Sonic hedgehog signaling regulates mode of cell division of early cerebral cortex progenitors and increases

The morphogen Sonic Hedgehog (SHH) plays a critical role in the development of different tissues. In the central nervous system, SHH is well known to contribute to the patterning of the spinal cord and separation of the brain hemispheres. In addition, it has recently been shown that SHH signaling also contributes to the patterning of the telencephalon and establishment of adult neurogenic niches. In this work, we investigated whether SHH signaling influences the behavior of neural progenitors isolated from the dorsal telencephalon, which generate excitatory neurons and macroglial cells in vitro. We observed that SHH increases proliferation of cortical progenitors and generation of astrocytes, whereas blocking SHH signaling with cyclopamine has opposite effects. In both cases, generation of neurons did not seem to be affected. However, cell survival was broadly affected by blockade of SHH signaling. SHH effects were related to three different cell phenomena: mode of cell division, cell cycle length and cell growth. Together, our data in vitro demonstrate that SHH signaling controls cell behaviors that are important for proliferation of cerebral cortex progenitors, as well as differentiation and survival of neurons and astroglial cells.

Developmental programming of polycystic ovary syndrome (PCOS): prenatal androgens establish pancreatic islet α/β cell ratio and subsequent insulin secretion

Exogenous androgenic steroids applied to pregnant sheep programmes a PCOS-like phenotype in female offspring. Via ultrasound guidance we applied steroids directly to ovine fetuses at d62 and d82 of gestation, and examined fetal (day 90 gestation) and postnatal (11 months old) pancreatic structure and function. Of three classes of steroid agonists applied (androgen - Testosterone propionate (TP), estrogen - Diethystilbesterol (DES) and glucocorticoid - Dexamethasone (DEX)), only androgens (TP) caused altered pancreatic development. Beta cell numbers were significantly elevated in prenatally androgenised female fetuses (P=0.03) (to approximately the higher numbers found in male fetuses), whereas alpha cell counts were unaffected, precipitating decreased alpha:beta cell ratios in the developing fetal pancreas (P=0.001), sustained into adolescence (P=0.0004). In adolescence basal insulin secretion was significantly higher in female offspring from androgen-excess pregnancies (P=0.045), and an exaggerated, hyperinsulinaemic response to glucose challenge (P=0.0007) observed, whereas prenatal DES or DEX treatment had no effects upon insulin secretion. Postnatal insulin secretion correlated with beta cell numbers (P=0.03). We conclude that the pancreas is a primary locus of androgenic stimulation during development, giving rise to postnatal offspring whose pancreas secreted excess insulin due to excess beta cells in the presence of a normal number of alpha cells.

Lead (pb) toxicity in saccharomyces cerevisiae: the role of cell wall

O chumbo é utilizado em muitos produtos, tais como baterias, gasolina, tintas e corantes, resultando na sua libertação no meio ambiente. Neste trabalho, foi examinado o papel da parede celular da levedura Saccharomyces cerevisiae como uma barreira ou como alvo da toxicidade do chumbo. A biodisponibilidade do Pb é muito reduzida pelos componentes do meio de cultura YEPD, o que dificulta a avaliação da toxicidade deste elemento em concentrações ambientalmente realistas. Para avaliar a toxicidade de Pb em S. cerevisiae, em condições de crescimento, foram efetuadas diferentes diluições (10-100 vezes) do meio YEPD, as quais foram misturadas com várias concentrações de Pb (0,1-1,0 mmol/l). Observou-se que o YEPD diluído 25 vezes constituía a melhor condição de compromisso entre o crescimento celular e a precipitação de Pb. Os genes CWP1 e CWP2 codificam para duas grandes manoproteínas da parede celular da levedura S. cerevisiae; a deleção destes genes CWP aumenta a permeabilidade da parede celular. A suscetibilidade de células de levedura interrompidas no gene CWP1 (estirpe cwp1Δ) ou CWP2 (estirpe cwp2Δ) foi comparada com a da estirpe, isogénica, selvagem (WT). Verificou-se que o crescimento das estirpes cwp1Δ e cwp2Δ, no meio de cultura YEPD 25 vezes diluído, na presença de Pb, não diferiu do crescimento da estirpe WT. Este resultado sugere que a alteração da permeabilidade da parede celular não altera a sensibilidade de células de levedura ao Pb. Foi investigada o impacto do Pb na parede celular de levedura. Para este efeito, comparou-se a suscetibilidade ao dodecil sulfato de sódio (SDS), ao calcofluor (CFW) e a uma enzima que degrada a parede da célula (liticase), em células da estirpe WT não expostas ou expostas a Pb durante 4, 8 ou 24 h. Além disso, o conteúdo de quitina da parede celular de levedura foi investigada por coloração das células com CFW. Os resultados não mostraram uma alteração da suscetibilidade ao SDS e ao CFW, nas células tratadas com Pb; contudo, nas células tratadas durante 24 h com Pb, observou-se um aumento da sensibilidade à liticase e um aumento da coloração com CFW. Estes resultados sugerem que o chumbo interage com a parede celular da levedura e influencia a sua composição. Deve ser levado a cabo trabalho adicional a fim de confirmar estes resultados.

Sonic hedgehog signaling regulates mode of cell division of early cerebral cortex progenitors and increases

The morphogen Sonic Hedgehog (SHH) plays a critical role in the development of different tissues. In the central nervous system, SHH is well known to contribute to the patterning of the spinal cord and separation of the brain hemispheres. In addition, it has recently been shown that SHH signaling also contributes to the patterning of the telencephalon and establishment of adult neurogenic niches. In this work, we investigated whether SHH signaling influences the behavior of neural progenitors isolated from the dorsal telencephalon, which generate excitatory neurons and macroglial cells in vitro. We observed that SHH increases proliferation of cortical progenitors and generation of astrocytes, whereas blocking SHH signaling with cyclopamine has opposite effects. In both cases, generation of neurons did not seem to be affected. However, cell survival was broadly affected by blockade of SHH signaling. SHH effects were related to three different cell phenomena: mode of cell division, cell cycle length and cell growth. Together, our data in vitro demonstrate that SHH signaling controls cell behaviors that are important for proliferation of cerebral cortex progenitors, as well as differentiation and survival of neurons and astroglial cells.

Leccinum vulpinum Watling induces DNA damage, decreases cell proliferation and induces apoptosis on the human MCF-7 breast cancer cell line

The current work aimed to study the antitumour activity of a phenolic extract of the edible mushroom Leccinum vulpinum Watling, rich essentially in hydroxybenzoic acids. In a first approach, the mushroom extract was tested against cancer cell growth by using four human tumour cell lines. Given the positive results obtained in these initial screening experiments and the evidence of some studies for an inverse relationship between mushroom consumption and breast cancer risk, a detailed study of the bioactivity of the extract was carried out on MCF-7 cells. Once the selected cell line to precede the work was the breast adenocarcinoma cell line, the human breast non-malignant cell line MCF-10A was used as control. Overall, the extract decreased cellular proliferation and induced apoptosis. Furthermore, the results also suggest that the extract causes cellular DNA damage. Data obtained highlight the potential of mushrooms as a source of biologically active compounds, particularly with antitumour activity.

Inhibition of proliferation, migration and invasion of human non-small cell lung cancer cell line A549 by phlomisoside F from Phlomis younghusbandii Mukerjee

Purpose: To determine the effect of phlomisoside F (PMF) on the proliferation, migration and invasion of human non-small cell lung cancer cell line A549 and explore the possible mechanisms. Methods: The anti-proliferative effect of PMF on A549 cells was determined by CCK-8. Subsequently, migration and invasion were evaluated by Transwell and Transwell with matrigel assays, respectively. Furthermore, cell cycle and apoptosis were assessed by flow cytometry, while the mechanisms of action were determined by Western blotting. Results: PMF exhibited significant anti-proliferative effect on A549 cells in concentration-dependent and time-dependent manners, with half maximal inhibitory concentration (IC50) of 54.51 μM. Treatment with PMF (10, 20 and 40 μM) for 48 h resulted in significantly decreased migration and invasion in A549 cells. In addition, PMF at concentrations of 25, 50 and 75 μM induced cell cycle arrest in G0/G1phase and enhanced cell apoptosis in A549 cells. Furthermore, caspase-3, caspase-9 and Bax protein expressions were up-regulated while Bacl-2 and COX-2 protein expressions were significantly downregulated at 10, 20 and 40 μM concentrations of PMF. Conclusion: PMF suppresses A549 cell growth, migration and invasion. The mechanism may be related to the induction of mitochondria-mediated apoptosis pathway via regulation of caspase-3, caspase-9, Bcl-2 and Bax expressions, and inhibition of PGE2 synthesis by reducing COX-2 expression.

Cell Pattern in Adult Human Corneal Endothelium

A review of the current data on the cell density of normal adult human endothelial cells was carried out in order to establish some common parameters appearing in the different considered populations. From the analysis of cell growth patterns, it is inferred that the cell aging rate is similar for each of the different considered populations. Also, the morphology, the cell distribution and the tendency to hexagonallity are studied. The results are consistent with the hypothesis that this phenomenon is analogous with cell behavior in other structures such as dry foams and grains in polycrystalline materials. Therefore, its driving force may be controlled by the surface tension and the mobility of the boundaries.

HydroxyprolineO-arabinosyltransferase mutants oppositely alter tip growth inArabidopsis thalianaandPhyscomitrella patens

Hydroxyproline O-arabinosyltransferases (HPATs) are members of a small, deeply conserved family of plant-specific glycosyltransferases that add arabinose sugars to diverse proteins including cell wall-associated extensins and small signaling peptides. Recent genetic studies in flowering plants suggest that different HPAT homologs have been co-opted to function in diverse species-specific developmental contexts. However, nothing is known about the roles of HPATs in basal plants. We show that complete loss of HPAT function in Arabidopsis thaliana and the moss Physcomitrella patens results in a shared defect in gametophytic tip cell growth. Arabidopsis hpat1/2/3 triple knockout mutants suffer from a strong male sterility defect as a consequence of pollen tubes that fail to fully elongate following pollination. Knocking out the two HPAT genes of Physcomitrella results in larger multicellular filamentous networks due to increased elongation of protonemal tip cells. Physcomitrella hpat mutants lack cell-wall associated hydroxyproline arabinosides and can be rescued with exogenous cellulose, while global expression profiling shows that cell wall-associated genes are severely misexpressed, implicating a defect in cell wall formation during tip growth. Our findings point to a major role for HPATs in influencing cell elongation during tip growth in plants.

Characterization of the molecular mechanisms mediated by the insulin-like growth factor-2 mRNA-binding proteins and Ephrin receptor A2 (EPHA2) signaling in the malignancy of CIC-DUX4 sarcomas

Ewing sarcoma (EWS) and CIC-DUX4 sarcoma (CDS) are pediatric fusion gene-driven tumors of mesenchymal origin characterized by an extremely stable genome and limited clinical solutions. Post-transcriptional regulatory mechanisms are crucial for understanding the development of this class of tumors. RNA binding proteins (RBPs) play a crucial role in the aggressiveness of these tumors. Numerous RBP families are dysregulated in cancer, including IGF2BPs. Among these, IGF2BP3 is a negative prognostic factor in EWS because it promotes cell growth, chemoresistence, and induces the metastatic process. Based on preliminary RNA sequencing data from clinical samples of EWS vs CDS patients, three major axes that are more expressed in CDS have been identified, two of which are dissected in this PhD work. The first involves the transcription factor HMGA2, IGF2BP2-3, and IGF2; the other involves the ephrin receptor system, particularly EphA2. EphA2 is involved in numerous cellular functions during embryonic stages, and its increased expression in adult tissues is often associated with pathological conditions. In tumors, its role is controversial because it can be associated with both pro- and anti-tumoral mechanisms. In EWS, it has been shown to play a role in promoting cell migration and neoangiogenesis. Our study has confirmed that the HMGA2/IGF2BPs/IGF2 axis contributes to CDS malignancy, and Akt hyperactivation has a strong impact on migration. Using loss/gain of function models for EphA2, we confirmed that it is a substrate of Akt, and Akt hyperactivation in CDS triggers ligand-independent activation of EphA2 through phosphorylation of S897. Moreover, the combination of Trabectedin and NVP/BEZ235 partially inhibits Akt/mTOR activation, resulting in reduced tumor growth in vivo. Inhibition of EphA2 through ALWII 41_27 significantly reduces migration in vitro. The project aim is the identification of target molecules in CDS that can distinguish it from EWS and thus develop new targeted therapeutic strategies.

The Crosslinking Degree Controls The Mechanical, Rheological, And Swelling Properties Of Hyaluronic Acid Microparticles.

Viscosupplements, used for treating joint and cartilage diseases, restore the rheological properties of synovial fluid, regulate joint homeostasis and act as scaffolds for cell growth and tissue regeneration. Most viscosupplements are hydrogels composed of hyaluronic acid (HA) microparticles suspended in fluid HA. These microparticles are crosslinked with chemicals to assure their stability against enzyme degradation and to prolong the action of the viscosupplement. However, the crosslinking also modifies the mechanical, swelling and rheological properties of the HA microparticle hydrogels, with consequences on the effectiveness of the application. The aim of this study is to correlate the crosslinking degree (CD) with these properties to achieve modulation of HA/DVS microparticles through CD control. Because divinyl sulfone (DVS) is the usual crosslinker of HA in viscosupplements, we examined the effects of CD by preparing HA microparticles at 1:1, 2:1, 3:1, and 5:1 HA/DVS mass ratios. The CD was calculated from inductively coupled plasma spectrometry data. HA microparticles were previously sized to a mean diameter of 87.5 µm. Higher CD increased the viscoelasticity and the extrusion force and reduced the swelling of the HA microparticle hydrogels, which also showed Newtonian pseudoplastic behavior and were classified as covalent weak. The hydrogels were not cytotoxic to fibroblasts according to an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2014.

Impaired Incretin Secretion And Pancreatic Dysfunction With Older Age And Diabetes.

To estimate the impact of aging and diabetes on insulin sensitivity, beta-cell function, adipocytokines, and incretin production. Hyperglycemic clamps, arginine tests and meal tolerance tests were performed in 50 non-obese subjects to measure insulin sensitivity (IS) and insulin secretion as well as plasma levels of glucagon, GLP-1 and GIP. Patients with diabetes and healthy control subjects were divided into the following groups: middle-aged type 2 diabetes (MA-DM), aged Type 2 diabetes (A-DM) and middle-aged or aged subjects with normal glucose tolerance (MA-NGT or A-NGT). IS, as determined by the homeostasis model assessment, glucose infusion rate, and oral glucose insulin sensitivity, was reduced in the aged and DM groups compared with MA-NGT, but it was similar in the MA-DM and A-DM groups. Insulinogenic index, first and second phase insulin secretion and the disposition indices, but not insulin response to arginine, were reduced in the aged and DM groups. Postprandial glucagon production was higher in MA-DM compared to MA-NGT. Whereas the GLP-1 production was reduced in A-DM, no differences between groups were observed in GIP production. In non-obese subjects, diabetes and aging impair insulin sensitivity. Insulin production is reduced by aging, and diabetes exacerbates this condition. Aging associated defects superimposed diabetic physiopathology, particularly regarding GLP-1 production. On the other hand, the glucose-independent secretion of insulin was preserved. Knowledge of the complex relationship between aging and diabetes could support the development of physiopathological and pharmacological based therapies.

Platinum Blue Staining Of Cells Grown In Electrospun Scaffolds.

Fibroblast cells grown in electrospun polymer scaffolds were stained with platinum blue, a heavy metal stain, and imaged using scanning electron microscopy. Good contrast on the cells was achieved compared with samples that were gold sputter coated. The cell morphology could be clearly observed, and the cells could be distinguished from the scaffold fibers. Here we optimized the required concentration of platinum blue for imaging cells grown in scaffolds and show that a higher concentration causes platinum aggregation. Overall, platinum blue is a useful stain for imaging cells because of its enhanced contrast using scanning electron microscopy (SEM). In the future it would be useful to investigate cell growth and morphology using three-dimensional imaging methods.

Oxidative Stress And Susceptibility To Mitochondrial Permeability Transition Precedes The Onset Of Diabetes In Autoimmune Non-obese Diabetic Mice.

Beta cell destruction in type 1 diabetes (TID) is associated with cellular oxidative stress and mitochondrial pathway of cell death. The aim of this study was to determine whether oxidative stress and mitochondrial dysfunction are present in T1D model (non-obese diabetic mouse, NOD) and if they are related to the stages of disease development. NOD mice were studied at three stages: non-diabetic, pre-diabetic, and diabetic and compared with age-matched Balb/c mice. Mitochondria respiration rates measured at phosphorylating and resting states in liver and soleus biopsies and in isolated liver mitochondria were similar in NOD and Balb/c mice at the three disease stages. However, NOD liver mitochondria were more susceptible to calcium-induced mitochondrial permeability transition as determined by cyclosporine-A-sensitive swelling and by decreased calcium retention capacity in all three stages of diabetes development. Mitochondria H2O2 production rate was higher in non-diabetic, but unaltered in pre-diabetic and diabetic NOD mice. The global cell reactive oxygen species (ROS), but not specific mitochondria ROS production, was significantly increased in NOD lymphomononuclear and stem cells in all disease stages. In addition, marked elevated rates of 2',7'-dichlorodihydrofluorescein (H2DCF) oxidation were observed in pancreatic islets from non-diabetic NOD mice. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and lipidomic approach, we identified oxidized lipid markers in NOD liver mitochondria for each disease stage, most of them being derivatives of diacylglycerols and phospholipids. These results suggest that the cellular oxidative stress precedes the establishment of diabetes and may be the cause of mitochondrial dysfunction that is involved in beta cell death.

Effect Of Atorvastatin On Wound Healing In Rats.

Skin-wound healing is a complex and dynamic biological process involving inflammation, proliferation, and remodeling. Recent studies have shown that statins are new therapeutical options because of their actions, such as anti-inflammatory and antioxidant activity, on vasodilation, endothelial dysfunction and neoangiogenesis, which are independent of their lipid-lowering action. Our aim was to investigate the effect of atorvastatin on tissue repair after acute injury in healthy animals. Rats were divided into four groups: placebo-treated (P), topical atorvastatin-treated (AT), oral atorvastatin-treated (AO), topical and oral atorvastatin-treated (ATO). Under anesthesia, rats were wounded with an 8-mm punch in the dorsal region. Lesions were photographed on Days 0, 1, 3, 7, 10, 12, and 14 post-injury and samples taken on Days 1, 3, 7, and 14 for protein-expression analysis of insulin receptor substrate (IRS)-1, phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), glycogen synthase kinase (GSK)-3, endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), extracellular signal-regulated kinase (ERK), interleukin (IL)-10, IL-1β, IL-6, and tumor necrosis factor (TNF)-α. Upon macroscopic examination, we observed significant reductions of lesion areas in groups AT, AO, and ATO compared to the P group. Additionally, AT and AO groups showed increased expression of IRS-1, PI3K, Akt, GSK-3, and IL-10 on Days 1 and 3 when compared with the P group. All atorvastatin-treated groups showed higher expression of IRS-1, PI3K, Akt, GSK-3, IL-10, eNOS, VEGF, and ERK on Day 7. On Days 1, 3, and 7, all atorvastatin-treated groups showed lower expression of IL-6 and TNF-α when compared with the P group. We conclude that atorvastatin accelerated tissue repair of acute lesions in rats and modulated expressions of proteins and cytokines associated with cell-growth pathways.

Surgical Treatment Of Type 2 Diabetes In Subjects With Mild Obesity: Mechanisms Underlying Metabolic Improvements.

This study aims to assess the clinical and physiological effects of Roux-en-Y gastric bypass (RYGBP) on type 2 diabetes associated with mild obesity (body mass index [BMI] 30-34.9 kg/m(2)) over 24 months postsurgery. In this prospective trial, 36 mildly obese subjects (19 males) with type 2 diabetes using oral antidiabetic drugs with (n = 24) or without insulin (n = 12) underwent RYGBP. Follow-up was conducted at baseline and 3, 6, 12, and 24 months postsurgery. The following endpoints were considered: changes in HbA1c, fasting glucose and insulin, antidiabetic therapy, BMI, oral glucose insulin sensitivity [OGIS, from meal tolerance test (MTT)], beta-cell secretory function [ΔCP(0-30)/ΔGlu(0-30) (ΔC-peptide/Δglucose ratio, MTT 0-30 min), disposition index (DI = OGIS [Symbol: see text] ΔCP(0-30)/ΔGlu(0-30)], glucagon-like peptide (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) [incremental area under the curve (AUCi)], adiponectin, C-reactive protein, and lipids. All subjects achieved normal-to-overweight BMI after 3 months. Over 24 months, 31/36 (86 %) subjects presented HbA1c <7 % [complete and partial remission of diabetes in 9/36 (22 %) and 1/36 (3 %), respectively]. Since 3 months postsurgery, improvements were observed in OGIS [290 (174) to 373 (77) ml/min/m(2), P = 0.009], ΔCP(0-30)/ΔGlu(0-30) [0.24 (0.19) to 0.52 (0.34) ng/mg, P = 0.001], DI [7.16 (8.53) to 19.8 (15.4) (ng/mg) (ml/min/m(2)), P = 0.001], GLP-1 AUCi [0.56 (0.64) to 3.97 (3.86) ng/dl [Symbol: see text] 10 min [Symbol: see text] 103, P = 0.000], and GIP AUCi [30.2 (12.6) to 27.0 (20.2) ng/dl [Symbol: see text] 10 min [Symbol: see text] 103, P = 0.004]. At baseline and after 12 months, subjects with diabetes nonremission had longer diabetes duration, higher HbA1c, lower beta-cell secretory function, and higher first 30-min GIP AUCi, compared with those with remission. RYGBP improves the glucose metabolism in subjects with type 2 diabetes and mild obesity. This effect is associated with improvement of insulin sensitivity, beta-cell secretory function, and incretin secretion.