Cation-chromatin binding as shown by ion microscopy is essential for the structural integrity of chromosomes.

Mammalian interphase and mitotic cells were analyzed for their cation composition using a three-dimensional high resolution scanning ion microprobe. This instrument maps the distribution of bound and unbound cations by secondary ion mass spectrometry (SIMS). SIMS analysis of cryofractured interphase and mitotic cells revealed a cell cycle dynamics of Ca2+, Mg2+, Na+, and K+. Direct analytical images showed that all four, but no other cations, were detected on mitotic chromosomes. SIMS measurements of the total cation content for diploid chromosomes imply that one Ca2+ binds to every 12.5-20 nucleotides and one Mg2+ to every 20-30 nucleotides. Only Ca2+ was enriched at the chromosomal DNA axis and colocalized with topoisomerase IIalpha (Topo II) and scaffold protein II (ScII). Cells depleted of Ca2+ and Mg2+ showed partially decondensed chromosomes and a loss of Topo II and ScII, but not hCAP-C and histones. The Ca2+-induced inhibition of Topo II catalytic activity and direct binding of Ca2+ to Topo II by a fluorescent filter-binding assay supports a regulatory role of Ca2+ during mitosis in promoting solely the structural function of Topo II. Our study directly implicates Ca2+, Mg2+, Na+, and K+ in higher order chromosome structure through electrostatic neutralization and a functional interaction with nonhistone proteins.

Assessment of pituitary and steroid hormones and members of the TGF-beta superfamily for ovarian function in patients with congenital uterus and vaginal aplasia (MRKH syndrome).

Patients with Mayer-Rokitanski-Kuster-Hauser (MRKH) syndrome have congenital uterine and vaginal aplasia. The main question of this study was, if the absence of a uterus along with other genital and organ malformations could contribute to hormone or other growth factor protein fluctuations involved in communication between the hypothalamus-pituitary axis, ovaries and uterus. Serum from 56 MRKH patients (mean 27.6 years) and 22 female controls (mean 30.7 years) were analyzed using ELISA to determine levels of pituitary and steroid hormones (LH, FSH, estradiol, progesterone), growth factors of the TGF-beta superfamily like activin A, inhibin B, and anti-Müllerian hormone (AMH). All serum levels were analyzed in relation to other organ malformations. Compared to controls, all 56 patients, including 5% with streak ovaries or unilateral ovarian aplasia, were generally similar in hormone and growth factor levels and could be grouped into hormonal phases. However, compared to controls LH/FSH and FSH/LH ratios of patients had significantly higher and lower mean values, of 2.75-fold (p=0.015) and 1.9-fold (p=0.002), respectively. Undetectable inhibin B levels of<10 pg/ml (p=0.05) were noted in 41.1% of MRKH patients, resulting in significantly higher activin A/inhibin B ratios (p<0.001). MRKH patients have hormonal phases supporting ovarian function, but patients with low FSH/LH ratios and undetectable inhibin B levels (<10 pg/ml) could represent cycle phasing irregularities. A model is discussed regarding our findings and the loss of ovarian-uterine communication.

Polymorphisms in estrogen metabolism and estrogen pathway genes and the risk of miscarriage.

PURPOSE: This study investigated genetic variations in the estrogen pathway and their association with miscarriages. METHODS: A total of 483 patients were recruited from a comprehensive control group for case-control studies. Three variants of the CYP19A1 gene (rs10046, rs4646 and rs700519) and one variant each of the estrogen (ESR1) and progesterone (PGR) receptor genes (rs3020314 and rs1042838) were investigated using polymorphism genotyping. The chi-squared test and one-way analysis of variation (ANOVA) were used for statistical analysis. RESULTS: For rs10046 (CYP19A1), the C/C genotype was associated with a greater frequency of miscarriages (P = 0.017). The other genotypes were not found to be associated with recurrent miscarriage. CONCLUSIONS: This is the first study that has identified a single-nucleotide polymorphism in the aromatase gene that suggests a significant association between genotypes and miscarriage. As aromatase is an essential enzyme in the estrogen pathway, it may be speculated that variations in the aromatase gene in some way give rise to different conditions in the endocrine environment that can lead to impaired fertility.

Higher incidence of linked malformations in siblings of Mayer-Rokitansky-Küster-Hauser-syndrome patients.

BACKGROUND: Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is a malformation of the female genital tract (vaginal aplasia, rudimentary uterus, normal fallopian tubes and high ovaries). The incidence is one in 4000 female newborns. The aim of the present study was to record genital and associated malformations among siblings and relatives of MRKH patients in order to draw possible conclusions regarding the etiology of the syndrome: heredity (dominant versus recessive) or spontaneous malformation. METHODS: Using a standardized questionnaire, affected MRKH patients were asked about other cases of MRKH and/or associated malformations among siblings and relatives. RESULTS: No other cases of MRKH syndrome had occurred among the siblings or relatives of 73 MRKH patients; however, 13 associated malformations were recorded among a total of 103 siblings. Musculoskeletal malformations were markedly increased (3.27 times higher) in comparison with the prevalence of congenital malformations among newborns in the normal population. CONCLUSIONS: This study shows that dominant inheritance cannot play a role in the etiology of MRKH syndrome, as no further cases of MRKH syndrome occurred among any of the siblings. The study provides support for the view that the syndrome has a multifactorial pathogenesis. Siblings/relatives of MRKH patients should be examined for associated musculoskeletal/urogenital malformations.

Polymorphisms in the novel serotonin receptor subunit gene HTR3C show different risks for acute chemotherapy-induced vomiting after anthracycline chemotherapy.

The aim of this study was to correlate chemotherapy-induced nausea and vomiting (CINV) with commonly occurring single nucleotide polymorphisms (SNP) in the 5-hydroxytryptamine receptor 3 genes (HTR3). Women with breast cancer without previous chemotherapy were eligible for this prospective study. All patients received epirubicin, with or without cyclophosphamide, and preventive medication with ondansetron and dexamethasone. The patients documented every vomiting event on an hourly basis. Real-time polymerase chain reaction (PCR) analysis was performed for the following nonsynonymous SNPs: p.Y129S (HTR3B), p.K163N (HTR3C) and p.A405G (HTR3C). The overall proportion of patients (total n = 110) who reported vomiting in the first 24 h after chemotherapy was 31.8%. The variant genotype of K163N (HTR3C) was associated with vomiting, which occurred in 50.0% (P = 0.009). Polymorphisms in the HTR3C gene could serve as a predictive factor for CINV in patients undergoing moderately emetogenic chemotherapy.

Detyrosinated tubulin is decreased in fetal vessels of preeclampsia placentas.

INTRODUCTION: Preeclampsia is a hypertensive, gestational disease, which is still the leading cause of pregnancy related morbidity and mortality. The impairment of placental angiogenesis and vascularization is discussed to be of etiopathologic relevance. Deytrosination and tyrosination of α-tubulin is important for the stability and dynamics of microtubules. An increase of α-tubulin detyrosination leads to microtubule stabilization, which is an essential prerequisite for physiologic vascular tube morphogenesis during angiogenesis. So far, little is known about the specific localization of detyrosinated (detyr) and tyrosinated (tyr) tubulin in the placenta and its relevance for preeclampsia. METHODS: Placental expression of detyr- and tyr-tubulin was analyzed by immunohistochemistry, immunofluorescence and western blot. For western blot quantification we used biopsies from healthy placentas (n = 21) and placentas from pregnancies complicated with small for gestational age (n = 5), preeclampsia (n = 5) or both (n = 5). RESULTS: Specific placental localization of detyr-tubulin was detected in the fetal endothelial cells of the placenta. Villous and extravillous trophoblasts as well as villous stroma cells were tyr-tubulin positive. Detyr-tubulin protein expression was significantly decreased in placentas complicated by preeclampsia. CONCLUSIONS: In summary, we report an accumulation of detyr-tubulin in villous vessels of the placenta and a significantly reduced level of detyr-tubulin in placental biopsies of preeclampsia cases. The reduction of placental detyr-tubulin in preeclampsia could suggest a deficit in villous vascular plasticity and might be associated with the impaired arborization of the disease.

Risk and risk assessment for breast cancer: molecular and clinical aspects.

Chemoprevention, prophylactic surgery and intensified screening programs are options which can be offered the patients with an increased lifetime risk (p(life)) for breast cancer (BC). Estimation of p(life) includes BRCA mutation analysis and risk estimation based on individual risk factors and family history. MENDEL and BRCAPRO are models which can estimate mutation carrier status probability (p(mut)), p(life) and p(mut) can be estimated using Cyrillic3 software which incorporates BRCAPRO and MENDEL. To integrate age, hormonal factors and benign breast biopsies in risk assessment the Tyrer-Cuzick model can be used. These models support the decision pro or contra genetic analysis and improve the number of positive gene testing results. Estimations of p(life) and p(mut), based on a mathematical model, should deal with algorithms and penetrance/frequency data adequate to the population counselled. Being the main modulatory factors, reproductive/hormonal data should be incorporated like the Tyrer-Cuzick model does.

Correlations between anti-müllerian hormone, inhibin B, and activin A in follicular fluid in IVF/ICSI patients for assessing the maturation and developmental potential of oocytes.

OBJECTIVE: The objective of the present study was to evaluate the correlation between anti-müllerian hormone (AMH), inhibin B, and activin A in follicular fluid from patients receiving treatment with in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), to identify a parameter to assess the maturation and developmental potential of oocytes. - MATERIALS AND METHODS: AMH, inhibin B, and activin A were measured in follicular fluid from 27 patients undergoing IVF/ICSI treatment for male-factor infertility, tubal occlusion, endometriosis, or anovulation. The values were correlated with the serum estradiol level, the numbers and maturation of the oocytes, and the outcome of IVF/ICSI. - RESULTS: A positive correlation was found between AMH in follicular fluid and the number of oocytes retrieved. High inhibin B levels in follicular fluid and high serum E subset2 levels indicated a normal ovarian response to stimulation, corresponding to the oocyte numbers, while low inhibin B and 17-beta-estradiol (E subset2) levels indicated poor responders to stimulation. An activin A/inhibin B ratio of less than 1 and very high inhibin B levels correlated with large numbers of oocytes, while a ratio of 1-2 and high inhibin levels correlated with regular numbers of oocytes. An activin/inhibin ratio of more than 3 and low inhibin levels were found in poor responders. Pregnancies occurred predominantly in the group with a normal or high response. Patients with elevated ratios for 17-beta-estradiol/AMH, oocyte numbers/AMH, and metaphase II oocyte numbers/AMH had the best chances of becoming pregnant, indicating an inverse correlation between AMH and the maturation and developmental potential of the oocytes. - CONCLUSIONS: In IVF/ICSI patients, a positive correlation was found between AMH, inhibin B, and the activin A/inhibin B ratio in follicular fluid, on the one hand; and between serum 17-beta-estradiol levels and the numbers of oocytes retrieved, on the other. The activin A/inhibin B ratio correlated with the number of oocytes retrieved. The ratio for 17-beta-estradiol, oocyte numbers, and metaphase II oocytes relative to AMH indicated the best developmental potential, and it can therefore be assumed that there is a negative correlation between AMH levels and the maturation and quality of oocytes.

Hypermethylation and loss of retinoic acid receptor responder 1 expression in human choriocarcinoma.

BACKGROUND: Human placental development resembles tumorigenesis, due to the invasive and fusogenic potential of trophoblasts. However, these features are tightly controlled in trophoblasts. Disturbance of this spatial and temporal regulation is thought to contribute to the rare formation of choriocarcinomas. Promoter hypermethylation and loss of the tumor suppressor Retinoic acid receptor responder 1 (RARRES1) were shown to contribute to cancer progression. Our study investigated the epigenetic and transcriptional regulation of RARRES1 in healthy human placenta in comparison to choriocarcinoma cell lines and cases. METHODS: Three choriocarcinoma cell lines (Jeg-3, JAR and BeWo) were treated with three different retinoic acid derivates (Am580, Tazarotene and all-trans retinoic acid) and 5-aza-2'-deoxycytidine. We analyzed RARRES1 promoter methylation by pyrosequencing and performed realtime-PCR quantification to determine RARRES1 expression in placental tissue and trophoblastic cell lines. Additionally, RARRES1 was stained in healthy placentas and in biopsies of choriocarcinoma cases (n = 10) as well as the first trimester trophoblast cell line Swan71 by immunofluorescence and immunohistochemistry. RESULTS: In the choriocarcinoma cell lines, RARRES1 expression could not be induced by sole retinoic acid treatment. Stimulation with 5-aza-2'-deoxycytidine significantly induced RARRES1 expression, which then could be further increased with Am580, Tazarotene and all-trans retinoic acid. In comparison to healthy placenta, choriocarcinoma cell lines showed a hypermethylation of the RARRES1 promoter, which correlated with a reduced RARRES1 expression. In concordance, RARRES1 protein expression was lost in choriocarcinoma tissue. Additionally, in the trophoblastic cell line Swan71, we found a significant induction of RARRES1 expression with increased cell density, during mitosis and in syncytial knots. CONCLUSIONS: Our findings showed that RARRES1 expression is absent in choriocarcinoma due to promoter methylation. Based on our analysis, we hypothesize that RARRES1 might exert tumor suppressive functions in multiple cellular processes (e.g. cell cycle regulation, adhesion, invasion and apoptosis).

DNA binding properties and processive proofreading of herpes simplex virus type 1 DNA polymerase.

The DNA binding properties of herpes simplex virus type 1 DNA polymerase (HSV pol), an alpha-like DNA polymerase, were investigated using an optimized band-shift assay. With linear double-stranded DNA (dsDNA), HSV pol formed two complexes. The favored DNA template was dsDNA with protruding 5'-phosphoryl termini. Stable binding of HSV pol was observed with a DNA hairpin containing a primer region of 9 bp of dsDNA, a 6-base loop and a 12-base 5'-terminal single-stranded extension. For the polymerization activity of HSV pol on poly(dT) an optimal primer length of 8 to 10 nucleotides was determined. The DNA binding event could be clearly separated from the enzymatic activities by its unique response to divalent cations and salt. Under ionic strength conditions where HSV pol exerts optimal polymerization activity in vitro, novel polymerase-DNA complexes were detected by band-shift analysis. These new complexes were similar while either in DNA polymerase or 3',5' exonuclease mode. Using a polymerase trap method and high-resolution polyacrylamide gel electrophoresis, HSV pol demonstrated internal switching from 3',5' exonuclease to polymerase-active mode during one DNA binding event. These results support the role of HSV pol as a true replicase, which proofreads without dissociating from the DNA template.

Improved band shift assay for the simultaneous analysis of protein-DNA interactions and enzymatic functions of DNA polymerases.

A simple method to assay the major properties of DNA polymerases such as template binding, polymerase and exonuclease activities in one step is exemplified with the DNA polymerases of E. coli, bacteriophage T4 and herpes simplex virus. Combining the advantages of the band-shift assay with the resolving power of polyacrylamide gradient gel electrophoresis, the procedure is particularly useful for a rapid functional analysis of mutant polymerases as well as inhibitors of DNA replication.

Hypersensitivity to aloe.

Hypersensitivity, manifested by generalized nummular eczematous and papular dermatitis, and presumably by contact urticaria, developed in a 47-year-old man after four years of using oral and topical aloe. Patch tests for aloe were positive in this patient.

Sclerosing agents in the treatment of telangiectasia. Comparison of the clinical and histologic effects of intravascular polidocanol, sodium tetradecyl sulfate, and hypertonic saline in the dorsal rabbit ear vein model.

A 0.25-mL quantity of 0.25%, 0.5%, and 1.0% polidocanol (Aethoxysclerol [France]), 0.5% sodium tetradecyl sulfate (Sotradecol injection), and 23.4% hypertonic saline was injected into the dorsal marginal rabbit ear vein; clinical and histologic thrombosis resulted that lasted between four and eight days. The lowest concentration of polidocanol (0.25%) demonstrated immediate thrombosis; however, no clinical or histologic changes occurred eight days after injection. With all other agents, histologic fibrosis of the vessel correlating with clinical disappearance occurred after eight days. However, 0.5% polidocanol and sodium tetradecyl sulfate developed recanalization through the initially sclerosed vessel between eight and 14 days, with clinical reappearance of the 0.5% polidocanol-injected vessel at 30 days, after injection. Cutaneous necrosis was noted clinically and histologically in three of ten vessels injected with 1.0% polidocanol and in two of ten vessels injected with hypertonic saline. Clinical and histologic evidence of necrosis occurred with and without extravasation of the sclerosants.

Treatment of acne vulgaris.

Acne vulgaris is a common skin disorder that is socially disabling to some degree in the majority of adolescents. Effective management of this disease has two major components: a strong physician-patient relationship and a sound medical regimen. The former is necessary to foster patient compliance with the long-term therapeutic regimen required, as well as to reduce the disease's potential for emotional scarring. The rational usage of the various topical and systemic medications available will serve to decrease the frequency and severity of exacerbations as well as to minimize possible scarring. Specific recommendations as to moisturizers and cosmetics are included.

Contribution of different placental cells to the expression and stimulation of antimicrobial proteins (AMPs).

The placenta is a major barrier that prevents potentially infectious agents from causing fetal diseases or related complications during pregnancy. Therefore, we postulated that the placenta might express a broad repertoire of antimicrobial proteins as well as inflammatory chemokines and cytokines to combat invading microorganisms. Here we demonstrate that placental cells indeed express a wide range of AMPs (antimicrobial peptides and proteins) including bactericidal/permeability-increasing protein (BPI), secretory leukocyte protease inhibitor (SLPI), human ß-defensin 2 (hBD2), acyloxyacyl hydrolase (AOAH), and cathelicidin (CAP18). In addition, these cells also secrete pro-inflammatory cytokines and chemokines upon stimulation with bacterial ligands. Notably, we show that BPI expression by placental cells could be completely attributed to granulocytes while highly purified placental trophoblasts expressed only a subset of the AMPs like SLPI. Unexpectedly, trophoblast AMPs did not exhibit inducible secretion in response to various TLR ligands and further investigations showed that the unresponsiveness of trophoblasts to lipopolysaccharide (LPS) was due to a lack of TLR4 expression. In summary, we have shown that the expression of different AMPs can be allocated to various cells in the placenta and the repertoire of the AMPs expressed by placental cells is a result of a cooperation of leukocytes as well as cells from embryonic origin.

Regulation of the NRSF/REST gene by methylation and CREB affects the cellular phenotype of small-cell lung cancer.

The neuron-restrictive silencer factor/RE1-silencing transcription factor (NRSF/REST) is a negative regulator of gene expression restricting the expression of neuronal genes to the nervous system. NRSF/REST is highly expressed in non-neuronal tissues like the lung. In previous work, we identified small-cell lung cancer (SCLC) cell lines with no detectable NRSF/REST expression that, as a consequence, expressed neuronal markers like L1-cell adhesion molecule (L1-CAM) and neural cell adhesion molecule (NCAM). The loss of NRSF/REST expression was linked to malignant progression; however, its mechanistic role remained elusive. Here, we show that NRSF/REST itself, rather than one of its regulated genes, acts like a classic tumour suppressor, being in part regulated by methylation. In SCLCs, NRSF/REST is positively regulated by CREB, with an NRSF/REST promoter fragment showing cell type specificity. Downstream, NRSF/REST directly regulates AKT2, in which NRSF/REST loss leads to an epidermal growth factor-mediated de-regulation of AKT-Serine473 phosphorylation, important for cellular proliferation and survival. Assaying anchorage-independent growth, we observed that with reduced NRSF/REST expression, proliferation was significantly enhanced, whereas NRSF/REST rescue decreased the potential of cells to grow anchorage independently. Our observations support the fact that NRSF/REST may act as an important modulator of malignant progression in SCLC.

Single nucleotide polymorphism D1853N of the ATM gene may alter the risk for breast cancer.

PURPOSE: Various ATM (ataxia telangiectasia-mutated) mutations and polymorphisms have been reported to be associated with an increased breast cancer risk. Recent studies have produced contradictory results regarding the association between ATM genetic variants and breast cancer risk. MATERIALS AND METHODS: The common ATM polymorphism 5557G>A (p.D1853N) (rs1801516), previously suggested to be associated with bilateral breast cancer, was analyzed using real-time PCR in 514 unselected patients with breast cancer and 511 age-matched healthy control individuals. DNA was obtained from peripheral blood draw. RESULTS: The ATM genotype was weakly associated with the risk for breast cancer (P = 0.04 for the overall test). The odds ratio for women with a heterozygous genotype was 0.70 (95% CI, 0.52-0.94) and for the homozygous variant 0.63 (95% CI, 0.27-1.49). Disease-free survival and overall survival showed no significant association with specific genotypes. CONCLUSIONS: The results of this study might suggest a minor association between polymorphism 5557G>A and a reduced risk of breast cancer.

Evaluation of clinical parameters and estrogen receptor alpha gene polymorphisms for patients with endometriosis.

Endometriosis is a chronic inflammatory disease, which is especially found in women with subfertility problems with an incidence of up to 30%. The disease is considered an estrogen-dependent disorder, where DNA polymorphisms of the estrogen receptor alpha (ERalpha) in connection with endometriosis are controversially discussed. From a German population of women, clinical data associated with the disease, including the American Fertility Society (AFS) I-IV classification, and non-clinical parameters were evaluated statistically in endometriosis patients (n = 98) and in control women (n = 98) without endometriosis. Using a multivariate statistical analysis, significant associations of endometriosis with dysmenorrhea (P < 0.001) and allergies against medicaments (P = 0.042) were found. A positive trend between first grade family history of endometriosis and allergies against medicaments was also observed, suggesting a genetic relationship. From both collectives, DNA from peripheral blood was analyzed for the frequency of the ERalpha DNA polymorphisms Xba1 (A/G) and PvuII (T/C) in intron 1 and the ERalpha exonic DNA polymorphism (G229A) with an amino acid exchange (Gly77Ser) in the transactivation domain. DNA samples from endometriosis lesions and control tissues from the same collectives were also analyzed for the exonic G229A polymorphism. Only homozygote wild-type alleles for the polymorphism G229A were found, making it a rare polymorphism in mid-European individuals. Allele types for the PvuII and Xba1 polymorphisms were analyzed with the observed statistically significant clinical parameters and showed no significant association with endometriosis; however a trend with AFS IV was noted, which could contribute to lesion severity. In conclusion, the analyzed polymorphisms in the ERalpha do not have a functional role concerning specific clinical parameters associated with endometriosis.

DNA sequence variations of the entire anti-Mullerian hormone (AMH) gene promoter and AMH protein expression in patients with the Mayer-Rokitanski-Kuster-Hauser syndrome.

BACKGROUND: The etiology of the Mayer-Rokitanski-Kuster-Hauser (MRKH) syndrome, where congenitally the Mullerian ducts fail to develop into the uterus, cervix and upper vagina, along with other malformations, is unresolved. Anti-Mullerian hormone (AMH) signal transduction inducing the degradation of Mullerian ducts in males is implicated in the MRKH syndrome. This study examined if DNA sequence variations are responsible for the activation of AMH and aberrant hormone levels in MRKH patients. METHODS: The entire AMH promoter and 3' regulatory elements of the constitutively expressed splicing factor SF3a2 were sequenced in 30 MRKH patients and genotyped in 48 control individuals using matrix-assisted laser desorption/ionization-time-of-flight mass spectronomy. Ovarian AMH promoter function was correlated with protein expression in plasma and peritoneal fluid of MRKH patients. RESULTS: Of six identified AMH promoter variations, two at positions -639 (SP1-binding site) and -210 [steroidogenic factor (SF)1-binding site] were homozygote in 73% of patients, and 69% of control individuals, destroying the SP1-binding site. AMH protein levels in plasma and peritoneal fluid from patients were equivalent to control individuals, however in three patients plasma levels were abnormally high. CONCLUSIONS: AMH is an important indicator for ovarian function. AMH promoter sequence variations or the previously proposed SF3a2-AMH fusion co-transcripts cannot be responsible for aberrant AMH expression leading to Mullerian duct degradation.