Allele frequency of two intragenic microsatellite loci of SEL1L gene in Northern Italy population

Two cytosine-adenine (CA) repeats CAR/CAL and RepIN20 occur in the human SEL1L gene, which is regarded as a candidate gene for insulin-dependent diabetes mellitus (IDDM) and Grave's disease. We have characterized these repeats to determine if they might serve as effective microsatellite markers for linkage analysis to clarify whether SEL1L gene plays a role in the pathogenesis of these autoimmune diseases. The allele frequencies and average heterozygosity of the microsatellite repeats were analysed in 94 DNA samples from peripheral blood mononuclear (PBMC) cells from adults of Northern Italy. The average heterozygosity was 0.68 for CAR/CAL polymorphism and 0.85 for RepIN20. The size of PCR fragments of CAR/CAL ranged from 207\u2013225 bp and the most frequent allele was 207 bp (40.4%). The size of the fragments of RepIN20 ranged from 237\u2013255 bp and the most frequent allele was 249 bp (30.8%). In the light of the highly polymorphic nature of both microsatellites and their intragenic location in SEL1L gene, we suggest that they could provide a means for linkage analysis to clarify the potential role of SEL1L in conferring susceptibility to IDDM or Grave's disease

Insights in 17β-HSD1 Enzyme Kinetics and Ligand Binding by Dynamic Motion Investigation

BACKGROUND: Bisubstrate enzymes, such as 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), exist in solution as an ensemble of conformations. 17beta-HSD1 catalyzes the last step of the biosynthesis of estradiol and, thus, it is a potentially attractive target for breast cancer treatment. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the conformational transitions of its catalytic cycle, a structural analysis of all available crystal structures was performed and representative conformations were assigned to each step of the putative kinetic mechanism. To cover most of the conformational space, all-atom molecular dynamic simulations were performed using the four crystallographic structures best describing apoform, opened, occluded and closed state of 17beta-HSD1 as starting structures. With three of them, binary and ternary complexes were built with NADPH and NADPH-estrone, respectively, while two were investigated as apoform. Free energy calculations were performed in order to judge more accurately which of the MD complexes describes a specific kinetic step. CONCLUSIONS/SIGNIFICANCE: Remarkably, the analysis of the eight long range trajectories resulting from this multi-trajectory/-complex approach revealed an essential role played by the backbone and side chain motions, especially of the betaF alphaG'-loop, in cofactor and substrate binding. Thus, a selected-fit mechanism is suggested for 17beta-HSD1, where ligand-binding induced concerted motions of the FG-segment and the C-terminal part guide the enzyme along its preferred catalytic pathway. Overall, we could assign different enzyme conformations to the five steps of the random bi-bi kinetic cycle of 17beta-HSD1 and we could postulate a preferred pathway for it. This study lays the basis for more-targeted biochemical studies on 17beta-HSD1, as well as for the design of specific inhibitors of this enzyme. Moreover, it provides a useful guideline for other enzymes, also characterized by a rigid core and a flexible region directing their catalysis

Membrane fluidity matters: Hyperthermia from the aspects of lipids and membranes

Hyperthermia is a promising treatment modality for cancer in combination both with radio- and chemotherapy. In spite of its great therapeutic potential, the underlying molecular mechanisms still remain to be clarified. Due to lipid imbalances and 'membrane defects' most of the tumour cells possess elevated membrane fluidity. However, further increasing membrane fluidity to sensitise to chemo-or radiotherapy could have some other effects. In fact, hyperfluidisation of cell membrane induced by membrane fluidiser initiates a stress response as the heat shock protein response, which may modulate positively or negatively apoptotic cell death. Overviewing some recent findings based on a technology allowing direct imaging of lipid rafts in live cells and lipidomics, novel aspects of the intimate relationship between the 'membrane stress' of tumour cells and the cellular heat shock response will be highlighted. Our findings lend support to both the importance of membrane remodelling and the release of lipid signals initiating stress protein response, which can operate in tandem to control the extent of the ultimate cellular thermosensitivity. Overall, we suggest that the fluidity variable of membranes should be used as an independent factor for predicting the efficacy of combinational cancer therapies

Joining S100 proteins and migration:for better or for worse, in sickness and in health

The vast diversity of S100 proteins has demonstrated a multitude of biological correlations with cell growth, cell differentiation and cell survival in numerous physiological and pathological conditions in all cells of the body. This review summarises some of the reported regulatory functions of S100 proteins (namely S100A1, S100A2, S100A4, S100A6, S100A7, S100A8/S100A9, S100A10, S100A11, S100A12, S100B and S100P) on cellular migration and invasion, established in both culture and animal model systems and the possible mechanisms that have been proposed to be responsible. These mechanisms involve intracellular events and components of the cytoskeletal organisation (actin/myosin filaments, intermediate filaments and microtubules) as well as extracellular signalling at different cell surface receptors (RAGE and integrins). Finally, we shall attempt to demonstrate how aberrant expression of the S100 proteins may lead to pathological events and human disorders and furthermore provide a rationale to possibly explain why the expression of some of the S100 proteins (mainly S100A4 and S100P) has led to conflicting results on motility, depending on the cells used. © 2013 Springer Basel

The in vitro modulation of steroidogenesis by inflammatory cytokines and insulin in TM3 Leydig cells

BACKGROUND: Cytokines and hormones, including insulin, are known to modulate the hypothalamic-pituitary-testes axis and steroidogenesis, both centrally and peripherally. In the context of chronic inflammation and hyperinsulinaemia mediating male hypogonadism associated with obesity, metabolic syndrome and type 2 diabetes mellitus, these mechanisms are poorly understood and the impact of cytokines and insulin on Leydig cell steroidogenesis has not been fully elicited. This study aimed to further investigate the in vitro impact of TNFα, IL1ß, IL6, IL8 and insulin on Leydig cell function and steroidogenesis. METHODS: hCG-stimulated TM3 Leydig cells were exposed to various concentrations of TNFα, IL1ß, IL6, IL8 (100 ng/ ml, 10 ng/ml, 1 ng/ml and 0.1 ng/ml) and insulin (10 ng/ml, 1 ng/ml, 0.1 ng/ml and 0.01 ng/ml) in optimal cell culture conditions over 48 h. Cell viability (XTT) and testosterone and progesterone concentrations (ELISA) were assessed using standardised laboratory techniques. RESULTS: TNFα significantly decreased cell viability and progesterone and testosterone concentrations in a dosedependent relationship. IL1ß and IL6 had a subtle but significant negative effect on cell viability and testosterone concentrations, with a marked significant decrease in progesterone concentration at all concentrations investigated. IL8 showed an increase in cell viability, with no significant effect on testosterone concentrations alongside a significant decrease in progesterone concentrations. Insulin significantly increased cell viability and testosterone concentrations in a dose dependent relationship, but interestingly significantly decreased progesterone concentrations. CONCLUSIONS: The inflammatory cytokines TNFα, IL1β and IL6 cause a dose dependent decline in steroidogenesis in TM3 Leydig cells. These results suggest that chronic inflammation may downregulate steroidogenesis in males via direct modulation of Leydig cell function. However, IL8 may stimulate TM3 Leydig cell growth. Insulin is associated with a dose-dependent increase in testosterone synthesis, with a significant decline in progesterone synthesis. With the phenomenon of insulin resistance, the literature is unclear on the potential role of hyperinsulinaemia in steroidogenesis. Further studies are warranted in order to fully elicit the molecular mechanisms and interactions of these molecules on male steroidogenesis