for trend=0.0007) and lycopene (p for trend=0.007) were inversely associated with endometrial cancer risk across tertiles. Women in the highest tertile of plasma beta-carotene and lycopene had a 0.12-fold (95% confidence intervals (CIs) 0.03-0.48) and 0.15-fold (95% CIs 0.04-0.61) decreased risk of endometrial cancer compared to women in the lowest tertile, respectively. Other micronutrients such as zeaxanthin plus lutein (p for trend=0.142), retinol (p for trend=0.108), alpha-tocopherol (p for trend=0.322), and gamma-tocopherol (p for trend=0.087) showed no association with endometrial cancer risk. CONCLUSIONS: Plasma levels of beta-carotene and lycopene are inversely associated with the risk of endometrial cancer in Korean women.

Oxidative inactivation of key mitochondrial proteins leads to dysfunction and injury in hepatic ischemia reperfusion.

BACKGROUND & AIMS: Ischemia-reperfusion (I/R) is a major mechanism of liver injury following hepatic surgery or transplantation. Despite numerous reports on the role of oxidative/nitrosative stress and mitochondrial dysfunction in hepatic I/R injury, the proteins that are oxidatively modified during I/R damage are poorly characterized. This study was aimed at investigating the oxidatively modified proteins underlying the mechanism for mitochondrial dysfunction in hepatic I/R injury. We also studied the effects of a superoxide dismutase mimetic/peroxynitrite scavenger metalloporphyrin (MnTMPyP) on oxidatively modified proteins and their functions. METHODS: The oxidized and/or S-nitrosylated mitochondrial proteins from I/R-injured mouse livers with or without MnTMPyP pretreatment were labeled with biotin-N-maleimide, purified with streptavidin-agarose, and resolved by 2-dimensional gel electrophoresis. The identities of the oxidatively modified proteins were determined using mass spectrometric analysis. Liver histopathology, serum transaminase levels, nitrosative stress markers, and activities of oxidatively modified mitochondrial proteins were measured. RESULTS: Comparative 2-dimensional gel analysis revealed markedly increased numbers of oxidized and S-nitrosylated mitochondrial proteins following hepatic I/R injury. Many key mitochondrial enzymes involved in cellular defense, fat metabolism, energy supply, and chaperones were identified as being oxidatively modified proteins. Pretreatment with MnTMPyP attenuated the I/R-induced increased serum transaminase levels, histologic damage, increased inducible nitric oxide synthase expression, and S-nitrosylation and/or nitration of various key mitochondrial proteins. MnTMPyP pretreatment also restored I/R-induced suppressed activities of mitochondrial aldehyde dehydrogenase, 3-ketoacyl-CoA thiolases, and adenosine triphosphate synthase. CONCLUSIONS: These results suggest that increased nitrosative stress is critically important in promoting S-nitrosylation and nitration of various mitochondrial proteins, leading to mitochondrial dysfunction with decreased energy supply and increased hepatic injury.

Developing quantitative, multiscale models for microgravity crystal growth.

Crystal growth conducted under microgravity conditions has had a profound impact on improving our understanding of melt crystal growth processes. Here, we present a brief history of microgravity crystal growth and discuss the development of appropriate models to interpret and optimize these growth experiments. The need for increased model realism and predictive capability demands new approaches for describing phenomena important at several disparate length scales. Of special importance is the ability to represent three-dimensional and transient continuum transport (flows, heat, and mass transfer), phase-change phenomena (thermodynamics and kinetics), and system design (such as furnace heat transfer during melt growth). An overview of mathematical models and numerical algorithms employed to represent such multiscale effects is presented.