Mutations in the Leucine Reach Repeat Kinase 2 (LRRK2) gene are the most frequent cause of familial Parkinson's disease (PD). Although the precise physiological and pathological role of LRRK2 is unclear, a direct link between mutant LRRK2 and apoptosis has been suggested. Using flow cytometric analysis (PI+Annexin V(FITC)) we showed increased spontaneous apoptosis of peripheral blood lymphocytes in patients with LRRK.2-associated PD compared to controls after 24 (P
Petersburg Nuclear Physics Institute named by B.P. Konstantinov of National Research Centre «Kurchatov Institute», Gatchina, Russia; Pavlov First Saint Petersburg State Medical University, Saint-Petersburg, Russia. Electronic address: usenko_ts@pnpi.nrcki.ru.
Lysosomal integral membrane protein-2 (LIMP-2), encoded by the SCARB2 gene, is the specific lysosomal receptor for glucocerebrosidase enzyme. Association between rs6812193 and rs68250047 of SCARB2 with PD has been shown in genetic studies, including large genome-wide association studies. The aim of the current study was to determine whether rs6812193 and rs8475 are associated with PD in Russia. rs6812193 and rs8475 were genotyped in a total of 604 PD patients (65 PD patients with positive (fPD) and 539 PD patients with negative family history (sPD)) and 413 controls and also in 17 patients with PD associated with GBA mutations (PD-GBA) and 18 asymptomatic GBA mutation carriers (GBA-Carriers). SCARB2 expression was measured by real-time PCR in CD45+ blood cells in part of individuals in the studied groups. No linkage disequilibrium was shown between rs6812193 and rs8475 in Russian population. Increased PD risk for TT variant of rs8475 (OR = 2.02; p
AIM: The aim of this study was to analyze the levels 70 kDa ribosomal protein S6 kinase 1 (S6K1) and 70 kDa ribosomal protein S6 kinase 2 (S6K2) expression and S6 ribosomal protein phosphorylation in endometrial adenocarcinomas. METHODS: S6K1/2 expression and phosphorylated ribosomal S6 protein (phS6) content have been detected in formalin fixed, paraffin embedded sections of 50 human endometrial adenocarcinomas with different grade of differentiation and in 13 normal endometrial tissues using immunohistochemical approach with following semiquantitative analysis. RESULTS: In normal endometrial epithelial cells both S6K1 and S6K2 were expressed on the low level. S6K1 and S6K2 has been detected predominantly in stromal elements. Increased phS6 level was found in superficial epithelial cells. In deeper parts of endometrial glands and vessels phS6 was discovered occasionally. In endometrial adenocarcinoma's tissues, overexpression of S6K1 was found in cytoplasm and nuclei in 8.0% of cases, overexpression of S6K2--in cytoplasm in 12.0% of cases and in nuclei in 18.0% of cases. Overexpression of S6K1 in endothelial cells of vessels was discovered in 58% of cases. Positive correlation has been determined between: 1) tumor stage and intensity of stromal staining for S6K1 (p = 0.027); 2) tumor differentiation grade and intensity of cytoplasm staining of cancer cells for S6K1 (p = 0.039); 3) intensity of stromal staining and vessel's staining for S6K1 (p = 0.019); 4) vessel's staining for S6K1 and staining for phS6 (p = 0.028). CONCLUSION: Overexpression of S6K1 and S6K2 is a characteristic feature of parenchyma and vessels of endometrial adenocarcinomas. Phosphorylation of ribosomal S6 protein is not dependent from expression level of S6K1 and S6K2.
AIM: To express recombinant S6K2 in baculovirus expression system; to purify large quantities of recombinant S6K2 for biochemical studies; to generate and characterise specific MABs against recombinant S6K2; to study the patterns S6K1 and S6K2 expression and subcellular localization in normal, benign and malignant breast tissues. METHODS: Recombinant baculovirus, expressing wild type S6K2 was generated using Bac-to-Bac system (Invitrogen); recombinant S6K was purified from infected Sf9 cells using affinity purification approach; monoclonal antibodies against recombinant S6K2 were generated; the specificity of generated MABs towards recombinant and endogenous S6K2 were examined by ELISA, Western blotting, immunoprecipitation and immuhohistochemical staining; immunohistochemical detection of S6K1 and S6K2 in human breast tissues was performed using specific monoclonal antibodies towards S6K1 and S6K2. RESULTS: Large amounts of enzymatically active S6K2 were purified using baculovirus expression system; highly purified preparations of S6K2 were used to generate and characterize anti-S6K2 MABs; elevated levels of S6K1 and S6K2 were found in breast tumors when compared to normal breast tissues; S6K2 is frequently localized in the nuclei of adenocarcinoma tissues, but rarely in fibroadenoma or "normal" breast tissues. CONCLUSION: Production of recombinant S6K2 in large amount and generation of specific monoclonal antibodies towards S6K2 has provided us with excellent tools to study the function and regulation of this important signalling molecule in normal and cancer cells. Immunnohistochemical analysis of S6K1 and S6K2 expression in normal and malignant breast clearly indicates that both kinases are overexpressed in breast tumors, when compared to "normal" tissues. The retention of S6K2 in the nuclei of malignant cells may be caused by disregulation of nucleocytoplasmic shuttling and could subsequently affect cell growth and proliferation.
AIM: To perform an immunohistochemical analysis of human breast adenomas and adenocarcinomas as well as normal breast tissues in respect of S6 ribosomal protein kinase (S6K) expression and localization in normal and transformed cells. METHODS: The expression level and localization of S6K have been detected in formalin fixed, paraffin embedded sections of normal human breast tissues, adenomas and adenocarcinomas with different grade of differentiation. Immunohistochemical detection of S6K1 and S6K2 in normal human breast tissues and breast tumors were performed using specific monoclonal and polyclonal antibodies against S6K1 and S6K2 with following semiquantitative analysis. RESULTS: The increase of S6K content in the cytoplasm of epithelial cells in benign and malignant tumors has been detected. Nuclear accumulation of S6K1 and to a greater extend S6K2 have been found in breast adenocarcinomas. About 80% of breast adenocarcinomas cases revealed S6K2 nuclear staining comparing to normal tissues. In 31% of cases more then 50% of cancer cells had strong nuclear staining. Accumulation of S6K1 in the nucleus of neoplastic cells has been demonstrated in 25% of cases. Nuclear localization of S6K in the epithelial cells in normal breast tissues has not been detected. CONCLUSION: Immunohistochemical analysis of S6K1 and S6K2 expression in normal human breast tissues, benign and malignant breast tumors clearly indicates that both kinases are overexpressed in breast tumors. Semiquantitative analysis of peculiarities of S6K localization in normal tissues and tumors revealed that nucleoplasmic accumulation of S6K (especially S6K2) is a distinguishing feature of cancer cells.
The aim of this work was to study the effect of a dose of 150 microCi 131I on the barrier properties of the thyroid epithelium in pregnant female rats. Thirty-five female Wistar rats were divided into a control and four experimental groups (each distinguished by the time of 131I injection: group I--no less then 12 days before mating; groups II, III, and IV--on 5th, 10th, and 16th days of gestation, respectively). The thyroid glands were fixed in Bouin's fluid, embedded in paraffin, and stained immunohistochemically for thyroglobulin and fibronectin. In group IV the appearance of follicles with fibronectin-positive colloid demonstrates the penetration of blood plasma into the follicular lumen. There are more fibronectin positive follicles in group III. Regardless of the nature of the follicles' contents, numerous thyrocytes with an intensive fibronectin positive reaction begin to appear in the follicles. In group II the number of fibronectin positive follicles and thyrocytes is clearly reduced, and in group I only a few remain. In group IV there is a noticeable reduction in the quantity of colloid inside the follicles and often an absence of any thyroglobulin positive reaction. There are thyrocytes in which thyroglobulin positive granules localized in the basal zone. There is thyroglobulin positive staining in the stroma and blood vessels. In group II thyroglobulin is no longer found in the stroma. Small doses of 131I provoke a serious breakdown in the thyroid epithelium's barrier properties, although these changes are of a transient nature. The central zone of the thyroid gland reacts more actively and dynamically to exposure to radioactive iodine than the peripheral zone.
There have been no works devoted to the study of the influence of (131)I and maternal (131)I-induced hypothyroidism on the state of the C-cells in the thyroid gland of the developing embryos. A study was made on the effect of a dose of 150 microCi (131)I (0.5 Gy) leading to hypothyroidism in rats, on 35 mother rats and 168 newborn pups. The mother rats were divided into control and four treated groups which were injected with (131)I before pregnancy, on gestation days 5, 10, and 16, respectively. Immunohistochemically, the thyroid gland was examined for calcitonin-positive cells. Maternal hypothyroidism induced by (131)I leads to the development of hyperplasia and hyperthrophy of calcitonin-positive cells in the pups at the time of birth. The discovery of separate C-cells in the peripheral zone of the thyroid lobe may be evidence of an unbalance in the development of the medial and lateral source of the thyroid. There is a verifiable increase in the quantity of C-cells per 1 mm(2) field of the localization in the central zone of the gestation days 10 and 16 groups. This might be a compensatory mechanism for regulating the activity of the thyroid gland under induced hypothyroidism. Thus, in cases when there is a breakdown in the normal external regulation of the embryonic morphogenesis, a reduction in the level of maternal thyroid hormones and also direct exposure to (131)I, there is also a change in the foetus' internal regulatory systems. A change in C-cell system could lead to the appearance of endocrinological disorders later in life.
To quantify the contributions of fossil and biomass sources to the wintertime Arctic aerosol burden source apportionment is reported for elemental (EC) and organic carbon (OC) fractions of six PM10 samples collected during a wintertime (2012-2013) campaign in Barrow, AK. Radiocarbon apportionment of EC indicates that fossil sources contribute an average of 68 ± 9% (0.01-0.07 µg m(-3)) in midwinter decreasing to 49 ± 6% (0.02 µg m(-3)) in late winter. The mean contribution of fossil sources to OC for the campaign was stable at 38 ± 8% (0.04-0.32 µg m(-3)). Samples were also analyzed for organic tracers, including levoglucosan, for use in a chemical mass balance (CMB) source apportionment model. The CMB model was able to apportion 24-53% and 99% of the OC and EC burdens, respectively, during the campaign, with fossil OC contributions ranging from 25 to 74% (0.02-0.09 µg m(-3)) and fossil EC contributions ranging from 73 to 94% (0.03-0.07 µg m(-3)). Back trajectories identified two major wintertime source regions to Barrow: the Russian and North American Arctic. Atmospheric lifetimes of levoglucosan, ranging from 50 to 320 h, revealed variability in wintertime atmospheric processing of this biomass burning tracer. This study allows for unambiguous apportionment of EC to fossil fuel and biomass combustion sources and intercomparison with CMB modeling.
