Study on the zona pellucida 4 (ZP4) gene sequence and its expression in the ovaries of patients with polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common endocrine disorder of unknown pathology, involving reproductive and metabolic abnormalities. Oocyte-specific genes are a group of genes expressed exclusively in ovarian tissue; therefore, they can play an important role in ovarian pathologies such as PCOS. The zona pellucida 4 (ZP4) gene encodes glycoprotein which is a part of the extracellular matrix of oocyte. MATERIALS AND METHODS: We analyzed 87 patients with PCOS, which were divided into four groups depending on their phenotype. In each patient, we performed profound clinical and biochemical analysis, including the measurement of serum androgens. The ovarian tissue samples were used to perform a real-time polymerase chain reaction and immunohistochemical staining using anti-ZP4 monoclonal antibodies. The ZP4 gene was sequenced from peripheral lymphocytes. RESULTS: The expression of ZP4 was present in early antral follicles and was stronger in mature follicles. The subgroup of patients with eumenorrhea and without hyperandrogenism presented the highest expression of ZP4 in ovarian tissue. In one case, we found a mutation of the ZP4 gene. No correlations were found between the ZP4 expression level and biochemical or clinical indices. CONCLUSIONS: Data from this and animal studies suggest a possible relationship between androgens and ZP4 expression. ZP4 expression is highest among patients with PCOS and a regular cycle, and this is a consequence of the presence of mature follicles in this group. In some patients with PCOS and infertility, ZP4 mutation can be found.

Alterations in mtDNA: a qualitative and quantitative study associated with cervical cancer development.

OBJECTIVE: High-risk human papillomaviruses are the causative agent of cervical carcinogenesis. Additionally, a number of other unknown factors are also instrumental in the development of cancer. The aim of this present study was the analysis of the mutations in the D-loop region of mitochondrial DNA, and 4.997 bp deletion during cervical cancer development. Our research also extended to the relationship between mtDNA copy number, ROS (reactive oxygen species) production and the MnSOD (manganese superoxide dismutase) expression level. METHODS: The study group consisted of postoperative tissues from patients diagnosed with L-SIL, H-SIL and squamous cell cervical carcinomas. A quantitative real-time polymerase chain reaction was used to determine the copy number of the mitochondrial DNA, and MnSOD mRNA expression levels. A PCR amplification and a sequencing of DNA were used for the identification of HPV DNA and mtDNA mutations. RESULTS: A total of 62 point mutations in the D-loop region of mtDNA were found in study patients. The mitochondrial DNA copy number increased during cervical cancer development when compared to the corresponding tissues in the control samples. About 70% of the mtDNA copy number have a 4.997 bp deletion in L-SIL. We also observed an increase in ROS generation during cervical cancer development. CONCLUSION: Alterations in mtDNA both qualitatively (by mutations) and quantitatively (by mtDNA copy number) are associated with cervical cancer developments. High levels of mtDNA copy with a 4.997 bp deletion in L-SIL cells can be associated with the susceptibility of cells to HPV persistent infection and cervical cancer development.

Human Eb peptide: not just a by-product of pre-pro-IGF1b processing?

Several physiological activities have been assigned to E-peptides derived from pre-pro-insulin-like growth factor (IGF1) processing; however, the whole range of the E-peptides' functions is still unknown. The objective of this study was to investigate human Eb peptide (hEb) in terms of its bioactivity, cellular localization, and intracellular trafficking using human cancer cells. Human Eb fused with red fluorescence protein (RFP) or green fluorescence protein (GFP) localizes strongly to nucleoli and to a lesser extent to nuclei of HeLa and U2-OS cells. Mutagenesis of hEb nucleolus localization sequence (NoLS) leads to its partial delocalization from nuclei and nucleoli to cytoplasm of transfected cells. Thus, NoLS is not sufficient for the hEb to be localized in nucleoli of the cells and a different mechanism may be involved in hEb targeting. A BrdU ELISA showed that the proliferation index of cells expressing hEb hybrid proteins increased up to 28%. For comparison, the same assay was performed using HeLa cells treated extracellularly with synthetic hEb. A significant increase in the proliferation index was observed (41-58% for concentrations ranging from 10-100 nM, respectively). Additionally, a cell migration assay was performed using stable U2-OS cell lines expressing hEb fused with RFP or RFP alone as a negative control. The migration index of hEb expressing cells was 38.3% greater. The increase in cell proliferation index and in motile properties of hEb expressing cells demonstrate that hEb is more than a pre-pro-IGF1b processing product, and has intrinsic activity of biological significance.

Association of the IGF-I promoter P1 polymorphism with risk of cervical cancer.

Human papillomaviruses (HPV16, HPV18, HPV31, HPV33) are etiological agents in the development of cervical cancer. HPVs infect epithelial cells and depend on epithelial differentiation for the completion of their life cycle. Insulin-like growth factor I (IGF-I) is a potent mitogen involved in the regulation of cell proliferation and apoptosis of many cell types including normal and transformed epithelial cells. Deregulation of IGF-I expression and action is linked to diverse pathologies including cancer. A polymorphism in the P1 promoter region of the IGF-I gene may directly influence its expression. Using the PCR-SSCP method and sequencing of DNA, we identified a single nucleotide polymorphism (SNP) at -383(C>T) position of promoter P1 of the IGF-I in 16% of the study HPV-positive women with precancerous and cancerous lesions. In vitro, we observed that the SNP at-383(C>T) site significantly increased the reporter gene expresion in the HepG2 cell line, but not in the HeLa cell line relative to the wild type promoter. It suggests that the studied SNP can change expression of the IGF-I gene in distinct ways in different types of tissues. Deregulation of expression of the IGF-I gene can affect normal epithelium development and in case of HPV infection can potentially disrupt the virus life cycle and stimulate its passage into the oncogenic life cycle or persistent viral infections. Therefore, we propose that SNP C>T at the -383 position of P1 promoter may be one of the helpful prognostic markers in the diagnosis of cervical cancer development of women with persistent infection in the ectocervical epithelium. We have not found any association between the polymorphism CA repeats in the promoter P1 region of the IGF-I gene and suceptibility to HPV infection and cervical cancer development. The (CA)19 allele was the most common in the study of this group of women.

The prevalence of leptotrichia amnionii in cervical swabs of HPV positive and negative women.

UNLABELLED: The aim of the present study was to determine the prevalence of Leptotrichia amnionii in cervical swabs of women and its possible correlation with HPV infection and the stage of cervical cancer. MATERIAL AND METHODS: A total of 139 cervical swabs from healthy women with normal cytology, with dysplastic changes and with cervical cancer were tested for the presence of L.amnionii and high-risk HPV DNA by PCR methods. RESULTS: L. amnionii was found in normal vaginal flora and in women with bacterial vaginosis (BV), which suggests that it may be oportunistic pathogen. L. amnionii infection was diagnosed in 13.7% (19/139). Statistical analysis showed that there was positive association (p < 0.01) between the presence of L.amnionii in women with cervical cancer (38.5%) and its presence in women without cancer (11.1%). On the other hand, there was no statistically significant association between L.amnionii and HPV infections. CONCLUSION: The data presented in this study show for the first time the prevalence of L. amnionii infection in cervical specimens collected from 2004-2006 in Poznan and Lublin, Poland, and its association with HPV infection and the stage of carcinogenesis of the cervix.

Prevalence of Chlamydia trachomatis and herpes simplex virus 2 in cervical carcinoma associated with human papillomavirus detected in paraffin-sectioned samples.

PURPOSE: The aim of the study was to evaluate the frequency of occurrence of HPV and co-infection: Chlamydia (C.) trachomatis and HSV-2 in cervical cancer. MATERIAL AND METHODS: The study group consisted of 570 paraffin-sectioned samples of patients with cervical cancer. In order to identify viral and bacterial DNA in DNA isolated from archival, postoperative material, PCR analysis was performed using starters complementary to various types of HPV, HSV-2 and C. trachomatis. RESULTS: In patients with squamous cell cervical cancer the presence of 33 types of HPV was found in 90% (468/520). HPV 16 infections occurred in 69.4% (325/468), while HPV 18 infections were present in 30.5% (143/468) of cases. In the control group C. trachomatis and HSV-2 were observed in four cases (4/50), which constitute 8.0%. In the tissue sections from patients with squamous cell cervical carcinoma, C. trachomatis was identified in 26% (135/520) and HSV-2 in 28% (145/520). In the group of patients with adenocarcinoma C. trachomatis infections were found in 24% (12/50) and herpes virus was identified in 30% (15/50). Statistically significantly higher frequency of occurrence of HSV-2 and C. trachomatis was observed in paraffin-sectioned samples for patients with invasive cervical cancer compared to the control group, without neoplastic lesions (p < 0.05). No correlation was found between frequency of occurrence of HPV and C. trachomatis and of HPV and HSV-2 detected in paraffin-sectioned samples for cervical carcinoma.

The association between socioeconomic status and health-related quality of life among Polish postmenopausal women from urban and rural communities.

In recent years, more scholarly attention has been paid to a growing range of geographic characteristics as antecedents of inequalities in women's health and well-being. The purpose of this study was to evaluate differences in health-related quality of life between rural and urban Polish postmenopausal women. Using a data set from a reproductive health preventive screening of 660 postmenopausal women aged 48-60 years, inhabitants of Wielkopolska and Lublin provinces, the association of place of residence, socioeconomic status and lifestyle factors with health-related quality of life (the SF-36 instrument) was evaluated using ANCOVA models and multiple logistic regression analysis with backward elimination steps. A consistent rural-to-urban gradient was found in all indices of physical health functioning and well-being but not in vitality, social functioning, emotional role and mental health scales with women in large cities being likely to enjoy the highest and those in villages the lowest quality of life. The rural-urban disparities in health-related quality of life were mediated by women's socioeconomic status. The likelihood of worse physical and mental functioning and well-being was 2-3 times greater for the low socioeconomic status rural women than their counterparts from more affluent urban areas. The educational attainment and employment status were the most powerful independent risk factors for health-related quality of life in both rural and urban women. Better understanding of the role of socioeconomic status that acts as a mediator in the association between area of residence and health-related quality of life may be useful in developing public health policies on health inequalities among women at midlife.

Genetic variants in the promoter region of the IGF-I gene as a reason for short stature.

DNA obtained from the blood cells of 88 adolescent patients with short stature, with low blood serum IGF-I concentrations, normal growth hormone (GH) secretion and normal GH receptor (GHR) structure, was analyzed in the promoter region for the IGF-I gene. A total of 24 genetic variants was detected in the DNA of 13 patients. An attempt was also made to analyze the impact of identified mutations on DNA-protein interactions using EMSA.

Chlamydia trachomatis and herpes simplex virus 2 infection in vulvar intraepithelial neoplasia associated with human papillomavirus.

BACKGROUND: The role of viral and bacterial co-infection is stressed in VIN. A view that VIN is a sexually transmitted disease made the area of research larger and stimulated scientists to seek other sexually transmitted factors, among which Chlamydia trachomatis and Herpes simplex are frequently examined. PURPOSE: The aim of the study was to evaluate the frequency of occurrence of HPV DNA and the frequency of co-infection with Herpes virus type 2 and Chlamydia trachomatis in VIN. MATERIAL AND METHODS: We identified archival diagnostic phase tissue specimens from 41 cases of vulvar intraepithelial neoplasia III. From the same paraffin blocks containing material from the margins of surgical sections during vulvectomy, normal epithelial tissue fragments were collected. They constituted the control group. Lesion characteristics were examined in comparison with the presence of HPV DNA, HSV-2 and Chlamydia trachomatsis. Identification was performed using PCR. RESULTS: In the study group HPV infection was found in 75.6% of cases. In 73% of cases it was HPV 16. In the control group we found HPV 16 DNA in only one case (2.43%). In the HPV positive study group HPV 16 was found in 30 (30/31) cases. In only one case (1/31) it was HPV 18 type. In the study group of 41 cases with VIN, HSV-2 infection was found in six cases (14.63%). In comparison with the control group (9.75%) the difference was not statistically significant. The frequency of occurrence of Chlamydia trachomatis in the analyzed study material was 14.63% (6/41) and in the control group it was 9.75% (4/41). The difference was not statistically significant. Statistical analyses of correlations between the occurrence of DNA HPV and HSV-2 as well as of HPV and Chlamydia trachomatis showed no correlation in either case. CONCLUSION: No correlation was found between the frequency of occurrence of HPV and HSV-2 and HPV and Chlamydia trachomatis in either group.

The level of antibody against E6 HPV 16 oncoprotein in blood sera of women with chronic HPV 16 infection and cervical cancer.

UNLABELLED: The aim of this study was to estimate of the role of chronic HPV 16 infection and the presence of anti E6 HPV 16 in the initiation of the cancerogenesis process of cervical cancer. MATERIAL AND METHODS: The study included two groups of patients. The first group comprised 323 women observed for three consecutive years (1998-2000), in whom the presence of HPV 16 viruses was estimated by PCR, and the level of anti E6 HPV 16 antibodies was estimated in the plasma with ELISA. A similar test was performed in a group of 46 patients with cervical intraepithelial neoplasia (CIN), 91 patients with invasive cervical cancer and 22 women after hysterectomy and RTG-therapy. RESULTS: In 32 patients, chronic HPV 16 infection showed a steady rise in the mean absorbance level of anti E6 HPV 16 antibodies from 0.04 in 1998 to 0.06 in 2000, while in HPV-negative women the mean absorbance value was 0.03-0.04. Mean absorbance value in patients with CIN III and invasive cancer rose with advancing stage of the cancer process and lowered after completion of oncological treatment. The values were 0.14, 0.33 and 0.13, respectively. CONCLUSION: The persistence of chronic HPV 16 infection and accompanying steady rise in absorbance index caused by an increase in the level of antiviral antibodies are a clear warning signal preceding in time the histological process of cancerogenesis.

No evidence of HTLV-I infection in patients with mycosis fungoides and Sezary syndrome.

The involvement of human T-cell lymphotropic virus type I (HTLV-I) in the etiology of cutaneous T-cell lymphomas (CTCL) is still controversial. The aim of the study was to evaluate the role of HTLV-I in the pathogenesis of mycosis fungoides (MF) and Sezary syndrome (SS) in Polish patients. The studied group consisted of 42 patients with MF, 5 with SS and 25 with chronic dermatitis. DNA was extracted from snap-frozen and paraffin-embedded skin biopsies and from peripheral blood. Polymerase chain reaction (PCR or nested PCR) was carried out for amplification of different regions of HTLV-I genome. Primer sets flanking pX, p 19, U5, tax and pol genes were used in the investigation. The presence of HTLV-I antibody was examined in 46 sera samples with the use of anti-HTLV-I/II EIA test. HTLV-I antibodies were not detected in any collected sera samples. PCR with two primer sets homologous to the pX region of HTLV-I showed negative results in all samples investigated. To confirm these results two other primer pairs specific for U5 and gag regions were designed. With these primer pairs no PCR product, except that in positive control, was observed. For more sensitive amplification a nested-PCR with pol and tax specific primers was performed. HTLV-I probably does not play an important role in the pathogenesis of MF in Polish patients.

Gherlin expression in women with polycystic ovary syndrome--a preliminary study.

The etiology and pathogenesis of polycystic ovary syndrome (PCOS) is still unknown. Using real-time PCR, we detected that polycystic ovaries showed almost ten times lower expression of ghrelin mRNA than normal ovaries, whereas the mRNA levels in blood cells were similar in both study groups. This suggests that the presence of ghrelin in PCOS and normal ovaries may have an autocrine/paracrine modulatory effect on ovary functions and local significance in the etiology of PCOS.

Intratype HPV16 sequence variation within LCR of isolates from asymptomatic carriers and cervical cancers.

BACKGROUND: HPV16 is a predominant type of virus identified in genital lesions and strongly associated with the development of genital cancers. Infection with the virus is considered to be the main risk factor in the development of cervical cancer. Based on HPV16 DNA isolated from invasive cancers, a classification of intratype genetic variants was established and the strains were designated according to geographical regions. The HPV16 variants classification was based on isolates derived from cancers. OBJECTIVES: Analysis of HPV16 LCR variants isolated from asymptomatic carriers for comparison with cervical cancer isolates to examine whether a correlation can be found between cervical epithelium state and variant of HPV16 it carries. MATERIALS AND METHODS: The HPV16 LCR fragments were amplified by PCR using DNA isolated from cervical swabs and tissue sections then screened for nucleotide changes by SSCP. Polymorphic sites were analysed for regulatory protein binding properties by EMSA. RESULTS: Comparison of the two groups revealed that isolates from cervical cancers predominantly carry changes in sequences of YY1 binding sites (especially at nucleotide 7519), while variants from asymptomatic carriers contained nucleotide changes within or close to transcription binding sites for AP-1, Oct-1, NF1, Tef-1, Tef-2, Sp1, YY1 and viral E2. EMSA study showed that sequence changes in the segment alter binding and formation of transcriptional complexes in quantitative and/or qualitative manner and so they may inflict viral activity. CONCLUSION: The results of our study show that there might be HPV16 variants of decreased oncogenic potential therefore infection with such variants can recede.

Valyl-tRNA and leucyl-tRNA synthetases in wheat germ and seedlings.

Valyl- and leucyl-tRNA synthetases (Val-RS and Leu-RS) isolated from wheat germ and seedlings were separated by chromatography on hydroxylapatite into organellar (Val-RS I and Leu-RS I) and cytoplasmic (Val-RS II and Leu-RS II) enzymes; the enzyme extracted from isolated chloroplasts and mitochondria corresponded to the RS I fractions. It was proved by RPC-5 chromatography of tRNA Val that Val-RS I and Val-RS II recognized all five isoacceptor tRNA Val species both from wheat germ and leaves, as well as tRNA Val from E. coli. However, out of the six isoacceptor tRNA Leu species, Leu-RS II aminoacylated two cytoplasmic species only, while Leu-RS I, the remaining four organellar tRNA Leu fractions. Both leucyl-tRNA synthetases charged E. coli tRNA, Leu-RS I more effectively than Leu-RS II. The absence of fraction RS I in cytosol seems to indicate that valyl- and leucyl-tRNA synthetases (coded for by the nuclear genome) were modified to the organellar forms after (or during) passage into organelles.

Comparative biochemical analysis of lectin and nuclease from Chelidonium majus L.

It has been recently recognized that lectins exhibit other activities besides hemagglutination. Previously we have found that purified lectin from Chelidonium majus showed DNase activity (Fik, Gozdzicka-Józefiak & Kedzia, 1995, Herba Polon. 41, 84-95). Comparison of lectin and DNase from the sap from leaves and roots of Chelidonium majus proved that both these compounds are composed of 24 kDa monomer subunits which have an identical N-terminal sequence but differ in amino-acid composition and degree of glycosylation. Possible interrelationship between lectin and DNase is discussed.

Effect of lectin from Chelidonium majus L. on normal and cancer cells in culture.

Lectin from Chelidonium majus L. (CML) significantly stimulates the proliferation of human lymphocytes and has hemagglutination activity towards group B human erythrocytes and potent antimicrobial properties against multiresistant enterococci and staphylococci. In the present work we describe the effect of lectin from Chelidonium majus L on normal and cancercells in culture in vitro. The studies were performed on three types of cells: CHO, R2C and on normal mouse fibroblasts. Effects on the cultures were examined 24 h after addition of CML. Exposure to CML resulted in growth inhibition of CHO and R2C cells but not of fibroblasts. Moreover, evident apoptotic lesions were observed in CHO cells and less well marked apoptotic lesions in R2C cells. In contrast, only insignificant numbers of fibroblasts reacted to the applied lectin.

Production of human papillomavirus type 16 early proteins in Bac-To-Bac Expression System (GibcoBRL).

Human papillomavirus type 16 (HPV16) is a major agent in cervical cancer etiology. Its early proteins are responsible for virus persistence, replication and initiation of neoplastic disease. In the present study we describe a use of baculovirus-insect cell expression system for production and study of HPV16 E2 and E4 proteins. The E2 protein binds specifically to viral DNA and E4 protein shows characteristic cytopathic effects on cells.

Human papillomavirus type 16 in ovarian serous surface papilloma.

DNA of HPV 16 was detected in the DNA isolated from the papillary lesion which was found on the right ovary of a pregnant women during cesarean section. The epithelium covering the papilloma stained by an indirect immunoperoxidase technique demonstrated progesterone specific receptors. Therefore, we suggest that the virus and hormone can increase the risk of malignant conversion in study cells.

Human papillomavirus 16 in the blood of women treated for cervical cancer.

In the present paper we study the behaviors of HPV 16 DNA in the blood of women during oncological treatment. Disappearance of HPV from the blood depends on the clinical Stage of cancer. It is possible that HPV remaining in the blood for a long time after oncological treatment might be some marker showing that the cancer tissue was not completely removed. There is some suggestion that detecting HPV in the cervix or in the blood of women without morphological lesions but with CIN in the past might indicate higher risk of cervical cancer recurrence.

Human papillomavirus (HPV) in upper genital tract carcinomas of women.

Using nucleic acid hybridization and polymerase chain reaction (PCR) HPV-DNA sequence was detected in endometrial and ovarian carcinomas. This sequence was amplified with primer specific for E1 region of DNA-HPV 18 and hybridized with DNA-HPV 18. The presence of HPV-DNA in cancer studies suggests that human papillomavirus can also be involved in carcinogenesis of the upper genital tract of women.