The many faces of estrogen signaling

Estrogens have long been known as important regulators of the female reproductive functions; however, our understanding of the role estrogens play in the human body has changed significantly over the past years. It is now commonly accepted that estrogens and androgens have important functions in both female and male physiology and pathology. This is in part due to the local synthesis and action of estrogens that broadens the role of estrogen signaling beyond that of the endocrine system. Furthermore, there are several different mechanisms through which the three estrogen receptors (ERs), ERα, ERβ and G protein-coupled estrogen receptor 1 (GPER1) are able to regulate target gene transcription. ERα and ERβ are mostly associated with the direct and indirect genomic signaling pathways that result in target gene expression. Membrane-bound GPER1 is on the other hand responsible for the rapid non-genomic actions of estrogens that activate various protein-kinase cascades. Estrogen signaling is also tightly connected with another important regulatory entity, i.e. epigenetic mechanisms. Posttranslational histone modifications, microRNAs (miRNAs) and DNA methylation have been shown to influence gene expression of ERs as well as being regulated by estrogen signaling. Moreover, several coregulators of estrogen signaling also exhibit chromatin-modifying activities further underlining the importance of epigenetic mechanisms in estrogen signaling. This review wishes to highlight the newer aspects of estrogen signaling that exceed its classical endocrine regulatory role, especially emphasizing its tight intertwinement with epigenetic mechanisms

Development of a new DHPLC assay for genotyping UGT1A1(TA)n polymorphism associated with Gilbert’s syndrome

Introduction: Gilbert’s syndrome is the most common hereditary disorder of bilirubin metabolism. The causative mutation in Caucasians is almost exclusively a (TA) dinucleotide insertion in the UGT1A1 promoter. Affected individuals are homozygous for the variant promoter and have 7 TA repeats in-stead of 6. Promoters with 5 and 8 TA repeats also exist but are extremely rare in Caucasians. The aim of our study was to develop denaturing high-performance liquid chromatography (DHPLC) assay for genotyping UGT1A1(TA)n polymorphism and to compare it with a previously described single-strand conformation polymorphism (SSCP) assay. Materials and methods: Fifty DNA samples with common genotypes ((TA)6/6, (TA)6/7, (TA)7/7) as well as 7 samples with one of the following rare genotypes - (TA)5/6, (TA)5/7, (TA)6/8 or (TA)7/8 were amplifi-ed by polymerase chain reaction (PCR) and genotyped by DHPLC using sizing mode. All samples were previously genotyped by SSCP assay which was validated by sequencing analysis. Results: All samples with either common or rare genotypes showed completely concordant results between DHPLC and SSCP assays. Our results show that sizing DHPLC assay is more efficient compared to classical SSCP assay due to shorter time of genotyping analysis, ability of genotyping increased number of samples per day, higher robustness, reproducibility and cost-effectiveness with no loss of accuracy in detection of all UGT1A1(TA)n genotypes. Conclusions: We developed a new DHPLC assay which is suitable for accurate, automated, highthroughput, robust genotyping of all UGT1A1(TA)n polymorphism variants, compared to a labour intensive and time-consuming SSCP assay

The expansion of CD4(+)CD28(- )T cells in patients with rheumatoid arthritis

Clonal expansion of CD4(+)CD28(- )T cells is a characteristic finding in patients with rheumatoid arthritis (RA). Expanded CD4(+ )clonotypes are present in the peripheral blood, infiltrate into the joints, and persist for years. CD4(+)CD28(- )T cells are oligoclonal lymphocytes that are rare in healthy individuals but are found in high percentages in patients with chronic inflammatory diseases. The size of the peripheral blood CD4(+)CD28(- )T-cell compartment was determined in 42 patients with RA and 24 healthy subjects by two-color FACS analysis. The frequency of CD4(+)CD28(- )T cells was significantly higher in RA patients than in healthy subjects. Additionally, the number of these cells was significantly higher in patients with extra-articular manifestations and advanced joint destruction than in patients with limited joint manifestations. The results suggest that the frequency of CD4(+)CD28(- )T cells may be a marker correlating with extra-articular manifestations and joint involvement

Association of redox status and lifestyle factors with telomere length in healthy subjects

Telomeres are DNA-protein structures located at the ends of eukaryotic chromosomes. Since their shortening is a natural process, which repeats with each cell division, telomere lengths have been proposed as a biomarker of aging. Also, there is evidence that telomere shortening and dysfunction can be accelerated by oxidative-stress and unhealthy lifestyle factors, including physical inactivity and inadequate nutrition, Thus, we aimed to evaluate the association of redox status and selected lifestyle factors with telomere length. The study included 94 apparently healthy adults, both genders with average age 46±12 years. Before anthropometric measurements and venous blood sampling, participants were asked to complete a lifestyle questionnaire. Serum antioxidant defense markers (total sulfhydryl groups, paraoxonase activity and total antioxidant status) and prooxidants and products of its activity (malondialdehyde, superoxide anion and total oxidant status) were determined. The Prooxidative score, Antioxidative score and Oxy score were calculated from measured redox status markers by using z-score statistics. Telomere length was determined by qPCR method using genomic DNA from peripheral blood leukocytes. A positive relation was found among telomere length and a moderate level of physical activity, intake of fruits and vegetables, especially for females. Increasing abdominal fat, alcohol and fried food intake, as well the Oxy score (difference between Proxidative and Antioxidative scores) were inversely associated with telomere length. Overall, these results support the impact of healthy lifestyle on healthy aging and redox control in the term of prevention of major age-related diseases.Nutraceuticals in balancing redox status in ageing and age-related diseases WGs Meeting of the NutRedOx COST Action CA16112 Belgrade, March 2-3, 202

Association between 11β-hydroxysteroid dehydrogenase type 1 gene polymorphisms and metabolic syndrome

ntroduction: The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyzes the con-version of the hormonally inactive cortisone to active cortisol, thus facilitating glucocorticoid receptor activation in target tissues. Increased expression of 11β-HSD1 in adipose tissue has been associated with obesity and insulin resistance. In this study, we investigated the association of two 11β-HSD1 gene (HSD11B1) polymorphisms with the metabolic syndrome (MetS) and its characteristics in the Bosnian population. Materials and methods: The study included 86 participants: 43 patients diagnosed with MetS and 43 healthy controls. Subjects were genotyped for two HSD11B1 gene polymorphisms: rs846910: G>A and rs45487298: insA, by the high resolution melting curve analysis. Genotype distribution and an influence of genotypes on clinical and biochemical parameters were assessed. Results: There was no significant difference in the mutated allele frequencies for the two HSD11B1 gene polymorphisms between MetS patients and controls. In MetS patients, no significant associati-ons between disease-associated traits and rs45487298: insA were found. Regarding rs846910: G>A variant, heterozygous patients (G/A) had significantly lower systolic (P = 0.017) and diastolic blood pressure (P = 0.015), lower HOMA-IR index (P = 0.011) and higher LDL-cholesterol levels (P = 0.049), compared to the wild-type homozygotes. In the control group, rs45487298: insA polymorphism was associated with lower fasting plasma insulin levels (P = 0.041), lower homeostasis model asses-sment insulin resistance (HOMA-IR) index (P = 0.041) and lower diastolic blood pressure (P = 0.048). Significant differences between rs846910: G>A genotypes in controls were not detected. Haplotype analysis confirmed the association of rs45487298: insA with markers of insulin resistance in the con-trol subjects. Conclusions: Our results indicate that a common rs45487298: insA polymorphism in HSD11B1 gene may have a protective effect against insulin resistance

Telomere-telomerase system status in patients with acute myocardial infarction with ST-segment elevation – relationship with oxidative stress

Introduction: Telomeres are protective chromosomal ends. Short telomeres are a proven biomarker of biological aging. We aimed to find an association of telomere length and telomerase activity in circulating leukocytes and thromboaspirates of patients with acute myocardial infarction. Furthermore, association of the telomere-telomerase system with oxidative stress markers (as common risk factors for coronary artery disease (CAD)) was tested. Material and methods: Patients were selected from the patients admitted to the intensive care unit with acute myocardial infarction with ST-segment elevation (STEMI), with the following inclusion criteria – STEMI patients between 18 and 80 years old of both genders and candidates for primary percutaneous coronary intervention, with infarction pain present for a maximum of 12 h. In all the patients leukocyte telomere length, telomerase activity and scores related to oxidative-stress status (Protective, Damage and OXY) were evaluated. Results: Patients were divided into different groups: with stable angina pectoris (AP) (n = 22), acute myocardial infarction with: STEMI (n = 93), non-obstructive coronary arteries (MINOCA) (n = 7), blood vessel rupture (n = 6) at three time points, and compared to the group of 84 healthy subjects. Telomerase activity was significantly higher in all CAD sub-groups compared to the control group (AP = 0.373 (0.355–0.386), STEMI = 0.375 (0.349–0.395), MINOCA = 0.391 (0.366–0.401), blood vessel rupture = 0.360 (0.352–0.385) vs. CG = 0.069 (0.061–0.081), p < 0.001), while telomeres were significantly shorter in STEMI, MINOCA and blood vessel rupture groups compared to the control group (STEMI = 1.179 (0.931–1.376), MINOCA = 1.026 (0.951–1.070), blood vessel rupture = 1.089 (0.842–1.173) vs. CG = 1.329 (1.096–1.624), p = 0.030]. Values of OXY score were significantly higher in STEMI and MINOCA patients compared to the control group and AP patients (5.83 (4.55–7.54) and 10.28 (9.19–10.72) vs. 4.94 (3.29–6.18) and 4.18 (2.58–4.86), p < 0.001). Longer telomeres and higher telomerase activity were found in thromboaspirates, compared to the peripheral blood leukocytes in the same patients (1.25 (1.01–1.84) vs. 1.18 (0.909–1.516), p = 0.036; and 0.366 (0.367–0.379) vs. 0.366 (0.367–0.379), p < 0.001, respectively). In addition, telomere length and telomerase activity had good diagnostic ability to separate STEMI patients from healthy persons. Conclusions: Leukocyte telomere length and telomerase activity can differentiate CAD patients from healthy persons, and relate CAD to oxidative stress

Nishodna regulacija MAPK/MAK/MRK preklapajuće kinaze u mononuklearnim ćelijama periferne krvi pedijatrijskih pacijenata sa tip1 diiabetes mellitus-om

Background: Type 1 diabetes mellitus (T1DM) is one of the most common endocrine diseases in children. T-cell autore- activity toward b-cells is controlled by significant changes in metabolism of T cells. Mammalian target of rapamycin (mTOR) is an important intracellular regulator of metabolism and cell growth. MAPK/MAK/MRK overlapping kinase 1 (MOK1) is one of the less known regulators of mTOR. We sought to investigate if MOK1 and mTOR mRNA levels in peripheral blood mononuclear cells (PBMCs) of T1DM pedi- atric patients are different compared to healthy subjects. Methods: This study included 172 adolescents with T1DM and 36 healthy adolescent volunteers designated for control group (CG). MOK1 and mTOR mRNA levels were deter- mined in PBMCs by qPCR. Results: T1DM patients have significant downregulation of MOK1 mRNA levels in PBMCs compared CG (P=0.018), while there was no significant difference in mTOR mRNA levels (P=0.891). Furthermore, in T1DM patients, MOK1 significantly correlated with age, triglycerides and mTOR, while mTOR correlated significantly with BMI and systolic blood pressure. Overweight T1DM subjects had significantly lower MOK1 (P=0.034) and mTOR (P=0.017) mRNA levels, together with significantly higher levels of systolic blood pressure (P<0.001), total cholesterol (P=0.001), LDL-cholesterol (P=0.001) and CRP (P<0.001). Multi- variate analysis showed that MOK1 was independently negatively associated with T1DM when adjusted for sex, age, HDL-C and CRP (OR=0.417 (95%CI: 0.175–0.997), p=0.049). Conclusions: Our study demonstrated for the first time that T1DM is associated with MOK1 downregulation. In addition, downregulation of both mTOR and MOK1 gene expressions was associated with cardiovascular risk factors in overweight T1DM patients.Uvod: Dijabetes melitus tip 1 (T1DM) jedno je od najčešćih endokrinih oboljenja kod dece. Autoreaktivnost T-ćelija prema b-ćelijama kontroliše se značajnim promenama u metabolizmu T ćelija. Cilj delovanja rapamicina kod sisara je važan unutarćelijski regulator metabolizma i rasta ćelija. MAPK/MAK/MRK preklapajuće kinaze koja se preklapa (MOK1) jedan je od manje poznatih regulatora mTOR-a. Cilj istraživanja je bio da se ispita da li su nivoi iRNK MOK1 i mTOR u mononuklearnim ćelijama periferne krvi različiti kod pedijatrijskih pacijenata sa T1DM u odnosu na zdrave ispitanike. Metode: Ovo istraživanje je obuhvatilo 172 adolescenta sa T1DM i 36 zdravih adolescenata dobrovoljaca koji su činili kontrolnu grupu (CG). Nivoi MOK1 i mTOR mRNA određeni su u PBMC-ima pomoću qPCR-a. Rezultati: Pacijenti sa T1DM imali su značajno niže nivoe iRNK MOK1 u PBMC u odnosu na CG, dok razlike u nivoima iRNK mTOR nisu bile značajne (P = 0,891). Štaviše, kod pacijenata sa T1DM, MOK1 je značajno korelirao sa godinama, trigliceri dima i mTOR, dok je mTOR značajno korelirao sa BMI i sistolnim krvnim pritiskom. Ispitanici sa prekomernom težinom T1DM imali su značajno niže nivoe iRNK MOK1 (P = 0,034) i mTOR (P = 0,017), zajedno sa značajno većim nivoima sistolnog krvnog pritiska (P <0,001), ukupnog holesterola (P = 0,001), LDL-holesterola (P = 0,001) i CRP (P <0,001). Multivarijantna analiza je pokazala da je MOK1 nezavisno negativno povezan sa T1DM kada je prilagođen polu, starosti, HDL-C i CRP (OR = 0,417 (95%CI: 0,175-0,997), p = 0,049). Zaključak: Naša studija je prva koja je pokazala da je T1DM udružen sa nishodnom regulacijom MOK1. Pored toga, snižena regulacija ekspresije gena mTOR i MOK1 bila je povezana sa kardiovaskularnim faktorima rizika kod pacije nata sa T1DM sa prekomernom težinom

Epigenetic enzymes influenced by oxidative stress and hypoxia mimetic in osteoblasts are differentially expressed in patients with osteoporosis and osteoarthritis

Epigenetic mechanisms including posttranslational histone modifications and DNA methylation are emerging as important determinants of bone homeostasis. With our case-control study we aimed to identify which chromatin-modifying enzymes could be involved in the pathology of postmenopausal osteoporosis and osteoarthritis while co-regulated by estrogens, oxidative stress and hypoxia. Gene expression of HAT1, KAT5, HDAC6, MBD1 and DNMT3A affected by oxidative stress and hypoxia in an in vitro qPCR screening step performed on an osteoblast cell line was analysed in trabecular bone tissue samples from 96 patients. Their expression was significantly reduced in patients with postmenopausal osteoporosis and osteoarthritis as compared to autopsy controls and significantly correlated with bone mineral density and several bone histomorphometry-derived parameters of bone quality and quantity as well as indicators of oxidative stress, RANK/RANKL/OPG system and angiogenesis. Furthermore, oxidative stress increased DNA methylation levels at the RANKL and OPG promoters while decreasing histone acetylation levels at these two genes. Our study is the first to show that higher expression of HAT1, HDAC6 and MBD1 is associated with superior quantity as well as quality of the bone tissue having a more favourable trabecular structure