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Sample records for cell enolase level

  1. Genetic and proteomic evidences support the localization of yeast enolase in the cell surface

    DEFF Research Database (Denmark)

    López-Villar, Elena; Monteoliva, Lucía; Larsen, Martin Røssel

    2006-01-01

    protein (GFP) as reporter proteins, proved that the 169 N-terminal amino acids are sufficient to target the protein to the cell surface. Furthermore, the enolase-GFP fusion co-localized with a plasma membrane marker. Enolase was also identified among membrane proteins obtained by a purification protocol...... that different experimental approaches (genetics, cellular biology and proteomics) show that yeast enolase can reach the cell surface and describe the protein regions involved in its cell surface targeting. Hybrid enolase truncates, fused at their C terminus with the yeast internal invertase or green fluorescent...

  2. Serum Levels of Neuron-Specific Enolase in Children With Diabetic Ketoacidosis.

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    Hamed, Sherifa; Metwalley, Kotb Abbass; Farghaly, Hekma Saad; Sherief, Tahra

    2017-04-01

    Neuron-specific enolase is a sensitive marker of neuronal damage in various neurologic disorders. This study aimed to measure serum neuron-specific enolase levels at different time points and severities of diabetic ketoacidosis. This study included 90 children (age 9.2 ± 3.4 years) with diabetic ketoacidosis. Neuron-specific enolase was measured at 3 time points (baseline and after 12 and 24 hours of starting treatment). Among patients, 74.4% had diagnosis of new diabetes, 60% had Glasgow Coma Scale score diabetic ketoacidosis. Compared with controls (n = 30), children with diabetic ketoacidosis had higher neuron-specific enolase levels at the 3 time points ( P = .0001). In multiple regression analysis, the factors associated with higher neuron-specific enolase levels were younger age, higher glucose, lower pH, and bicarbonate values. This study indicates that serum neuron-specific enolase is elevated in diabetic ketoacidosis and correlated with the severity of hyperglycemia, ketosis, and acidosis. This study indicates that diabetic ketoacidosis may cause neuronal injury from which the patients recovered partially but not completely.

  3. Alpha-enolase on apical surface of renal tubular epithelial cells serves as a calcium oxalate crystal receptor

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    Fong-Ngern, Kedsarin; Thongboonkerd, Visith

    2016-10-01

    To search for a strategy to prevent kidney stone formation/recurrence, this study addressed the role of α-enolase on apical membrane of renal tubular cells in mediating calcium oxalate monohydrate (COM) crystal adhesion. Its presence on apical membrane and in COM crystal-bound fraction was confirmed by Western blotting and immunofluorescence staining. Pretreating MDCK cells with anti-α-enolase antibody, not isotype-controlled IgG, dramatically reduced cell-crystal adhesion. Immunofluorescence staining also confirmed the direct binding of purified α-enolase to COM crystals at {121} > {100} > {010} crystal faces. Coating COM crystals with urinary proteins diminished the crystal binding capacity to cells and purified α-enolase. Moreover, α-enolase selectively bound to COM, not other crystals. Chemico-protein interactions analysis revealed that α-enolase interacted directly with Ca2+ and Mg2+. Incubating the cells with Mg2+ prior to cell-crystal adhesion assay significantly reduced crystal binding on the cell surface, whereas preincubation with EDTA, a divalent cation chelator, completely abolished Mg2+ effect, indicating that COM and Mg2+ competitively bind to α-enolase. Taken together, we successfully confirmed the role of α-enolase as a COM crystal receptor to mediate COM crystal adhesion at apical membrane of renal tubular cells. It may also serve as a target for stone prevention by blocking cell-crystal adhesion and stone nidus formation.

  4. Calcium oxalate crystals increased enolase-1 secretion from renal tubular cells that subsequently enhanced crystal and monocyte invasion through renal interstitium.

    Science.gov (United States)

    Chiangjong, Wararat; Thongboonkerd, Visith

    2016-04-05

    Calcium oxalate monohydrate (COM) crystals cause kidney stone disease by still unclear mechanisms. The present study aimed to characterize changes in secretion of proteins from basolateral compartment of renal tubular epithelial cells after exposure to COM crystals and then correlated them with the stone pathogenesis. Polarized MDCK cells were cultivated in serum-free medium with or without 100 μg/ml COM crystals for 20 h. Secreted proteins collected from the lower chamber (basolateral compartment) were then resolved in 2-D gels and visualized by Deep Purple stain (n = 5 gels/group). Spot matching and intensity analysis revealed six protein spots with significantly altered levels in COM-treated samples. These proteins were then identified by tandem mass spectrometry (Q-TOF MS/MS), including enolase-1, phosphoglycerate mutase-1, actinin, 14-3-3 protein epsilon, alpha-tubulin 2, and ubiquitin-activating enzyme E1. The increased enolase-1 level was confirmed by Western blot analysis. Functional analysis revealed that enolase-1 dramatically induced COM crystal invasion through ECM migrating chamber in a dose-dependent manner. Moreover, enolase-1 bound onto U937 monocytic cell surface markedly enhanced cell migration through the ECM migrating chamber. In summary, our data indicated that the increased secretory enolase-1 induced by COM crystals played an important role in crystal invasion and inflammatory process in renal interstitium.

  5. Mobility of creatine phosphokinase and beta-enolase in cultured muscle cells

    OpenAIRE

    Arrio-Dupont, M.; Foucault, G.; Vacher, M.; Douhou, A.; Cribier, S.

    1997-01-01

    The diffusion of beta-enolase and creatine phosphokinase in muscle cells has been studied by modulated fringe pattern photobleaching. Beta-enolase is mobile in the sarcoplasm. At 20 degrees C, the diffusion coefficient is 13.5 +/- 2.5 microm2 s(-1) in the cytosol and 56 microm2 s(-1) in aqueous media. As in the case of dextrans of the same hydrodynamic radius, its mobility is hindered by both the crowding of the fluid phase of the cytoplasm and the screening effect due to myofilaments. A frac...

  6. Neuron-specific enolase is a useful maker of neuroendocrine origin in pheochromocytoma cell culture

    International Nuclear Information System (INIS)

    Abelin, N.; Dahia, P.L.M.; Martin, R.; Kato, S.; Toledo, S.P.A.

    1994-01-01

    Neuron-specific enolase (NSE) has been used as a marker for neuroendocrine tumors either in immunocytochemical studies or in serum measurements. In this paper NSE levels were determined in cultured pheochromocytoma cells to test whether it is also a useful marker in cell culture of tumors derived from neuroendocrine system. Cultured pheochromocytoma cells came from a primary explant and were grown in RPMI supplemented with 20% fetal calf serum, 100 μg/mL ampicillin and 100 μ/mL streptomycin. NSE was measured in culture medium and cell homogenates. Samples from different pheochromocytoma cultures were analyzed and compared to normal cultured fibroblast cells derived from human skin. NSE was measured by a commercially available radioimmunoassay kit. NSE levels were higher in cell homogenates as compared to those in culture medium, reaching levels as high as 6-fold in the former in TE cell line (26.46 ng/mL and 4.39 ng/mL, respectively). Serial measurements in culture medium from TE cell line evidenced decreasing values in subsequential subcultures (from 9.24 ng/mL during primary explant to 1.7 ng/mL in the tenth subculture). In cultured normal fibroblasts, NSE levels in cultured media were definitely lower than those obtained from pheochromocytoma cultures. These preliminary data suggest that NSE may be a useful marker of neuroendocrine derived tumors, such as pheochromocytoma, in culture. Thus, the simplicity and availability of NSE radioimmunoassay provides an alternative to catecholamine measurement to better characterize pheochromocytoma cell lines in culture, with the advantage of faster result at lower costs. (author). 18 refs, 2 tabs

  7. Correlative study between neuron-specific enolase and blood sugar level in ischemic stroke patients

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    Aparna Pandey

    2011-01-01

    Full Text Available Background: A study to investigate the level of the neurobiochemical marker, Neuron-Specific Enolase (NSE, at the time of admission and its correlation with the blood sugar level in ischemic stroke patients. Patients and Methods: We investigated 90 patients with complete stroke who were admitted to the Stroke Unit of the Department of Neurology at Sri Aurobindo Institute of Medical Sciences. NSE was measured with commercially available quantitative ′sandwich′ enzyme-linked immunosorbent assay kits obtained from R and D Systems. Hyperglycemia was defined as blood glucose concentration ≥ 7 mmol / L, and measured using the glucose oxidase method immediately. Results: Significantly increased NSE and lipid profile levels were found in ischemic stroke patients as compared to the control. Hyperglycemic ischemic stroke patients had increased levels of NSE, lipid profile, and National Institute of Health stroke scale scores (NIHSS score compared to normoglycemic ischemic stroke patients. In addition the serum NSE level of hyperglycemic stroke patients was also positively correlated with the blood sugar level (r = 0.734 P < 0.001. Conclusions: Hyperglycemia predicts an increased risk of poor outcome after ischemic stroke and it is reflected by a significantly increased level of Neuron-Specific Enolase.

  8. A T4SS Effector Targets Host Cell Alpha-Enolase Contributing to Brucella abortus Intracellular Lifestyle.

    Science.gov (United States)

    Marchesini, María I; Morrone Seijo, Susana M; Guaimas, Francisco F; Comerci, Diego J

    2016-01-01

    Brucella abortus , the causative agent of bovine brucellosis, invades and replicates within cells inside a membrane-bound compartment known as the Brucella containing vacuole (BCV). After trafficking along the endocytic and secretory pathways, BCVs mature into endoplasmic reticulum-derived compartments permissive for bacterial replication. Brucella Type IV Secretion System (VirB) is a major virulence factor essential for the biogenesis of the replicative organelle. Upon infection, Brucella uses the VirB system to translocate effector proteins from the BCV into the host cell cytoplasm. Although the functions of many translocated proteins remain unknown, some of them have been demonstrated to modulate host cell signaling pathways to favor intracellular survival and replication. BPE123 (BAB2_0123) is a B. abortus VirB-translocated effector protein recently identified by our group whose function is yet unknown. In an attempt to identify host cell proteins interacting with BPE123, a pull-down assay was performed and human alpha-enolase (ENO-1) was identified by LC/MS-MS as a potential interaction partner of BPE123. These results were confirmed by immunoprecipitation assays. In bone-marrow derived macrophages infected with B. abortus , ENO-1 associates to BCVs in a BPE123-dependent manner, indicating that interaction with translocated BPE123 is also occurring during the intracellular phase of the bacterium. Furthermore, ENO-1 depletion by siRNA impaired B. abortus intracellular replication in HeLa cells, confirming a role for α-enolase during the infection process. Indeed, ENO-1 activity levels were enhanced upon B. abortus infection of THP-1 macrophagic cells, and this activation is highly dependent on BPE123. Taken together, these results suggest that interaction between BPE123 and host cell ENO-1 contributes to the intracellular lifestyle of B. abortus .

  9. Mobility of creatine phosphokinase and beta-enolase in cultured muscle cells.

    Science.gov (United States)

    Arrio-Dupont, M; Foucault, G; Vacher, M; Douhou, A; Cribier, S

    1997-11-01

    The diffusion of beta-enolase and creatine phosphokinase in muscle cells has been studied by modulated fringe pattern photobleaching. Beta-enolase is mobile in the sarcoplasm. At 20 degrees C, the diffusion coefficient is 13.5 +/- 2.5 microm2 s(-1) in the cytosol and 56 microm2 s(-1) in aqueous media. As in the case of dextrans of the same hydrodynamic radius, its mobility is hindered by both the crowding of the fluid phase of the cytoplasm and the screening effect due to myofilaments. A fraction of creatine phosphokinase is mobile in the sarcoplasm. Its diffusion coefficient in the cytosol, 4.5 +/- 1 microm2 s(-1), is lower than that of the dextran of equivalent size. The other fraction (20 to 50%) is very slightly mobile, with an apparent diffusion coefficient varying from 0.0035 to 0.043 microm2 s(-1). This low mobility might be attributed to exchange between free and bound creatine phosphokinase. The bound fraction of the endogenous enzyme was localized by immunocytofluorescence on the cultured muscle cells. Our results favor a localization of bound cytosolic creatine phosphokinase on the M-line and a diffuse distribution in all myotubes.

  10. In silico-based identification of human α-enolase inhibitors to block cancer cell growth metabolically

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    Lung J

    2017-11-01

    Full Text Available Jrhau Lung,1 Kuan-Liang Chen,2 Chien-Hui Hung,3 Chih-Cheng Chen,4,5 Ming-Szu Hung,5–7 Yu-Ching Lin,5–7 Ching-Yuan Wu,8 Kuan-Der Lee,9 Neng-Yao Shih,10 Ying Huang Tsai11,12 1Department of Medical Research and Development, Chang Gung Memorial Hospital, Chiayi, 2Department of Endodontics, ChiMei Medical Center, Tainan, 3Graduate Institute of Clinical Medical Sciences, Chang Gung University, Taoyuan, 4Division of Hematology and Oncology, Chang Gung Memorial Hospital, Chiayi, 5Department of Medicine, Chang Gung University, Taoyuan, 6Department of Respiratory Care, Chang Gung University of Science and Technology, Chiayi, 7Division of Thoracic Oncology, Department of Pulmonary and Critical Care Medicine, 8Department of Chinese Medicine; Chang Gung Memorial Hospital, Chiayi, 9Department of Hematology and Oncology, Taipei Medical University Hospital, Taipei, 10National Institute of Cancer Research, National Health Research Institutes, Tainan, 11Department of Respiratory Care, College of Medicine, Chang Gung University, Taoyuan, 12Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Kaohsiung, Taiwan Abstract: Unlimited growth of cancer cells requires an extensive nutrient supply. To meet this demand, cancer cells drastically upregulate glucose uptake and metabolism compared to normal cells. This difference has made the blocking of glycolysis a fascinating strategy to treat this malignant disease. α-enolase is not only one of the most upregulated glycolytic enzymes in cancer cells, but also associates with many cellular processes or conditions important to cancer cell survival, such as cell migration, invasion, and hypoxia. Targeting α-enolase could simultaneously disturb cancer cells in multiple ways and, therefore, is a good target for anticancer drug development. In the current study, more than 22 million chemical structures meeting the criteria of Lipinski

  11. The level of neuron-specific enolase and S-100 protein in the cerebrospinal fluid of patients with acute bacterial meningitis

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    A. V. Sokhan

    2016-08-01

    Full Text Available Aim. To evaluate the diagnostic and prognostic role of neuron-specific enolase (NSE and S-100 protein levels in cerebrospinal fluid (CSF of patients with acute bacterial meningitis in the course of the disease. Materials and Methods. 54 cases of acute bacterial meningitis were analyzed, among them – 26 with pneumococcal and 28 with meningococcal etiology. Patients were divided into groups depending on the severity and etiology of disease. In addition to routine laboratory methods, we analyzed the CSF levels of S-100 protein and NSE at admission and after 10 – 12 days of treatment. 12 patients with acute respiratory infections and meningism were examined as a comparison group. Results. In all patients with acute bacterial meningitis CSF NSE and protein S-100 levels were significantly higher than in the control group (P <0,05. CSF neuro specific proteins level was in direct dependence on severity of the disease, and in patients with severe disease was significantly higher than in patients with moderate severity and in the control group (P <0,01. After 10 – 12 days of treatment, the level of the NSE and S-100 protein decreased, but in severe cases was still higher than in the control group (P <0,05. Conclusions. Increased cerebrospinal fluid NSE and S – 100 protein levels shows the presence and value of neurons and glial cells damage in patients with acute bacterial meningitis. CSF S-100 protein and neuron-specific enolase levels help to determine the severity of neurons destruction and glial cells in patients with acute bacterial meningitis. Level of neurospecific protein is in direct proportion to the severity of the disease and is the highest in patients with severe cases (P<0,05. It confirms the diagnostic and prognostic value of CSF neurospecific protein determination in patients with bacterial meningitis.

  12. Acute sleep deprivation increases serum levels of neuron-specific enolase (NSE) and S100 calcium binding protein B (S-100B) in healthy young men.

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    Benedict, Christian; Cedernaes, Jonathan; Giedraitis, Vilmantas; Nilsson, Emil K; Hogenkamp, Pleunie S; Vågesjö, Evelina; Massena, Sara; Pettersson, Ulrika; Christoffersson, Gustaf; Phillipson, Mia; Broman, Jan-Erik; Lannfelt, Lars; Zetterberg, Henrik; Schiöth, Helgi B

    2014-01-01

    To investigate whether total sleep deprivation (TSD) affects circulating concentrations of neuron-specific enolase (NSE) and S100 calcium binding protein B (S-100B) in humans. These factors are usually found in the cytoplasm of neurons and glia cells. Increasing concentrations of these factors in blood may be therefore indicative for either neuronal damage, impaired blood brain barrier function, or both. In addition, amyloid β (Aβ) peptides 1-42 and 1-40 were measured in plasma to calculate their ratio. A reduced plasma ratio of Aβ peptides 1-42 to 1-40 is considered an indirect measure of increased deposition of Aβ 1-42 peptide in the brain. Subjects participated in two conditions (including either 8-h of nocturnal sleep [22:30-06:30] or TSD). Fasting blood samples were drawn before and after sleep interventions (19:30 and 07:30, respectively). Sleep laboratory. 15 healthy young men. TSD increased morning serum levels of NSE (P = 0.002) and S-100B (P = 0.02) by approximately 20%, compared with values obtained after a night of sleep. In contrast, the ratio of Aβ peptides 1-42 to 1-40 did not differ between the sleep interventions. Future studies in which both serum and cerebrospinal fluid are sampled after sleep loss should elucidate whether the increase in serum neuron-specific enolase and S100 calcium binding protein B is primarily caused by neuronal damage, impaired blood brain barrier function, or is just a consequence of increased gene expression in non-neuronal cells, such as leukocytes.

  13. Functional and Structural Characterization of a Novel HLA-DRB1*04:01-Restricted α-Enolase T Cell Epitope in Rheumatoid Arthritis

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    Christina Gerstner

    2016-11-01

    Full Text Available Antibodies to citrullinated proteins, common in rheumatoid arthritis (RA patients, are strongly associated to a specific set of HLA-DR alleles including HLA-DRB1*04:01, *04:04, and *01:01. Here, we first demonstrate that autoantibody levels toward the dominant citrullinated B cell epitope from α-enolase are significantly elevated in HLA-DRB1*04:01-positive RA patients. Furthermore, we identified α-enolase-derived T cell epitopes and demonstrated that native and citrullinated versions of several peptides bind with different affinities to HLA-DRB1*04:01, *04:04, and *01:01. The citrulline residues in the eight identified peptides are distributed throughout the entire length of the presented epitopes and more specifically, localized at peptide positions p-2, p2, p4, p6, p7, p10, and p11. Importantly, in contrast to its native version peptide 26 (TSKGLFRAAVPSGAS, the HLA-DRB1*04:01-restricted citrullinated peptide Cit26 (TSKGLFCitAAVPSGAS elicited significant functional T cell responses in primary cells from RA patients. Comparative analysis of the crystal structures of HLA-DRB1*04:01 in complex with peptide 26 or Cit26 demonstrated that the posttranslational modification did not alter the conformation of the peptide. And since citrullination is the only structural difference between the two complexes, this indicates that the neo-antigen Cit26 is recognized by T cells with high specificity to the citrulline residue.

  14. Early detection of response in small cell bronchogenic carcinoma by changes in serum concentrations of creatine kinase, neuron specific enolase, calcitonin, ACTH, serotonin and gastrin releasing peptide

    DEFF Research Database (Denmark)

    Bork, E; Hansen, M; Urdal, P

    1988-01-01

    Creatine kinase (CK-BB), neuron specific enolase (NSE), ACTH, calcitonin, serotonin and gastrin releasing peptide (GRP) were measured in serum or plasma before and immediately after initiation of treatment in patients with small cell lung cancer (SCC). Pretherapeutic elevated concentrations of CK...

  15. Research of the serum level of neuron-specific enolase in children with various types of seizure

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    WANG Chun

    2012-10-01

    Full Text Available Objective To explore the relevance between the level changes of serum neuron-specific enolase (NSE and neuronal damage in various seizure types of children with epilepsy. Methods According to the classification criteria of seizure types formulated by International League Against Epilepsy (ILAE in 1981, 190 children with epilepsy were enrolled including tonic-clonic seizure group (41 cases, tonic seizure group (34 cases, clonic seizure group (22 cases, myoclonic seizure group (12 cases, atonic seizure group (17 cases, absence seizure group (22 cases, simple partial seizure group (21 cases and complex partial seizure group (21 cases, and 64 healthy children were enrolled as control group. The long-range vedio-electroencephalogram (VEEG was operated and the blood samples were collected from these cases within 72 h after their seizures. Results The serum NSE levels of epileptic children were significantly higher than control group (P = 0.000. Among these seizure groups, serum NSE in myoclonic seizure group [(32.42 ± 6.62 ng/ml] was significantly higher than the other types, except for tonic-clonic seizure group (P = 0.062. There was no significant difference among the other types (P > 0.05, for all. According to rank correlation analysis, there was positive corrlation between serum NSE levels and VEEG abnormal intensity (rs = 0.613, P = 0.000. Conclusion The serum NSE were markedly increased in children with epilepsy after seizures, suggesting that a certain degree of neuronal damage may result from seizures; the higher NSE levels were, the more serious neuronal damage caused by epileptiform discharges was. The serum NSE levels in myoclonic seizure group and tonic-clonic seizure group were significantly higher than other seizure types, indicating the two kinds of seizures may result in greater neuronal damage.

  16. Enolase 1 (ENO1 and protein disulfide-isomerase associated 3 (PDIA3 regulate Wnt/β-catenin-driven trans-differentiation of murine alveolar epithelial cells

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    Kathrin Mutze

    2015-08-01

    Full Text Available The alveolar epithelium represents a major site of tissue destruction during lung injury. It consists of alveolar epithelial type I (ATI and type II (ATII cells. ATII cells are capable of self-renewal and exert progenitor function for ATI cells upon alveolar epithelial injury. Cell differentiation pathways enabling this plasticity and allowing for proper repair, however, are poorly understood. Here, we applied proteomics, expression analysis and functional studies in primary murine ATII cells to identify proteins and molecular mechanisms involved in alveolar epithelial plasticity. Mass spectrometry of cultured ATII cells revealed a reduction of carbonyl reductase 2 (CBR2 and an increase in enolase 1 (ENO1 and protein disulfide-isomerase associated 3 (PDIA3 protein expression during ATII-to-ATI cell trans-differentiation. This was accompanied by increased Wnt/β-catenin signaling, as analyzed by qRT-PCR and immunoblotting. Notably, ENO1 and PDIA3, along with T1α (podoplanin; an ATI cell marker, exhibited decreased protein expression upon pharmacological and molecular Wnt/β-catenin inhibition in cultured ATII cells, whereas CBR2 levels were stabilized. Moreover, we analyzed primary ATII cells from mice with bleomycin-induced lung injury, a model exhibiting activated Wnt/β-catenin signaling in vivo. We observed reduced CBR2 significantly correlating with surfactant protein C (SFTPC, whereas ENO1 and PDIA3 along with T1α were increased in injured ATII cells. Finally, siRNA-mediated knockdown of ENO1, as well as PDIA3, in primary ATII cells led to reduced T1α expression, indicating diminished cell trans-differentiation. Our data thus identified proteins involved in ATII-to-ATI cell trans-differentiation and suggest a Wnt/β-catenin-driven functional role of ENO1 and PDIA3 in alveolar epithelial cell plasticity in lung injury and repair.

  17. The Saccharomyces cerevisiae enolase-related regions encode proteins that are active enolases.

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    Kornblatt, M J; Richard Albert, J; Mattie, S; Zakaib, J; Dayanandan, S; Hanic-Joyce, P J; Joyce, P B M

    2013-02-01

    In addition to two genes (ENO1 and ENO2) known to code for enolase (EC4.2.1.11), the Saccharomyces cerevisiae genome contains three enolase-related regions (ERR1, ERR2 and ERR3) which could potentially encode proteins with enolase function. Here, we show that products of these genes (Err2p and Err3p) have secondary and quaternary structures similar to those of yeast enolase (Eno1p). In addition, Err2p and Err3p can convert 2-phosphoglycerate to phosphoenolpyruvate, with kinetic parameters similar to those of Eno1p, suggesting that these proteins could function as enolases in vivo. To address this possibility, we overexpressed the ERR2 and ERR3 genes individually in a double-null yeast strain lacking ENO1 and ENO2, and showed that either ERR2 or ERR3 could complement the growth defect in this strain when cells are grown in medium with glucose as the carbon source. Taken together, these data suggest that the ERR genes in Saccharomyces cerevisiae encode a protein that could function in glycolysis as enolase. The presence of these enolase-related regions in Saccharomyces cerevisiae and their absence in other related yeasts suggests that these genes may play some unique role in Saccharomyces cerevisiae. Further experiments will be required to determine whether these functions are related to glycolysis or other cellular processes. Copyright © 2012 John Wiley & Sons, Ltd.

  18. Early detection of response in small cell bronchogenic carcinoma by changes in serum concentrations of creatine kinase, neuron specific enolase, calcitonin, ACTH, serotonin and gastrin releasing peptide

    DEFF Research Database (Denmark)

    Bork, E; Hansen, M; Urdal, P

    1988-01-01

    determined within 4-8 weeks. The results indicate that serum CK-BB and NSE are potential markers for SCC at the time of diagnosis and that changes in the concentrations during the first course of cytostatic therapy are promising as biochemical tests for early detection of response to chemotherapy.......Creatine kinase (CK-BB), neuron specific enolase (NSE), ACTH, calcitonin, serotonin and gastrin releasing peptide (GRP) were measured in serum or plasma before and immediately after initiation of treatment in patients with small cell lung cancer (SCC). Pretherapeutic elevated concentrations of CK...

  19. Carbon Partitioning in Green Algae (Chlorophyta and the Enolase Enzyme

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    Jürgen E. W. Polle

    2014-08-01

    Full Text Available The exact mechanisms underlying the distribution of fixed carbon within photoautotrophic cells, also referred to as carbon partitioning, and the subcellular localization of many enzymes involved in carbon metabolism are still unknown. In contrast to the majority of investigated green algae, higher plants have multiple isoforms of the glycolytic enolase enzyme, which are differentially regulated in higher plants. Here we report on the number of gene copies coding for the enolase in several genomes of species spanning the major classes of green algae. Our genomic analysis of several green algae revealed the presence of only one gene coding for a glycolytic enolase [EC 4.2.1.11]. Our predicted cytosolic localization would require export of organic carbon from the plastid to provide substrate for the enolase and subsequent re-import of organic carbon back into the plastids. Further, our comparative sequence study of the enolase and its 3D-structure prediction may suggest that the N-terminal extension found in green algal enolases could be involved in regulation of the enolase activity. In summary, we propose that the enolase represents one of the crucial regulatory bottlenecks in carbon partitioning in green algae.

  20. Surface-expressed enolases of Plasmodium and other pathogens

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    Anil Kumar Ghosh

    2011-08-01

    Full Text Available Enolase is the eighth enzyme in the glycolytic pathway, a reaction that generates ATP from phosphoenol pyruvate in cytosolic compartments. Enolase is essential, especially for organisms devoid of the Krebs cycle that depend solely on glycolysis for energy. Interestingly, enolase appears to serve a separate function in some organisms, in that it is also exported to the cell surface via a poorly understood mechanism. In these organisms, surface enolase assists in the invasion of their host cells by binding plasminogen, an abundant plasma protease precursor. Binding is mediated by the interaction between a lysine motif of enolase with Kringle domains of plasminogen. The bound plasminogen is then cleaved by specific proteases to generate active plasmin. Plasmin is a potent serine protease that is thought to function in the degradation of the extracellular matrix surrounding the targeted host cell, thereby facilitating pathogen invasion. Recent work revealed that the malaria parasite Plasmodium also expresses surface enolase, and that this feature may be essential for completion of its life cycle. The therapeutic potential of targeting surface enolases of pathogens is discussed.

  1. Determination of serum neuron specific enolase and glutathion S transferases levels in patients with acute cerebral infarction and its clinical significance

    International Nuclear Information System (INIS)

    Guo Jianyi; Lu Tianhe; Bao Yanmei

    2002-01-01

    Objective: To evaluate the variation of serum neuron specific enolase (NSE) and glutathion S transferases (GST) levels in patients with cerebral infarction and its clinical significance. Methods: The serum levels of NSE in cerebral infarction patients were determined with immunoradiometric assay (IRMA), and the serum level of GST were determined by enzyme immuno sandwich assay (ELISA). Results: Serum NSE levels linked in patients were significantly higher (p<0.01) and GST serum levels were significantly lower (p < 0.01) within 3 days after onset of disease than those at two weeks and those in the controls. There was a positive correlation between serum NSE levels and neurological deficit scores (p < 0.001) and a negative correlation with serum GST levels (p < 0.05). There was also a close relationship between the serum NSE levels and the volume of infarction (p < 0.001). Conclusion: There was a close relationship between the Serum levels of NSE, GST and clinical features of Patients in the early stage of cerebral infarction

  2. rEnolase maternal immunization confers caries protection on offspring.

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    Dinis, M; Trigo, G; Chaves, N; Fonseca, A J M M; Ribeiro, A; Tavares, D; Cabrita, A M S; Ferreira, P

    2011-03-01

    Therapeutic vaccination with Streptococcus sobrinus recombinant enolase (rEnolase) protects rats from dental caries. Here, we investigated the effect that maternal rEnolase vaccination before pregnancy had on the offspring's immune response to S. sobrinus oral infection and dental caries progression. Female Wistar rats were immunized by intranasal and subcutaneous routes with rEnolase adsorbed onto aluminum hydroxide as adjuvant or similarly treated with the adjuvant alone (sham-immunized). Ten days after the last administration, the immunized females were paired with a male rat. The oral immune responses to S. sobrinus infection and dental caries in the offspring were evaluated. The results showed that pups born from rEnolase-immunized mothers had higher levels of rEnolase-specific salivary IgA and IgG antibodies (indicating a placental antibody transfer) and lower sulcal and proximal enamel caries scores than rats born from sham-immunized mothers. In conclusion, rEnolase maternal immunization before pregnancy provides offspring with protection against S. sobrinus-induced dental caries.

  3. Pseudomonas aeruginosa enolase influences bacterial tolerance to oxidative stresses and virulence

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    Yuding Weng

    2016-12-01

    Full Text Available Pseudomonas aeruginosa is a Gram negative opportunistic pathogenic bacterium, which causes acute and chronic infections. Upon entering host, bacteria alter global gene expression to adapt to host environment and avoid clearance by host. Enolase is a glycolytic enzyme involved in carbon metabolism. It is also a component of RNA degradosome, which is involved in RNA processing and gene regulation. Here we report that enolase is required for the pathogenesis of P. aeruginosa in a murine acute pneumonia model. Mutation of enolase coding gene (eno increased bacterial susceptibility to neutrophil mediated killing, which is due to reduced tolerance to oxidative stress. Catalases and alkyl hydroperoxide reductases play a major role in protecting the cell from oxidative damages. In the eno mutant, the expression levels of catalases (KatA, KatB were similar as those in the wild type strain in the presence of H2O2, however, the expression levels of alkyl hydroperoxide reductases (AhpB, AhpC were significantly reduced. Overexpression of ahpB but not ahpC in the eno mutant fully restored the bacterial resistance to H2O2 as well as neutrophil mediated killing, and partially restored bacterial virulence in the murine acute pneumonia model. Therefore, we have identified a novel role of enolase in the virulence of P. aeruginosa.

  4. Denervation of rabbit gastrocnemius and soleus muscles: effect on muscle-specific enolase.

    Science.gov (United States)

    Nozais, M; Merkulova, T; Keller, A; Janmot, C; Lompré, A M; D'Albis, A; Lucas, M

    1999-07-01

    We report here, for the first time, the expression of the muscle-specific isoform of the glycolytic enzyme, enolase (EC 4.2.1. 11) (beta enolase), in rabbit skeletal muscles. We have analysed the fast-twitch gastrocnemius and the slow-twitch soleus muscles during normal postnatal development and following denervation. We show that, in rabbit, as already described in rodents, beta enolase gene expression behaves as a good marker of the fast-twitch fibers. In soleus muscle, the beta enolase transcript level is 10-20% of that found in gastrocnemius. Denervation, performed at 8 postnatal days, induces an important drop of beta enolase transcript levels in both developing soleus and gastrocnemius muscles, with a 80% decrease observed 1 week after denervation in the operated muscles, as compared to the corresponding contralateral muscles. Thereafter, the beta enolase transcript level continues to decrease in the fast-twitch muscle, with the beta enolase subunit being detectable only in the atrophic fast-twitch fibers. In contrast, the beta transcript level tends to increase in the denervated slow-twitch muscle, reaching about 50% of that in contralateral soleus, at 7 weeks after surgery. The level of beta enolase transcripts still expressed after denervation seems to stabilize at the same low level in both types of inactive muscles. This suggests that the small fraction of beta enolase expression which is not controlled by the nerve, or by the contractile activity imposed by it, is independent of the muscle phenotype.

  5. Metal ion effects on enolase activity

    International Nuclear Information System (INIS)

    Lee, M.E.; Nowak, T.

    1986-01-01

    Most metal binding studies with yeast enolase suggest that two metals per monomer are required for catalytic activity. The functions of metal I and metal II have not been unequivocally defined. In a series of kinetic experiments where the concentration of MgII is kept constant at subsaturating levels (1mM), the addition of MnII or of ZnII gives a hyperbolic decrease in activity. The final velocity of these mixed metal systems is the same velocity obtained with either only MnII or ZnII respectively. The concentration of MnII (40 μM) or of Zn (2μM) which gives half maximal effect in the presence of (1mM) MgII is approximately the same as the Km' value for MnII (9μM) or ZnII (3μM) respectively. Direct binding of MnII to enolase in the absence and presence of MgII shows that MnII and MgII compete for the same metal site on enolase. In the presence of 2-phosphoglycerate (2-PGA) and MgII, only a single site is occupied by MnII. Results suggest MnII at site I and MgII at site II. PRR and high resolution 1 H and 31 P NMR studies of enzyme-ligand complexes containing MnII and MgII and MnII are consistent with this model. 31 P measurements allow a measure of the equilibrium constant (0.36) for enolase. Saturation transfer measurements yield net rate constants (k/sub f/ = 0.49s -1 ; k/sub r/ = 1.3s -1 ) for the overall reaction. These values are smaller than k/sub cat/ (38s -1 ) measured under analogous conditions. The cation at site I appears to determine catalytic activity

  6. Co-purification of arrestin like proteins with alpha-enolase from bovine myocardial tissues and the possible role in heart diseases as an autoantigen

    Energy Technology Data Exchange (ETDEWEB)

    Mirshahi, M., E-mail: massoud.mirshahi@inserm.fr; Le Marchand, S.

    2015-05-08

    Aim: Previously, we reported that visual arrestin co-purified with glycolytic enzymes. The aim of this study was to analyze the co-purification of arrestin like proteins (ALP) in bovine cardiac tissues with enolases. Methods: The soluble extract of bovine myocardial tissues from different regions such as left and right atriums and ventricles of the bovine heart (n = 3) was analyzed by ACA-34 gel filtration, immuno-affinity column, SDS-PAGE, ELISA, western blot and a sandwich immune assay for quantification of ALP and sequence analysis. Results: We observed that; 1) The cardiac muscle contained a 50 kDa ALP at a concentration of 751 pg/mg of soluble protein extract, 2) ALP purified, by immunoaffinity, contained alpha-enolase of 48 kDa confirmed by protein sequence analysis; 3) Cardiomyocyte cells exposed to anti arrestin and anti enolase monoclonal antibodies showed decreased proliferation in vitro, 4) High level of autoantibodies were detected by ELISA (3.57% for arrestin and 9.12% for α-enolase) in serum of patients with infarcted heart disease. Conclusion: We suggest a possible interaction between ALP and alpha-enolases yielding a complex that may be involved in the induction of cardiac autoimmune diseases. - Highlights: • We examine a possible interaction between arrestin like protein and alpha-enolases in cardiomyocyte. • We demonstrated the effect of antibodies against arrestin and enolase on cardiomyocyte cell proliferation. • We suggest that this proteins complex may be involved in the induction of cardiac autoimmune diseases.

  7. Phosphorylated alpha-enolase induces autoantibodies in HLA-DR8 pancreatic cancer patients and triggers HLA-DR8 restricted T-cell activation.

    Science.gov (United States)

    Capello, Michela; Caorsi, Cristiana; Bogantes Hernandez, Pablo Josè; Dametto, Ennia; Bertinetto, Francesca Eleonora; Magistroni, Paola; Rendine, Sabina; Amoroso, Antonio; Novelli, Francesco

    2015-09-01

    Pancreatic ductal adenocarcinoma (PDAC) is the fourth cause of cancer-induced death in the Western World. In PDAC patients, alpha-enolase (ENOA), a glycolytic enzyme that also acts as plasminogen receptor, is up-regulated and elicits the production of autoantibodies. Our previous studies revealed that most PDAC patients specifically produce antibodies to Serine(419)phosphorylated ENOA (Ser(419)P-ENOA) isoforms (ENOA1,2), and that this humoral response correlates with a better clinical outcome. Since autoantibody production can be influenced by HLA polymorphisms, and the ENOA sequence presents multiple peptides predicted to preferentially bind HLA-DR molecules, including the peptide containing Ser(419), we hypothesized that the presence of autoantibodies against ENOA1,2 is associated with specific HLA-DRB1 alleles. Here, we demonstrate that the HLA-DRB1*08 allele is significantly more frequent in PDAC patients with autoantibodies to ENOA1,2 (ENOA1,2(+), 8%) compared to healthy controls (3%, p=0.0112). We observed that a Ser(419)P-ENOA peptide, bioinformatically predicted to bind with high affinity to the HLA-DR8 allele coded by HLA-DRB1*08:01 or *08:04 alleles, was able to activate specific CD4(+) T cell clones derived from a HLA-DRB1*08:01. Thus complexes of the Ser(419)P-ENOA peptide with the HLA that trigger T-cell signaling might be relevant for induction of anti-tumor immune response. Copyright © 2015 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  8. Oral therapeutic vaccination with Streptococcus sobrinus recombinant enolase confers protection against dental caries in rats.

    Science.gov (United States)

    Dinis, Márcia; Tavares, Delfina; Veiga-Malta, Isabel; Fonseca, António J M M; Andrade, Elva Bonifácio; Trigo, Gabriela; Ribeiro, Adília; Videira, Arnaldo; Cabrita, António M Silvério; Ferreira, Paula

    2009-01-01

    Dental caries is among the more prevalent chronic human infections for which an effective human vaccine has not yet been achieved. Enolase from Streptococcus sobrinus has been identified as an immunomodulatory protein. In the present study, we used S. sobrinus recombinant enolase (rEnolase) as a target antigen and assessed its therapeutic effect in a rat model of dental caries. Wistar rats that were fed a cariogenic solid diet on day 18 after birth were orally infected with S. sobrinus on day 19 after birth and for 5 consecutive days thereafter. Five days after infection and, again, 3 weeks later, rEnolase plus alum adjuvant was delivered into the oral cavity of the rats. A sham-immunized group of rats was contemporarily treated with adjuvant alone. In the rEnolase-immunized rats, increased levels of salivary IgA and IgG antibodies specific for this recombinant protein were detected. A significant decrease in sulcal, proximal enamel, and dentin caries scores was observed in these animals, compared with sham-immunized control animals. No detectable histopathologic alterations were observed in all immunized animals. Furthermore, the antibodies produced against bacterial enolase did not react with human enolase. Overall, these results indicate that rEnolase could be a promising and safe candidate for testing in trials of vaccines against dental caries in humans.

  9. Crystal structure of enolase from Drosophila melanogaster.

    Science.gov (United States)

    Sun, Congcong; Xu, Baokui; Liu, Xueyan; Zhang, Zhen; Su, Zhongliang

    2017-04-01

    Enolase is an important enzyme in glycolysis and various biological processes. Its dysfunction is closely associated with diseases. Here, the enolase from Drosophila melanogaster (DmENO) was purified and crystallized. A crystal of DmENO diffracted to 2.0 Å resolution and belonged to space group R32. The structure was solved by molecular replacement. Like most enolases, DmENO forms a homodimer with conserved residues in the dimer interface. DmENO possesses an open conformation in this structure and contains conserved elements for catalytic activity. This work provides a structural basis for further functional and evolutionary studies of enolase.

  10. The multifaceted roles of Leptospira enolase.

    Science.gov (United States)

    Salazar, Natália; Souza, Matilde Costa Lima de; Biasioli, Amanda Gameiro; Silva, Ludmila Bezerra da; Barbosa, Angela Silva

    A previous study had demonstrated that Leptospira enolase is secreted extracellularly by a yet unknown mechanism and reassociates with the bacterial membrane. Surface-anchored leptospiral enolase displays plasminogen binding activity. In this work, we explored the consequences of this interaction and also assessed whether Leptospira enolase might display additional moonlighting functions by interacting with other host effector proteins. We first demonstrated that enolase-bound plasminogen is converted to its active form, plasmin. The protease plasmin targets human fibrinogen and vitronectin, but not the complement proteins C3b and C5. Leptospira enolase also acts as an immune evasion protein by interacting with the negative complement regulators C4b binding protein and factor H. Once bound to enolase, both regulators remain functional as cofactors of factor I, mediating cleavage of C4b and C3b. In conclusion, enolase may facilitate leptospiral survival and dissemination, thus contributing to bacterial virulence. The identification and characterization of moonlighting proteins is a growing field of bacterial pathogenesis, as these multifaceted proteins may represent potential future therapeutic targets to fight bacterial infections. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  11. Naegleria fowleri: enolase is expressed during cyst differentiation.

    Science.gov (United States)

    Chávez-Munguía, Bibiana; Segovia-Gamboa, Norma; Salazar-Villatoro, Lizbeth; Omaña-Molina, Maritza; Espinosa-Cantellano, Martha; Martínez-Palomo, Adolfo

    2011-01-01

    Cysts of Naegleria fowleri present an external single-layered cyst wall. To date, little information exists on the biochemical components of this cyst wall. Knowledge of the cyst wall composition is important to understand its resistance capacity under adverse environmental conditions. We have used of a monoclonal antibody (B4F2 mAb) that specifically recognizes enolase in the cyst wall of Entamoeba invadens. By Western blot assays this antibody recognized in soluble extracts of N. fowleri cysts a 48-kDa protein with similar molecular weight to the enolase reported in E. invadens cysts. Immunofluorescence with the B4F2 mAb revealed positive cytoplasmic vesicles in encysting amebas, as well as a positive reaction at the cell wall of mature cysts. Immunoelectron microscopy using the same monoclonal antibody confirmed the presence of enolase in the cell wall of N. fowleri cysts and in cytoplasmic vesicular structures. In addition, the B4F2 mAb had a clear inhibitory effect on encystation of N. fowleri. © 2011 The Author(s). Journal of Eukaryotic Microbiology © 2011 International Society of Protistologists.

  12. Schistosomes Enhance Plasminogen Activation: The Role of Tegumental Enolase.

    Directory of Open Access Journals (Sweden)

    Barbara C Figueiredo

    2015-12-01

    Full Text Available Schistosoma mansoni is a blood fluke parasite that causes schistosomiasis, a debilitating disease of global public health importance. These relatively large parasites are able to survive prolonged periods in the human vasculature without inducing stable blood clots around them. We show here that the intravascular life stages (schistosomula and adult males and females can all promote significant plasminogen (PLMG activation in the presence of tissue plasminogen activator (tPA. This results in the generation of the potent fibrinolytic agent plasmin which could degrade blood clots forming around the worms in vivo. We demonstrate that S. mansoni enolase (SmEno is a host-interactive tegumental enzyme that, in recombinant form, can bind PLMG and promote its activation. Like classical members of the enolase protein family, SmEno can catalyze the interconversion of 2-phospho-D-glycerate (2-PGA and phosphoenolpyruvate (PEP. The enzyme has maximal activity at pH 7.5, requires Mg2+ for optimal activity and can be inhibited by NaF but not mefloquin. Suppressing expression of the SmEno gene significantly diminishes enolase mRNA levels, protein levels and surface enzyme activity but, surprisingly, does not affect the ability of the worms to promote PLMG activation. Thus, while SmEno can enhance PLMG activation, our analysis suggests that it is not the only contributor to the parasite's ability to perform this function. We show that the worms possess several other PLMG-binding proteins in addition to SmEno and these may have a greater importance in schistosome-driven PLMG activation.

  13. NEUROSPECIFIC ENOLASE IN DIAGNOSTICS FOR PERINATAL DAMAGE TO THE CENTRAL NERVOUS SYSTEM IN PREMATURE INFANTS

    Directory of Open Access Journals (Sweden)

    E.G. Novopol'tseva

    2010-01-01

    Full Text Available Neurospecific enolase is an endoenzyme of the central nervous system (CNS present in neurons of the brain and peripheral neuraltissue. This is currently the only known general marker of all differentiated neurons. The article illustrates the results of determining this enzyme in premature infants with fetal infections and assessment of their importance as a marker of damage to CNS in this group of children. A high level of neurospecific enolase in children with infectious and inflammatory diseases is not only the marker of damage to blood-brain barrier, but also reflects the nature of damage (hypoxia, intoxication, inflammation. This parameter in premature infants with various pathologies may serve as a degree of perinatal damage severity, and along with other parameters, determine the performed therapy tactics. Key words: neurospecific enolase, marker of CNS damage, perinatal damage, children. (Pediatric Pharmacology. – 2010; 7(3:66-70

  14. Role of Caveolin 1, E-Cadherin, Enolase 2 and PKCalpha on resistance to methotrexate in human HT29 colon cancer cells

    DEFF Research Database (Denmark)

    Selga, Elisabet; Morales Torres, Christina; Noé, Véronique

    2008-01-01

    ABSTRACT: BACKGROUND: Methotrexate is one of the earliest cytotoxic drugs used in cancer therapy, and despite the isolation of multiple other folate antagonists, methotrexate maintains its significant role as a treatment for different types of cancer and other disorders. The usefulness of treatment...... validations were performed either by siRNAs or by transfection of an expression plasmid. RESULTS: Genes adjacent to the dhfr locus and included in the 5q14 amplicon were overexpressed in HT29 MTX-resistant cells. Treatment with siRNAs against those genes caused a slight reduction in cell viability in both HT...... viability and caused a decreased MTX resistance capacity. Moreover, overexpression of E-cadherin, which was found underexpressed in MTX-resistant cells, also sensitized the cells toward the chemotherapeutic agent. Combined treatments targeting siRNA inhibition of caveolin 1 and overexpression of E...

  15. Oral Immunization Against Candidiasis Using Lactobacillus casei Displaying Enolase 1 from Candida albicans

    OpenAIRE

    Shibasaki, Seiji; Karasaki, Miki; Tafuku, Senji; Aoki, Wataru; Sewaki, Tomomitsu; Ueda, Mitsuyoshi

    2014-01-01

    Abstract Candidiasis is a common fungal infection that is prevalent in immunocompromised individuals. In this study, an oral vaccine against Candida albicans was developed by using the molecular display approach. Enolase 1 protein (Eno1p) of C. albicans was expressed on the Lactobacillus casei cell surface by using poly-gamma-glutamic acid synthetase complex A from Bacillus subtilis as an anchoring protein. The Eno1p-displaying L. casei cells were used to immunize mice, which were later chall...

  16. Association of levels of antibodies against citrullinated cyclic peptides and citrullinated α-enolase in chronic and aggressive periodontitis as a risk factor of Rheumatoid arthritis: a case control study.

    Science.gov (United States)

    Reichert, Stefan; Schlumberger, Wolfgang; Dähnrich, Cornelia; Hornig, Nora; Altermann, Wolfgang; Schaller, Hans-Günter; Schulz, Susanne

    2015-08-29

    Periodontal disease could be a risk factor for rheumatoid arthritis (RA). It is assumed that the bacterial strain Porphyromonas gingivalis mediates citrullination of host peptides and thereby the generation of RA-associated autoantibodies in genetically predisposed individuals. For that reason non-RA individuals who suffered from generalized aggressive (GAgP, N = 51) and generalized chronic periodontitis (GChP, N = 50) were investigated regarding the occurrence of antibodies against citrullinated cyclic peptides (anti-CCP) and citrullinated α-enolase peptide-1 (anti-CEP-1) in comparison to non-RA non-periodontitis controls (N = 89). Furthermore, putative associations between infections with five periodontopathic bacteria or expression of certain human leucocyte antigens (HLA) to these autoantibodies were investigated. The presence of anti-CCP and anti-CEP-1 in plasma samples was conducted with enzyme linked immunosorbent assay. Subgingival plaque specimens were taken from the deepest pocket of each quadrant and pooled. For detection of DNA of five periodontopathic bacteria PCR with sequence specific oligonucleotides was carried out. Low resolution HLA typing was carried out with PCR with sequence specific primers. Differences between patients and controls were assessed using Chi square test with Yates correction or Fisher`s exact test if the expected number n in one group was periodontitis and RA should be investigated in further studies.

  17. Mapping of antigenic sites in human neuron-specific enolase by expression subcloning.

    Science.gov (United States)

    Quinn, G B; Reeves, I G; Day, I N

    1994-05-01

    Human serum neuron-specific enolase (NSE) is a marker of neurons and of small-cell carcinoma of the lung; improved immunoassays of NSE remain an important goal. Here, we used overlapping complementary DNA (cDNA) clones for reconstruction to express full-length recombinant NSE, and also to express a set of cloned subfragments through the prokaryotic expression vectors pUEX and pUBEX. Subfragments expressed as fusion proteins were used to characterize immunogenic and antigenic regions and epitopes and, expressed as affinity matrices, to derive purified, fractionated polyclonal antibodies. NSE epitope data can be visualized with yeast enolase-1 crystal structure coordinates: The two protein sequences align almost perfectly and are 61% identical. This approach demonstrates the complementarity of cDNA expression with techniques of polyclonal antiserum and monoclonal antibody production and with chemical peptide synthesis in the refinement of immunodiagnostic reagents.

  18. [Blood neuronal specific enolase in newborns with perinatal asphyxia].

    Science.gov (United States)

    Verdú Pérez, A; Falero, M P; Arroyos, A; Estévez, F; Félix, V; López, Y; Pantoja, A; Ureta, A

    Neuron-specific enolase (NSE) is a sensitive marker of brain injury after hypoxia or ischemia. There are few studies about its usefulness in asphyctic newborns. To study the correlation between blood NSE levels and neurological outcome in newborns with hypoxic ischemic encephalopathy. We have determined the blood values of NSE by radioimmunoassay in 25 asphyctic term-newborns with clinical encephalopathy (of mild, moderate and severe degree) and in 22 healthy term newborns (control group). Blood samples were obtained between 24 and 72 hours after birth in all neonates. Surviving infants were followed for a variable time (median: 3.5 years; range: 1-6) and the neurological status was determined. NSE levels in the group of asphyctic neonates who died or developed neurological sequelae (n= 6; mean: 116.4 ng/ml; range: 42-226) were significantly higher than NSE values in the group of asphyctic neonates who were neurologically normal at follow-up (n= 19; mean: 21.3 ng/ml; range: 7.4-40), with p< 0.001. There were not differences between NSE values in the group of asphyctic neonates who developed neurologically normal and the control group (mean: 7.6 ng/ml; range: 10.3-28.3). Sensitivity and specificity of blood NSE as predictor of poor outcome were, respectively, 100% and 78%. The combined specificity for blood NSE together with a moderate/severe encephalopathy was 95%. The presence of elevated NSE values in blood after perinatal asphyxia can be a sensitive indicator of conspicuous brain damage. The combined information provided by the severity of the encephalopathy together with the blood NSE values have shown a high predictive value for neurological outcome.

  19. Nanostructured materials detect epidermal growth factor receptor, neuron specific enolase and carcinoembryonic antigen

    Science.gov (United States)

    Stefan-van Staden, Raluca-Ioana; Comnea-Stancu, Ionela Raluca; Surdu-Bob, Carmen Cristina; Badulescu, Marius

    2015-09-01

    New nanostructured materials based on thin films of Cu and Ni deposited on textile material (veil), as well as gold nanostructured microspheres were used for the design of new stochastic sensors. The stochastic sensors were able to detect simultaneously a panel of biomarkers comprising epidermal growth factor receptor, neuron specific enolase, and carcinoembryonic antigen from whole blood samples with high reliabilities - recovery tests higher than 97.00%, with a RSD (%) lower than 0.1%. The stochastic sensors had shown high sensitivities and low determination levels for the detection of the proposed panel of biomarkers making early detection of lung cancer possible by fast screening of whole blood.

  20. S-100b and neuron-specific enolase in patients with fulminant hepatic failure

    DEFF Research Database (Denmark)

    Strauss, Gitte Irene; Christiansen, Michael; Møller, Kirsten

    2001-01-01

    , the cerebral flux of S-100b and NSE was measured. We included 35 patients with FHF, 6 patients with acute on chronic liver disease (AOCLD), 13 patients with cirrhosis of the liver without hepatic encephalopathy, and 8 healthy subjects. Blood samples were obtained from catheters placed in the radial artery......Patients with fulminant hepatic failure (FHF) frequently develop cerebral edema and intracranial hypertension. The aim of this study was to evaluate circulating S-100b and neuron-specific enolase (NSE) levels as markers of neurological outcome in patients with FHF. In a subgroup of patients...

  1. Stability of the Octameric Structure Affects Plasminogen-Binding Capacity of Streptococcal Enolase

    Science.gov (United States)

    Law, Ruby H. P.; Casey, Lachlan W.; Valkov, Eugene; Bertozzi, Carlo; Stamp, Anna; Jovcevski, Blagojce; Aquilina, J. Andrew; Whisstock, James C.; Walker, Mark J.; Kobe, Bostjan

    2015-01-01

    Group A Streptococcus (GAS) is a human pathogen that has the potential to cause invasive disease by binding and activating human plasmin(ogen). Streptococcal surface enolase (SEN) is an octameric α-enolase that is localized at the GAS cell surface. In addition to its glycolytic role inside the cell, SEN functions as a receptor for plasmin(ogen) on the bacterial surface, but the understanding of the molecular basis of plasmin(ogen) binding is limited. In this study, we determined the crystal and solution structures of GAS SEN and characterized the increased plasminogen binding by two SEN mutants. The plasminogen binding ability of SENK312A and SENK362A is ~2- and ~3.4-fold greater than for the wild-type protein. A combination of thermal stability assays, native mass spectrometry and X-ray crystallography approaches shows that increased plasminogen binding ability correlates with decreased stability of the octamer. We propose that decreased stability of the octameric structure facilitates the access of plasmin(ogen) to its binding sites, leading to more efficient plasmin(ogen) binding and activation. PMID:25807546

  2. Stability of the octameric structure affects plasminogen-binding capacity of streptococcal enolase.

    Directory of Open Access Journals (Sweden)

    Amanda J Cork

    Full Text Available Group A Streptococcus (GAS is a human pathogen that has the potential to cause invasive disease by binding and activating human plasmin(ogen. Streptococcal surface enolase (SEN is an octameric α-enolase that is localized at the GAS cell surface. In addition to its glycolytic role inside the cell, SEN functions as a receptor for plasmin(ogen on the bacterial surface, but the understanding of the molecular basis of plasmin(ogen binding is limited. In this study, we determined the crystal and solution structures of GAS SEN and characterized the increased plasminogen binding by two SEN mutants. The plasminogen binding ability of SENK312A and SENK362A is ~2- and ~3.4-fold greater than for the wild-type protein. A combination of thermal stability assays, native mass spectrometry and X-ray crystallography approaches shows that increased plasminogen binding ability correlates with decreased stability of the octamer. We propose that decreased stability of the octameric structure facilitates the access of plasmin(ogen to its binding sites, leading to more efficient plasmin(ogen binding and activation.

  3. Purification, crystallization and preliminary crystallographic study of the putative enolase MJ0232 from the hyperthermophilic archaeon Methanococcus jannaschii.

    Science.gov (United States)

    Yamamoto, Hitoshi; Kunishima, Naoki

    2008-11-01

    Enolase is a glycolytic enzyme that catalyzes the interconversion of phosphoenolpyruvate and 2-phosphoglycerate. In order to gain insight into the biological significance of the oligomeric state of this enzyme, the putative enolase MJ0232 from the hyperthermophilic archaeon Methanococcus jannaschii was cloned, overexpressed and purified. Crystals were obtained by the oil-microbatch method at 291 K using PEG 4000 as a precipitant. A native data set was collected to 1.85 A resolution. The crystal belonged to the tetragonal space group I4, with unit-cell parameters a = 148.8, c = 91.2 A. An initial model was obtained by molecular replacement, which revealed an octameric subunit association (a tetramer of dimers). This result is consistent with that from a dynamic light-scattering experiment, suggesting biological relevance of the octameric state of MJ0232 in solution.

  4. Diagnostic utility of neuron specific enolase (NSE) in serum and pleural fluids from patients with lung cancer and tuberculosis

    International Nuclear Information System (INIS)

    Alam, J.M.; Baig, J.A.; Asghar, S.S.; Mahmood, S.R.; Ansari, M.A.; Jamil, S.

    2010-01-01

    Several past and recent investigations have focused on the determination of tumor markers in pleural fluids to assess their Usefulness as less invasive replacement method of diagnosis. In this regard, few studies have dealt with the determination of the tumor marker, neuron specific enolase (NSE), in pleural fluids of patients suffering from both benign and malignant diseases such as non small cell lung carcinoma( NSCLC), small cell lung carcinoma( SCLC) and tuberculosis. Therefore, the present study was undertaken to establish the diagnostic utility of NSE in malignant condition by assessing levels in serum and pleural fluids of patients with lung cancer and by comparing it with a benign pulmonary disease of tuberculosis. Pleural fluids were obtained from 22 patients with carcinomatous pleurisy due to SCLC, 11 patients with carcinomatous pleurisy due to non-small cell lung cancer, and 30 patients with tuberculosis pleurisy for comparison purpose. Determination of NSE levels was performed by ECL technology according to the manufacturer's instructions. NSE levels of pleural fluids from SCLC patients were significantly elevated( P<0.0001) when compared with pleural fluids from NSCLC and tuberculosis patients. Moreover, pleural fluids of all 30 tuberculosis patients and 11 NSCLC patients showed moderate significance ( P< O.05 and P < 0.01, respectively) when compared with each other. In addition, cumulative results of NSE levels from SCLC and NSCLC combined also showed high significance (P<0.001) as compared to pleural fluids of tuberculosis patients and moderate significance (P<0.01) when compared with serum levels of both malignant and benign groups. It is concluded that determination of NSE levels in pleural fluids of lung cancer patients noted to be an effective diagnostic tool to differentiate carcinomatous pleurisy due to SCLC from those occurring due to NSCLC and tuberculosis. Further studies with larger group of patients are under progress to further establish

  5. Oral Immunization Against Candidiasis Using Lactobacillus casei Displaying Enolase 1 from Candida albicans.

    Science.gov (United States)

    Shibasaki, Seiji; Karasaki, Miki; Tafuku, Senji; Aoki, Wataru; Sewaki, Tomomitsu; Ueda, Mitsuyoshi

    2014-01-01

    Candidiasis is a common fungal infection that is prevalent in immunocompromised individuals. In this study, an oral vaccine against Candida albicans was developed by using the molecular display approach. Enolase 1 protein (Eno1p) of C. albicans was expressed on the Lactobacillus casei cell surface by using poly-gamma-glutamic acid synthetase complex A from Bacillus subtilis as an anchoring protein. The Eno1p-displaying L. casei cells were used to immunize mice, which were later challenged with a lethal dose of C. albicans. The data indicated that the vaccine elicited a strong IgG response and increased the survival rate of the vaccinated mice. Furthermore, L. casei acted as a potent adjuvant and induced high antibody titers that were comparable to those induced by strong adjuvants such as the cholera toxin. Overall, the molecular display method can be used to rapidly develop vaccines that can be conveniently administered and require minimal processing.

  6. Plasma autoantibodies against heat shock protein 70, enolase 1 and ribonuclease/angiogenin inhibitor 1 as potential biomarkers for cholangiocarcinoma.

    Directory of Open Access Journals (Sweden)

    Rucksak Rucksaken

    Full Text Available The diagnosis of cholangiocarcinoma (CCA is often challenging, leading to poor prognosis. CCA arises via chronic inflammation which may be associated with autoantibodies production. This study aims to identify IgG antibodies directed at self-proteins and tumor-associated antigens. Proteins derived from immortalized cholangiocyte cell line (MMNK1 and CCA cell lines (M055, M214 and M139 were separated using 2-dimensional electrophoresis and incubated with pooled plasma of patients with CCA and non-neoplastic controls by immunoblotting. Twenty five immunoreactive spots against all cell lines-derived proteins were observed on stained gels and studied by LC-MS/MS. Among these, heat shock protein 70 (HSP70, enolase 1 (ENO1 and ribonuclease/angiogenin inhibitor 1 (RNH1 obtained the highest matching scores and were thus selected for further validation. Western blot revealed immunoreactivity against HSP70 and RNH1 in the majority of CCA cases and weakly in healthy individuals. Further, ELISA showed that plasma HSP70 autoantibody level in CCA was significantly capable to discriminate CCA from healthy individuals with an area under the receiver operating characteristic curve of 0.9158 (cut-off 0.2630, 93.55% sensitivity and 73.91% specificity. Plasma levels of IgG autoantibodies against HSP70 were correlated with progression from healthy individuals to cholangitis to CCA (r = 0.679, P<0.001. In addition, circulating ENO1 and RNH1 autoantibodies levels were also significantly higher in cholangitis and CCA compared to healthy controls (P<0.05. Moreover, the combinations of HSP70, ENO1 or RNH1 autoantibodies positivity rates improved specificity to over 78%. In conclusion, plasma IgG autoantibodies against HSP70, ENO1 and RNH1 may represent new diagnostic markers for CCA.

  7. Correlation between serum neuron specific enolase and functional neurological outcome in patients of acute ischemic stroke.

    Science.gov (United States)

    Zaheer, Sana; Beg, Mujahid; Rizvi, Imran; Islam, Najmul; Ullah, Ekram; Akhtar, Nishat

    2013-10-01

    The use of biomarkers to predict stroke prognosis is gaining particular attention nowadays. Neuron specific enolase (NSE), which is a dimeric isoenzyme of the glycolytic enzyme enolase and is found mainly in the neurons is one such biomarker. This study was carried out on patients of acute ischemic stroke with the aims to determine the correlation between NSE levels on the day of admission with infarct volume, stroke severity, and functional neurological outcome on day 30. Seventy five patients of acute ischemic stroke admitted in the Department of Medicine were included in the study. Levels of NSE were determined on day 1 using the human NSE ELISA kit (Alpha Diagnostic International Texas 78244, USA). Volume of infarct was measured by computed tomography (CT) scan using the preinstalled software Syngo (version A40A) of Siemen's medical solutions (Forchheim, Germany). Stroke severity at admission was assessed using Glasgow coma scale (GCS) and functional neurological outcome was assessed using modified Rankin scale (mRS) on day 30. Statistical analysis was performed using the SPSS software for windows version 15.0 (SPSS). A positive correlation was found between concentration of NSE on day 1 and infarct volume determined by CT scan (r = 0.955, P < 0.001). A strong negative correlation was found between GCS at presentation and concentration of NSE on day 1 (r = -0.806, P < 0.001). There was a positive correlation between NSE levels at day 1 and functional neurological outcome assessed by mRS at day 30 (r = 0.744, P < 0.001). Serum levels of NSE in first few days of ischemic stroke can serve as a useful marker to predict stroke severity and early functional outcome. However, larger studies with serial estimation of NSE are needed to establish these observations more firmly.

  8. Mefloquine interferes with glycolysis in schistosomula of Schistosoma mansoni via inhibition of enolase.

    Science.gov (United States)

    Manneck, Theresia; Keiser, Jennifer; Müller, Joachim

    2012-04-01

    The antimalarial drug mefloquine has promising antischistosomal properties killing haematophagous adult schistosomes as well as schistosomula. The mode of action and involved drug targets of mefloquine in Schistosoma mansoni schistosomula are unknown. In order to identify mefloquine-binding proteins and thus potential drug targets, mefloquine affinity chromatography with S. mansoni schistosomula crude extracts was performed. We found one specific mefloquine-binding protein that was identified by mass spectrometry as the glycolytic enzyme enolase (Q27877). Enolase activity assays were performed on schistosomula crude extracts and on the recombinant enolase Q27877 expressed in Escherichia coli. In schistosomula crude extracts enolase activity was inhibited by mefloquine and by the enolase inhibitor sodium fluoride, while activity of the recombinant enolase was not affected. In contrast to enolase from crude extracts, recombinant Q27877 did not bind to mefloquine-agarose. Using isothermal microcalorimetry, we next investigated the metabolic inhibition of mefloquine and 3 known glycolytic inhibitors in Schistosoma spp., namely sodium fluoride, 3-bromopyruvate and menadione on schistosomula in the presence or absence of glucose. We found that in the presence of glucose, schistosomula were less affected by mefloquine, sodium fluoride and 3-bromopyruvate, whereas glucose had no protective effect when schistosomula had been exposed to menadione. These results suggest a potential role of mefloquine as an inhibitor of glycolysis, at least in stages where other targets like haem degradation are not relevant.

  9. The interaction of streptococcal enolase with canine plasminogen: the role of surfaces in complex formation.

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    Vinod Balhara

    Full Text Available The enolase from Streptococcus pyogenes (Str enolase F137L/E363G is a homo-octamer shaped like a donut. Plasminogen (Pgn is a monomeric protein composed of seven discrete separated domains organized into a lock washer. The enolase is known to bind Pgn. In past work we searched for conditions in which the two proteins would bind to one another. The two native proteins in solution would not bind under any of the tried conditions. We found that if the structures were perturbed binding would occur. We stated that only the non-native Str enolase or Pgn would interact such that we could detect binding. We report here the results of a series of dual polarization interferometry (DPI experiments coupled with atomic force microscopy (AFM, isothermal titration calorimetry (ITC, dynamic light scattering (DLS, and fluorescence. We show that the critical condition for forming stable complexes of the two native proteins involves Str enolase binding to a surface. Surfaces that attract Str enolase are a sufficient condition for binding Pgn. Under certain conditions, Pgn adsorbed to a surface will bind Str enolase.

  10. Proinflammatory Cytokines, Enolase and S-100 as Early Biochemical Indicators of Hypoxic-Ischemic Encephalopathy Following Perinatal Asphyxia in Newborns.

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    Chaparro-Huerta, Verónica; Flores-Soto, Mario Eduardo; Merin Sigala, Mario Ernesto; Barrera de León, Juan Carlos; Lemus-Varela, María de Lourdes; Torres-Mendoza, Blanca Miriam de Guadalupe; Beas-Zárate, Carlos

    2017-02-01

    Estimation of the neurological prognosis of infants suffering from perinatal asphyxia and signs of hypoxic-ischemic encephalopathy is of great clinical importance; however, it remains difficult to satisfactorily assess these signs with current standard medical practices. Prognoses are typically based on data obtained from clinical examinations and neurological tests, such as electroencephalography (EEG) and neuroimaging, but their sensitivities and specificities are far from optimal, and they do not always reliably predict future neurological sequelae. In an attempt to improve prognostic estimates, neurological research envisaged various biochemical markers detectable in the umbilical cord blood of newborns (NB). Few studies examining these biochemical factors in the whole blood of newborns exist. Thus, the aim of this study was to determine the expression and concentrations of proinflammatory cytokines (TNF-α, IL-1β and IL-6) and specific CNS enzymes (S-100 and enolase) in infants with perinatal asphyxia. These data were compared between the affected infants and controls and were related to the degree of HIE to determine their utilities as biochemical markers for early diagnosis and prognosis. The levels of the proinflammatory cytokines and enzymes were measured by enzyme-linked immunosorbent assay (ELISA) and Reverse Transcription polymerase chain reaction (RT-PCR). The expression and serum levels of the proinflammatory cytokines, enolase and S-100 were significantly increased in the children with asphyxia compared with the controls. The role of cytokines after hypoxic-ischemic insult has been determined in studies of transgenic mice that support the use of these molecules as candidate biomarkers. Similarly, S-100 and enolase are considered promising candidates because these markers have been correlated with tissue damage in different experimental models. Copyright © 2016. Published by Elsevier B.V.

  11. Delirium tremens: some clinico-chemical features. A study of alanine-aminotransferase, alcaline phosphatase, prothrombine and enolase.

    Science.gov (United States)

    Hemmingsen, R; Kramp, P; Dissing, J

    1980-11-01

    The relationship between variables reflecting liver disease (serum-alanine-aminotransferase (SGPT), serum alcaline phosphatase and plasma prothrombine) and the clinical signs and symptoms during delirium tremens (DT; grade 3) and related clinical states (grade 2) was studied. Furthermore, it was investigated whether the two isoenzymes of enolase which predominante in brain tissue were present in plasma or cerebrospinal fluid (CSF) in DT patients. A correlation between SGPT and clinical state was not observed, which indicates that a causal relationship does not exist between acute liver cell damage and clinical state during DT of grade 3 or 2. In grade 2 patients, but not in grade 3 patients, both SGPT and serum alcaline phosphatase decreased between admission and recovery. This difference between the groups may be due to a higher alcohol consumption and a shorter interval between last drink and admission in grade 3. The difference in recent drinking history may also account for the finding of a higher plasma prothrombine index in grade 3 compared with grade 2, because chronic ethanol intoxication may be accompanied by enhanced hepatic protein synthesis. "Brain-enolase" was not present in detectable amounts in blood or CSF during DT thus suggesting that brain cell damage resulting in leakage of this enzyme from the cells did not prevail during DT.

  12. Elevated Squamous Cell Carcinoma Antigen, Cytokeratin 19 Fragment, and Carcinoembryonic Antigen Levels in Diabetic Nephropathy

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    Jianzhong Chen

    2017-01-01

    Full Text Available Objective. We aimed to explore whether squamous cell carcinoma antigen (SCC, cytokeratin 19 fragment (Cyfra21-1, neuron-specific enolase (NSE, and carcinoembryonic antigen (CEA are elevated in diabetic nephropathy (DN and the association between urinary albumin-to-creatinine ratio (UACR and tumor markers in diabetic patients. Methods. Nondialysis patients with diabetes (n=261 and 90 healthy controls were enrolled. DN was defined as an UACR ≥ 30 mg/g in the absence of a urinary tract infection or other renal abnormalities. Results. Patients with DN had significantly higher serum SCC, Cyfra21-1, and CEA levels than those with normoalbuminuria and healthy controls. The rates of positive SCC, Cyfra21-1, and CEA significantly increased with increasing urinary albumin excretion (all P for trend < 0.001. In contrast, NSE was not affected by DN. SCC, Cyfra21-1, and CEA were significantly and positively correlated with UACR. In logistic regression, after multivariable adjustment, increased UACR was associated with increased odds ratio of elevated tumor marker levels (all P for trend < 0.05. Conclusions. Serum levels of SCC, Cyfra21-1, and CEA are markedly increased with increasing urinary albumin excretion, which affects the specificity for diagnosis for lung cancer. Appropriate interpretation of tumor markers in diabetic patients is mandatory to avoid unnecessary and even hazardous biopsies.

  13. Characterization of squid enolase mRNA: sequence analysis, tissue distribution, and axonal localization.

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    Chun, J T; Gioio, A E; Crispino, M; Giuditta, A; Kaplan, B B

    1995-08-01

    Enolase is a glycolytic enzyme whose amino acid sequence is highly conserved across a wide range of animal species. In mammals, enolase is known to be a dimeric protein composed of distinct but closely related subunits: alpha (non-neuronal), beta (muscle-specific), and gamma (neuron-specific). However, little information is available on the primary sequence of enolase in invertebrates. Here we report the isolation of two overlapping cDNA clones and the putative primary structure of the enzyme from the squid (Loligo pealii) nervous system. The composite sequence of those cDNA clones is 1575 bp and contains the entire coding region (1302 bp), as well as 66 and 207 bp of 5' and 3' untranslated sequence, respectively. Cross-species comparison of enolase primary structure reveals that squid enolase shares over 70% sequence identity to vertebrate forms of the enzyme. The greatest degree of sequence similarity was manifest to the alpha isoform of the human homologue. Results of Northern analysis revealed a single 1.6 kb mRNA species, the relative abundance of which differs approximately 10-fold between various tissues. Interestingly, evidence derived from in situ hybridization and polymerase chain reaction experiments indicate that the mRNA encoding enolase is present in the squid giant axon.

  14. The TFG-TEC oncoprotein induces transcriptional activation of the human β-enolase gene via chromatin modification of the promoter region.

    Science.gov (United States)

    Kim, Ah-Young; Lim, Bobae; Choi, JeeHyun; Kim, Jungho

    2016-10-01

    Recurrent chromosome translocations are the hallmark of many human cancers. A proportion of human extraskeletal myxoid chondrosarcomas (EMCs) are associated with the characteristic chromosomal translocation t(3;9)(q11-12;q22), which results in the formation of a chimeric protein in which the N-terminal domain of the TRK-fused gene (TFG) is fused to the translocated in extraskeletal chondrosarcoma (TEC; also called CHN, CSMF, MINOR, NOR1, and NR4A3) gene. The oncogenic effect of this translocation may be due to the higher transactivation ability of the TFG-TEC chimeric protein; however, downstream target genes of TFG-TEC have not yet been identified. The results presented here, demonstrate that TFG-TEC activates the human β-enolase promoter. EMSAs, ChIP assays, and luciferase reporter assays revealed that TFG-TEC upregulates β-enolase transcription by binding to two NGFI-B response element motifs located upstream of the putative transcription start site. In addition, northern blot, quantitative real-time PCR, and Western blot analyses showed that overexpression of TFG-TEC up-regulated β-enolase mRNA and protein expression in cultured cell lines. Finally, ChIP analyses revealed that TFG-TEC controls the activity of the endogenous β-enolase promoter by promoting histone H3 acetylation. Overall, the results presented here indicate that TFG-TEC triggers a regulatory gene hierarchy implicated in cancer cell metabolism. This finding may aid the development of new therapeutic strategies for the treatment of human EMCs. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  15. Serum neuron specific enolase - a novel indicator for neuropsychiatric systemic lupus erythematosus?

    Science.gov (United States)

    Hawro, T; Bogucki, A; Krupińska-Kun, M; Maurer, M; Woźniacka, A

    2015-12-01

    Neuropsychiatric (NP) lupus, a common manifestation of systemic lupus erythematosus (SLE), is still insufficiently understood, in part, because of the lack of specific biomarkers. Neuron specific enolase (NSE), an important neuronal glycolytic enzyme, shows increased serum levels following acute brain injury, and decreased serum levels in several chronic disorders of the nervous system, including multi infarct dementia, multiple sclerosis and depression. The aim of the study was to evaluate serum NSE levels in SLE patients with and without nervous system involvement, and in healthy controls, and to assess the correlation of NSE serum levels of patients with neuropsychiatric systemic lupus erythematosus (NPSLE) with clinical parameters. The study comprised 47 SLE patients and 28 controls. SLE activity was assessed using the Systemic Lupus Activity Measure (SLAM). A neurologist and a psychiatrist examined all patients. NP involvement was diagnosed according to strict NPSLE criteria proposed by Ainiala and coworkers, as modification to American College of Rheumatology (ACR) nomenclature and case definitions. NSE serum levels were determined by use of an immunoassay. Mean NSE serum concentrations in patients with NPSLE were significantly lower than in non-NPSLE patients (6.3 ± 2.6 µg/L vs. 9.7 ± 3.3 µg/L, p manifestations diagnosed (-0.37; p = 0.001). Decreased serum concentrations of NSE may reflect chronic neuronal damage with declined metabolism of the nervous tissue in patients with NPSLE. © The Author(s) 2015.

  16. The relationship between neuron-specific enolase and prognosis of patients with acute traumatic brain injury

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    Yun-yang LIU

    2015-03-01

    Full Text Available Objective To investigate the relationship between neuron-specific enolase (NSE levels in serum and cerebrospinal fluid (CSF of patients with acute traumatic brain injury (TBI and the prognosis of TBI patients.  Methods A total of 89 patients with acute TBI were divided into light, medium, heavy and severe TBI groups based on admission Glasgow Coma Scale (GCS score. Serum NSE expression levels were detected in all cases and NSE levels in CSF were detected in 18 cases within 12 h after TBI. The expression levels of serum NSE in 20 normal people, except cases of lung disease and nervous system damage, were detected as a control group. Results Compared with the control group, serum NSE expression levels of patients in each TBI group were elevated (P < 0.05, for all, and the NSE levels in severe and heavy TBI groups were higher than that in medium and light groups (P < 0.05, for all. The serum NSE expression levels of patients with cerebral contusion were higher than that of patients with diffuse axonal injury (DAI, P = 0.025, subdural hematoma (P = 0.031 and epidural hematoma (P = 0.021. Serum NSE expression levels were negatively correlated with GCS score (rs = - 0.327, P = 0.024 and Glasgow Outcome Scale (GOS score (rs = - 0.252, P = 0.049. The NSE expression levels of CSF in severe and heavy TBI patients were higher than that of serum (P = 0.039, 0.031.  Conclusions NSE expression changes can be evaluated as an auxiliary indicator in reflecting the degree of acute TBI, typing diagnosis and prognostic evaluation, and NSE levels of CSF is more sensitive than that of serum. DOI: 10.3969/j.issn.1672-6731.2015.03.013

  17. Homology modeling of Leishmania donovani enolase and its molecular interaction with novel inhibitors

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    Jay Prakash Mahato

    2017-01-01

    Full Text Available Introduction: The treatment of Indian tropical disease such as kala-azar is likely to be troublesome to the clinicians as AmpB- and miltefosine-resistant Leishmania donovani has been reported. The rationale behind designed a novel inhibitors of model of L. donovani enolase and performing a binding study with its inhibitors to gain details of the interaction between protein residues and ligand molecules. Methods and Materials: The L. donovani enolase model consists of two typical domains. The N-terminal one contains three-stranded antiparallel β-sheets, followed by six α-helices. The C-terminal domain composes of eleven-stranded mixed α/β-barrel with connectivity. The first α-helix within the C-terminal domain, H7, and the second β-strand, S7, of the barrel domain was arranged in an antiparallel fashion compared to all other α-helices and β-strands. The root-mean-square deviation between predicted model and template is 0.4 Å. The overall conformation of L. donovani enolase model is similar to those of Trypanosoma cruzi enolase and Streptococcus pneumoniae enolase crystal structures. Result: The key amino acid residues within the docking complex model involved in the interaction between model enolase structure and ligand molecule are Lys70, Asn165, Ala168, Asp17, and Asn213. Conclusion: Our theoretical prediction may lead to the establishment of prophylactic and therapeutic approaches for the treatment of kala-azar. This biomedical informatics analysis will help us to combat future kala-azar.

  18. Disaccharide combinations and the expression of enolase3 and plasma membrane Ca2+ATPase isoform in sturgeon sperm cryopreservation.

    Science.gov (United States)

    Xi, M D; Li, P; Du, H; Qiao, X M; Liu, Z G; Wei, Q W

    2018-01-19

    Acipenser sinensis and Acipenser dabryanus are critically endangered species, so germplasm conservation via cryopreservation of sperm is necessary. Disaccharides can act as membrane-impermeable cryoprotectants, and enolase3 (ENO3) and plasma membrane Ca 2+ ATPase isoform (PMCA2) are proteins associated with sperm quality. We considered seven characteristics of sperm quality in cultured brood stock from A. sinensis and A. dabryanus. We tested use of sucrose or trehalose alone and in combination at different concentrations for cryopreservation of A. dabryanus sperm. A low concentration of sucrose plus trehalose (S 15 T 15 ) was optimal. Mixing of the extender with sucrose, lactose, or trehalose alone or with pairwise mixtures revealed that a mixture of lactose and trehalose (L 15 T 15 ) gave the best results for both A. sinensis and A. dabryanus. Enolase3 and PMCA2 expression levels were measured in cryopreserved A. sinensis sperm via Western blotting. Relative ENO3 and PMCA2 expression levels were examined, and the relationship between disaccharide composition, sperm quality and protein expression was explored in A. sinensis. The results showed that relative ENO3 and PMCA2 expression levels were the highest at L 15 T 15 in cryopreserved A. sinensis sperm. There were significant positive correlations between ENO3 expression and percentage membrane integrity, and between PMCA2 expression and sperm motility parameters (percentage of motile sperm, curvilinear velocity, straight-line velocity and average path velocity; p < .05) in cryopreserved A. sinensis sperm. Our results indicate the optimal disaccharide combination and concentrations for cryopreservation of A. sinensis and A. dabryanus sperm and suggest that ENO3 and PMCA2 expression levels could serve as a valuable indicator of sperm quality in A. sinensis. © 2018 Blackwell Verlag GmbH.

  19. Neurone-specific enolase and N-acetyl-aspartate as potential peripheral markers of ischaemic stroke

    NARCIS (Netherlands)

    Stevens, H; Jakobs, C; de Jager, AEJ; Cunningham, RT; Korf, J

    Background After stroke, brain-specific proteins (including neurone-specific enolase) leak into the blood. The question addressed in the present study was whether N-acetyl-aspartate (amino acid derivative localized in cerebral neurones) could also serve as a peripheral marker of ischaemic damage.

  20. Increased serum neuron specific enolase concentrations in patients with hyperglycemic cortical ischemic stroke

    NARCIS (Netherlands)

    Elting, JW; De Keyser, J; Sulter, G.

    1998-01-01

    A detrimental effect of hyperglycemia in ischemic brain has been demonstrated in laboratory experiments and it has been found that hyperglycemia in ischemic stroke is a predictor of poor outcome. We determined serum neuron specific enolase (NSE) concentrations in 41 consecutive patients with a

  1. The structure of bradyzoite-specific enolase from Toxoplasma gondii reveals insights into its dual cytoplasmic and nuclear functions

    Energy Technology Data Exchange (ETDEWEB)

    Ruan, Jiapeng [Northwestern University, 320 E. Superior Street, Morton 7-601, Chicago, IL 60611 (United States); Mouveaux, Thomas [Université Lille Nord de France, (France); Light, Samuel H.; Minasov, George; Anderson, Wayne F. [Northwestern University, 320 E. Superior Street, Morton 7-601, Chicago, IL 60611 (United States); Tomavo, Stanislas [Université Lille Nord de France, (France); Ngô, Huân M., E-mail: h-ngo@northwestern.edu [Northwestern University, 320 E. Superior Street, Morton 7-601, Chicago, IL 60611 (United States); BrainMicro LLC, 21 Pendleton Street, New Haven, CT 06511 (United States)

    2015-03-01

    The second crystal structure of a parasite protein preferentially enriched in the brain cyst of T. gondii has been solved at 2.75 Å resolution. Bradyzoite enolase 1 is reported to have differential functions as a glycolytic enzyme and a transcriptional regulator in bradyzoites. In addition to catalyzing a central step in glycolysis, enolase assumes a remarkably diverse set of secondary functions in different organisms, including transcription regulation as documented for the oncogene c-Myc promoter-binding protein 1. The apicomplexan parasite Toxoplasma gondii differentially expresses two nuclear-localized, plant-like enolases: enolase 1 (TgENO1) in the latent bradyzoite cyst stage and enolase 2 (TgENO2) in the rapidly replicative tachyzoite stage. A 2.75 Å resolution crystal structure of bradyzoite enolase 1, the second structure to be reported of a bradyzoite-specific protein in Toxoplasma, captures an open conformational state and reveals that distinctive plant-like insertions are located on surface loops. The enolase 1 structure reveals that a unique residue, Glu164, in catalytic loop 2 may account for the lower activity of this cyst-stage isozyme. Recombinant TgENO1 specifically binds to a TTTTCT DNA motif present in the cyst matrix antigen 1 (TgMAG1) gene promoter as demonstrated by gel retardation. Furthermore, direct physical interactions of both nuclear TgENO1 and TgENO2 with the TgMAG1 gene promoter are demonstrated in vivo using chromatin immunoprecipitation (ChIP) assays. Structural and biochemical studies reveal that T. gondii enolase functions are multifaceted, including the coordination of gene regulation in parasitic stage development. Enolase 1 provides a potential lead in the design of drugs against Toxoplasma brain cysts.

  2. Validating serum S100B and neuron-specific enolase as biomarkers for the human brain - a combined serum, gene expression and MRI study.

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    Daniel-Paolo Streitbürger

    Full Text Available INTRODUCTION: Former studies have investigated the potential of serum biomarkers for diseases affecting the human brain. In particular the glial protein S100B, a neuro- and gliotrophin inducing plasticity, seems to be involved in the pathogenesis and treatment of psychiatric diseases such as major depression and schizophrenia. Neuron-specific enolase (NSE is a specific serum marker for neuronal damage. However, the specificity of these biomarkers for cell type and brain region has not been investigated in vivo until now. METHODS: We acquired two magnetic resonance imaging parameters sensitive to changes in gray and white matter (T(1-weighted/diffusion tensor imaging and obtained serum S100B and NSE levels of 41 healthy subjects. Additionally, we analyzed whole brain gene expressions of S100B in another male cohort of three subjects using the Allen Brain Atlas. Furthermore, a female post mortal brain was investigated using double immunofluorescence labelling with oligodendrocyte markers. RESULTS: We show that S100B is specifically related to white matter structures, namely the corpus callosum, anterior forceps and superior longitudinal fasciculus in female subjects. This effect was observed in fractional anisotropy and radial diffusivity - the latest an indicator of myelin changes. Histological data confirmed a co-localization of S100B with oligodendrocyte markers in the human corpus callosum. S100B was most abundantly expressed in the corpus callosum according to the whole genome Allen Human Brain Atlas. In addition, NSE was related to gray matter structures, namely the amygdala. This effect was detected across sexes. CONCLUSION: Our data demonstrates a very high S100B expression in white matter tracts, in particular in human corpus callosum. Our study is the first in vivo study validating the specificity of the glial marker S100B for the human brain, and supporting the assumption that radial diffusivity represents a myelin marker. Our results

  3. Validating Serum S100B and Neuron-Specific Enolase as Biomarkers for the Human Brain – A Combined Serum, Gene Expression and MRI Study

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    Streitbürger, Daniel-Paolo; Arelin, Katrin; Kratzsch, Jürgen; Thiery, Joachim; Steiner, Johann; Villringer, Arno

    2012-01-01

    Introduction Former studies have investigated the potential of serum biomarkers for diseases affecting the human brain. In particular the glial protein S100B, a neuro- and gliotrophin inducing plasticity, seems to be involved in the pathogenesis and treatment of psychiatric diseases such as major depression and schizophrenia. Neuron-specific enolase (NSE) is a specific serum marker for neuronal damage. However, the specificity of these biomarkers for cell type and brain region has not been investigated in vivo until now. Methods We acquired two magnetic resonance imaging parameters sensitive to changes in gray and white matter (T1-weighted/diffusion tensor imaging) and obtained serum S100B and NSE levels of 41 healthy subjects. Additionally, we analyzed whole brain gene expressions of S100B in another male cohort of three subjects using the Allen Brain Atlas. Furthermore, a female post mortal brain was investigated using double immunofluorescence labelling with oligodendrocyte markers. Results We show that S100B is specifically related to white matter structures, namely the corpus callosum, anterior forceps and superior longitudinal fasciculus in female subjects. This effect was observed in fractional anisotropy and radial diffusivity – the latest an indicator of myelin changes. Histological data confirmed a co-localization of S100B with oligodendrocyte markers in the human corpus callosum. S100B was most abundantly expressed in the corpus callosum according to the whole genome Allen Human Brain Atlas. In addition, NSE was related to gray matter structures, namely the amygdala. This effect was detected across sexes. Conclusion Our data demonstrates a very high S100B expression in white matter tracts, in particular in human corpus callosum. Our study is the first in vivo study validating the specificity of the glial marker S100B for the human brain, and supporting the assumption that radial diffusivity represents a myelin marker. Our results open a new perspective

  4. System level modeling and component level control of fuel cells

    Science.gov (United States)

    Xue, Xingjian

    This dissertation investigates the fuel cell systems and the related technologies in three aspects: (1) system-level dynamic modeling of both PEM fuel cell (PEMFC) and solid oxide fuel cell (SOFC); (2) condition monitoring scheme development of PEM fuel cell system using model-based statistical method; and (3) strategy and algorithm development of precision control with potential application in energy systems. The dissertation first presents a system level dynamic modeling strategy for PEM fuel cells. It is well known that water plays a critical role in PEM fuel cell operations. It makes the membrane function appropriately and improves the durability. The low temperature operating conditions, however, impose modeling difficulties in characterizing the liquid-vapor two phase change phenomenon, which becomes even more complex under dynamic operating conditions. This dissertation proposes an innovative method to characterize this phenomenon, and builds a comprehensive model for PEM fuel cell at the system level. The model features the complete characterization of multi-physics dynamic coupling effects with the inclusion of dynamic phase change. The model is validated using Ballard stack experimental result from open literature. The system behavior and the internal coupling effects are also investigated using this model under various operating conditions. Anode-supported tubular SOFC is also investigated in the dissertation. While the Nernst potential plays a central role in characterizing the electrochemical performance, the traditional Nernst equation may lead to incorrect analysis results under dynamic operating conditions due to the current reverse flow phenomenon. This dissertation presents a systematic study in this regard to incorporate a modified Nernst potential expression and the heat/mass transfer into the analysis. The model is used to investigate the limitations and optimal results of various operating conditions; it can also be utilized to perform the

  5. Plasma neuronal specific enolase: a potential stage diagnostic marker in human African trypanosomiasis.

    Science.gov (United States)

    Sternberg, Jeremy M; Mitchell, Julia A

    2014-07-01

    This study was carried out to determine the potential of neuronal specific enolase (NSE) as a stage diagnostic marker in human African trypanosomiasis. Plasma and cerebrospinal fluid were obtained from a cohort of Trypanosoma brucei rhodesiense-infected patients and non-infected controls. Neuronal specific enolase concentrations were measured by ELISA and analysed in relation to diagnosis and disease-stage data. Plasma NSE concentration was significantly increased in late-stage patients (median 21 ng/ml), compared to the control (median 11 ng/ml), but not in early-stage patients (median 5.3 ng/ml). Cerebrospinal fluid NSE concentration did not vary between stages. Plasma NSE is a potential stage diagnostic in this cohort and merits further investigation. © The Author 2014. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. A new nuclear function of the Entamoeba histolytica glycolytic enzyme enolase: the metabolic regulation of cytosine-5 methyltransferase 2 (Dnmt2 activity.

    Directory of Open Access Journals (Sweden)

    Ayala Tovy

    2010-02-01

    Full Text Available Cytosine-5 methyltransferases of the Dnmt2 family function as DNA and tRNA methyltransferases. Insight into the role and biological significance of Dnmt2 is greatly hampered by a lack of knowledge about its protein interactions. In this report, we address the subject of protein interaction by identifying enolase through a yeast two-hybrid screen as a Dnmt2-binding protein. Enolase, which is known to catalyze the conversion of 2-phosphoglycerate (2-PG to phosphoenolpyruvate (PEP, was shown to have both a cytoplasmatic and a nuclear localization in the parasite Entamoeba histolytica. We discovered that enolase acts as a Dnmt2 inhibitor. This unexpected inhibitory activity was antagonized by 2-PG, which suggests that glucose metabolism controls the non-glycolytic function of enolase. Interestingly, glucose starvation drives enolase to accumulate within the nucleus, which in turn leads to the formation of additional enolase-E.histolytica DNMT2 homolog (Ehmeth complex, and to a significant reduction of the tRNA(Asp methylation in the parasite. The crucial role of enolase as a Dnmt2 inhibitor was also demonstrated in E.histolytica expressing a nuclear localization signal (NLS-fused-enolase. These results establish enolase as the first Dnmt2 interacting protein, and highlight an unexpected role of a glycolytic enzyme in the modulation of Dnmt2 activity.

  7. The important role of circulating CYFRA21-1 in metastasis diagnosis and prognostic value compared with carcinoembryonic antigen and neuron-specific enolase in lung cancer patients.

    Science.gov (United States)

    Zhang, Li; Liu, Dan; Li, Lei; Pu, Dan; Zhou, Ping; Jing, Yuting; Yu, He; Wang, Yanwen; Zhu, Yihan; He, Yanqi; Li, Yalun; Zhao, Shuang; Qiu, Zhixin; Li, Weimin

    2017-02-02

    The roles of carcinoembryonic antigen (CEA), cytokeratin 19 fragments (CYFRA21-1) and neuron-specific enolase (NSE) in metastases occurrence and poor diagnosis in specific histological classifications of lung cancer need further exploring. In this study, we investigated relationship between elevated levels of three biomarkers of CEA, CYFRA21-1 and NSE (individually and in combination) and metastasis, survival status and prognosis in lung cancer patients. Eight hundred and sixty eight lung cancer patients including adenocarcinoma (ADC, N = 445), squamous cell carcinoma (SCC, N = 215), small cell lung cancer (SCLC, N = 159) and other types (N = 49) were categorized into negative, moderate and high groups according to serum levels of biomarkers, and were then categorized into negative, single, double and triple groups according to any positive combination of three biomarkers. The cutoff values of three biomarkers for groupings were developed on the training group (N = 432) and verified in a validation group (N = 436). Clinical and laboratory characteristics were then assessed for correlation with occurrence of metastasis, survival status and prognosis between the two groups. Further correlation analyses were also conducted by different subtypes (ADC, SCC and SCLC) and tumor stages (I + II, III and IV) of lung cancers. The consistent results between training and validation group confirmed the rationality of grouping methods. CYFRA21-1 levels had stronger association with metastases and survival status than CEA and NSE in all lung cancer patients. When stratified by subtypes, these significances only existed in ADC patients for CYFRA21-1. Cox regression analyses showed that CYFRA21-1 and NSE were independent prognostic factors for lung cancer patients. However, only CYFRA21-1 was an independent prognostic factor in ADC and SCLC patients subtypes. Cox-regression results also indicated that CYFRA21-1 could act as independent prognostic factor

  8. Dual Immunomagnetic Nanobeads-Based Lateral Flow Test Strip for Simultaneous Quantitative Detection of Carcinoembryonic Antigen and Neuron Specific Enolase

    Science.gov (United States)

    Lu, Wenting; Wang, Kan; Xiao, Kun; Qin, Weijian; Hou, Yafei; Xu, Hao; Yan, Xinyu; Chen, Yanrong; Cui, Daxiang; He, Jinghua

    2017-01-01

    A novel immunomagnetic nanobeads -based lateral flow test strip was developed for the simultaneous quantitative detection of neuron specific enolase (NSE) and carcinoembryonic antigen (CEA), which are sensitive and specific in the clinical diagnosis of small cell lung cancer. Using this nanoscale method, high saturation magnetization, carboxyl-modified magnetic nanobeads were successfully synthesized. To obtain the immunomagnetic probes, a covalent bioconjugation of the magnetic nanobeads with the antibody of NSE and CEA was carried out. The detection area contained test line 1 and test line 2 which captured the immune complexes sensitively and formed sandwich complexes. In this assay, cross-reactivity results were negative and both NSE and CEA were detected simultaneously with no obvious influence on each other. The magnetic signal intensity of the nitrocellulose membrane was measured by a magnetic assay reader. For quantitative analysis, the calculated limit of detection was 0.094 ng/mL for NSE and 0.045 ng/mL for CEA. One hundred thirty clinical samples were used to validate the test strip which exhibited high sensitivity and specificity. This dual lateral flow test strip not only provided an easy, rapid, simultaneous quantitative detection strategy for NSE and CEA, but may also be valuable in automated and portable diagnostic applications. PMID:28186176

  9. Label-free electrochemical immunoassay for neuron specific enolase based on 3D macroporous reduced graphene oxide/polyaniline film.

    Science.gov (United States)

    Zhang, Qi; Li, Xiaoyan; Qian, Chunhua; Dou, Li; Cui, Feng; Chen, Xiaojun

    2018-01-01

    The content of neuron specific enolase (NSE) in serum is considered to be an essential indicator of small cell lung cancer (SCLC). Here, a novel label-free electrochemical immunoassay for the detection of NSE based on the three dimensionally macroporous reduced graphene oxide/polyaniline (3DM rGO/PANI) film has been proposed. The 3DM rGO/PANI film was constructed by electrochemical co-deposition of GO and aniline into the interspaces of a sacrificial silica opal template modified Au slice. During the co-deposition, GO was successfully reduced by aniline and PANI could be deposited on the surfaces of rGO sheets. The ratio of rGO and PANI in the composite was also optimized to achieve the maximum electrochemical performance. The 3DM rGO/PANI composite provided larger specific surface area for the antibody immobilization, exhibited enhanced conductivity for electron transfer, and more important was that PANI acted as the electroactive probe for indicating the NSE concentration. Under the optimal conditions, a linear current response of PANI to NSE concentration was obtained over 0.5 pg mL -1 -10.0 ng mL -1 with a detection limit of 0.1 pg mL -1 . Moreover, the immunosensor showed excellent selectivity, good stability, satisfactory reproducibility and regeneration, and was employed to detect NSE in clinical serum specimens. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Release of erythropoietin and neuron-specific enolase after breath holding in competing free divers

    DEFF Research Database (Denmark)

    Kjeld, Thomas; Jattu, T; Nielsen, Henrik

    2015-01-01

    , and troponin T. Venous blood samples were obtained from 17 competing free divers before and 3 h after sessions of static apnea and underwater swimming. The heart was evaluated by echocardiography. Static apnea for 293 ± 78 s (mean ± SD) and subsequent 88 ± 21 m underwater swimming increased plasma......Free diving is associated with extreme hypoxia. This study evaluated the combined effect of maximal static breath holding and underwater swimming on plasma biomarkers of tissue hypoxemia: erythropoietin, neuron-specific enolase and S100B, C-reactive protein, pro-atrial natriuretic peptide...

  11. Immune response induced by oral delivery of Bacillus subtilis spores expressing enolase of Clonorchis sinensis in grass carps (Ctenopharyngodon idellus).

    Science.gov (United States)

    Jiang, Hongye; Chen, Tingjin; Sun, Hengchang; Tang, Zeli; Yu, Jinyun; Lin, Zhipeng; Ren, Pengli; Zhou, Xinyi; Huang, Yan; Li, Xuerong; Yu, Xinbing

    2017-01-01

    Clonorchiasis, caused by the consumption of raw or undercooked freshwater fish containing infective metacercariae of Clonorchis sinensisis (C.sinensis), remains a common public health problem. New effective prevention strategies are still urgent to control this food-borne infectious disease. The previous studies suggested Bacillus subtilis (B. subtilis) spores was an ideal vaccines delivery system, and the C.sinensis enolase (CsENO) was a potential vaccine candidate against clonorchiasis. In the current study, we detected CsENO-specific IgM levels by ELISA in sera, intestinal mucus and skin mucus in grass carps (Ctenopharyngodon idella) through oral administration with B. subtilis spores surface expressing CsENO. In addition, immune-related genes expression was also measured by qRT-PCR. Grass carps orally treated with B. subtilis spores or normal forages were used as controls. The results of ELISA manifested that specific IgM levels of grass carps in CsENO group in sera, intestine mucus and skin mucus almost significantly increased from week 4 post the first oral administration when compared to the two control groups. The levels of specific IgM reached its peak in intestine mucus firstly, then in sera, and last in skin mucus. qRT-PCR results showed that 5 immune-related genes expression had different degree of rising trend in CsENO group when compared to the two control groups. Our study demonstrated that orally administrated with B. subtilis spores expressing CsENO induced innate and adaptive immunity, systemic and local mucosal immunity, and humoral and cellular immunity. Our work may pave the way to clarify the exact mechanisms of protective efficacy elicited by B. subtilis spores expressing CsENO and provide new ideas for vaccine development against C. sinensis infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Probing bacterial adhesion at the single-cell level

    DEFF Research Database (Denmark)

    Zeng, Guanghong; Müller, Torsten; Meyer, Rikke Louise

    Bacteria initiate attachment to surfaces with the aid of different extracellular proteins and polymeric adhesins. To quantitatively analyse the cell-cell and cell-surface interactions provided by bacterial adhesins, it is essential to go down to single cell level where cell-to-cell variation can...... be considered. We have developed a simple and versatile method to make single-cell bacterial probes for measuring single cell adhesion by force spectroscopy using atomic force microscopy (AFM). A single-cell probe was readily made by picking up a bacterial cell from a glass surface by approaching a tipless AFM...... cantilever coated with the commercial cell adhesive CellTakTM. We applied the method to study adhesion of living cells to abiotic surfaces at the single-cell level. Immobilisation of single bacterial cells to the cantilever was stable for several hours, and viability was confirmed by Live/Dead staining...

  13. [Prokaryotic expression, identification and histo-localization of the Taenia asiatica enolase gene].

    Science.gov (United States)

    Du, Wu-Ying; Dai, Jia-Ling; Huang, Jiang; Hu, Xu-Chu; Xu, Jin; Yu, Xin-Bing; Liao, Xing-Jiang; Lang, Shu-Yuan

    2009-12-01

    To express enolase gene of Taenia asiatica, investigate the immunoreactivity of the recombinant TaENO protein, and immuno-histo-localize the presence of the recombinant TaENO in adults of T. asiatica. The gene encoding enolase of T. asiatica (TaENO) was cloned by high throughput sequencing from the cDNA library of adult T. asiatica. The coding region of TaENO was amplified by PCR, and cloned into a prokaryotic expression vector pET-30a (+). The recombinant plasmid was transformed into E. coli BL-21/DE3 and followed by expression of the protein induced by IPTG. The protein was purified by Ni-IDA affinity chromatography, and tested by SDS-PAGE. Its immunoreactivity was examined by Western blotting. The mice were immunized subcutaneously with purified TaENO formulated in Freund's adjuvant. Serum samples were collected and analyzed for specific antibodies by ELISA. The localization of TaENO in adult worms was demonstrated by immunofluorescent technique. The recombinant expression plasmid was identified by PCR, double endonuclease digestion and sequencing. The recombinant TaENO was about Mr 47 000 with a concentration of 0.37 mg/ml. It was recognized by antisera of SD rats immunized with TaENO, sera of taeniasis patients and sera of infected swine. The immunofluorescence assay revealed that TaENO immune serum located in the tegument of T. asiatica adult. The TaENO gene has been expressed with immunoreactivity, and the recombinant TaENO is immunolocalized in the tegument of T. asiatica adult.

  14. Biology at a single cell level

    CSIR Research Space (South Africa)

    Mthunzi, P

    2012-10-01

    Full Text Available ://www.regenexx.com/wp-content/uploads/2011/05/IPS-cell-problems.jpg Induced pluripotent stem cells differentiated in culture http://www.youtube.com/watch?v=ECllrIzTKbA&feature=related Transfecting neuroblastomas Neuroblastoma ? Brain cells ? 80 ? 120 billion neurons in human... brain ? Non- renewing cell type ? Neurons difficult to transfect with established protocols ? Susceptible to degenerative disorders: - Parkinson?s disease - Multiple sclerosis - Alzheimer's disease http...

  15. Evolution of Enzymatic Activities in the Enolase Superfamily: L-Fuconate Dehydratase from Xanthomonas campestris

    Energy Technology Data Exchange (ETDEWEB)

    Yew,W.; Fedorov, A.; Fedorov, E.; Rakus, J.; Pierce, R.; Almo, S.; Gerlt, J.

    2006-01-01

    Many members of the mechanistically diverse enolase superfamily have unknown functions. In this report the authors use both genome (operon) context and screening of a library of acid sugars to assign the L-fuconate dehydratase (FucD) function to a member of the mandelate racemase (MR) subgroup of the superfamily encoded by the Xanthomonas campestris pv. campestris str. ATCC 33913 genome (GI: 21233491). Orthologues of FucD are found in both bacteria and eukaryotes, the latter including the rTS beta protein in Homo sapiens that has been implicated in regulating thymidylate synthase activity. As suggested by sequence alignments and confirmed by high-resolution structures in the presence of active site ligands, FucD and MR share the same active site motif of functional groups: three carboxylate ligands for the essential Mg2+ located at the ends of th third, fourth, and fifth-strands in the (/)7-barrel domain (Asp 248, Glu 274, and Glu 301, respectively), a Lys-x-Lys motif at the end of the second-strand (Lys 218 and Lys 220), a His-Asp dyad at the end of the seventh and sixth-strands (His 351 and Asp 324, respectively), and a Glue at the end of the eighth-strand (Glu 382). The mechanism of the FucD reaction involves initial abstraction of the 2-proton by Lys 220, acid catalysis of the vinylogous-elimination of the 3-OH group by His 351, and stereospecific ketonization of the resulting 2-keto-3-deoxy-L-fuconate product. Screening of the library of acid sugars revealed substrate and functional promiscuity: In addition to L-fuconate, FucD also catalyzes the dehydration of L-galactonate, D-arabinonate, D-altronate, L-talonate, and D-ribonate. The dehydrations of L-fuconate, L-galactonate, and D-arabinonate are initiated by abstraction of the 2-protons by Lys 220. The dehydrations of L-talonate and D-ribonate are initiated by abstraction of the 2-protons by His 351; however, protonation of the enediolate intermediates by the conjugate acid of Lys 220 yields L

  16. Anti-citrullinated alpha enolase antibodies, interstitial lung disease and bone erosion in rheumatoid arthritis.

    Science.gov (United States)

    Alunno, Alessia; Bistoni, Onelia; Pratesi, Federico; La Paglia, Giuliana Maria Concetta; Puxeddu, Ilaria; Migliorini, Paola; Gerli, Roberto

    2018-02-14

    RA is an articular chronic inflammatory disease that in a subgroup of patients can also present with extra-articular manifestations (EAMs). Despite intense investigation on this topic, reliable biomarkers for EAMs are lacking. In recent years several ACPAs, including those targeting anti-citrullinated alpha enolase peptide-1 (anti-CEP-1), have been identified in patients with RA. Data about the ability of anti-CEP-1 to predict the development of erosive disease are confliciting and no evidence concerning their possible association with EAMs in RA is currently available. The aim of this study was to investigate the prevalence and significance of anti-CEP-1 with regard to the association with erosive disease and EAMs in a large cohort of patients with RA. Anti-CCP and anti-CEP-1 antibodies have been assessed on serum samples of RA patients, healthy donors and patients with SpA using commercially available ELISA kits. Anti-CEP-1 antibodies are detectable in over 40% of RA patients and are associated with erosive RA and with RA-associated interstitial lung disease (ILD). Anti-CEP-1 antibodies may represent a useful biomarker for RA-associated ILD and erosive disease to be employed in clinical practice. © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  17. Immunohistochemical localization of gastrin-releasing peptide, neuronal nitric oxide synthase and neurone-specific enolase in the uterus of the North American opossum, Didelphis virginiana.

    Science.gov (United States)

    Kumano, A; Sasaki, M; Budipitojo, T; Kitamura, N; Krause, W J; Yamada, J

    2005-08-01

    The present study has demonstrated the immunohistochemical localization of gastrin-releasing peptide (GRP), neuronal nitric oxide synthase (nNOS) and neurone-specific enolase (NSE) in the uterus of the North American opossum. Although the presence of GRP, nNOS and NSE has been reported recently in the uterus of eutherian species this is the first description of these peptides in a metatherian species. Metatherian mammals are of interest because in these species it is the prolonged lactation phase of development that is the period of primary reproductive investment rather than intrauterine development as is true of eutherian mammals. The opossum, like other marsupial species, has a very abbreviated gestation period which in Didelphis lasts only 12.5 days. GRP was localized in the cytoplasm of cells forming the surface lining epithelium and the glandular epithelium of the opossum endometrium late in pregnancy, at 11.5 days of gestation. Likewise, immunoreactivities of nNOS and NSE were found primarily within the epithelial cells of the endometrium at 11.5 days of gestation. As these peptides and enzymes appear primarily at the time of establishment of the yolk sac placenta (between day 10 and day 12.5 gestation), the present results strongly suggest that these factors may play a fundamental role in the placentation of the opossum.

  18. [The changes of memory and their correlations to S100beta protein as well as neuron-specific enolase in patients with obstructive sleep apnea-hypopnea syndrome].

    Science.gov (United States)

    Feng, Huiwei; Fan, Xianliang; Jiang, Hong

    2011-02-01

    To explore the possible mechanism of brain damage and memory impairment in patients with obstructive sleep apnea hypopnea syndrome (OSAHS) by detecting the memory, serum S100beta protein, neuron specific enolase( NSE) and analyzing the relationship among them. Thirty patients with moderate or severe OSAHS (AHI > 20/h) and twenty normal controls were included in this study. All subjects were detected by polysomnography in the sleep laboratory and the memory of them were evaluated before PSG examination. Memory tests including point memory, association learning, picture free recall, meaningless picture recognition, face characters associated memory have been conducted. The serum S100beta protein was detected by ELISA and the serum NSE was detected by immunoradiometric assay. The relationship between memory and serum S100beta as well as NSE were analyzed in both experiment group and control group. The score of point memory, association learning, meaningless picture recognition, face characters associated memory and memory quotient in patients with OSAHS was significantly lower than control group (P Memory quotient correlated negatively with AHI, ODI, serum S100beta and NSE level; and correlated positively with LSaO2, MSaO2. Memory impairment were present in patients with OSAHS. The increased level of serum S100Beta and NSE may be one of the mechanisms of brain damage and memory impairment in with, OSAHS. And nocturnal hypoxia may contribute to the increased level of serum S100beta and NSE.

  19. Measurement of Heme Synthesis Levels in Mammalian Cells.

    Science.gov (United States)

    Hooda, Jagmohan; Alam, Maksudul; Zhang, Li

    2015-07-09

    Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-(14)C] 5-aminolevulinic acid ([(14)C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [(14)C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

  20. A wireless point-of-care testing system for the detection of neuron-specific enolase with microfluidic paper-based analytical devices.

    Science.gov (United States)

    Fan, Yan; Liu, Juntao; Wang, Yang; Luo, Jinping; Xu, Huiren; Xu, Shengwei; Cai, Xinxia

    2017-09-15

    Neuron-specific enolase (NSE) had clinical significance on diagnosis, staging, monitoring effect and judging prognosis of small cell lung cancer. Thus, there had a growing demand for the on-site testing of NSE. Here, a wireless point-of-care testing (POCT) system with electrochemical measurement for NSE detection was developed and verified. The wireless POCT system consisted of microfluidic paper-based analytical devices (μPADs), electrochemical detector and Android's smartphone. Differential pulse voltammetry (DPV) measurement was adopted by means of electrochemical detector which including a potentiostat and current-to-voltage converter. μPADs were modified with nanocomposites synthesized by Amino functional graphene, thionine and gold nanoparticles (NH 2 -G/Thi/AuNPs) as immunosensors for NSE detection. Combined with μPADs, the performance of the wireless POCT system was evaluated. The peak currents showed good linear relationship of the logarithm of NSE concentration ranging from 1 to 500ngmL -1 with the limit of detection (LOD) of 10pgmL -1 . The detection results were automatically stored in EEPROM memory and could be displayed on Android's smartphone through Bluetooth in real time. The detection results were comparable to those measured by a commercial electrochemical workstation. The wireless POCT system had the potential for on-site testing of other tumor markers. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Carbohydrate metabolism of Xylella fastidiosa: Detection of glycolytic and pentose phosphate pathway enzymes and cloning and expression of the enolase gene

    Directory of Open Access Journals (Sweden)

    Facincani Agda Paula

    2003-01-01

    Full Text Available The objective of this work was to assess the functionality of the glycolytic pathways in the bacterium Xylella fastidiosa. To this effect, the enzymes phosphoglucose isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase of the glycolytic pathway, and glucose 6-phosphate dehydrogenase of the Entner-Doudoroff pathway were studied, followed by cloning and expression studies of the enolase gene and determination of its activity. These studies showed that X. fastidiosa does not use the glycolytic pathway to metabolize carbohydrates, which explains the increased duplication time of this phytopatogen. Recombinant enolase was expressed as inclusion bodies and solubilized with urea (most efficient extractor, Triton X-100, and TCA. Enolase extracted from X. fastidiosa and from chicken muscle and liver is irreversibly inactivated by urea. The purification of enolase was partial and resulted in a low yield. No enzymatic activity was detected for either recombinant and native enolases, aldolase, and glyceraldehyde-3-phosphate dehydrogenase, suggesting that X. fastidiosa uses the Entner-Doudoroff pathway to produce pyruvate. Evidence is presented supporting the idea that the regulation of genes and the presence of isoforms with regulation patterns might make it difficult to understand the metabolism of carbohydrates in X. fastidiosa.

  2. Cell-level finite element studies of viscous cells in planar aggregates.

    Science.gov (United States)

    Chen, H H; Brodland, G W

    2000-08-01

    A new cell-level finite element formulation is presented and used to investigate how epithelia and other planar collections of viscous cells might deform during events such as embryo morphogenesis and wound healing. Forces arising from cytoskeletal components, cytoplasm viscosity, and cell-cell adhesions are included. Individual cells are modeled using multiple finite elements, and cell rearrangements can occur. Simulations of cell-sheet stretching indicate that the initial stages of sheet stretching are characterized by changes in cell shape, while subsequent stages are governed by cell rearrangement. Inferences can be made from the simulations about the forces that act in real cell sheets when suitable experimental data are available.

  3. Serum Antioxidant Vitamins Levels in Children with Sickle Cell ...

    African Journals Online (AJOL)

    Sickle cell anaemia is associated with elevated oxidative stress via increase generation of reactive oxygen species (ROS), and decline in antioxidant defences. Increased oxidative stress is thought to play a role in the development of sickle cell anaemic complications. In the current study, vitamins A, C, and E levels were ...

  4. Cell cycle progression in response to oxygen levels.

    Science.gov (United States)

    Ortmann, Brian; Druker, Jimena; Rocha, Sonia

    2014-09-01

    Hypoxia' or decreases in oxygen availability' results in the activation of a number of different responses at both the whole organism and the cellular level. These responses include drastic changes in gene expression, which allow the organism (or cell) to cope efficiently with the stresses associated with the hypoxic insult. A major breakthrough in the understanding of the cellular response to hypoxia was the discovery of a hypoxia sensitive family of transcription factors known as the hypoxia inducible factors (HIFs). The hypoxia response mounted by the HIFs promotes cell survival and energy conservation. As such, this response has to deal with important cellular process such as cell division. In this review, the integration of oxygen sensing with the cell cycle will be discussed. HIFs, as well as other components of the hypoxia pathway, can influence cell cycle progression. The role of HIF and the cell molecular oxygen sensors in the control of the cell cycle will be reviewed.

  5. MicroRNA-9 promotes the neuronal differentiation of rat bone marrow mesenchymal stem cells by activating autophagy

    Directory of Open Access Journals (Sweden)

    Guang-yu Zhang

    2015-01-01

    Full Text Available MicroRNA-9 (miR-9 has been shown to promote the differentiation of bone marrow mesenchymal stem cells into neuronal cells, but the precise mechanism is unclear. Our previous study confirmed that increased autophagic activity improved the efficiency of neuronal differentiation in bone marrow mesenchymal stem cells. Accumulating evidence reveals that miRNAs adjust the autophagic pathways. This study used miR-9-1 lentiviral vector and miR-9-1 inhibitor to modulate the expression level of miR-9. Autophagic activity and neuronal differentiation were measured by the number of light chain-3 (LC3-positive dots, the ratio of LC3-II/LC3, and the expression levels of the neuronal markers enolase and microtubule-associated protein 2. Results showed that LC3-positive dots, the ratio of LC3-II/LC3, and expression of neuron specific enolase and microtubule-associated protein 2 increased in the miR-9 + group. The above results suggest that autophagic activity increased and bone marrow mesenchymal stem cells were prone to differentiate into neuronal cells when miR-9 was overexpressed, demonstrating that miR-9 can promote neuronal differentiation by increasing autophagic activity.

  6. High epitope expression levels increase competition between T cells.

    Directory of Open Access Journals (Sweden)

    Almut Scherer

    2006-08-01

    Full Text Available Both theoretical predictions and experimental findings suggest that T cell populations can compete with each other. There is some debate on whether T cells compete for aspecific stimuli, such as access to the surface on antigen-presenting cells (APCs or for specific stimuli, such as their cognate epitope ligand. We have developed an individual-based computer simulation model to study T cell competition. Our model shows that the expression level of foreign epitopes per APC determines whether T cell competition is mainly for specific or aspecific stimuli. Under low epitope expression, competition is mainly for the specific epitope stimuli, and, hence, different epitope-specific T cell populations coexist readily. However, if epitope expression levels are high, aspecific competition becomes more important. Such between-specificity competition can lead to competitive exclusion between different epitope-specific T cell populations. Our model allows us to delineate the circumstances that facilitate coexistence of T cells of different epitope specificity. Understanding mechanisms of T cell coexistence has important practical implications for immune therapies that require a broad immune response.

  7. Serum Neuron-Specific Enolase, Biogenic Amino-Acids and Neurobehavioral Function in Lead-Exposed Workers from Lead-Acid Battery Manufacturing Process

    Directory of Open Access Journals (Sweden)

    K Ravibabu

    2015-01-01

    Full Text Available Background: The interaction between serum neuron-specific enolase (NSE, biogenic amino-acids and neurobehavioral function with blood lead levels in workers exposed to lead form lead-acid battery manufacturing process was not studied. Objective: To evaluate serum NSE and biogenic amino-acids (dopamine and serotonin levels, and neurobehavioral performance among workers exposed to lead from lead-acid storage battery plant, and its relation with blood lead levels (BLLs. Methods: In a cross-sectional study, we performed biochemical and neurobehavioral function tests on 146 workers exposed to lead from lead-acid battery manufacturing process. BLLs were assessed by an atomic absorption spectrophotometer. Serum NSE, dopamine and serotonin were measured by ELISA. Neurobehavioral functions were assessed by CDC-recommended tests—simple reaction time (SRT, symbol digit substitution test (SDST, and serial digit learning test (SDLT. Results: There was a significant correlation (r 0.199, p<0.05 between SDST and BLL. SDLT and SRT had also a significant positive correlation (r 0.238, p<0.01. NSE had a negative correlation (r –0.194, p<0.05 with serotonin level. Multiple linear regression analysis revealed that both SRT and SDST had positive significant associations with BLL. SRT also had a positive significant association with age. Conclusion: Serum NSE cannot be used as a marker for BLL. The only domain of neurobehavioral function tests that is affected by increased BLL in workers of lead-acid battery manufacturing process is that of the “attention and perception” (SDST.

  8. Human mast cells decrease SLPI levels in type II – like alveolar cell model, in vitro

    Directory of Open Access Journals (Sweden)

    Nyström Max

    2003-08-01

    Full Text Available Abstract Background Mast cells are known to accumulate at sites of inflammation and upon activation to release their granule content, e.g. histamine, cytokines and proteases. The secretory leukocyte protease inhibitor (SLPI is produced in the respiratory mucous and plays a role in regulating the activity of the proteases. Result We have used the HMC-1 cell line as a model for human mast cells to investigate their effect on SLPI expression and its levels in cell co-culture experiments, in vitro. In comparison with controls, we found a significant reduction in SLPI levels (by 2.35-fold, p Conclusion These results indicate that SLPI-producing cells may assist mast cell migration and that the regulation of SLPI release and/or consumption by mast cells requires interaction between these cell types. Therefore, a "local relationship" between mast cells and airway epithelial cells might be an important step in the inflammatory response.

  9. Serdemetan antagonizes the Mdm2-HIF1α axis leading to decreased levels of glycolytic enzymes.

    Directory of Open Access Journals (Sweden)

    Jason A Lehman

    Full Text Available Serdemetan (JNJ-26854165, an antagonist to Mdm2, was anticipated to promote the activation of p53. While regulation of p53 by Mdm2 is important, Mdm2 also regulates numerous proteins involved in diverse cellular functions. We investigated if Serdemetan would alter the Mdm2-HIF1α axis and affect cell survival in human glioblastoma cells independently of p53. Treatment of cells with Serdemetan under hypoxia resulted in a decrease in HIF1α levels. HIF1α downstream targets, VEGF and the glycolytic enzymes (enolase, phosphoglycerate kinase1/2, and glucose transporter 1, were all decreased in response to Serdemetan. The involvement of Mdm2 in regulating gene expression of glycolytic enzymes raises the possibility of side effects associated with therapeutically targeting Mdm2.

  10. Transgenic cells with increased plastoquinone levels and methods of use

    Energy Technology Data Exchange (ETDEWEB)

    Sayre, Richard T.; Subramanian, Sowmya; Cahoon, Edgar

    2016-12-27

    Disclosed herein are transgenic cells expressing a heterologous nucleic acid encoding a prephenate dehydrogenase (PDH) protein, a heterologous nucleic acid encoding a homogentisate solanesyl transferase (HST) protein, a heterologous nucleic acid encoding a deoxyxylulose phosphate synthase (DXS) protein, or a combination of two or more thereof. In particular examples, the disclosed transgenic cells have increased plastoquinone levels. Also disclosed are methods of increasing cell growth rates or production of biomass by cultivating transgenic cells expressing a heterologous nucleic acid encoding a PDH protein, a heterologous nucleic acid encoding an HST protein, a heterologous nucleic acid encoding a DXS protein, or a combination of two or more thereof under conditions sufficient to produce cell growth or biomass.

  11. Low Level Laser Irradiation of Nerve Cells In Vitro

    Science.gov (United States)

    1996-01-01

    Lim et al. used a GaAIAs low level laser to treat post orthodontic manipulation pain.46 Patients exposed to the laser reported a lower level of pain...1993-0-336-043 Standard Form 298 Back (Rev. 2-89) LOW LEVEL LASER IRRADIATION OF NERVE CELLS IN VITRO A Thesis Presented in Partial fulfillment of the...penetrates to the nerve. The present study does not support the hypothesis that there is a direct effect of the low level laser energy on perineural or

  12. Soluble CD163 levels in children with sickle cell disease

    DEFF Research Database (Denmark)

    Moller, Holger Jon; Nielsen, Marianne Jensby; Bartram, Jack

    2011-01-01

    Sickle cell disease (SCD) is characterized by vasculopathy, which has been causally linked to intravascular haemolysis and high levels of free plasma haemoglobin. Soluble CD163 (sCD163) is implicated in the clearance of free plasma haemoglobin and high plasma concentrations have been linked...... to arterial disease. We therefore investigated the value of sCD163 as a biomarker in children with SCD, and also measured haptoglobin levels in this population. We measured sCD163 in 25 control children with no haemoglobinopathy, 41 with sickle cell anaemia (HbSS) in the steady state, 27 with HbSS taking...

  13. Protein C and Antithrombin Levels in Patients with Sickle Cell ...

    African Journals Online (AJOL)

    Muktar HM. Protein C and antithrombin levels in patients with sickle cell anemia in Ahmadu Bello University Teaching Hospital Zaria, Nigeria. Niger. J Clin Pract 2017;20:998-1001. This is an open access article distributed under the terms of the Creative Commons. Attribution-Non Commercial-Share Alike 3.0 License, ...

  14. Enolasa específica de neurona en dos recién nacidos con depresión ligera al nacer Neuron- specific Enolase in two newborns presenting with moderate depression at birth

    Directory of Open Access Journals (Sweden)

    Raisa Bu-Coifiu Fanego

    2009-03-01

    in serum may be altered. The aim of present paper was to study Enolase specific of neuron in the serum of two newborns with low Apgar score and to determine if, during a one year follow-up, such patients presented disorders of psychomotor development. METHODS: We took serum samples at birth and at 72 hours. Levels of Enolase specific of neuron by immunoenzymatic method type ELISA were determined. Each sample was assessed by polymerase chain reaction (PCR to cytomegalovirus in Wuersburg Institute of Immunology (Germany. Also, we quantified antibodies to IgM and IgG cytomegalovirus in Neurochemistry Institute of Georg August of Goettingen University (Germany. Interesting clinical data of each newborn were collected and after a year, all these patients were cited for a physical examination to evaluate tits neurodevelopment. RESULTS: Enolase figures were increased both at birth and at 72 hours, with IgG anticytomegalovirus antibodies from mother through placenta. At birth there was not presence of this virus. At physical and neurological examination performed at 1 year it was possible to confirm that children evolved adequately until now. CONCLUSIONS: Study must to be prolonged until 3 years of life, and to increase number of study patients, emphasizing on those with an Apgar score lower than 5 at 5 minutes post-birth.

  15. Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining

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    Yomo Tetsuya

    2006-06-01

    Full Text Available Abstract Background Single-cell level studies are being used increasingly to measure cell properties not directly observable in a cell population. High-performance data acquisition systems for such studies have, by necessity, developed in synchrony. However, improvements in sample purification techniques are also required to reveal new phenomena. Here we assessed a cell sorter as a sample-pretreatment tool for a single-cell level assay. A cell sorter is routinely used for selecting one type of cells from a heterogeneous mixture of cells using specific fluorescence labels. In this case, we wanted to select cells of exactly the same size, shape, and cell-cycle stage from a population, without using a specific fluorescence label. Results We used four light scatter parameters: the peak height and area of the forward scatter (FSheight and FSarea and side scatter (SSheight and SSarea. The rat pheochromocytoma PC12 cell line, a neuronal cell line, was used for all experiments. The living cells concentrated in the high FSarea and middle SSheight/SSarea fractions. Single cells without cell clumps were concentrated in the low SS and middle FS fractions, and in the higher FSheight/FSarea and SSheight/SSarea fractions. The cell populations from these viable, single-cell-rich fractions were divided into twelve subfractions based on their FSarea-SSarea profiles, for more detailed analysis. We found that SSarea was proportional to the cell volume and the FSarea correlated with cell roundness and elongation, as well as with the level of DNA in the cell. To test the method and to characterize the basic properties of the isolated single cells, sorted cells were cultured in separate wells. The cells in all subfractions survived, proliferated and differentiated normally, suggesting that there was no serious damage. The smallest, roundest, and smoothest cells had the highest viability. There was no correlation between proliferation and differentiation. NGF increases

  16. Inactive enzymatic mutant proteins (phosphoglycerate mutase and enolase as sugar binders for ribulose-1,5-bisphosphate regeneration reactors

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    Giri Ashok

    2005-02-01

    Full Text Available Abstract Background Carbon dioxide fixation bioprocess in reactors necessitates recycling of D-ribulose1,5-bisphosphate (RuBP for continuous operation. A radically new close loop of RuBP regenerating reactor design has been proposed that will harbor enzyme-complexes instead of purified enzymes. These reactors will need binders enabling selective capture and release of sugar and intermediate metabolites enabling specific conversions during regeneration. In the current manuscript we describe properties of proteins that will act as potential binders in RuBP regeneration reactors. Results We demonstrate specific binding of 3-phosphoglycerate (3PGA and 3-phosphoglyceraldehyde (3PGAL from sugar mixtures by inactive mutant of yeast enzymes phosphoglycerate mutase and enolase. The reversibility in binding with respect to pH and EDTA has also been shown. No chemical conversion of incubated sugars or sugar intermediate metabolites were found by the inactive enzymatic proteins. The dissociation constants for sugar metabolites are in the micromolar range, both proteins showed lower dissociation constant (Kd for 3-phosphoglycerate (655–796 μM compared to 3-phosphoglyceraldehyde (822–966 μM indicating higher affinity for 3PGA. The proteins did not show binding to glucose, sucrose or fructose within the sensitivity limits of detection. Phosphoglycerate mutase showed slightly lower stability on repeated use than enolase mutants. Conclusions The sugar and their intermediate metabolite binders may have a useful role in RuBP regeneration reactors. The reversibility of binding with respect to changes in physicochemical factors and stability when subjected to repeated changes in these conditions are expected to make the mutant proteins candidates for in-situ removal of sugar intermediate metabolites for forward driving of specific reactions in enzyme-complex reactors.

  17. Protein Expression Analyses at the Single Cell Level

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    Masae Ohno

    2014-09-01

    Full Text Available The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level.

  18. System-level modeling and simulation of the cell culture microfluidic biochip ProCell

    DEFF Research Database (Denmark)

    Minhass, Wajid Hassan; Pop, Paul; Madsen, Jan

    2010-01-01

    Microfluidic biochips offer a promising alternative to a conventional biochemical laboratory. There are two technologies for the microfluidic biochips: droplet-based and flow-based. In this paper we are interested in flow-based microfluidic biochips, where the liquid flows continuously through pre......-defined micro-channels using valves and pumps. We present an approach to the system-level modeling and simulation of a cell culture microfluidic biochip called ProCell, Programmable Cell Culture Chip. ProCell contains a cell culture chamber, which is envisioned to run 256 simultaneous experiments (viewed...... and a comprehensive fault model that captures permanent faults occurring during chip operation. Using the proposed modeling and simulation framework, we perform an architectural level evaluation of two cell culture chamber implementations. A qualitative success metric is also proposed to evaluate chip performance...

  19. Identification of enolases and aldolases as important fish allergens in cod, salmon and tuna: component resolved diagnosis using parvalbumin and the new allergens.

    Science.gov (United States)

    Kuehn, A; Hilger, C; Lehners-Weber, C; Codreanu-Morel, F; Morisset, M; Metz-Favre, C; Pauli, G; de Blay, F; Revets, D; Muller, C P; Vogel, L; Vieths, S; Hentges, F

    2013-07-01

    The majority of fish-allergic patients are sensitized to parvalbumin, known to be the cause of important IgE cross-reactivity among fish species. Little is known about the importance of fish allergens other than parvalbumin. The aim of this study was to characterize hitherto undefined fish allergens in three commonly consumed fish species, cod, salmon and tuna, and to evaluate their importance for in vitro IgE-diagnosis in addition to parvalbumin and fish gelatin. Sixty-two patients were diagnosed by clinical history, skin prick tests and specific IgE to fish extracts. Two new fish allergens from cod, salmon and tuna were identified by microsequencing. These proteins were characterized by immunoblot, ELISA and mediator release assay. Purified parvalbumin, enolase, aldolase and fish gelatin were used for quantification of specific IgE in ELISA. Parvalbumin and two other allergens of 50 and 40 kDa were detected in IgE-immunoblots of cod, salmon and tuna extracts by most patient sera. The 50 and 40 kDa proteins were identified as beta-enolase and fructose-bisphosphate aldolase A respectively. Both purified enzymes showed allergenic activity in the mediator release assay. Indeed, 72.6% of the patients were sensitized to parvalbumin, 20% of these had specific IgE to salmon parvalbumin only. IgE to enolases were found in 62.9% (0.5-95.0 kUA /L), to aldolases in 50.0% (0.4-26.0 kUA /L) and to fish gelatin in 19.3% (0.4-20.0 kUA /L) of the patients. Inter-species cross-reactivity, even though limited, was found for enolases and aldolases by IgE-inhibition ELISA. Fish enolase and aldolase have been identified as important new fish allergens. In fish allergy diagnosis, IgE to enolase and aldolase are especially relevant when IgE to parvalbumin are absent. © 2013 John Wiley & Sons Ltd.

  20. Nutritional stress enhances cell viability of odontoblast-like cells subjected to low level laser irradiation

    Science.gov (United States)

    Tagliani, M. M.; Oliveira, C. F.; Lins, E. M. M.; Kurachi, C.; Hebling, J.; Bagnato, V. S.; de Souza Costa, C. A.

    2010-03-01

    In spite of knowing that cells under stress are biostimulated by low level laser (LLL) irradiation, the ideal condition of stress to different cell lines has not yet been established. Consequently, the aim of the present in vitro study was to evaluate the effects of a defined parameter of LLL irradiation applied on stressed odontoblast-like pulp cells (MDPC-23). The cells were seeded (12500 cells/cm2) in wells of 24-well plates using complete culture medium (DMEM) and incubated for 24 hours. Then, the DMEM was replaced by a new medium with low concentrations (nutritional stress condition) of fetal bovine serum (FBS) giving rise to the following experimental groups: G1: 2% FBS; G2: 5% FBS; and G3: 10% FBS. The cells were irradiated three times with LLL in specific parameters (808±3 nm, 100 mW, 1.5 J/cm2) every 24 hours. No irradiation was carried out in groups G4 (2% FBS-Control), G5 (5% FBS-Control), and G6 (10% FBS-Control). For all groups, the cell metabolism (MTT assay) and morphology (SEM) was evaluated. The experimental groups showed enhanced cell metabolism and normal cell morphology regardless of FBS concentration. A slight increase in the cell metabolism was observed only in group G2. It was concluded that cell nutritional stress caused by reducing the concentration of FBS to 5% is the most suitable method to assess the biostimulation of LLL irradiated MDPC-23 cells.

  1. Neutrophil Extracellular DNA Traps Induce Autoantigen Production by Airway Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Youngwoo Choi

    2017-01-01

    Full Text Available The hypothesis of autoimmune involvement in asthma has received much recent interest. Autoantibodies, such as anti-cytokeratin (CK 18, anti-CK19, and anti-α-enolase antibodies, react with self-antigens and are found at high levels in the sera of patients with severe asthma (SA. However, the mechanisms underlying autoantibody production in SA have not been fully determined. The present study was conducted to demonstrate that neutrophil extracellular DNA traps (NETs, cytotoxic molecules released from neutrophils, are a key player in the stimulation of airway epithelial cells (AECs to produce autoantigens. This study showed that NETs significantly increased the intracellular expression of tissue transglutaminase (tTG but did not affect that of CK18 in AECs. NETs induced the extracellular release of both tTG and CK18 in a concentration-dependent manner. Moreover, NETs directly degraded intracellular α-enolase into small fragments. However, antibodies against neutrophil elastase (NE or myeloperoxidase (MPO attenuated the effects of NETs on AECs. Furthermore, each NET isolated from healthy controls (HC, nonsevere asthma (NSA, and SA had different characteristics. Taken together, these findings suggest that AECs exposed to NETs may exhibit higher autoantigen production, especially in SA. Therefore, targeting of NETs may represent a new therapy for neutrophilic asthma with a high level of autoantigens.

  2. Rising cyclin-CDK levels order cell cycle events.

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    Catherine Oikonomou

    Full Text Available Diverse mitotic events can be triggered in the correct order and time by a single cyclin-CDK. A single regulator could confer order and timing on multiple events if later events require higher cyclin-CDK than earlier events, so that gradually rising cyclin-CDK levels can sequentially trigger responsive events: the "quantitative model" of ordering.This 'quantitative model' makes predictions for the effect of locking cyclin at fixed levels for a protracted period: at low cyclin levels, early events should occur rapidly, while late events should be slow, defective, or highly variable (depending on threshold mechanism. We titrated the budding yeast mitotic cyclin Clb2 within its endogenous expression range to a stable, fixed level and measured time to occurrence of three mitotic events: growth depolarization, spindle formation, and spindle elongation, as a function of fixed Clb2 level. These events require increasingly more Clb2 according to their normal order of occurrence. Events occur efficiently and with low variability at fixed Clb2 levels similar to those observed when the events normally occur. A second prediction of the model is that increasing the rate of cyclin accumulation should globally advance timing of all events. Moderate (<2-fold overexpression of Clb2 accelerates all events of mitosis, resulting in consistently rapid sequential cell cycles. However, this moderate overexpression also causes a significant frequency of premature mitoses leading to inviability, suggesting that Clb2 expression level is optimized to balance the fitness costs of variability and catastrophe.We conclude that mitotic events are regulated by discrete cyclin-CDK thresholds. These thresholds are sequentially triggered as cyclin increases, yielding reliable order and timing. In many biological processes a graded input must be translated into discrete outputs. In such systems, expression of the central regulator is likely to be tuned to an optimum level, as we

  3. The Energetics of Streptococcal Enolase Octamer Formation: The Quantitative Contributions of the Last Eight Amino Acids at the Carboxy-Terminus.

    Directory of Open Access Journals (Sweden)

    Jack A Kornblatt

    Full Text Available The enolase produced by Streptococcus pyogenes is a homo-octamer whose overall shape resembles that of a donut. The octamer is best described as a tetramer of dimers. As such, it contains two types of interfaces. The first is common to almost all enolases as most enolases that have been studied are dimers. The second is unique to the octamers and includes residues near the carboxy-terminus. The primary sequence of the enolase contains 435 residues with an added 19 as an N-terminal hexahistine tag. We have systematically truncated the carboxy-terminus, individually removing the first 8 residues. This gave rise to a series of eight structures containing respectively, 435, 434, 433, 432, 431, 430, 429 and 427 residues. The truncations cause the protein to gradually dissociate from octamers to enzymatically inactive monomers with very small amounts of intermediate tetramers and dimers. We have evaluated the contributions of the missing residues to the monomer/octamer equilibrium using a combination of analytical ultracentrifugation and activity assays. For the dissociation reaction, octamer 8 monomer truncation of all eight C-terminal residues resulted in a diminution in the standard Gibbs energy of dissociation of about 59 kJ/mole of octamer relative to the full length protein. Considering that this change is spread over eight subunits, this translates to a change in standard Gibbs interaction energy of less than 8 kJ/mole of monomer distributed over the eight monomers. The resulting proteins, containing 434, 433, 432, 431, 430, 429 and 427 residues per monomer, showed intermediate free energies of dissociation. Finally, three other mutations were introduced into our reference protein to establish how they influenced the equilibrium. The main importance of this work is it shows that for homo-multimeric proteins a small change in the standard Gibbs interaction energy between subunits can have major physiological effects.

  4. Immunoreactive neuron-specific enolase (NSE) is expressed in testicular carcinoma-in-situ

    DEFF Research Database (Denmark)

    Kang, J L; Rajpert-De Meyts, E; Skakkebaek, N E

    1996-01-01

    -seminomas, and a mixed germ cell tumour. As the co-existence of high NSE production and gene amplification of N-myc has been reported in some tumours, including germ cell tumours, the expression of the protein product of N-myc was also examined in this study, but only sporadic cases showed N-myc staining. These results...... are evidence against a relationship between NSE and N-myc in testicular germ cell tumours. The high expression of NSE in CIS and overt germ cell tumours may be due to the increased gene dosage effect associated with the overrepresentation of isochromosome 12p....

  5. Chasing the precursor of functional hematopoietic stem cells at the single cell levels in mouse embryos.

    Science.gov (United States)

    Wang, Xiaochen; Gong, Yuemin; Ema, Hideo

    2016-07-22

    Adult hematopoietic stem cells (HSCs), the ideal system for regenerative research, were isolated at single cell levels decades ago, whereas studies on embryonic HSCs are much more difficult. Zhou et al identified a new pre-HSC cell surface marker, CD201, by which they isolated pre-HSCs at single cell levels for further analyses. The novel expression pattern of HSC development is revealed, including the fundamental role of mammalian targets of rapamycin (mTOR) signaling pathway in HSCs emergence, and the repopulation potential of S/G2/M phase pre-HSCs. Deeper understandings of the cellular origin and developmental regulatory network of HSCs are essential to develop new strategies of generating HSCs in vitro for clinical application.

  6. Dose levels in the hot cells area ININ

    International Nuclear Information System (INIS)

    Torre, J. De la; Ramirez, J.M.; Solis, M.L.

    2004-01-01

    The Laboratory of Hot Cells (LCC) located in the National Institute of Nuclear Research (ININ) is an institution, it is an area where radioactive material is managed with different activity values, in function of its original design for 10,000 curies of Co-60. Managing this materials in the installation, it implies to measure and to analyze the dose levels that the POE will receive as well as the implementation of appropriate measures of radiological protection and radiological safety, so that that is completed settled down by the concept ALARA. In this work they are carried out mensurations of the levels of the dose to receive for the POE when managing radionuclides with maximum activities that can be allowed in function of the current conditions of the cells and an evaluation of the obtained results is made comparing them with the effective international norms as well as the application of the program of surveillance and radiological protection implemented for the development of the works that are carry out in the installation. (Author)

  7. Cardiac, renal, and neurological benefits of preoperative levosimendan administration in patients with right ventricular dysfunction and pulmonary hypertension undergoing cardiac surgery: evaluation with two biomarkers neutrophil gelatinase-associated lipocalin and neuronal enolase

    Directory of Open Access Journals (Sweden)

    Guerrero-Orriach JL

    2016-04-01

    Full Text Available José Luis Guerrero-Orriach,1 Daniel Ariza-Villanueva,1 Ana Florez-Vela,1 Lourdes Garrido-Sánchez,2,3 María Isabel Moreno-Cortés,1 Manuel Galán-Ortega,1 Alicia Ramírez-Fernández,1 Juan Alcaide Torres,3 Concepción Santiago Fernandez,3 Isabel Navarro Arce,1 José María Melero-Tejedor,4 Manuel Rubio-Navarro,1 José Cruz-Mañas1 1Department of Cardio-Anaesthesiology, University Hospital Virgen de la Victoria, Málaga, Spain; 2CIBER Fisiología de la Obesidad y Nutrición (CIBEROBN, Instituto de Salud Carlos III, Málaga, Spain; 3Department of Nutrition and Endocrinology, Instituto de Investigaciones Biomédicas de Málaga (IBIMA, University Hospital Virgen de la Victoria, Málaga, Spain; 4Department of Cardiovascular Surgery, University Hospital Virgen de la Victoria, Málaga, Spain Purpose: To evaluate if the preoperative administration of levosimendan in patients with right ventricular (RV dysfunction, pulmonary hypertension, and high perioperative risk would improve cardiac function and would also have a protective effect on renal and neurological functions, assessed using two biomarkers neutrophil gelatinase-associated lipocalin (N-GAL and neuronal enolase. Methods: This is an observational study. Twenty-seven high-risk cardiac patients with RV dysfunction and pulmonary hypertension, scheduled for cardiac valve surgery, were prospectively followed after preoperative administration of levosimendan. Levosimendan was administered preoperatively on the day before surgery. All patients were considered high risk of cardiac and perioperative renal complications. Cardiac function was assessed by echocardiography, renal function by urinary N-GAL levels, and the acute kidney injury scale. Neuronal damage was assessed by neuron-specific enolase levels. Results: After surgery, no significant variations were found in mean and SE levels of N-GAL (14.31 [28.34] ng/mL vs 13.41 [38.24] ng/mL, neuron-specific enolase (5.40 [0.41] ng/mL vs 4.32 [0.61] ng

  8. Depletion of host cell riboflavin reduces Wolbachia levels in cultured mosquito cells

    Science.gov (United States)

    Baldridge, Gerald D.; Carroll, Elissa M.; Kurtz, Cassandra M.

    2015-01-01

    Wolbachia is an obligate intracellular alphaproteobacterium that occurs in arthropod and nematode hosts. Wolbachia presumably provides a fitness benefit to its hosts, but the basis for its retention and spread in host populations remains unclear. Wolbachia genomes retain biosynthetic pathways for some vitamins, and the possibility that these vitamins benefit host cells provides a potential means of selecting for Wolbachia-infected cell lines. To explore whether riboflavin produced by Wolbachia is available to its host cell, we established that growth of uninfected C7–10 mosquito cells decreases in riboflavin-depleted culture medium. A well studied inhibitor of riboflavin uptake, lumiflavin, further inhibits growth of uninfected C7–10 cells with an LC50 of approximately 12 µg/ml. Growth of C/wStr1 mosquito cells, infected with Wolbachia from the planthopper, Laodelphax striatellus, was enhanced in medium containing low levels of lumiflavin, but Wolbachia levels decreased. Lumiflavin-enhanced growth thus resembled the improved growth that accompanies treatment with antibiotics that deplete Wolbachia, rather than a metabolic advantage provided by the Wolbachia infection. We used the polymerase chain reaction to validate the decrease in Wolbachia abundance and evaluated our results in the context of a proteomic analysis in which we detected nearly 800 wStr proteins. Our data indicate that Wolbachia converts riboflavin to FMN and FAD for its own metabolic needs, and does not provide a source of riboflavin for its host cell. PMID:24789726

  9. Temporal profile and clinical significance of serum neuron-specific enolase and S100 in ischemic and hemorrhagic stroke.

    Science.gov (United States)

    Brea, David; Sobrino, Tomás; Blanco, Miguel; Cristobo, Iván; Rodríguez-González, Raquel; Rodríguez-Yañez, Manuel; Moldes, Octavio; Agulla, Jesús; Leira, Rogelio; Castillo, José

    2009-01-01

    Neuron-specific enolase (NSE) and S100 protein are implicated in several brain injuries, including stroke. Our objective was to analyze the temporal profile and the clinical significance of NSE and S-100 in acute ischemic (IS) and intracerebral hemorrhage (ICH). We studied 224 patients with IS and 44 patients with ICH. Computerized tomography (CT) scans were performed to assess infarct volume. Stroke severity was evaluated using the National Institute of Health Stroke Scale (NIHSS), and functional outcome at 3 months with the modified Rankin Scale (mRS). Serum NSE and S100 protein were measured using an electrochemiluminescence-immunoassay. Peak values were found at 72 h for NSE and at 24 h for S100 in IS. For ICH, peak values were found at 24 h for both NSE and S100. At these time intervals S100 and NSE correlated with the NIHSS score and were independently associated with poor outcome. High serum NSE and S100 are associated with poor outcome in IS, and high serum NSE is associated with poor outcome in ICH. These findings suggest the potential utility of NSE and S100 as prognostic markers for acute stroke.

  10. Enolase1 (ENO1) and glucose-6-phosphate isomerase (GPI) are good markers to predict human sperm freezability.

    Science.gov (United States)

    Jiang, Xu-ping; Wang, Shang-qian; Wang, Wei; Xu, Yang; Xu, Zhen; Tang, Jing-yuan; Sun, Hong-yong; Wang, Zeng-jun; Zhang, Wei

    2015-08-01

    Sperm cryopreservation is a method to preserve sperm samples for a long period. However, the fertility of sperm decreases markedly after freezing and thawing in a certain amount of samples. The aim of the present study was to find useful and reliable predictive biomarkers of the capacity to withstand the freeze-thawing process in human ejaculates. Previous researches have shown that enolase1 (ENO1) and glucose-6-phosphate isomerase (GPI) are closely related to spermatozoa quality. We chose the two proteins as probable markers of sperm freezing capacity. Ejaculate samples were separated into good freezability ejaculates (GFE) and poor freezability ejaculates (PFE) according to progressive motility of the sperm after thawing. Before starting cryopreservation protocols, the two proteins from each group were compared using western blot analysis and immunofluorescence. Results showed that normalized content of ENO1 (P<0.05) and GPI (P<0.01) were both significantly higher in GFE than in PFE. The association of ENO1 and GPI with postthaw sperm viability and motility was confirmed using Pearson's linear correlation. In conclusion, ENO1 and GPI can be used as markers of human sperm freezability before starting the cryopreservation procedure. Copyright © 2015. Published by Elsevier Inc.

  11. [Circulating endothelial progenitor cell levels in treated hypertensive patients].

    Science.gov (United States)

    Maroun-Eid, C; Ortega-Hernández, A; Abad, M; García-Donaire, J A; Barbero, A; Reinares, L; Martell-Claros, N; Gómez-Garre, D

    2015-01-01

    Most optimally treated hypertensive patients still have an around 50% increased risk of any cardiovascular event, suggesting the possible existence of unidentified risk factors. In the last years there has been evidence of the essential role of circulating endothelial progenitor cells (EPCs) in the maintenance of endothelial integrity and function, increasing the interest in their involvement in cardiovascular disease. In this study, the circulating levels of EPCs and vascular endothelial growth factor (VEGF) are investigated in treated hypertensive patients with adequate control of blood pressure (BP). Blood samples were collected from treated hypertensive patients with controlled BP. Plasma levels of EPCs CD34+/KDR+ and CD34+/VE-cadherin+ were quantified by flow cytometry. Plasma concentration of VEGF was determined by ELISA. A group of healthy subjects without cardiovascular risk factors was included as controls. A total of 108 hypertensive patients were included (61±12 years, 47.2% men) of which 82.4% showed BP<140/90 mmHg, 91.7% and 81.5% controlled diabetes (HbA1c <7%) and cLDL (<130 or 100 mg/dL), respectively, and 85.2% were non-smokers. Around 45% of them were obese. Although patients had cardiovascular parameters within normal ranges, they showed significantly lower levels of CD34+/KDR+ and CD34+/VE-cadherin+ compared with healthy control group, although plasma VEGF concentration was higher in patients than in controls. Despite an optimal treatment, hypertensive patients show a decreased number of circulating EPCs that could be, at least in part, responsible for their residual cardiovascular risk, suggesting that these cells could be a therapeutic target. Copyright © 2015 SEHLELHA. Published by Elsevier España, S.L.U. All rights reserved.

  12. Immunohistochemical identification of neuron-specific enolase, synaptophysin, chromogranin and endocrine granule constituent in neuroendocrine tumours

    DEFF Research Database (Denmark)

    Vyberg, M; Horn, T; Francis, D

    1990-01-01

    Immunohistochemical identification of neuroendocrine tumour markers in paraffin embedded material from 22 tumours (5 small cell carcinomas of the lung (SCCL), 12 carcinoids, 2 medullary thyroid carcinomas, 2 pheochromocytomas and one paraganglioma) with electron microscopically verified dense...

  13. Amyloid β levels in human red blood cells.

    Directory of Open Access Journals (Sweden)

    Takehiro Kiko

    Full Text Available UNLABELLED: Amyloid β-peptide (Aβ is hypothesized to play a key role by oxidatively impairing the capacity of red blood cells (RBCs to deliver oxygen to the brain. These processes are implicated in the pathogenesis of Alzheimer's disease (AD. Although plasma Aβ has been investigated thoroughly, the presence and distribution of Aβ in human RBCs are still unclear. In this study, we quantitated Aβ40 and Aβ42 in human RBCs with ELISA assays, and provided evidence that significant amounts of Aβ could be detected in RBCs and that the RBC Aβ levels increased with aging. The RBC Aβ levels increased with aging. On the other hand, providing an antioxidant supplement (astaxanthin, a polar carotenoid to humans was found to decrease RBC Aβ as well as oxidative stress marker levels. These results suggest that plasma Aβ40 and Aβ42 bind to RBCs (possibly with aging, implying a pathogenic role of RBC Aβ. Moreover, the data indicate that RBC Aβ40 and Aβ42 may constitute biomarkers of AD. As a preventive strategy, therapeutic application of astaxanthin as an Aβ-lowering agent in RBCs could be considered as a possible anti-dementia agent. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN42483402.

  14. Autophagy Alleviates Melamine-Induced Cell Death in PC12 Cells Via Decreasing ROS Level.

    Science.gov (United States)

    Wang, Hui; Gao, Na; Li, Zhigui; Yang, Zhuo; Zhang, Tao

    2016-04-01

    Since melamine was illegally added to raw milk for increasing the apparent protein content, such a scandal has not been quite blown out. Previous studies showed that melamine induced apoptosis and oxidative damage in both in vivo and in vitro experiments. It is well known that autophagy is closely related to oxidative stress. In the present study, we examined whether autophagy played an important role in protecting PC12 cells, which were damaged by melamine. Immunofluorescence assay showed that melamine enhanced the number of punctuate dot, indicating the increase of autophagosomes. Western blot assay presented that melamine significantly elevated the expression level of autophagy markers including LC3-II/LC3-I ratio, beclin-1, and Atg 7. Rapamycin further enhanced the effect, whereas 3-methyadenine (3-MA) inhibited it. MTT assay exhibited that rapamycin significantly enhanced the cell viability (P PC12 cells (P cells (P PC12 cells (P cells (P PC12 cells from melamine-induced cell death via inhibiting the excessive generation of ROS. Regulating autophagy may become a new targeted therapy to relieve the damage induced by melamine.

  15. Application of low level laser on skin cell lines

    CSIR Research Space (South Africa)

    Ndhundhuma, IM

    2010-01-01

    Full Text Available Lasers have emerged as powerful tools for tissue engineering. To examine cellular growth, and cell to cell interactions, in vitro skin models have been developed combining two major cell types of skin, keratinocytes and fibroblasts. The main...

  16. Available Task-Level Parallelism on the Cell BE

    Directory of Open Access Journals (Sweden)

    Alejandro Rico

    2009-01-01

    Full Text Available There is a clear industrial trend towards chip multiprocessors (CMP as the most power efficient way of further increasing performance. Heterogeneous CMP architectures take one more step along this power efficiency trend by using multiple types of processors, tailored to the workloads they will execute. Programming these CMP architectures has been identified as one of the main challenges in the near future, and programming heterogeneous systems is even more challenging. High-level programming models which allow the programmer to identify parallel tasks, and the runtime management of the inter-task dependencies, have been identified as a suitable model for programming such heterogeneous CMP architectures. In this paper we analyze the performance of Cell Superscalar, a task-based programming model for the Cell Broadband Engine Architecture, in terms of its scalability to higher number of on-chip processors. Our results show that the low performance of the PPE component limits the scalability of some applications to less than 16 processors. Since the PPE has been identified as the limiting element, we perform a set of simulation studies evaluating the impact of out-of-order execution, branch prediction and larger caches on the task management overhead. We conclude that out-of-order execution is a very desirable feature, since it increases task management performance by 50%. We also identify memory latency as a fundamental aspect in performance, while the working set is not that large. We expect a significant performance impact if task management would run using a fast private memory to store the task dependency graph instead of relying on the cache hierarchy.

  17. Identification and characterisation in vitro of cells with a non-SCLC cell-like phenotype derived from a continuous SCLC cell line.

    Science.gov (United States)

    Khan, M Z; Freshney, R I; Murray, A M; Merry, S; Plumb, J A; McNicol, A M

    1991-01-01

    Two adherent sublines, H69V and H69VZ, have been isolated from the classic SCLC cell line NCI-H69. Significant morphological differences were observed between the parental and the derivative cell lines. While NCI-H69 grew as densely packed free floating cellular aggregates the derivative lines grew as a monolayer of epithelioid cells. The growth rates of both the derivative lines were faster than the parental line with doubling times closer to non-SCLC cell lines in the derivative lines. Both H69V and H69VZ either express very low levels or do not express neuroendocrine cell markers including L-dopa-decarboxylase (DDC), creatine kinase-BB isoenzyme (CK-BB), bombesin-like immunoreactivity (BLI), neuron specific enolase (NSE), and neurosecretory type dense core granules (DGCs), compared to the parental cell line. All the lines stained positive for epithelial markers such as CAM5.2. LDH isoenzyme and chromosome analyses confirmed the human origin of all the cell lines. Therefore, it appears that cell line NCI-H69 contains stem cell subpopulation capable of generating cells of both small and non-small cell like phenotypes.

  18. Transformation to small cell lung cancer after first-line afatinib treatment

    Directory of Open Access Journals (Sweden)

    Takayuki Shiroyama

    Full Text Available Acquiring resistance to epidermal growth factor receptor (EGFR-tyrosine kinase inhibitors (TKIs is inevitable. Transformation to small cell lung cancer (SCLC is reported as a possible mechanism of this acquired resistance. We describe the case of a 35-year-old man with lung adenocarcinoma harboring EGFR exon 19 deletion. After 7 months of successful treatment with afatinib, he experienced relapse and rebiopsy revealed SCLC with EGFR exon 19 deletion. Tumor marker tests at this point showed normal levels of serum neuron-specific enolase and pro-gastrin releasing peptide. Our case highlights the importance of rebiopsy for revealing SCLC transformation, a potential mechanism of acquired resistance to afatinib as with other EGFR-TKIs, and normal-range values of tumor markers for SCLC cannot exclude the possibility of SCLC transformation. Keywords: Transformation, Small cell lung cancer, Adenocarcinoma, Rebiopsy, Acquired resistance, Afatinib

  19. Mesenchymal stem cells-derived vascular smooth muscle cells release abundant levels of osteoprotegerin

    Directory of Open Access Journals (Sweden)

    M Vaccarezza

    2009-03-01

    Full Text Available Although several studies have shown that the serum levels of osteoprotegerin (OPG are significantly elevated in patients affected with atherosclerotic lesions in coronary and peripheral arteries, the cellular source and the role of OPG in the physiopathology of atherosclerosis are not completely defined. Therefore, we aimed to investigate the potential contribution of mesenchymal stem cells in the production/release of OPG. OPG was detectable by immunohistochemistry in aortic and coronary atherosclerotic plaques, within or in proximity of intimal vascular smooth muscle cells (SMC. In addition, bone marrow mesenchymal stem cell (MSC-derived vascular SMC as well as primary aortic SMC released in the culture supernatant significantly higher levels of OPG with respect to MSCderived endothelial cells (EC or primary aortic EC. On the other hand, in vitro exposure to full-length human recombinant OPG significantly increased the proliferation rate of aortic SMC cultures, as monitored by bromodeoxyuridine incorporation. Taken together, these data suggest that OPG acts as an autocrine/paracrine growth factor for vascular SMC, which might contribute to the progression of atherosclerotic lesions.

  20. Correlation of low levels of nitrite and high levels of fetal hemoglobin in patients with sickle cell disease at baseline

    Directory of Open Access Journals (Sweden)

    Darcielle Bruna Dias Elias

    2012-01-01

    Full Text Available BACKGROUND: Sickle cell disease is a hemoglobinopathy characterized by hemolytic anemia, increased susceptibility to infections and recurrent vaso-occlusive crises that reduces the quality of life of sufferers. OBJECTIVE: To evaluate the correlation of the levels of lactate dehydrogenase, malonaldehyde and nitrite to fetal hemoglobin in patients with sickle cell disease not under treatment with hydroxyurea in outpatients at a university hospital in Fortaleza, Ceará, Brazil. METHODS: Forty-four patients diagnosed with sickle cell disease were enrolled at baseline. Diagnosis was confirmed by evaluating the beta globin gene using polymerase chain reaction-restriction fragment length polymorphism. The concentration of fetal hemoglobin was obtained by high-performance liquid chromatography. Serum levels of nitrite, malonaldehyde and lactate dehydrogenase were measured by biochemical methods. RESULTS: Significantly higher levels of lactate dehydrogenase, nitrite and malonaldehyde were observed in patients with sickle cell disease compared to a control group. The study of the correlation between fetal hemoglobin levels and these variables showed a negative correlation with nitrite levels. No correlation was found between fetal hemoglobin and malonaldehyde or lactate dehydrogenase. When the study population was stratified according to fetal hemoglobin levels, a decrease in the levels of nitrite was observed with higher levels of fetal hemoglobin (p-value = 0.0415. CONCLUSION: The results show that, similar to fetal hemoglobin levels, the concentration of nitrite can predict the clinical course of the disease, but should not be used alone as a modulator of prognosis in patients with sickle cell disease.

  1. The biomarkers neuron-specific enolase and S100b measured the day following admission for severe accidental hypothermia have high predictive values for poor outcome

    DEFF Research Database (Denmark)

    Wiberg, Sebastian; Kjaergaard, Jesper; Kjærgaard, Benedict

    2017-01-01

    AIM: The aim of the present study was to assess the ability of the biomarkers neuron-specific enolase (NSE) and S100 calcium-binding protein b (S100b) to predict mortality and poor neurologic outcome after 30days in patients admitted with severe accidental hypothermia. METHODS: Consecutive patients...... was analyzed for NSE and S100b. Follow-up was conducted after 30days and poor neurologic outcome was defined as a Cerebral Performance Category (CPC) score of 3-5. The predictive value of NSE and S100b was assessed as the area under the receiver-operating characteristics curve (AUC). RESULTS: A total of 34...

  2. Influences of lamin A levels on induction of pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Bingfeng Zuo

    2012-09-01

    Lamin A is an inner nuclear membrane protein that maintains nuclear structure integrity, is involved in transcription, DNA damage response and genomic stability, and also links to cell differentiation, senescence, premature aging and associated diseases. Induced pluripotent stem (iPS cells have been successfully generated from various types of cells and used to model human diseases. It remains unclear whether levels of lamin A influence reprogramming of somatic cells to pluripotent states during iPS induction. Consistently, lamin A is expressed more in differentiated than in relatively undifferentiated somatic cells, and increases in expression levels with age. Somatic cells with various expression levels of lamin A differ in their dynamics and efficiency during iPS cell induction. Cells with higher levels of lamin A show slower reprogramming and decreased efficiency to iPS cells. Furthermore, depletion of lamin A by transient shRNA accelerates iPS cell induction from fibroblasts. Reduced levels of lamin A are associated with increased expression of pluripotent genes Oct4 and Nanog, and telomerase genes Tert and Terc. On the contrary, overexpression of lamin A retards somatic cell reprogramming to iPS-like colony formation. Our data suggest that levels of lamin A influence reprogramming of somatic cells to pluripotent stem cells and that artificial silencing of lamin A facilitates iPS cell induction. These findings may have implications in enhancing rejuvenation of senescent or older cells by iPS technology and manipulating lamin A levels.

  3. Decoding Signal Processing at the Single-Cell Level

    Energy Technology Data Exchange (ETDEWEB)

    Wiley, H. Steven

    2017-12-01

    The ability of cells to detect and decode information about their extracellular environment is critical to generating an appropriate response. In multicellular organisms, cells must decode dozens of signals from their neighbors and extracellular matrix to maintain tissue homeostasis while still responding to environmental stressors. How cells detect and process information from their surroundings through a surprisingly limited number of signal transduction pathways is one of the most important question in biology. Despite many decades of research, many of the fundamental principles that underlie cell signal processing remain obscure. However, in this issue of Cell Systems, Gillies et al present compelling evidence that the early response gene circuit can act as a linear signal integrator, thus providing significant insight into how cells handle fluctuating signals and noise in their environment.

  4. Comparison of the levels of inositol metabolites in transformed haemopoietic cells and their normal counterparts.

    OpenAIRE

    Bunce, C M; French, P J; Allen, P; Mountford, J C; Moor, B; Greaves, M F; Michell, R H; Brown, G

    1993-01-01

    We have compared the levels of inositol metabolites in three pairs of normal and transformed cells which have been matched with respect to their cell lineage, differentiation and proliferation status: (i) normal human myeloid blast cells and the human promyelocytic leukaemic cell line, HL60; (ii) human umbilical-cord T-helper cells and C8166 cells, a HTLV-1-transformed T-helper cell line; and (iii) an interleukin 3-dependent long-term culture of murine pro-B-cells (BAF3) and BAF3 cells transf...

  5. Drosophila syndecan regulates tracheal cell migration by stabilizing Robo levels

    Science.gov (United States)

    Schulz, Joachim G; Ceulemans, Helga; Caussinus, Emmanuel; Baietti, Maria F; Affolter, Markus; Hassan, Bassem A; David, Guido

    2011-01-01

    Here we identify a new role for Syndecan (Sdc), the only transmembrane heparan sulphate proteoglycan in Drosophila, in tracheal development. Sdc is required cell autonomously for efficient directed migration and fusion of dorsal branch cells, but not for dorsal branch formation per se. The cytoplasmic domain of Sdc is dispensable, indicating that Sdc does not transduce a signal by itself. Although the branch-specific phenotype of sdc mutants resembles those seen in the absence of Slit/Robo2 signalling, genetic interaction experiments indicate that Sdc also helps to suppress Slit/Robo2 signalling. We conclude that Sdc cell autonomously regulates Slit/Robo2 signalling in tracheal cells to guarantee ordered directional migration and branch fusion. PMID:21836636

  6. Low-level radiation effects on immune cells

    International Nuclear Information System (INIS)

    Makinodan, T.

    1995-01-01

    The purpose of this study was to characterize the effects of chronic low-dose ionizing radiation (LDR) on murine immune cells. Previously, it had been reported that LDR enhances the proliferative activity of T cells in vitro and delays the growth of transplantable immunogenic tumors in vivo. This suggests that LDR eliminates immune suppressor cells, which downregulates immune response and/or adoptively upregulates the responsiveness of immune effector cells. It had also been reported that human lymphocytes become refractive to high dose radiation-induced chromosomal aberrations by pretreating mitotically active lymphocytes in vitro with very low doses of ionizing radiation, and the adaptive effect can be abrogated by cycloheximide. This suggests that protein synthesis is required for lymphocytes to respond adoptively to LDR

  7. Hematopoietic stem cell transplant candidate and designated proxy distress levels prior to hematopoietic stem cell transplantation.

    Science.gov (United States)

    Duckworth, Katharine E; Forti, Allison M; Russell, Gregory B; Naik, Seema; Hurd, David D; McQuellon, Richard P

    2014-12-01

    Hematopoietic stem cell transplantation (HCT) is associated with a high risk of morbidity, making advance care planning (ACP) essential. The purpose of this study was to assess and compare proxy and HCT candidate distress levels (Distress Thermometer) before (T1) and after (T2) ACP question completion. 79 participants (40 HCT candidates, 39 proxies) rated their distress. The T1, T2 mean distress scores (SD) for HCT candidates were 3.13(2.27), 2.96(2.10); 43% and 38% endorsed clinically significant distress (≥4). Proxies reported 4.21(2.48), 4.33 (2.46); 62% endorsed significant distress at T1, T2. The majority of proxies endorsed distress levels that were clinically significant and comparatively higher (T1 (p = 0.047) and T2 (p = 0.009)) than their paired HCT recipients. Responding to questions about ACP did not increase overall distress ratings. © The Author(s) 2013.

  8. Levels of hepatic Th17 cells and regulatory T cells upregulated by hepatic stellate cells in advanced HBV-related liver fibrosis.

    Science.gov (United States)

    Li, Xiaoyan; Su, Yujie; Hua, Xuefeng; Xie, Chan; Liu, Jing; Huang, Yuehua; Zhou, Liang; Zhang, Min; Li, Xu; Gao, Zhiliang

    2017-04-11

    Liver fibrosis which mainly occurs upon chronic hepatitis virus infection potentially leads to portal hypertension, hepatic failure and hepatocellular carcinoma. However, the immune status of Th17 and Treg cells in liver fibrosis is controversial and the exact mechanisms remain largely elusive. Liver tissues and peripheral blood were obtained simultaneously from 32 hepatitis B virus infected patients undergoing surgery for hepatocellular carcinoma at the medical center of Sun Yat-sen University. Liver tissues at least 3 cm away from the tumor site were used for the analyses. Levels of Th17 cells and regulatory T cells were detected by flow cytometry analysis and immunohistochemistry. In vitro experiment, we adopted magnetic cell sorting to investigate how hepatic stellate cells regulate the levels of Th17 cells and regulatory T cells. We found that hepatic Th17 cells and regulatory T cells were increased in patients with advanced stage HBV-related liver fibrosis. Hepatic stellate cells upregulated the levels of Th17 cells and regulatory T cells via PGE2/EP2 and EP4 pathway. We found that the increased levels of Th17 cells and regulatory T cells were upregulated by hepatic stellate cells. These results may provide insight into the role of hepatic stellate cells and Th17 cells and regulatory T cells in the persistence of fibrosis and into the occurrence of hepatocellular carcinoma following cirrhosis.

  9. Levels Of Haemoglobin And Packed Cell Volumes In Umbilical Cord ...

    African Journals Online (AJOL)

    The Hb concentration was determined by the indirect colorimetric method, and the PCV by the microhaematocrit centrifuge method. The mean values obtained were Hb 12.13 ± 0.11 g/dl and PCV 37.95 ± 0.49%. The mean umbilical cord blood haemoglobin and packed cell volumes were compared with those of the mothers ...

  10. Restoration of Mitochondrial NAD+Levels Delays Stem Cell Senescence and Facilitates Reprogramming of Aged Somatic Cells.

    Science.gov (United States)

    Son, Myung Jin; Kwon, Youjeong; Son, Taekwon; Cho, Yee Sook

    2016-12-01

    The fundamental tenet that aging is irreversible has been challenged by the development of reprogramming technology that can restore molecular and cellular age by reversing the progression of aging. The use of cells from aged individuals as sources for reprogramming or transplantation creates a major barrier in stem cell therapy with respect to cell quality and quantity. Here, we investigated the molecular features underlying senescence and rejuvenation during aged cell reprogramming and identified novel factors that can overcome age-associated barriers. Enzymes, such as nicotinamide nucleotide transhydrogenase (NNT) and nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3), that control mitochondrial NAD + levels appear to be susceptible to aging. In aged cells, mitochondrial NAD + levels decrease, accompanied by reduced SIRT3 activity; these changes severely impede cell fate transition. However, in cells collected from aged p16 knockout mice, which exhibit delayed cellular senescence, no changes in NNT or NMNAT3 expression were found. Importantly, restoring mitochondrial NAD + levels by overexpressing NNT and NMNAT3 enhanced reprogramming efficiency of aged somatic cells and extended the lifespan of human mesenchymal stem cells by delaying replicative senescence. These results demonstrate that maintenance of mitochondrial NAD + levels is critical for reversing the mechanisms of aging and ensuring that cells collected from aged individuals are of high quality. Stem Cells 2016;34:2840-2851. © 2016 AlphaMed Press.

  11. Distinct gene expression signatures in human embryonic stem cells differentiated towards definitive endoderm at single-cell level

    DEFF Research Database (Denmark)

    Norrman, Karin; Strömbeck, Anna; Semb, Henrik

    2013-01-01

    Characterization of directed differentiation of pluripotent stem cells towards therapeutically relevant cell types, including pancreatic beta-cells and hepatocytes, depends on molecular markers and assays that resolve the signature of individual cells. Pancreas and liver both have a common origin...... for the three activin A based protocols applied. Our data provide novel insights in DE gene expression at the cellular level of in vitro differentiated human embryonic stem cells, and illustrate the power of using single-cell gene expression profiling to study differentiation heterogeneity and to characterize...

  12. Neuron-Specific Enolase as a Predictor of Death or Poor Neurological Outcome After Out-of-Hospital Cardiac Arrest and Targeted Temperature Management at 33°C and 36°C

    DEFF Research Database (Denmark)

    Stammet, Pascal; Collignon, Olivier; Hassager, Christian

    2015-01-01

    BACKGROUND: Neuron-specific enolase (NSE) is a widely-used biomarker for prognostication of neurological outcome after cardiac arrest, but the relevance of recommended cutoff values has been questioned due to the lack of a standardized methodology and uncertainties over the influence of temperatu...

  13. Elevated level of polysaccharides in a high level UV-B tolerant cell ...

    African Journals Online (AJOL)

    Jane

    2011-04-26

    Apr 26, 2011 ... Key words: Bupleurum scorzonerifolium Willd, polysaccharides, in vitro culture, UV-B tolerance, antioxidant. ..... The number of calli formed from protoplasts of the anti-UV-B cell line after different generation of subculture. Figure 4. ..... Tanaka A, Sakamoto A, Ishigaki Y, Nikaido O, Sun G, Hase Y, Shikazono.

  14. Sustained levels of FGF2 maintain undifferentiated stem cell cultures with biweekly feeding.

    Directory of Open Access Journals (Sweden)

    Steven Lotz

    Full Text Available An essential aspect of stem cell culture is the successful maintenance of the undifferentiated state. Many types of stem cells are FGF2 dependent, and pluripotent stem cells are maintained by replacing FGF2-containing media daily, while tissue-specific stem cells are typically fed every 3rd day. Frequent feeding, however, results in significant variation in growth factor levels due to FGF2 instability, which limits effective maintenance due to spontaneous differentiation. We report that stabilization of FGF2 levels using controlled release PLGA microspheres improves expression of stem cell markers, increases stem cell numbers and decreases spontaneous differentiation. The controlled release FGF2 additive reduces the frequency of media changes needed to maintain stem cell cultures, so that human embryonic stem cells and induced pluripotent stem cells can be maintained successfully with biweekly feedings.

  15. Cell-free fetal DNA and cell-free total DNA levels in spontaneous abortion with fetal chromosomal aneuploidy.

    Directory of Open Access Journals (Sweden)

    Ji Hyae Lim

    Full Text Available Cell-free fetal DNA and cell-free total DNA in maternal circulation have been proposed as potential markers for noninvasive monitoring of the placental condition during the pregnancy. However, the correlation of and change in cell-free fetal DNA and cell-free total DNA in spontaneous abortion (SA with fetal chromosomal aneuploidy have not yet been reported. Therefore, we investigated cell-free fetal DNA and cell-free total DNA levels in SA women with fetal chromosomal aneuploidy.A nested case-control study was conducted with maternal plasma collected from 268 women in their first trimester of pregnancy. Subjects included 41 SA with normal fetal karyotype, 26 SA with fetal chromosomal aneuploidy, and 201 normal controls. The unmethylated PDE9A gene was used to measure the maternal plasma levels of cell-free fetal DNA. The GAPDH gene was used to measure the maternal plasma levels of cell-free total DNA. The diagnostic accuracy was measured using receiver-operating characteristic (ROC curves. Levels of cell-free fetal DNA and cell-free total DNA were significantly higher in both SA women with normal fetal karyotype and SA women with fetal chromosomal aneuploidy in comparison with the normal controls (P<0.001 in both. The correlation between cell-free fetal DNA and cell-free total DNA levels was stronger in the normal controls (r = 0.843, P<0.001 than in SA women with normal karyotype (r = 0.465, P = 0.002 and SA women with fetal chromosomal aneuploidy (r = 0.412, P = 0.037. The area under the ROC curve for cell-free fetal DNA and cell-free total DNA was 0.898 (95% CI, 0.852-0.945 and 0.939 (95% CI, 0.903-0.975, respectively.Significantly high levels of cell-free fetal DNA and cell-free total DNA were found in SA women with fetal chromosomal aneuploidy. Our findings suggest that cell-free fetal DNA and cell-free total DNA may be useful biomarkers for the prediction of SA with fetal chromosomal aneuploidy, regardless of fetal

  16. The expression of SLAMF7 levels in malignant B cells: a novel ...

    African Journals Online (AJOL)

    Signalling lymphocyte activation molecule (SLAM) F7 is found on the surface of some immune cells including B-lymphocytes. Its activation leads to the proliferation or differentiation of immune cells. The objectives of the study were to measure SLAMF7 expression levels on B-CLL cells, and to upregulate the expression of ...

  17. Dose-dependent regulation of target gene expression and cell proliferation by c-Myc levels

    OpenAIRE

    Schuhmacher, Marino; Eick, Dirk

    2013-01-01

    The proto-oncogene c-myc encodes a basic helix-loop-helix leucine zipper transcription factor (c-Myc). c-Myc plays a crucial role in cell growth and proliferation. Here, we examined how expression of c-Myc target genes and cell proliferation depend on variation of c-Myc protein levels. We show that proliferation rates, the number of cells in S-phase, and cell size increased in a dose-dependent manner in response to increasing c-Myc levels. Likewise, the mRNA levels of c-Myc responsive genes s...

  18. Layout level design for testability strategy applied to a CMOS cell library

    NARCIS (Netherlands)

    Blom, F.C.; Oliver, J.; Rullan, M.; Ferrer, C.

    1993-01-01

    The layout level design for testability (LLDFT) rules used here allow to avoid some hard to detect faults or even undetectable faults on a cell library by modifying the cell layout without changing their behavior and achieving a good level of reliability. These rules avoid some open faults or reduce

  19. Different roles of prepubertal and postpubertal germ cells and Sertoli cells in the regulation of serum inhibin B levels

    DEFF Research Database (Denmark)

    Andersson, A M; Müller, J; Skakkebaek, N E

    1998-01-01

    To elucidate the role of germ cells in the regulation of inhibin B secretion, serum inhibin B levels in prepubertal boys and adult men whom had a concurrent testicular biopsy showing either normal or impaired testicular function were compared. In addition, by immunohistochemistry the cellular loc...... were expressed by different cell types. We speculate that during puberty Sertoli cell maturation induces a change in inhibin subunit expression. Thus, immature Sertoli cells express both alpha and betaB inhibin subunits, whereas fully differentiated Sertoli cells only express the alpha...

  20. Analysis of the inventory level in the production cell

    Directory of Open Access Journals (Sweden)

    2010-09-01

    Full Text Available Simulation in production management is becoming increasingly important for efficient and competitive manufacturing. Production planners have to focus on several main goals: scheduling the plan efficiently, keeping inventories low, satisfying customer demand and anticipating the impact of every decision on the supply chain. They should understand how much inventory is needed to support demand during the production cycle and how much is needed to support a desired level of customer service. They should know how to reshape plans smoothly and responsively, without breaking the rhythm of the plan. And they should be able to visualize the impact of a planning decision on the entire supply chain. But before making decisions about management of the production they have to organize manufacturing system. In this article we would like to present example how to organize the level of stock to shorten the time of executing orders and satisfy the needs of any given customer by using simulations in software application Arena.

  1. Modular Approach for Continuous Cell-Level Balancing to Improve Performance of Large Battery Packs: Preprint

    Energy Technology Data Exchange (ETDEWEB)

    Muneed ur Rehman, M.; Evzelman, M.; Hathaway, K.; Zane, R.; Plett, G. L.; Smith, K.; Wood, E.; Maksimovic, D.

    2014-10-01

    Energy storage systems require battery cell balancing circuits to avoid divergence of cell state of charge (SOC). A modular approach based on distributed continuous cell-level control is presented that extends the balancing function to higher level pack performance objectives such as improving power capability and increasing pack lifetime. This is achieved by adding DC-DC converters in parallel with cells and using state estimation and control to autonomously bias individual cell SOC and SOC range, forcing healthier cells to be cycled deeper than weaker cells. The result is a pack with improved degradation characteristics and extended lifetime. The modular architecture and control concepts are developed and hardware results are demonstrated for a 91.2-Wh battery pack consisting of four series Li-ion battery cells and four dual active bridge (DAB) bypass DC-DC converters.

  2. Low-level lasers alter mRNA levels from traditional reference genes used in breast cancer cells

    Science.gov (United States)

    Teixeira, A. F.; Canuto, K. S.; Rodrigues, J. A.; Fonseca, A. S.; Mencalha, A. L.

    2017-07-01

    Cancer is among the leading causes of mortality worldwide, increasing the importance of treatment development. Low-level lasers are used in several diseases, but some concerns remains on cancers. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a technique used to understand cellular behavior through quantification of mRNA levels. Output data from target genes are commonly relative to a reference that cannot vary according to treatment. This study evaluated reference genes levels from MDA-MB-231 cells exposed to red or infrared lasers at different fluences. Cultures were exposed to red and infrared lasers, incubated (4 h, 37 °C), total RNA was extracted and cDNA synthesis was performed to evaluate mRNA levels from ACTB, GUSB and TRFC genes by RT-qPCR. Specific amplification was verified by melting curves and agarose gel electrophoresis. RefFinder enabled data analysis by geNorm, NormFinder and BestKeeper. Specific amplifications were obtained and, although mRNA levels from ACTB, GUSB or TRFC genes presented no significant variation through traditional statistical analysis, Excel-based tools revealed that the use of these reference genes are dependent of laser characteristics. Our data showed that exposure to low-level red and infrared lasers at different fluences alter the mRNA levels from ACTB, GUSB and TRFC in MDA-MB-231 cells.

  3. Effects of physiological levels of the green tea extract Epigallocatechin gallate (EGCG on breast cancer cells

    Directory of Open Access Journals (Sweden)

    Li eZeng

    2014-05-01

    Full Text Available Physiological concentrations of the green tea extract EGCG caused growth inhibition in oestrogen receptor α (ERα-positive MCF7 cells, that was associated with down-regulation of the ERα and reduced insulin-like growth factor (IGF binding protein-2 (IGFBP-2 abundance and increased protein abundance of the tumour suppressor genes p53/p21. In contrast to MCF7 cells that have wt p53, EGCG alone did not change cell proliferation or death significantly in another ERα-positive cell line T47D that possesses mutant p53. EGCG increased ERα protein levels and as a consequence, the cells responded significantly better to an ERα antagonist Tamoxifen (TAM in the presence of EGCG. EGCG significantly increased cell death in an ERα-negative cell line, MDA-MB-231 that also possesses mutant p53. EGCG significantly increased the ERα and IGF-I receptor (IGF-IR levels and thereby enhanced the sensitivities of the cells to Tamoxifen and a blocking antibody targeting the IGF-1R (αIR3. In contrast to MCF7, T47D and MDA-MB-231 breast cancer cells that exhibited significant changes in key molecules involved in breast growth and survival upon treatment with physiological levels of EGCG, the growth, survival and levels of these proteins in non-malignant breast epithelial cells, MCF10A cells, were not affected.

  4. The effect of splenectomy on the levels of PCV-13-induced memory B- and T cells.

    Science.gov (United States)

    Gazi, Umut; Karasartova, Djursun; Sahiner, Ibrahim Tayfun; Gureser, Ayse Semra; Tosun, Ozgur; Derici, Mehmet Kursat; Dolapci, Mete; Taylan Ozkan, Aysegul

    2018-03-13

    Splenectomised patients are associated with lifelong risk of fatal overwhelming post-splenectomy infection (OPSI), which is mostly caused by Streptococcus pneumoniae. Today OPSI cases can still be reported even in patients with appropriate vaccination. In our study, the levels of vaccine-specific memory B- and T cells were compared between control and splenectomised patients to enlighten the underlying reason. Five healthy and 14 post-traumatic splenectomised individuals were vaccinated with 13-valent pneumococcal conjugate vaccine (PCV-13) followed by 23-valent pneumococcal polysaccharide vaccine (PPV-23). The levels of memory B- and T cells were compared by ELISPOT analysis. Splenectomised patients generated reduced levels of memory IgG B cells in response to PCV-13 vaccination, while the memory IFN-γ T-cell levels were undetectable in asplenic patients. This was despite the detection of vaccine-induced memory T-cell levels in control patients, which were analysed simultaneously following the same experimental protocol. Our results suggest that spleen is important, but not essential, for survival and/or generation of memory IgG B cells. In contrast, it seems to be indispensable for PCV-13-specific memory T H 1-cell levels. Studies enhancing the levels of vaccine-induced memory cells and further enlightening the immune responses in asplenic individuals are required to develop more effective vaccination strategies against OPSI. © 2018 John Wiley & Sons Ltd.

  5. Identifying EGFR-Expressed Cells and Detecting EGFR Multi-Mutations at Single-Cell Level by Microfluidic Chip

    Science.gov (United States)

    Li, Ren; Zhou, Mingxing; Li, Jine; Wang, Zihua; Zhang, Weikai; Yue, Chunyan; Ma, Yan; Peng, Hailin; Wei, Zewen; Hu, Zhiyuan

    2018-03-01

    EGFR mutations companion diagnostics have been proved to be crucial for the efficacy of tyrosine kinase inhibitor targeted cancer therapies. To uncover multiple mutations occurred in minority of EGFR-mutated cells, which may be covered by the noises from majority of un-mutated cells, is currently becoming an urgent clinical requirement. Here we present the validation of a microfluidic-chip-based method for detecting EGFR multi-mutations at single-cell level. By trapping and immunofluorescently imaging single cells in specifically designed silicon microwells, the EGFR-expressed cells were easily identified. By in situ lysing single cells, the cell lysates of EGFR-expressed cells were retrieved without cross-contamination. Benefited from excluding the noise from cells without EGFR expression, the simple and cost-effective Sanger's sequencing, but not the expensive deep sequencing of the whole cell population, was used to discover multi-mutations. We verified the new method with precisely discovering three most important EGFR drug-related mutations from a sample in which EGFR-mutated cells only account for a small percentage of whole cell population. The microfluidic chip is capable of discovering not only the existence of specific EGFR multi-mutations, but also other valuable single-cell-level information: on which specific cells the mutations occurred, or whether different mutations coexist on the same cells. This microfluidic chip constitutes a promising method to promote simple and cost-effective Sanger's sequencing to be a routine test before performing targeted cancer therapy.[Figure not available: see fulltext.

  6. Detection of circulating tumour cells on mRNA levels with established breast cancer cell lines.

    Science.gov (United States)

    Zebisch, Michael; Kölbl, Alexandra C; Andergassen, Ulrich; Hutter, Stephan; Neugebauer, Julia; Engelstädter, Verena; Günthner-Biller, Maria; Jeschke, Udo; Friese, Klaus; Rack, Brigitte

    2013-03-01

    Circulating tumour cells were detected and quantified by real-time polymerase chain reaction (PCR) in peripheral blood, based on the fact that the expression of certain genes is upregulated in tumour tissues in comparison to surrounding blood cells. Calibration curves showing gene expression as functions of the number of tumour cells within a blood sample were prepared. Blood samples were therefore spiked with cells of breast cancer cell lines, RNA was extracted, transcribed to complementary DNA (cDNA) and used in real-time PCR reaction on the Cytokeratins (CK) 8, 18 and 19. Calibration curves were generated by Microsoft™ Excel®. Relative quantification curves of gene expression in different breast cancer cell lines showed no unitary tendencies. The oscillations in the relative quantification curves of gene expression suggested an occurrence of immunological effects, leading to an apparent agglutination of added tumour cells together with the blood cells of the sample. Thus, strategies to obtain evaluable results should be considered.

  7. Low-level laser effects on simulated orthodontic tension side periodontal ligament cells.

    Science.gov (United States)

    Huang, Tsui-Hsien; Liu, Shiau-Lee; Chen, Chih-Lin; Shie, Ming-You; Kao, Chia-Tze

    2013-02-01

    The purpose of this study was to analyze proliferation, inflammation, and osteogenic effects on periodontal ligament (PDL) cells after low-level laser therapy (LLLT) under simulated orthodontic tension conditions. Low-level lasers affect fibroblast proliferation and collagen synthesis and reduce inflammation. Few studies have focused on the LLLT changes in the PDL caused by moving teeth. A human PDL cell line was cultured in a -100 kPa tension incubator. The PDL cells were treated with a 670 nm low-level diode laser, output power of 500 mW (continuous wave modus) for 2.5 or 5 sec, spot area 0.25 cm(2), corresponding to 1.25 and 2.5 J at an energy density of 5 or 10 J/cm(2), respectively. PDL cell viability was assayed by detecting the ability of the cells to cleave tetrazolium salt to formazan dye. Inflammation and osteogenic markers were analyzed by Western blot analysis. PDL cell viablity increased in the experimental group, based on the ability of the cells to cleave tetrazolium salt at day 7 (posteocalcin (OC) expression level was significantly higher at day 7 (p<0.05) than in the control cells. LLLT significantly increased PDL cell proliferation, decreased PDL cell inflammation, and increased PDL OC activity under the tension conditions used in this study.

  8. Changes in cytokine levels and NK cell activation associated with influenza.

    Directory of Open Access Journals (Sweden)

    Stephanie Jost

    Full Text Available Several studies have highlighted the important role played by murine natural killer (NK cells in the control of influenza infection. However, human NK cell responses in acute influenza infection, including infection with the 2009 pandemic H1N1 influenza virus, are poorly documented. Here, we examined changes in NK cell phenotype and function and plasma cytokine levels associated with influenza infection and vaccination. We show that absolute numbers of peripheral blood NK cells, and particularly those of CD56(bright NK cells, decreased upon acute influenza infection while this NK cell subset expanded following intramuscular influenza vaccination. NK cells exposed to influenza antigens were activated, with higher proportions of NK cells expressing CD69 in study subjects infected with seasonal influenza strains. Vaccination led to increased levels of CD25+ NK cells, and notably CD56(bright CD25+ NK cells, whereas decreased amounts of this subset were present in the peripheral blood of influenza infected individuals, and predominantly in study subjects infected with the 2009 pandemic H1N1 influenza virus. Finally, acute influenza infection was associated with low plasma concentrations of inflammatory cytokines, including IFN-γ, MIP-1β, IL-2 and IL-15, and high levels of the anti-inflammatory cytokines IL-10 and IL-1ra. Altogether, these data suggest a role for the CD56(bright NK cell subset in the response to influenza, potentially involving their recruitment to infected tissues and a local production and/or uptake of inflammatory cytokines.

  9. Low-level laser irradiation induces in vitro proliferation of mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Barboza, Carlos Augusto Galvão; Ginani, Fernanda [Universidade Federal do Rio Grande do Norte, Natal, RN (Brazil); Soares, Diego Moura [Universidade Federal de Pernambuco, Recife, PE (Brazil); Henriques, Águida Cristina Gomes; Freitas, Roseana de Almeida [Universidade Federal do Rio Grande do Norte, Natal, RN (Brazil)

    2014-07-01

    To evaluate the effect of low-level laser irradiation on the proliferation and possible nuclear morphological changes of mouse mesenchymal stem cells. Mesenchymal stem cells derived from bone marrow and adipose tissue were submitted to two applications (T0 and T48 hours) of low-level laser irradiation (660nm; doses of 0.5 and 1.0J/cm{sup 2}). The trypan blue assay was used to evaluate cell viability, and growth curves were used to analyze proliferation at zero, 24, 48, and 72 hours. Nuclear alterations were evaluated by staining with DAPI (4'-6-diamidino-2-phenylindole) at 72 hours. Bone marrow-derived mesenchymal stem cells responded to laser therapy in a dose-dependent manner. Higher cell growth was observed when the cells were irradiated with a dose of 1.0J/cm{sup 2}, especially after 24 hours (p<0.01). Adipose-derived mesenchymal stem cells responded better to a dose of 1.0J/cm{sup 2}, but higher cell proliferation was observed after 48 hours (p<0.05) and 72 hours (p<0.01). Neither nuclear alterations nor a significant change in cell viability was detected in the studied groups. Low-level laser irradiation stimulated the proliferation of mouse mesenchymal stem cells without causing nuclear alterations. The biostimulation of mesenchymal stem cells using laser therapy might be an important tool for regenerative therapy and tissue engineering.

  10. Low-level laser irradiation induces in vitro proliferation of mesenchymal stem cells

    International Nuclear Information System (INIS)

    Barboza, Carlos Augusto Galvão; Ginani, Fernanda; Soares, Diego Moura; Henriques, Águida Cristina Gomes; Freitas, Roseana de Almeida

    2014-01-01

    To evaluate the effect of low-level laser irradiation on the proliferation and possible nuclear morphological changes of mouse mesenchymal stem cells. Mesenchymal stem cells derived from bone marrow and adipose tissue were submitted to two applications (T0 and T48 hours) of low-level laser irradiation (660nm; doses of 0.5 and 1.0J/cm 2 ). The trypan blue assay was used to evaluate cell viability, and growth curves were used to analyze proliferation at zero, 24, 48, and 72 hours. Nuclear alterations were evaluated by staining with DAPI (4'-6-diamidino-2-phenylindole) at 72 hours. Bone marrow-derived mesenchymal stem cells responded to laser therapy in a dose-dependent manner. Higher cell growth was observed when the cells were irradiated with a dose of 1.0J/cm 2 , especially after 24 hours (p<0.01). Adipose-derived mesenchymal stem cells responded better to a dose of 1.0J/cm 2 , but higher cell proliferation was observed after 48 hours (p<0.05) and 72 hours (p<0.01). Neither nuclear alterations nor a significant change in cell viability was detected in the studied groups. Low-level laser irradiation stimulated the proliferation of mouse mesenchymal stem cells without causing nuclear alterations. The biostimulation of mesenchymal stem cells using laser therapy might be an important tool for regenerative therapy and tissue engineering

  11. Intercellular Variability in Protein Levels from Stochastic Expression and Noisy Cell Cycle Processes.

    Directory of Open Access Journals (Sweden)

    Mohammad Soltani

    2016-08-01

    Full Text Available Inside individual cells, expression of genes is inherently stochastic and manifests as cell-to-cell variability or noise in protein copy numbers. Since proteins half-lives can be comparable to the cell-cycle length, randomness in cell-division times generates additional intercellular variability in protein levels. Moreover, as many mRNA/protein species are expressed at low-copy numbers, errors incurred in partitioning of molecules between two daughter cells are significant. We derive analytical formulas for the total noise in protein levels when the cell-cycle duration follows a general class of probability distributions. Using a novel hybrid approach the total noise is decomposed into components arising from i stochastic expression; ii partitioning errors at the time of cell division and iii random cell-division events. These formulas reveal that random cell-division times not only generate additional extrinsic noise, but also critically affect the mean protein copy numbers and intrinsic noise components. Counter intuitively, in some parameter regimes, noise in protein levels can decrease as cell-division times become more stochastic. Computations are extended to consider genome duplication, where transcription rate is increased at a random point in the cell cycle. We systematically investigate how the timing of genome duplication influences different protein noise components. Intriguingly, results show that noise contribution from stochastic expression is minimized at an optimal genome-duplication time. Our theoretical results motivate new experimental methods for decomposing protein noise levels from synchronized and asynchronized single-cell expression data. Characterizing the contributions of individual noise mechanisms will lead to precise estimates of gene expression parameters and techniques for altering stochasticity to change phenotype of individual cells.

  12. Unique responses of stem cell-derived vascular endothelial and mesenchymal cells to high levels of glucose.

    Directory of Open Access Journals (Sweden)

    Emily Keats

    Full Text Available Diabetes leads to complications in selected organ systems, and vascular endothelial cell (EC dysfunction and loss is the key initiating and perpetuating step in the development of these complications. Experimental and clinical studies have shown that hyperglycemia leads to EC dysfunction in diabetes. Vascular stem cells that give rise to endothelial progenitor cells (EPCs and mesenchymal progenitor cells (MPCs represent an attractive target for cell therapy for diabetic patients. Whether these vascular stem/progenitor cells succumb to the adverse effects of high glucose remains unknown. We sought to determine whether adult vascular stem/progenitor cells display cellular activation and dysfunction upon exposure to high levels of glucose as seen in diabetic complications. Mononuclear cell fraction was prepared from adult blood and bone marrow. EPCs and MPCs were derived, characterized, and exposed to either normal glucose (5 mmol/L or high glucose levels (25 mmol/L. We then assayed for cell activity and molecular changes following both acute and chronic exposure to high glucose. Our results show that high levels of glucose do not alter the derivation of either EPCs or MPCs. The adult blood-derived EPCs were also resistant to the effects of glucose in terms of growth. Acute exposure to high glucose levels increased caspase-3 activity in EPCs (1.4x increase and mature ECs (2.3x increase. Interestingly, MPCs showed a transient reduction in growth upon glucose challenge. Our results also show that glucose skews the differentiation of MPCs towards the adipocyte lineage while suppressing other mesenchymal lineages. In summary, our studies show that EPCs are resistant to the effects of high levels of glucose, even following chronic exposure. The findings further show that hyperglycemia may have detrimental effects on the MPCs, causing reduced growth and altering the differentiation potential.

  13. Tools for Genomic and Transcriptomic Analysis of Microbes at Single-Cell Level

    Directory of Open Access Journals (Sweden)

    Zixi Chen

    2017-09-01

    Full Text Available Microbiologists traditionally study population rather than individual cells, as it is generally assumed that the status of individual cells will be similar to that observed in the population. However, the recent studies have shown that the individual behavior of each single cell could be quite different from that of the whole population, suggesting the importance of extending traditional microbiology studies to single-cell level. With recent technological advances, such as flow cytometry, next-generation sequencing (NGS, and microspectroscopy, single-cell microbiology has greatly enhanced the understanding of individuality and heterogeneity of microbes in many biological systems. Notably, the application of multiple ‘omics’ in single-cell analysis has shed light on how individual cells perceive, respond, and adapt to the environment, how heterogeneity arises under external stress and finally determines the fate of the whole population, and how microbes survive under natural conditions. As single-cell analysis involves no axenic cultivation of target microorganism, it has also been demonstrated as a valuable tool for dissecting the microbial ‘dark matter.’ In this review, current state-of-the-art tools and methods for genomic and transcriptomic analysis of microbes at single-cell level were critically summarized, including single-cell isolation methods and experimental strategies of single-cell analysis with NGS. In addition, perspectives on the future trends of technology development in the field of single-cell analysis was also presented.

  14. Comparing the level of bystander effect in a couple of tumor and normal cell lines

    OpenAIRE

    Soleymanifard, Shokouhozaman; Bahreyni, Mohammad T. Toossi

    2012-01-01

    Radiation-induced bystander effect refers to radiation responses which occur in non-irradiated cells. The purpose of this study was to compare the level of bystander effect in a couple of tumor and normal cell lines (QU-DB and MRC5). To induce bystander effect, cells were irradiated with 0.5, 2, and 4 Gy of 60Co gamma rays and their media were transferred to non-irradiated (bystander) cells of the same type. Cells containing micronuclei were counted in bystander subgroups, non-irradiated, and...

  15. Hydrogen peroxide production is affected by oxygen levels in mammalian cell culture.

    Science.gov (United States)

    Maddalena, Lucas A; Selim, Shehab M; Fonseca, Joao; Messner, Holt; McGowan, Shannon; Stuart, Jeffrey A

    2017-11-04

    Although oxygen levels in the extracellular space of most mammalian tissues are just a few percent, under standard cell culture conditions they are not regulated and are often substantially higher. Some cellular sources of reactive oxygen species, like NADPH oxidase 4, are sensitive to oxygen levels in the range between 'normal' physiological (typically 1-5%) and standard cell culture (up to 18%). Hydrogen peroxide in particular participates in signal transduction pathways via protein redox modifications, so the potential increase in its production under standard cell culture conditions is important to understand. We measured the rates of cellular hydrogen peroxide production in some common cell lines, including C2C12, PC-3, HeLa, SH-SY5Y, MCF-7, and mouse embryonic fibroblasts (MEFs) maintained at 18% or 5% oxygen. In all instances the rate of hydrogen peroxide production by these cells was significantly greater at 18% oxygen than at 5%. The increase in hydrogen peroxide production at higher oxygen levels was either abolished or substantially reduced by treatment with GKT 137831, a selective inhibitor of NADPH oxidase subunits 1 and 4. These data indicate that oxygen levels experienced by cells in culture influence hydrogen peroxide production via NADPH oxidase 1/4, highlighting the importance of regulating oxygen levels in culture near physiological values. However, we measured pericellular oxygen levels adjacent to cell monolayers under a variety of conditions and with different cell lines and found that, particularly when growing at 5% incubator oxygen levels, pericellular oxygen was often lower and variable. Together, these observations indicate the importance, and difficulty, of regulating oxygen levels experienced by cells in culture. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Foetal Leydig cells and the neuroendocrine system.

    Science.gov (United States)

    Sklebar, Duska; Semanjski, Kristina; Kos, Marina; Sklebar, Ivan; Jezek, Davor

    2008-01-01

    It has been shown that adult human Leydig cells express a number of neuroendocrine markers, and, therefore, could be considered as a part of the neuroendocrine system in the adult. A limited number of studies have dealt with the dynamics of development of human foetal Leydig cells, whereas studies on their neuroendocrine nature are still extremely rare. Therefore, the aim of our study was to investigate the development of human foetal Leydig cells in different weeks of gestation (wg) and to check if these cells express certain markers characteristic of the diffuse neuroendocrine system (DNS). Qualitative, quantitative histological studies and immunohistochemical analyses of human foetal testicular tissue have been performed. According to our data, Leydig cells formed a dynamic population of cells within the interstitum of testes in the period between 15 and 36 wg. The total number of Leydig cells of human foetal testes changed through different stages of gestation by means of 'pulsatile' dynamics (most likely, by following the variable level of gonadotropins). At early stages of development (15-17 wg) immunohistochemical reactions for the expression of neuron specific enolase (NSE) were positive within sex cords and between them, in the interstitum. Pro-spermatogonia in the sex cords were positive, as well as elongated spindle-shaped cells of the interstitum (very likely, precursors of Leydig cells). During the later stages of development (28-36 wg), excluding the pro-spermatogonia, the interstitial Leydig cells were also positive. The results of the immunohistochemical analyses (the expression of NSE) confirmed the hypothesis that human foetal Leydig cells were of neuroendocrine nature.

  17. Radiation level analysis for the port cell of the ITER electron cyclotron-heating upper launcher

    Energy Technology Data Exchange (ETDEWEB)

    Weinhorst, Bastian, E-mail: bastian.weinhorst@kit.edu [KIT, Institute for Neutron Physics and Reactor Technology, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen (Germany); Fischer, Ulrich; Lu, Lei [KIT, Institute for Neutron Physics and Reactor Technology, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen (Germany); Strauss, Dirk; Spaeh, Peter; Scherer, Theo [KIT, Institute for Applied Materials, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen (Germany); Leichtle, Dieter [F4E, Analysis & Codes/Technical Support Services, Josep Pla 2, Torres Diagonal Litoral B3, 08019 Barcelona (Spain)

    2016-11-01

    Highlights: • First detailed neutronic modelling of the ECHUL port cell with ECHUL equipment (including beam lines with diamond windows, the beam lines mounting box, conduit boxes and rails). • Three different bioshield port plug configurations and two different neutron source configurations are investigated. • Radiation Levels are calculated in the port cell, focusing on the position of the diamond window. • The dose rate in the port cell is below the limit for maintenance in the port cell. • The radiation level at the diamond window is very low and should not influence its performance. - Abstract: The electron cyclotron-heating upper launcher (ECHUL) will be installed in four upper ports of the ITER tokamak thermonuclear fusion reactor. Each ECHUL is able to deposit 8 MW power into the plasma for plasma mode stabilization via microwave beam lines. An essential part of these beam lines are the diamond windows. They are located in the upper port cell behind the bioshield to reduce the radiation levels to a minimum. The paper describes the first detailed neutronic modelling of the ECHUL port cell with ECHUL equipment. The bioshield plug is modelled including passageways for the microwave beam lines, piping and cables looms as well as rails and openings for ventilation. The port cell is equipped with the beam lines including the diamond windows, the beam lines mounting box, conduit boxes and rails. The neutrons are transported into the port cell starting from a surface source in front of the bioshield. Neutronic results are obtained for radiation levels in the port cell at different positions, mainly focusing on the diamond windows position. It is shown that the radiation level is below the limit for maintenance in the port cell. The radiation level at the diamond window is very low and should not influence its performance.

  18. Vitamin E-Mediated Modulation of Glutamate Receptor Expression in an Oxidative Stress Model of Neural Cells Derived from Embryonic Stem Cell Cultures

    Directory of Open Access Journals (Sweden)

    Afifah Abd Jalil

    2017-01-01

    Full Text Available Glutamate is the primary excitatory neurotransmitter in the central nervous system. Excessive concentrations of glutamate in the brain can be excitotoxic and cause oxidative stress, which is associated with Alzheimer’s disease. In the present study, the effects of vitamin E in the form of tocotrienol-rich fraction (TRF and alpha-tocopherol (α-TCP in modulating the glutamate receptor and neuron injury markers in an in vitro model of oxidative stress in neural-derived embryonic stem (ES cell cultures were elucidated. A transgenic mouse ES cell line (46C was differentiated into a neural lineage in vitro via induction with retinoic acid. These cells were then subjected to oxidative stress with a significantly high concentration of glutamate. Measurement of reactive oxygen species (ROS was performed after inducing glutamate excitotoxicity, and recovery from this toxicity in response to vitamin E was determined. The gene expression levels of glutamate receptors and neuron-specific enolase were elucidated using real-time PCR. The results reveal that neural cells derived from 46C cells and subjected to oxidative stress exhibit downregulation of NMDA, kainate receptor, and NSE after posttreatment with different concentrations of TRF and α-TCP, a sign of neurorecovery. Treatment of either TRF or α-TCP reduced the levels of ROS in neural cells subjected to glutamate-induced oxidative stress; these results indicated that vitamin E is a potent antioxidant.

  19. The effect of gamma radiation and neocarzinostatin on NAD and ATP levels in mouse leukaemia cells

    International Nuclear Information System (INIS)

    Goodwin, P.M.; Lewis, P.J.; Davies, M.I.; Skidmore, C.J.; Shall, S.

    1978-01-01

    When mouse leukaemia cells are treated with γ-radiation or neocarzinostatin the intracelluiar NAD and ATP levels fall rapidly. It is shown that the ATP response is a consequence of the decreased NAD level. It is suggested that this low NAD level results in decreased glycolytic activity and that there is a subsequent accumulation of phosphorylated sugars associated with the fall in ATP. Under these extreme conditions, therefore, the NAD level probably regulates the rate of glycolysis in cells which are utilising a rapidly metabolisable sugar as their energy source. (Auth.)

  20. Raised serum squamous cell carcinoma antigen levels in malignant transformation of mature cystic ovarian teratoma

    NARCIS (Netherlands)

    DeBruijn, HWA; Hollema, H; Willemse, PHB; TenHoor, KA; Boonstra, J.

    1996-01-01

    Mature cystic ovarian teratoma with malignant transformation to squamous cell carcinoma was diagnosed in four patients. Squamous cell carcinoma (SCC) antigen serum levels were elevated at diagnosis and during progression of the disease, but normal in complete remission. Elevated serum SCC antigen

  1. Multiweek Cell Culture Project for Use in Upper-Level Biology Laboratories

    Science.gov (United States)

    Marion, Rebecca E.; Gardner, Grant E.; Parks, Lisa D.

    2012-01-01

    This article describes a laboratory protocol for a multiweek project piloted in a new upper-level biology laboratory (BIO 426) using cell culture techniques. Human embryonic kidney-293 cells were used, and several culture media and supplements were identified for students to design their own experiments. Treatments included amino acids, EGF,…

  2. Elevated levels of homocysteine increase IL-6 production in monocytic Mono Mac 6 cells

    NARCIS (Netherlands)

    van Aken, B. E.; Jansen, J.; van Deventer, S. J.; Reitsma, P. H.

    2000-01-01

    Hyperhomocysteinemia is a risk factor for atherosclerosis and thrombosis. The aim of this study was to analyze if exposure of monocytic cells to increased levels of homocysteine (HCY) induces the accumulation of inflammatory mediators. Interleukin (IL)-6 production by monocytic cell line Mono Mac 6

  3. Residential Proximity to Major Roadways Is Associated With Increased Levels of AC133+ Circulating Angiogenic Cells.

    Science.gov (United States)

    DeJarnett, Natasha; Yeager, Ray; Conklin, Daniel J; Lee, Jongmin; O'Toole, Timothy E; McCracken, James; Abplanalp, Wes; Srivastava, Sanjay; Riggs, Daniel W; Hamzeh, Ihab; Wagner, Stephen; Chugh, Atul; DeFilippis, Andrew; Ciszewski, Tiffany; Wyatt, Brad; Becher, Carrie; Higdon, Deirdre; Ramos, Kenneth S; Tollerud, David J; Myers, John A; Rai, Shesh N; Shah, Jasmit; Zafar, Nagma; Krishnasamy, Sathya S; Prabhu, Sumanth D; Bhatnagar, Aruni

    2015-11-01

    Previous studies have shown that residential proximity to a roadway is associated with increased cardiovascular disease risk. Yet, the nature of this association remains unclear, and its effect on individual cardiovascular disease risk factors has not been assessed. The objective of this study was to determine whether residential proximity to roadways influences systemic inflammation and the levels of circulating angiogenic cells. In a cross-sectional study, cardiovascular disease risk factors, blood levels of C-reactive protein, and 15 antigenically defined circulating angiogenic cell populations were measured in participants (n=316) with moderate-to-high cardiovascular disease risk. Attributes of roadways surrounding residential locations were assessed using geographic information systems. Associations between road proximity and cardiovascular indices were analyzed using generalized linear models. Close proximity (proximity to a major roadway (CD31(+)/AC133(+), AC133(+), CD34(+)/AC133(+), and CD34(+)/45(dim)/AC133(+) cells) and positively associated with road segment distance (CD31(+)/AC133(+), AC133(+), and CD34(+)/AC133(+) cells), traffic intensity (CD31(+)/AC133(+) and AC133(+) cells), and distance-weighted traffic intensity (CD31(+)/34(+)/45(+)/AC133(+) cells). Living close to a major roadway is associated with elevated levels of circulating cells positive for the early stem marker AC133(+). This may reflect an increased need for vascular repair. Levels of these cells in peripheral blood may be a sensitive index of cardiovascular injury because of residential proximity to roadways. © 2015 American Heart Association, Inc.

  4. Monocytes and granulocytes reduce CD38 expression levels on myeloma cells in patients treated with daratumumab

    DEFF Research Database (Denmark)

    Krejcik, Jakub; Frerichs, Kristine A; Nijhof, Inger S

    2017-01-01

    of daratumumab alone or in combination with lenalidomide-dexamethasone, on CD38 levels of MM cells and non-tumor immune cells in the GEN501 study (daratumumab monotherapy) and the GEN503 study (daratumumab combined with lenalidomide-dexamethasone). In vitro assays were also performed. RESULTS: In both trials...

  5. Cannabidiol (CBD) induces functional Tregs in response to low-level T cell activation.

    Science.gov (United States)

    Dhital, Saphala; Stokes, John V; Park, Nogi; Seo, Keun Seok; Kaplan, Barbara L F

    2017-02-01

    Many effects of the non-psychoactive cannabinoid, cannabidiol (CBD), have been described in immune responses induced by strong immunological stimuli. It has also been shown that CBD enhances IL-2 production in response to low-level T cell stimulation. Since IL-2, in combination with TGF-β1, are critical for Treg induction, we hypothesized that CBD would induce CD4 + CD25 + FOXP3 + Tregs in response to low-level stimulation. Low-level T cell stimulation conditions were established based on minimal CD25 expression in CD4 + cells using suboptimal PMA/Io (4nM/0.05μM, S/o), ultrasuboptimal PMA/Io (1nM/0.0125μM, Us/o) or soluble anti-CD3/28 (400-800ng each, s3/28). CBD increased CD25 + FOXP3 + cells from CD4 + , CD4 + CD25 + , and CD4 + CD25 - T cells, as well as in CD4 + T cells derived from FOXP3-GFP mice. Most importantly, the Us/o+CBD-induced CD4 + CD25 + Tregs robustly suppressed responder T cell proliferation, demonstrating that the mechanism by which CBD is immunosuppressive under low-level T cell stimulation involves induction of functional Tregs. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Low-Level Laser Effects on Simulated Orthodontic Tension Side Periodontal Ligament Cells

    OpenAIRE

    Huang, Tsui-Hsien; Liu, Shiau-Lee; Chen, Chih-Lin; Shie, Ming-You; Kao, Chia-Tze

    2013-01-01

    Objective: The purpose of this study was to analyze proliferation, inflammation, and osteogenic effects on periodontal ligament (PDL) cells after low-level laser therapy (LLLT) under simulated orthodontic tension conditions. Background data: Low-level lasers affect fibroblast proliferation and collagen synthesis and reduce inflammation. Few studies have focused on the LLLT changes in the PDL caused by moving teeth. Materials and methods: A human PDL cell line was cultured in a −100 kPa tensio...

  7. Decreased level of phosphatidylcholine (16:0/20:4) in multiple myeloma cells compared to plasma cells: a single-cell MALDI-IMS approach.

    Science.gov (United States)

    Hossen, Md Amir; Nagata, Yasuyuki; Waki, Michihiko; Ide, Yoshimi; Takei, Shiro; Fukano, Hana; Romero-Perez, Gustavo A; Tajima, Shogo; Yao, Ikuko; Ohnishi, Kazunori; Setou, Mitsutoshi

    2015-07-01

    Lipid metabolic changes under diseased conditions, particularly in solid tumors, are attracting increased attention. However, in non-solid tumors, including most hematopoietic tumors, lipid analyses are scarce. Multiple myeloma (MM) is a plasma cell disorder arising from bone marrow, and the lipid status of MM cells has not been reported yet. In this study, we analyzed flow cytometry-sorted single MM cells and normal plasma cells (NPCs) using matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS), a two-dimensional label-free mass spectrometry technique for biomolecular analysis, to obtain specific lipid information. We isolated 1.31-5.77% of MM cells and 0.03-0.24% of NPCs using fluorescence-activated cell sorting (FACS). Analysis of purified cells using MALDI-IMS at the single-cell level revealed that the peak intensity and ion signals of phosphatidylcholine [PC (16:0/20:4) + H](+) at m/z 782.5 were significantly decreased in MM cells compared to NPCs. By examining particular cell populations rather than cell mixtures, our method can become a suitable tool for the analysis of rare cell populations at the single-cell level and advance the understanding of MM progression.

  8. Evaluating cytoplasmic and nuclear levels of inflammatory cytokines in cancer cells by western blotting.

    Science.gov (United States)

    Gatla, Himavanth R; Singha, Bipradeb; Persaud, Valerie; Vancurova, Ivana

    2014-01-01

    Increased expression and cellular release of inflammatory cytokines, interleukin-8 (IL-8; CXCL8), and high mobility group box-1 (HMGB1) are associated with increased cell proliferation, angiogenesis, and metastasis during cancer progression. In prostate and ovarian cancer cells, increased levels of IL-8 and HMGB1 correlate with poor prognosis. We have recently shown that proteasome inhibition by bortezomib (BZ) specifically increases IL-8 release from metastatic prostate and ovarian cancer cells. In this chapter, we describe a protocol to analyze the cytoplasmic and nuclear levels of IL-8 and HMGB1 in prostate and ovarian cancer cells by western blotting. IL-8 is localized in the cytoplasm in both cell types, and its protein levels are significantly increased by BZ. In contrast, HMGB1 is localized in the nucleus, and BZ increases its nuclear levels only in ovarian cancer cells. The protocol includes isolation of cytoplasmic and nuclear extracts, followed by SDS electrophoresis and western blotting, and can be easily modified to analyze the cytoplasmic and nuclear cytokine levels in other cell types.

  9. Automatic Detection of Cervical Cancer Cells by a Two-Level Cascade Classification System

    Directory of Open Access Journals (Sweden)

    Jie Su

    2016-01-01

    Full Text Available We proposed a method for automatic detection of cervical cancer cells in images captured from thin liquid based cytology slides. We selected 20,000 cells in images derived from 120 different thin liquid based cytology slides, which include 5000 epithelial cells (normal 2500, abnormal 2500, lymphoid cells, neutrophils, and junk cells. We first proposed 28 features, including 20 morphologic features and 8 texture features, based on the characteristics of each cell type. We then used a two-level cascade integration system of two classifiers to classify the cervical cells into normal and abnormal epithelial cells. The results showed that the recognition rates for abnormal cervical epithelial cells were 92.7% and 93.2%, respectively, when C4.5 classifier or LR (LR: logical regression classifier was used individually; while the recognition rate was significantly higher (95.642% when our two-level cascade integrated classifier system was used. The false negative rate and false positive rate (both 1.44% of the proposed automatic two-level cascade classification system are also much lower than those of traditional Pap smear review.

  10. Signaling profiling at the single-cell level identifies a distinct signaling signature in murine hematopoietic stem cells.

    Science.gov (United States)

    Du, Juan; Wang, Jinyong; Kong, Guangyao; Jiang, Jing; Zhang, Jingfang; Liu, Yangang; Tong, Wei; Zhang, Jing

    2012-07-01

    Hematopoietic stem cell (HSC) function is tightly regulated by cytokine signaling. Although phospho-flow cytometry allows us to study signaling in defined populations of cells, there has been tremendous hurdle to carry out this study in rare HSCs due to unrecoverable critical HSC markers, low HSC number, and poor cell recovery rate. Here, we overcame these difficulties and developed a "HSC phospho-flow" method to analyze cytokine signaling in murine HSCs at the single-cell level and compare HSC signaling profile to that of multipotent progenitors (MPPs), a cell type immediately downstream of HSCs, and commonly used Lin(-) cKit(+) cells (LK cells, enriched for myeloid progenitors). We chose to study signaling evoked from three representative cytokines, stem cell factor (SCF) and thrombopoietin (TPO) that are essential for HSC function and granulocyte macrophage-colony-stimulating factor (GM-CSF) that is dispensable for HSCs. HSCs display a distinct TPO and GM-CSF signaling signature from MPPs and LK cells, which highly correlates with receptor surface expression. In contrast, although majority of LK cells express lower levels of cKit than HSCs and MPPs, SCF-evoked ERK1/2 activation in LK cells shows a significantly increased magnitude for a prolonged period. These results suggest that specific cellular context plays a more important role than receptor surface expression in SCF signaling. Our study of HSC signaling at the homeostasis stage paves the way to investigate signaling changes in HSCs under conditions of stress, aging, and hematopoietic diseases. Copyright © 2012 AlphaMed Press.

  11. Basal HIF-1a expression levels are not predictive for radiosensitivity of human cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Schilling, D.; Multhoff, G. [Klinikum rechts der Isar der Technischen Univ. Muenchen (Germany). Dept. of Radiation Oncology; Helmholtz Center Munich, CCG - Innate Immunity in Tumor Biology, Munich (Germany). German Research Center for Environmental Health - Inst. of Pathology; Bayer, C.; Emmerich, K.; Molls, M.; Vaupel, P. [Klinikum rechts der Isar der Technischen Univ. Muenchen (Germany). Dept. of Radiation Oncology; Huber, R.M. [Klinikum der Univ. Muenchen (Germany). Dept. of Pneumology

    2012-04-15

    High levels of hypoxia inducible factor (HIF)-1a in tumors are reported to be associated with tumor progression and resistance to therapy. To examine the impact of HIF-1a on radioresistance under normoxia, the sensitivity towards irradiation was measured in human tumor cell lines that differ significantly in their basal HIF-1a levels. HIF-1a levels were quantified in lysates of H1339, EPLC-272H, A549, SAS, XF354, FaDu, BHY, and CX- tumor cell lines by ELISA. Protein levels of HIF-1a, HIF-2a, carbonic anhydrase IX (CA IX), and GAPDH were assessed by Western blot analysis. Knock-down experiments were performed using HIF-1a siRNA. Clonogenic survival after irradiation was determined by the colony forming assay. According to their basal HIF-1a status, the tumor cell lines were divided into low (SAS, XF354, FaDu, A549, CX-), intermediate (EPLC-272H, BHY), and high (H1339) HIF-1a expressors. The functionality of the high basal HIF-1a expression in H1339 cells was proven by reduced CA IX expression after knocking-down HIF-1a. Linear regression analysis revealed no correlation between basal HIF-1a levels and the survival fraction at either 2 or 4 Gy in all tumor cell lines investigated. Our data suggest that basal HIF-1a levels in human tumor cell lines do not predict their radiosensitivity under normoxia. (orig.)

  12. Relationship of visfatin level to pancreatic endocrine hormone level, HOMA-IR index, and HOMA β-cell index in overweight women who performed hydraulic resistance exercise.

    Science.gov (United States)

    Ha, Chang Ho; Swearingin, Brenda; Jeon, Yong Kyun

    2015-09-01

    [Purpose] This study aimed to examine the correlation of visfatin level to pancreatic endocrine hormone level, homeostasis model assessment of insulin resistance (HOMA-IR) index, and HOMA β-cell index in hydraulic resistance exercise. Furthermore, it investigated the relationship between visfatin level and other variables affected by exercise in overweight women. [Subjects and Methods] The exercise group trained for 12 weeks, 70 minutes/day, 5 days/week. Visfatin level, pancreatic endocrine hormone level, HOMA-IR index, and HOMA β-cell index were measured before and after the intervention. Based on the blood insulin and glucose concentrations, HOMA-IR index, the indicator of insulin resistance, and HOMA β-cell index, the indicator of insulin secretion level, were assessed. [Results] Interaction effects on visfatin level, insulin level, HOMA-IR index, and HOMA β-cell index were observed. Interaction effects on glucagon and glucose levels were not observed between the intervention groups. The correlations of visfatin level to insulin, glucagon, and glucose levels, and HOMA-IR and HOMA β-cell indexes were not significant for any of the subjects. [Conclusion] Therefore, the 12-week resistance exercise affected body composition, visfatin level, insulin level, HOMA-IR index, and HOMA β-cell index. Finally, visfatin was not related to insulin, glucagon, and glucose levels, and HOMA-IR and HOMA β-cell indexes.

  13. Analysis of gene expression levels in individual bacterial cells without image segmentation

    Energy Technology Data Exchange (ETDEWEB)

    Kwak, In Hae; Son, Minjun [Physics Department, University of Florida, P.O. Box 118440, Gainesville, FL 32611-8440 (United States); Hagen, Stephen J., E-mail: sjhagen@ufl.edu [Physics Department, University of Florida, P.O. Box 118440, Gainesville, FL 32611-8440 (United States)

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer We present a method for extracting gene expression data from images of bacterial cells. Black-Right-Pointing-Pointer The method does not employ cell segmentation and does not require high magnification. Black-Right-Pointing-Pointer Fluorescence and phase contrast images of the cells are correlated through the physics of phase contrast. Black-Right-Pointing-Pointer We demonstrate the method by characterizing noisy expression of comX in Streptococcus mutans. -- Abstract: Studies of stochasticity in gene expression typically make use of fluorescent protein reporters, which permit the measurement of expression levels within individual cells by fluorescence microscopy. Analysis of such microscopy images is almost invariably based on a segmentation algorithm, where the image of a cell or cluster is analyzed mathematically to delineate individual cell boundaries. However segmentation can be ineffective for studying bacterial cells or clusters, especially at lower magnification, where outlines of individual cells are poorly resolved. Here we demonstrate an alternative method for analyzing such images without segmentation. The method employs a comparison between the pixel brightness in phase contrast vs fluorescence microscopy images. By fitting the correlation between phase contrast and fluorescence intensity to a physical model, we obtain well-defined estimates for the different levels of gene expression that are present in the cell or cluster. The method reveals the boundaries of the individual cells, even if the source images lack the resolution to show these boundaries clearly.

  14. Analysis of gene expression levels in individual bacterial cells without image segmentation

    International Nuclear Information System (INIS)

    Kwak, In Hae; Son, Minjun; Hagen, Stephen J.

    2012-01-01

    Highlights: ► We present a method for extracting gene expression data from images of bacterial cells. ► The method does not employ cell segmentation and does not require high magnification. ► Fluorescence and phase contrast images of the cells are correlated through the physics of phase contrast. ► We demonstrate the method by characterizing noisy expression of comX in Streptococcus mutans. -- Abstract: Studies of stochasticity in gene expression typically make use of fluorescent protein reporters, which permit the measurement of expression levels within individual cells by fluorescence microscopy. Analysis of such microscopy images is almost invariably based on a segmentation algorithm, where the image of a cell or cluster is analyzed mathematically to delineate individual cell boundaries. However segmentation can be ineffective for studying bacterial cells or clusters, especially at lower magnification, where outlines of individual cells are poorly resolved. Here we demonstrate an alternative method for analyzing such images without segmentation. The method employs a comparison between the pixel brightness in phase contrast vs fluorescence microscopy images. By fitting the correlation between phase contrast and fluorescence intensity to a physical model, we obtain well-defined estimates for the different levels of gene expression that are present in the cell or cluster. The method reveals the boundaries of the individual cells, even if the source images lack the resolution to show these boundaries clearly.

  15. Effect of oestradiol on mast cell number and histamine level in the mammary glands of rat.

    Science.gov (United States)

    Jing, H; Wang, Z; Chen, Y

    2012-06-01

    Variations of mast cell number, histamine concentration and oestrogen receptor (ER) expression in mammary glands with the fluctuation of plasma oestradiol level were identified either in the intact rats at different oestrous stages or in the ovary-ectomized rats administrated with different doses of oestradiol benzoate. The results showed that the number of mast cells and histamine concentration fluctuated concomitantly with plasma oestradiol level during the oestrous cycle. More mast cell number and higher histamine concentrations were observed in the oestrous stage than that in the prooestrous and dioestrous stages. Ovariectomy decreased the mast cell number and histamine concentration, which were reconstituted by exogenous oestradiol. ER was mainly found in the nuclear of epithelial cells and interstitial cells of mammary glands. In addition, ER was also expressed in the cytoplasm of some stromal cells. These stromal cells were verified to be mast cells. In conclusion, our results suggested that oestradiol modulated mast cell number and its degranulation in the mammary gland through the ERs pathway. © 2011 Blackwell Verlag GmbH.

  16. Correlation between residual level of DNA double-strand breaks and the radiosensitivity of cancer cells

    International Nuclear Information System (INIS)

    Sun Jianxiang; Sun Weijian; Sui Jianli; Zhou Pingkun

    2008-01-01

    Objective: To understand the variation of the DNA double-strand break rejoining capacity among different cultured cancer cell lines and the primary cancer cells from brain cancer patients, and to explore the predictor of radiotherapy responses of cancers. Methods: DNA double-strand breaks (DSBs) were induced by 60 Co γ-irradiation. Pulsed-field gel electrophoresis was used to analyze the initial production and rejoining of DNA DSBs. Radiosensitivity was determined by in vitro assay of clonogenic-forming capacity. Results: A wide variation of radiosensitivity, e.g. the survival parameter of Do varied from 0.65 to 2.15 Gy, was displayed among the eight cell lines derived from different type of cancers. Although differential level of initial DNA DSBs induced by 20 Gy γ-rays was observed among various cell lines, it was not correlated with the radiosensitivity. The deficiency of DNA DSB rejoining in radiosensitive cell lines was shown either in the early rapid-rejoining phase (SX-10 cells) or in the late slow-rejoining phase (A2780 cells). A significant relationship was observed between the residual level of DNA DSBs measured at 2 h post-20 Gy irradiation and the cellular radiosensitivity (D 0 or SF 2 ). The kinetic curves of rejoining DNA DSBs in the primary human brain tumor cells indicated a variation on DSB rejoining capacity among different individual tumor. The residual level of DNA DSBs after 2 h of rejoining post 20 Gy irradiation in primary human brain tumor cells is compatible to the results obtained in vitro culture cancer cell lines. Conclusions: The residual level of DNA DSBs is correlated with radioresistance of cancer cells, and the residual DNA damage is a useful parameter in predicting the response of tumor tissue to radiotherapy. (authors)

  17. A two-step strategy for neuronal differentiation in vitro of human dental follicle cells.

    Science.gov (United States)

    Völlner, Florian; Ernst, Wolfgang; Driemel, Oliver; Morsczeck, Christian

    2009-06-01

    Human dental follicle cells (DFCs) derived from wisdom teeth are precursor cells for cementoblasts. In this study, we recognized that naïve DFCs express constitutively the early neural cell marker beta-III-tubulin. Interestingly, DFCs formed beta-III-tubulin-positive neurosphere-like cell clusters (NLCCs) on low-attachment cell culture dishes in serum-replacement medium (SRM). For a detailed examination of the neural differentiation potential, DFCs were cultivated in different compositions of SRM containing supplements such as N2, B27, G5 and the neural stem cell supplement. Moreover, these cell culture media were combined with different cell culture substrates such as gelatin, laminin, poly-L-ornithine or poly-L-lysine. After cultivation in SRM, DFCs differentiated into cells with small cell bodies and long cellular extrusions. The expression of nestin, beta-III-tubulin, neuron-specific enolase (NSE) and neurofilament was up-regulated in SRM supplemented with G5, a cell culture supplement for glial cells, and the neural stem cell supplement. DFCs formed NLCCs and demonstrated an increased gene expression of neural cell markers beta-III-tubulin, NSE, nestin and for small neuron markers such as neuropeptides galanin (GAL) and tachykinin (TAC1) after cultivation on poly-L-lysine. For a further neural differentiation NLCC-derived cells were sub-cultivated on laminin and poly-L-ornithine cell culture substrate. After 2 weeks of differentiation, DFCs exposed neural-like cell morphology with small neurite-like cell extrusions. These cells differentially express neurofilament and NSE, but only low levels of beta-III-tubulin and nestin. In conclusion, we demonstrated the differentiation of human DFCs into neuron-like cells after a two-step strategy for neuronal differentiation.

  18. GLP-1 receptor regulates cell growth through regulating IDE expression level in Aβ1-42-treated PC12 cells.

    Science.gov (United States)

    Li, Huajie; Cao, Liping; Ren, Yi; Jiang, Ying; Xie, Wei; Li, Dawen

    2017-12-20

    This study aimed to validate whether glucagon-like peptide-1 receptor (GLP-1R) /cyclic adenosine monophosphate (cAMP) /protein kinase (PKA) / insulin-degrading enzyme (IDE) signaling pathway was associated with neuronal apoptosis.We developed an animal model presenting both Alzheimer's disease (AD) and type 2 diabetes (T2D), bycrossing APP/PS1 mice (AD model) with streptozotocin(STZ)-treated mice (a T2D model). Neuronal apoptosis was detected by TUNEL staining and the expression levels of apoptosis-related proteins were examined by Western blotting. The viability of PC12 cells was analyzed by MTT assay and apoptosis of PC12 cells was detected by flow cytometry. The mRNA expression level was detected by qRT-PCR.T2D contributes to AD progress by prompting neuronal apoptosis and increasing expression of pro-apoptotic protein. β-amyloid peptide1-42 (Aβ1-42) was shown to exerteffects on inhibiting cell viability and prompting cell apoptosis of PC12 cells. However, GLP-1R agonist geniposide(Gen) significantly reversed them, exerting a protective role on PC12 cells. And insulin-degrading enzyme (IDE)antagonistbacitracin (Bac) markedly reversed the protective effects of Gen on Aβ1-42-treated PC12 cells. Besides, Gensignificantly reversed the effects of Aβ1-42 treatment on IDEexpression, and the inhibitor of cAMP/PKA signaling pathway markedly reversed the effects of Gen on IDE expression level in Aβ1-42-treated PC12 cells.In conclusion,GLP-1R regulates cellgrowth, at least partially,through regulating cAMP/PKA/IDE signaling pathway in Aβ1-42-treated PC12 cells. ©2017 The Author(s).

  19. Heterogeneity of Metazoan Cells and Beyond: To Integrative Analysis of Cellular Populations at Single-Cell Level.

    Science.gov (United States)

    Barteneva, Natasha S; Vorobjev, Ivan A

    2018-01-01

    In this paper, we review some of the recent advances in cellular heterogeneity and single-cell analysis methods. In modern research of cellular heterogeneity, there are four major approaches: analysis of pooled samples, single-cell analysis, high-throughput single-cell analysis, and lately integrated analysis of cellular population at a single-cell level. Recently developed high-throughput single-cell genetic analysis methods such as RNA-Seq require purification step and destruction of an analyzed cell often are providing a snapshot of the investigated cell without spatiotemporal context. Correlative analysis of multiparameter morphological, functional, and molecular information is important for differentiation of more uniform groups in the spectrum of different cell types. Simplified distributions (histograms and 2D plots) can underrepresent biologically significant subpopulations. Future directions may include the development of nondestructive methods for dissecting molecular events in intact cells, simultaneous correlative cellular analysis of phenotypic and molecular features by hybrid technologies such as imaging flow cytometry, and further progress in supervised and non-supervised statistical analysis algorithms.

  20. Activated T cells can induce high levels of CTLA-4 expression on B cells

    NARCIS (Netherlands)

    Kuiper, H. M.; Brouwer, M.; Linsley, P. S.; van Lier, R. A.

    1995-01-01

    Engagement of the TCR/CD3 complex together with ligation of CD28 by its counterstructures B7-1 (CD80) and B7-2 (CD86) on APC are required for mitogenic T cell activation. After activation, T cells not only express B7-1 and B7-2 molecules, but a second receptor for the B7 ligands, CTLA-4, can be

  1. [Effect of trastuzumab on tumor cell lines shedding high or low level of HER-2 ECD].

    Science.gov (United States)

    Liu, Cai-Yun; Yang, Wei; Li, Jin-Feng; Sun, Su-Lian; Shou, Cheng-Chao

    2007-02-01

    To examine the effect of trastuzumab on cell proliferation, colony formation and changes of HER-2 proteins in human breast cancer cell line SKBR3 and human ovarian cancer cell line SKOV3 cells which overexpress p185 HER-2 but shed high or low HER-2 extracellular domain (ECD) levels. SKBR3 cells and SKOV3 cells were treated with or without trastuzumab. Cell number and the rate of colony formation were calculated. Western blot analysis was used to detect p185 HER-2, HER-2 ECD and phospho-HER-2. Two-site ELISA assay was used for the detection of HER-2 ECD. Trastuzumab inhibited cell proliferation, colony formation, and decreased or eliminated the levels of two uncharacterized phospho-proteins (molar weight about 90 000 and 40 000) in SKBR3 cells shedding high level of HER-2 ECD expression. These responses were not observed in SKOV3 cells shedding low level of HER-2 ECD expression. But total p185, phospho-p185 and phospho-p95 proteins did not appear to change in SKBR3 and SKOV3 cells after treatment with trastuzumab. Trastuzumab reacts not only with proteolytic cleavage HER-2 ECD containing HER-2 ECD I , II , III and IV subdomains of p185 HER-2 extracellular domain, but also with the secreted autoinhibitor p68/ECD III a specifying 340 residues, identical to subdomains I and II from the extracellular domain of p185 HER-2, followed by a unique C-terminal sequence of 79 aa encoded by intron 8, which suggested that there may be a trastuzumab binding site on p68/ECD III a protein. Comparing with HER-2 ECD levels of the same number of SKBR3 cells, there was no significant decrease of HER-2 ECD shedding level after treatment with or without trastuzumab for 4 days in serum-free medium. Antitumor effects of trastuzumab may be related to the two uncharacterized phospho-p90 and/or phospho-p40 proteins. There is probably a trastuzumab epitope on p68/ECD III a. The decrease of HER-2 ECD levels may be positively correlated with the number of SKBR3 cells.

  2. Nanoparticle uptake and their co-localization with cell compartments - a confocal Raman microscopy study at single cell level

    Science.gov (United States)

    Estrela-Lopis, I.; Romero, G.; Rojas, E.; Moya, S. E.; Donath, E.

    2011-07-01

    Confocal Raman Microscopy, a non-invasive, non-destructive and label-free technique, was employed to study the uptake and localization of nanoparticles (NPs) in the Hepatocarcinoma human cell line HepG2 at the level of single cells. Cells were exposed to carbon nanotubes (CNTs) the surface of which was engineered with polyelectrolytes and lipid layers, aluminium oxide and cerium dioxide nanoparticles. Raman spectra deconvolution was applied to obtain the spatial distributions of NPs together with lipids/proteins in cells. The colocalization of the NPs with different intracellular environments, lipid bodies, protein and DNA, was inferred. Lipid coated CNTs associated preferentially with lipid rich regions, whereas polyelectrolyte coated CNTs were excluded from lipid rich regions. Al2O3 NPs were found in the cytoplasm. CeO2 NPs were readily taken up and have been observed all over the cell. Raman z-scans proved the intracellular distribution of the respective NPs.

  3. Mass Cytometry for Detection of Silver at the Bacterial Single Cell Level

    Directory of Open Access Journals (Sweden)

    Yuting Guo

    2017-07-01

    Full Text Available Background: Mass cytometry (Cytometry by Time of Flight, CyTOF allows single-cell characterization on the basis of specific metal-based cell markers. In addition, other metals in the mass range such as silver can be detected per cell. Bacteria are known to be sensible to silver and a protocol was developed to measure both the number of affected cells per population and the quantities of silver per cell.Methods: For mass cytometry ruthenium red was used as a marker for all cells of a population while parallel application of cisplatin discriminated live from dead cells. Silver quantities per cell and frequencies of silver containing cells in a population were measured by mass cytometry. In addition, live/dead subpopulations were analyzed by flow cytometry and distinguished by cell sorting based on ruthenium red and propidium iodide double staining. Verification of the cells’ silver load was performed on the bulk level by using ICP-MS in combination with cell sorting. The protocol was developed by conveying both, fast and non-growing Pseudomonas putida cells as test organisms.Results: A workflow for labeling bacteria in order to be analyzed by mass cytometry was developed. Three different parameters were tested: ruthenium red provided counts for all bacterial cells in a population while consecutively applied cisplatin marked the frequency of dead cells. Apparent population heterogeneity was detected by different frequencies of silver containing cells. Silver quantities per cell were also well measurable. Generally, AgNP-10 treatment caused higher frequencies of dead cells, higher frequencies of silver containing cells and higher per-cell silver quantities. Due to an assumed chemical equilibrium of free and bound silver ions live and dead cells were associated with silver in equal quantities and this preferably during exponential growth. With ICP-MS up to 1.5 fg silver per bacterial cell were detected.Conclusion: An effective mass cytometry

  4. Emergence of cytotoxic resistance in cancer cell populations: Single-cell mechanisms and population-level consequences

    International Nuclear Information System (INIS)

    Lorenzi, Tommaso; Chisholm, Rebecca H.; Lorz, Alexander; Neves de Almeida, Luís; Clairambault, Jean; Larsen, Annette K.; Escargueil, Alexandre

    2016-01-01

    We formulate an individual-based model and a population model of phenotypic evolution, under cytotoxic drugs, in a cancer cell population structured by the expression levels of survival-potential and proliferation-potential. We apply these models to a recently studied experimental system. Our results suggest that mechanisms based on fundamental laws of biology can reversibly push an actively-proliferating, and drug-sensitive, cell population to transition into a weakly-proliferative and drug-tolerant state, which will eventually facilitate the emergence of more potent, proliferating and drug-tolerant cells.

  5. Emergence of cytotoxic resistance in cancer cell populations: Single-cell mechanisms and population-level consequences

    Energy Technology Data Exchange (ETDEWEB)

    Lorenzi, Tommaso [Centre de Mathématiques et de Leurs Applications, ENS Cachan, CNRS, Cachan 94230 Cedex, France & INRIA-Paris-Rocquencourt, MAMBA Team, Domaine de Voluceau, BP105, 78153 Le Chesnay Cedex (France); Chisholm, Rebecca H. [School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney NSW 2052 (Australia); Lorz, Alexander; Neves de Almeida, Luís; Clairambault, Jean [Sorbonne Universités, UPMC Univ Paris 06, UMR 7598, Laboratoire Jacques-Louis Lions, F-75005, Paris (France); CNRS, UMR 7598, Laboratoire Jacques-Louis Lions, F-75005, Paris (France); INRIA-Paris-Rocquencourt, MAMBA Team, Domaine de Voluceau, BP105, 78153 Le Chesnay Cedex (France); Larsen, Annette K.; Escargueil, Alexandre [Sorbonne Universités, UPMC Univ Paris 06, F-75005, Paris (France); INSERM, UMR-S 938, Laboratory of “Cancer Biology and Therapeutics”, F-75012, Paris (France)

    2016-06-08

    We formulate an individual-based model and a population model of phenotypic evolution, under cytotoxic drugs, in a cancer cell population structured by the expression levels of survival-potential and proliferation-potential. We apply these models to a recently studied experimental system. Our results suggest that mechanisms based on fundamental laws of biology can reversibly push an actively-proliferating, and drug-sensitive, cell population to transition into a weakly-proliferative and drug-tolerant state, which will eventually facilitate the emergence of more potent, proliferating and drug-tolerant cells.

  6. Baseline levels of influenza-specific CD4 memory T-cells affect T-cell responses to influenza vaccines.

    Science.gov (United States)

    He, Xiao-Song; Holmes, Tyson H; Sasaki, Sanae; Jaimes, Maria C; Kemble, George W; Dekker, Cornelia L; Arvin, Ann M; Greenberg, Harry B

    2008-07-02

    Factors affecting immune responses to influenza vaccines have not been studied systematically. We hypothesized that T-cell and antibody responses to the vaccines are functions of pre-existing host immunity against influenza antigens. During the 2004 and 2005 influenza seasons, we have collected data on cellular and humoral immune reactivity to influenza virus in blood samples collected before and after immunization with inactivated or live attenuated influenza vaccines in healthy children and adults. We first used cross-validated lasso regression on the 2004 dataset to identify a group of candidate baseline correlates with T-cell and antibody responses to vaccines, defined as fold-increase in influenza-specific T-cells and serum HAI titer after vaccination. The following baseline parameters were examined: percentages of influenza-reactive IFN-gamma(+) cells in T and NK cell subsets, percentages of influenza-specific memory B-cells, HAI titer, age, and type of vaccine. The candidate baseline correlates were then tested with the independent 2005 dataset. Baseline percentage of influenza-specific IFN-gamma(+) CD4 T-cells was identified as a significant correlate of CD4 and CD8 T-cell responses, with lower baseline levels associated with larger T-cell responses. Baseline HAI titer and vaccine type were identified as significant correlates for HAI response, with lower baseline levels and the inactivated vaccine associated with larger HAI responses. Previously we reported that baseline levels of CD56(dim) NK reactivity against influenza virus inversely correlated with the immediate T-cell response to vaccination, and that NK reactivity induced by influenza virus depended on IL-2 produced by influenza-specific memory T-cells. Taken together these results suggest a novel mechanism for the homeostasis of virus-specific T-cells, which involves interaction between memory helper T-cells, CD56(dim) NK and DC. These results demonstrate that assessment of baseline biomarkers may

  7. Baseline levels of influenza-specific CD4 memory T-cells affect T-cell responses to influenza vaccines.

    Directory of Open Access Journals (Sweden)

    Xiao-Song He

    Full Text Available BACKGROUND: Factors affecting immune responses to influenza vaccines have not been studied systematically. We hypothesized that T-cell and antibody responses to the vaccines are functions of pre-existing host immunity against influenza antigens. METHODOLOGY/PRINCIPAL FINDINGS: During the 2004 and 2005 influenza seasons, we have collected data on cellular and humoral immune reactivity to influenza virus in blood samples collected before and after immunization with inactivated or live attenuated influenza vaccines in healthy children and adults. We first used cross-validated lasso regression on the 2004 dataset to identify a group of candidate baseline correlates with T-cell and antibody responses to vaccines, defined as fold-increase in influenza-specific T-cells and serum HAI titer after vaccination. The following baseline parameters were examined: percentages of influenza-reactive IFN-gamma(+ cells in T and NK cell subsets, percentages of influenza-specific memory B-cells, HAI titer, age, and type of vaccine. The candidate baseline correlates were then tested with the independent 2005 dataset. Baseline percentage of influenza-specific IFN-gamma(+ CD4 T-cells was identified as a significant correlate of CD4 and CD8 T-cell responses, with lower baseline levels associated with larger T-cell responses. Baseline HAI titer and vaccine type were identified as significant correlates for HAI response, with lower baseline levels and the inactivated vaccine associated with larger HAI responses. Previously we reported that baseline levels of CD56(dim NK reactivity against influenza virus inversely correlated with the immediate T-cell response to vaccination, and that NK reactivity induced by influenza virus depended on IL-2 produced by influenza-specific memory T-cells. Taken together these results suggest a novel mechanism for the homeostasis of virus-specific T-cells, which involves interaction between memory helper T-cells, CD56(dim NK and DC

  8. Assessment of flhDC mRNA levels in Serratia liquefaciens swarm cells

    DEFF Research Database (Denmark)

    Tolker-Nielsen, Tim; Christensen, Allan Beck; Holmstrøm, K.

    2000-01-01

    We reported previously that artificial overexpression of the flhDC operon in liquid-grown Serratia liquefaciens resulted in the formation of filamentous, multinucleated, and hyperflagellated cells that were indistinguishable from surface-induced swarm cells (L. Eberl, G. Christiansen, S. Molin......, and M. Givskov, J. Bacteriol. 178:554-559, 1996). In the present report we show by means of reporter gene measurements, Northern analysis, and in situ reverse transcription-PCR that the amount of flhDC mRNA in surface-grown swarm cells does not exceed the maximum level found in nondifferentiated......, vegetative cells. This suggests that surface-induced S. liquefaciens swarm cell differentiation, although dependent on flhDC gene expression, does not occur through elevated flhDC mRNA levels....

  9. The calculation of deep levels in semiconductors by using a recursion method for super-cells

    International Nuclear Information System (INIS)

    Wong Yongliang.

    1987-01-01

    The paper presents the theory of deep levels in semiconductors, the super-cell approach to the theory of deep level impurities, the calculation of band structure by using the tight-binding method and the recursion method used to study the defects in the presence of lattice relaxation and extended defect complexes. 47 refs

  10. Restoring E-cadherin-mediated cell-cell adhesion increases PTEN protein level and stability in human breast carcinoma cells

    International Nuclear Information System (INIS)

    Li Zengxia; Wang Liying; Zhang Wen; Fu Yi; Zhao Hongbo; Hu Yali; Prins, Bram Peter; Zha Xiliang

    2007-01-01

    The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a well-characterized tumor suppressor that negatively regulates cell growth and survival. Despite the critical role of PTEN in cell signaling, the mechanisms of its regulation are still under investigation. We reported here that PTEN expression could be controlled by overexpression or knock-down of E-cadherin in several mammary carcinoma cell lines. Furthermore, we showed that the accumulation of PTEN protein in E-cadherin overexpressing cells was due to increased PTEN protein stability rather than the regulation of its transcription. The proteasome-dependent PTEN degradation pathway was impaired after restoring E-cadherin expression. Moreover, maintenance of E-cadherin mediated cell-cell adhesion was necessary for its regulating PTEN. Altogether, our results suggested that E-cadherin mediated cell-cell adhesion was essential for preventing the proteasome degradation of PTEN, which might explain how breast carcinoma cells which lost cell-cell contact proliferate rapidly and are prone to metastasis

  11. Effect of low-level laser-treated mesenchymal stem cells on myocardial infarction.

    Science.gov (United States)

    El Gammal, Zaynab H; Zaher, Amr M; El-Badri, Nagwa

    2017-09-01

    Cardiovascular disease is the leading cause of death worldwide. Although cardiac transplantation is considered the most effective therapy for end-stage cardiac diseases, it is limited by the availability of matching donors and the complications of the immune suppressive regimen used to prevent graft rejection. Application of stem cell therapy in experimental animal models was shown to reverse cardiac remodeling, attenuate cardiac fibrosis, improve heart functions, and stimulate angiogenesis. The efficacy of stem cell therapy can be amplified by low-level laser radiation. It is well established that the bio-stimulatory effect of low-level laser is influenced by the following parameters: wavelength, power density, duration, energy density, delivery time, and the type of irradiated target. In this review, we evaluate the available experimental data on treatment of myocardial infarction using low-level laser. Eligible papers were characterized as in vivo experimental studies that evaluated the use of low-level laser therapy on stem cells in order to attenuate myocardial infarction. The following descriptors were used separately and in combination: laser therapy, low-level laser, low-power laser, stem cell, and myocardial infarction. The assessed low-level laser parameters were wavelength (635-804 nm), power density (6-50 mW/cm 2 ), duration (20-150 s), energy density (0.96-1 J/cm 2 ), delivery time (20 min-3 weeks after myocardial infarction), and the type of irradiated target (bone marrow or in vitro-cultured bone marrow mesenchymal stem cells). The analysis focused on the cardioprotective effect of this form of therapy, the attenuation of scar tissue, and the enhancement of angiogenesis as primary targets. Other effects such as cell survival, cell differentiation, and homing are also included. Among the evaluated protocols using different parameters, the best outcome for treating myocardial infarction was achieved by treating the bone marrow by one dose of low-level

  12. Structure of the first representative of Pfam family PF04016 (DUF364) reveals enolase and Rossmann-like folds that combine to form a unique active site with a possible role in heavy-metal chelation

    International Nuclear Information System (INIS)

    Miller, Mitchell D.; Aravind, L.; Bakolitsa, Constantina; Rife, Christopher L.; Carlton, Dennis; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Chiu, Hsiu-Ju; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Feuerhelm, Julie; Grant, Joanna C.; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Kumar, Abhinav; Marciano, David; McMullan, Daniel; Morse, Andrew T.; Nigoghossian, Edward; Okach, Linda; Reyes, Ron; Bedem, Henry van den; Weekes, Dana; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2010-01-01

    The crystal structure of the first representative of DUF364 family reveals a combination of enolase N-terminal-like and C-terminal Rossmann-like folds. Analysis of the interdomain cleft combined with sequence and genome context conservation among homologs, suggests a unique catalytic site likely involved in the synthesis of a flavin or pterin derivative. The crystal structure of Dhaf4260 from Desulfitobacterium hafniense DCB-2 was determined by single-wavelength anomalous diffraction (SAD) to a resolution of 2.01 Å using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). This protein structure is the first representative of the PF04016 (DUF364) Pfam family and reveals a novel combination of two well known domains (an enolase N-terminal-like fold followed by a Rossmann-like domain). Structural and bioinformatic analyses reveal partial similarities to Rossmann-like methyltransferases, with residues from the enolase-like fold combining to form a unique active site that is likely to be involved in the condensation or hydrolysis of molecules implicated in the synthesis of flavins, pterins or other siderophores. The genome context of Dhaf4260 and homologs additionally supports a role in heavy-metal chelation

  13. Determination of gamma radiation lethal dose (LD50) and resveratrol cytotoxicity level in tumor cells line

    International Nuclear Information System (INIS)

    Magalhaes, Vanessa D.; Rogero, Sizue O.; Rogero, Jose R.; Cruz, Aurea S.

    2011-01-01

    Cancer is a disease with high incidence and it is considered a worldwide public health problem. Resveratrol is a polyphenol occurring naturally in a wide variety of plants according to response of ultraviolet radiation (UV) exposition or according to mechanical stress resulting of pathogens or chemical and physical agents. This polyphenol possesses a pharmacological activity of carcinogenesis inhibition in multiple levels. It also protects cells by scavenging the free radicals which are considered toxic products. These free radicals are formed of natural process of cell aging and also by incidence of ionizing radiation in the organism. Thus, resveratrol is considered as a cell radioprotector. On the other hand, in some elevated concentrations resveratrol may be considered as a radiosensitizing. The aim of this work was the determination of radiation lethal dose (LD 50 ) and also verifies the cytotoxicity level of resveratrol in tumor cells line: muco epidermoid pulmonary carcinoma cells (NCI-H292) and rhabdomyosarcoma cells (RD). The cytotoxicity test was performed by neutral red uptake assay. The results of resveratrol IC 50% in NCI-H292 cells was 192μM and in RD cells was 128μM; and RD cells gamma radiation LD 50 was 435Gy. (author)

  14. How low-level laser therapy can change mechanical properties of cells

    Science.gov (United States)

    de Magalhães, Ana Carolina; Martinez, Diana; Ferreira, Marcia Z. J.; Yoshimura, Ellisabeth M.; Alencar, Adriano M.; Chavantes, Maria Cristina

    2013-03-01

    Low level laser therapy is used as a treatment of several conditions, including inflammatory processes and wound healing. Possible changes in mechanical properties of cells, caused by illumination, are investigated with optical magnetic twisting cytometry (OMTC), which is a technique used to evaluate mechanical properties in cell culture. Ferromagnetic micro beads are bound to cell cytoskeleton, the beads are magnetized vertically and a horizontal twisting magnetic field is applied causing a torque that moves the beads and deforms the cell, the beads rotate and displace. Based on the lateral displacement of the beads, elastic shear and loss moduli are obtained. Samples of human bronchial epithelial cell culture were divided in two groups: one was illuminated with a 660 nm red laser, 30 mW power, 0.75 W/cm2 irradiance, during different time intervals, and the other one, the control group, was not illuminated. The values of the mechanical constants of the cells of the control group showed a tendency of increasing with the time out of the incubator. On the other hand, the illuminated group showed constancy on the behavior of both moduli, keeping the normal conditions of the cell culture. Those results indicate that illumination can induce cells to homeostasis, and OMTC is sensitive to observe departures from the steady conditions. Hence, OMTC is an important technique which can be used to aggregate knowledge on the light effect in cell cytoskeleton and even on the low level laser therapy mechanisms in inflammatory processes and/or wound healing.

  15. Regulation of myogenesis and skeletal muscle regeneration: effects of oxygen levels on satellite cell activity.

    Science.gov (United States)

    Chaillou, Thomas; Lanner, Johanna T

    2016-12-01

    Reduced oxygen (O 2 ) levels (hypoxia) are present during embryogenesis and exposure to altitude and in pathologic conditions. During embryogenesis, myogenic progenitor cells reside in a hypoxic microenvironment, which may regulate their activity. Satellite cells are myogenic progenitor cells localized in a local environment, suggesting that the O 2 level could affect their activity during muscle regeneration. In this review, we present the idea that O 2 levels regulate myogenesis and muscle regeneration, we elucidate the molecular mechanisms underlying myogenesis and muscle regeneration in hypoxia and depict therapeutic strategies using changes in O 2 levels to promote muscle regeneration. Severe hypoxia (≤1% O 2 ) appears detrimental for myogenic differentiation in vitro, whereas a 3-6% O 2 level could promote myogenesis. Hypoxia impairs the regenerative capacity of injured muscles. Although it remains to be explored, hypoxia may contribute to the muscle damage observed in patients with pathologies associated with hypoxia (chronic obstructive pulmonary disease, and peripheral arterial disease). Hypoxia affects satellite cell activity and myogenesis through mechanisms dependent and independent of hypoxia-inducible factor-1α. Finally, hyperbaric oxygen therapy and transplantation of hypoxia-conditioned myoblasts are beneficial procedures to enhance muscle regeneration in animals. These therapies may be clinically relevant to treatment of patients with severe muscle damage.-Chaillou, T. Lanner, J. T. Regulation of myogenesis and skeletal muscle regeneration: effects of oxygen levels on satellite cell activity. © FASEB.

  16. Ten-eleven translocation 1 dysfunction reduces 5-hydroxymethylcytosine expression levels in gastric cancer cells

    OpenAIRE

    Wang, Kuo-Chiang; Kang, Chi-Hsiang; Tsai, Chung-Yu; Chou, Nan-Hua; Tu, Ya-Ting; Li, Guan-Cheng; Lam, Hing-Chung; Liu, Shiuh-Inn; Chang, Po-Min; Lin, Yan-Hwai; Tsai, Kuo-Wang

    2017-01-01

    A sixth base, 5-hydroxymethylcytosine (5hmC), is formed by the oxidation of 5-methylcytosine (5mC) via the catalysis of the ten-eleven translocation (TET) protein family in cells. Expression levels of 5hmC are frequently depleted during carcinogenesis. However, the detailed mechanisms underlying the depletion of 5hmC expression in gastric cancer cells remains unclear, and further research is required. The present study examined the expression levels of 5mC and 5hmC and the expression levels o...

  17. Laminin increases both levels and activity of tyrosine hydroxylase in calf adrenal chromaffin cells

    OpenAIRE

    1986-01-01

    We have investigated the effects of substrate-bound laminin on levels of enzymes of the catecholamine biosynthetic pathway in primary cultures of calf adrenal chromaffin cells. Laminin increases the levels of the enzymes tyrosine hydroxylase, dopamine-beta-hydroxylase, and phenylethanolamine-N-methyl-transferase. This effect is selective, in that levels of other enzymes (lactate dehydrogenase, aromatic amino acid decarboxylase, and acetylcholinesterase) are not increased. The effect of lamini...

  18. Serum Copper Level Significantly Influences Platelet Count, Lymphocyte Count and Mean Cell Hemoglobin in Sickle Cell Anemia

    Directory of Open Access Journals (Sweden)

    Okocha Chide

    2015-12-01

    Full Text Available Background Changes in serum micro nutrients levels affect a number of critically important metabolic processes; these could potentially influence blood counts and ultimately disease presentation in patients with sickle cell anemia (SCA. Objectives To evaluate the influence of serum micro-nutrients levels; zinc, copper, selenium and magnesium on blood counts in steady state SCA patients. Methods A cross sectional study that involved 28 steady state adult SCA subjects. Seven milliliters (mls of blood was collected; 3 mls was for hemoglobin electrophoresis and full blood count determination while 4 mls was for measurement of serum micro nutrients levels, by the atomic absorption spectrophotometry. Correlation between serum micro-nutrient levels and blood counts was done by the Pearson’s linear regression. Ethical approval was obtained from the institutional review board and each participant gave informed consent. All data was analyzed by SPSS software version 20. Results There was a significant correlation between serum copper levels and mean cell hemoglobin (MCH, platelet and lymphocyte counts (r = 0.418; P = 0.02, r = -0.376; P = 0.04 and r = -0.383; P = 0.04, respectively. There were no significant correlations between serum levels of other micro nutrients (selenium, zinc and magnesium and blood counts. Conclusions Copper influences blood count in SCA patients probably by inducing red cell haemolysis, oxidant tissue damage and stimulating the immune system.

  19. No impact on P-gp level in radio-resistant Mcf-7 cells

    International Nuclear Information System (INIS)

    Madhu, L.N.; Rao, Shama; Sarojini, B.K.

    2016-01-01

    Cancer has become the leading cause of human death worldwide. One possible cause for therapeutic failure is that residual tumor cells are reminiscent of stem cells, which ultimately give rise to secondary tumors or distant metastasis. The property of resistance to radiation therapy or chemotherapy might be the major clinical criterion to characterize 'cancer stem cells (CSCs)'. In the process of radiotherapy, the radiosensitive cancer will become a radioresistant one. Such radio-resistance cells might also show the characters of multi drug resistance (MRD) properties which may affect the chemotherapy process. The present study was carried out to know the expression level of P-gp, a MRD protein in radioresistance breast cancer cells. The study conducted by exposing the MCF-7 cells to 4Gy of gamma radiation

  20. Probing single- to multi-cell level charge transport in Geobacter sulfurreducens DL-1.

    Science.gov (United States)

    Jiang, Xiaocheng; Hu, Jinsong; Petersen, Emily R; Fitzgerald, Lisa A; Jackan, Charles S; Lieber, Alexander M; Ringeisen, Bradley R; Lieber, Charles M; Biffinger, Justin C

    2013-01-01

    Microbial fuel cells, in which living microorganisms convert chemical energy into electricity, represent a potentially sustainable energy technology for the future. Here we report the single-bacterium level current measurements of Geobacter sulfurreducens DL-1 to elucidate the fundamental limits and factors determining maximum power output from a microbial fuel cell. Quantized stepwise current outputs of 92(±33) and 196(±20) fA are generated from microelectrode arrays confined in isolated wells. Simultaneous cell imaging/tracking and current recording reveals that the current steps are directly correlated with the contact of one or two cells with the electrodes. This work establishes the amount of current generated by an individual Geobacter cell in the absence of a biofilm and highlights the potential upper limit of microbial fuel cell performance for Geobacter in thin biofilms.

  1. Microarray analysis of genes associated with cell surface NIS protein levels in breast cancer.

    Science.gov (United States)

    Beyer, Sasha J; Zhang, Xiaoli; Jimenez, Rafael E; Lee, Mei-Ling T; Richardson, Andrea L; Huang, Kun; Jhiang, Sissy M

    2011-10-11

    Na+/I- symporter (NIS)-mediated iodide uptake allows radioiodine therapy for thyroid cancer. NIS is also expressed in breast tumors, raising potential for radionuclide therapy of breast cancer. However, NIS expression in most breast cancers is low and may not be sufficient for radionuclide therapy. We aimed to identify biomarkers associated with NIS expression such that mechanisms underlying NIS modulation in human breast tumors may be elucidated. Published oligonucleotide microarray data within the National Center for Biotechnology Information Gene Expression Omnibus database were analyzed to identify gene expression tightly correlated with NIS mRNA level among human breast tumors. NIS immunostaining was performed in a tissue microarray composed of 28 human breast tumors which had corresponding oligonucleotide microarray data available for each tumor such that gene expression associated with cell surface NIS protein level could be identified. NIS mRNA levels do not vary among breast tumors or when compared to normal breast tissues when detected by Affymetrix oligonucleotide microarray platforms. Cell surface NIS protein levels are much more variable than their corresponding NIS mRNA levels. Despite a limited number of breast tumors examined, our analysis identified cysteinyl-tRNA synthetase as a biomarker that is highly associated with cell surface NIS protein levels in the ER-positive breast cancer subtype. Further investigation on genes associated with cell surface NIS protein levels within each breast cancer molecular subtype may lead to novel targets for selectively increasing NIS expression/function in a subset of breast cancers patients.

  2. Fetal microchimerism persists at high levels in c-kit stem cells in sensitized mothers.

    Science.gov (United States)

    Dutta, Partha; Dart, Melanie L; Schumacher, Steve M; Burlingham, William J

    2010-10-01

    We previously showed that fetal and maternal exposure to non-inherited maternal antigens (NIMA) during gestation and nursing resulted in lifelong tolerance to NIMA in some offspring. This NIMA-specific tolerance was mediated by regulatory T cells (Tregs) and was correlated with the level of multi-lineage maternal microchimerism (Mc) indicating a causative link between Mc and Treg development. To determine if transfer of fetal cells into mothers resulted in a similar tolerance to fetal cells, we used qPCR to detect rare fetal derived cells and a delayed type hypersensitivity (DTH) assay to detect fetal alloantigen-specific effector and regulatory T cells in mothers. We found that 5/8 B6 mothers of H2(b/d) offspring were sensitized to the alloantigens H2(d) and HY, indicating a dominance of alloantigen-specific effector T cells. Though these sensitized mothers did not have detectable fetal Mc (FMc) in any of the organs tested, they had very high levels of fetus-derived c-kit(+) stem cells in their bone marrow. The remaining 3/8 B6 mothers that were not sensitized to the fetal antigens had detectable FMc found mostly in heart, lungs and liver, and in 2/3, we could detect alloantigen-specific regulatory T cells. This data indicates that, as in NIMA-specific tolerance, tolerance in multiparous females to inherited paternal antigens (IPA) expressed by the fetus is associated with the presence of fetal Mc in differentiated cell subsets. Surprisingly, robust lin(-)c-kit(+) bone marrow cell fetal Mc can occur in sensitized mothers. This suggests a continuous source of allospecific priming, coupled with active elimination of mature IPA-expressing lin(+) cells by effector T cells of the maternal host.

  3. Hydrogen gas alleviates oxygen toxicity by reducing hydroxyl radical levels in PC12 cells.

    Directory of Open Access Journals (Sweden)

    Junchao Yu

    Full Text Available Hyperbaric oxygen (HBO therapy through breathing oxygen at the pressure of above 1 atmosphere absolute (ATA is useful for varieties of clinical conditions, especially hypoxic-ischemic diseases. Because of generation of reactive oxygen species (ROS, breathing oxygen gas at high pressures can cause oxygen toxicity in the central nervous system, leading to multiple neurological dysfunction, which limits the use of HBO therapy. Studies have shown that Hydrogen gas (H2 can diminish oxidative stress and effectively reduce active ROS associated with diseases. However, the effect of H2 on ROS generated from HBO therapy remains unclear. In this study, we investigated the effect of H2 on ROS during HBO therapy using PC12 cells. PC12 cells cultured in medium were exposed to oxygen gas or mixed oxygen gas and H2 at 1 ATA or 5 ATA. Cells viability and oxidation products and ROS were determined. The data showed that H2 promoted the cell viability and inhibited the damage in the cell and mitochondria membrane, reduced the levels of lipid peroxidation and DNA oxidation, and selectively decreased the levels of •OH but not disturbing the levels of O2•-, H2O2, or NO• in PC12 cells during HBO therapy. These results indicated that H2 effectively reduced •OH, protected cells against oxygen toxicity resulting from HBO therapy, and had no effect on other ROS. Our data supported that H2 could be potentially used as an antioxidant during HBO therapy.

  4. Increased reactive oxygen species levels cause ER stress and cytotoxicity in andrographolide treated colon cancer cells.

    Science.gov (United States)

    Banerjee, Aditi; Banerjee, Vivekjyoti; Czinn, Steven; Blanchard, Thomas

    2017-04-18

    Chemotherapy continues to play an essential role in the management of many cancers including colon cancer, the third leading cause of death due to cancer in the United States. Many naturally occurring plant compounds have been demonstrated to possess anti-cancer cell activity and have the potential to supplement existing chemotherapy strategies. The plant metabolite andrographolide induces cell death in cancer cells and apoptosis is dependent upon the induction of endoplasmic reticulum stress (ER stress) leading to the unfolded protein response (UPR). The goal of the present study was to determine the mechanism by which andrographolide induces ER stress and to further evaluate its role in promoting cell death pathways. The T84 and COLO 205 cancer cell lines were used to demonstrate that andrographolide induces increased ROS levels, corresponding anti-oxidant response molecules, and reduced mitochondrial membrane potential. No increases in ROS levels were detected in control colon fibroblast cells. Andrographolide-induced cell death, UPR signaling, and CHOP, Bax, and caspase 3 apoptosis elements were all inhibited in the presence of the ROS scavenger NAC. Additionally, andrographolide-induced suppression of cyclins B1 and D1 were also reversed in the presence of NAC. Finally, Akt phosphorylation and phospho-mTOR levels that are normally suppressed by andrographolide were also expressed at normal levels in the absence of ROS. These data demonstrate that andrographolide induces ER stress leading to apoptosis through the induction of ROS and that elevated ROS also play an important role in down-regulating cell cycle progression and cell survival pathways as well.

  5. High levels of SOX5 decrease proliferative capacity of human B cells, but permit plasmablast differentiation.

    Directory of Open Access Journals (Sweden)

    Mirzokhid Rakhmanov

    Full Text Available Currently very little is known about the differential expression and function of the transcription factor SOX5 during B cell maturation. We identified two new splice variants of SOX5 in human B cells, encoding the known L-SOX5B isoform and a new shorter isoform L-SOX5F. The SOX5 transcripts are highly expressed during late stages of B-cell differentiation, including atypical memory B cells, activated CD21low B cells and germinal center B cells of tonsils. In tonsillar sections SOX5 expression was predominantly polarized to centrocytes within the light zone. After in vitro stimulation, SOX5 expression was down-regulated during proliferation while high expression levels were permissible for plasmablast differentiation. Overexpression of L-SOX5F in human primary B lymphocytes resulted in reduced proliferation, less survival of CD138neg B cells, but comparable numbers of CD138+CD38hi plasmablasts compared to control cells. Thus, our findings describe for the first time a functional role of SOX5 during late B cell development reducing the proliferative capacity and thus potentially affecting the differentiation of B cells during the germinal center response.

  6. Functional importance of GLP-1 receptor species and expression levels in cell lines.

    Science.gov (United States)

    Knudsen, Lotte Bjerre; Hastrup, Sven; Underwood, Christina Rye; Wulff, Birgitte Schjellerup; Fleckner, Jan

    2012-04-10

    Of the mammalian species, only the GLP-1 receptors of rat and human origin have been described and characterized. Here, we report the cloning of the homologous GLP-1 receptors from mouse, rabbit, pig, cynomolgus monkey and chimp. The GLP-1 receptor is highly conserved across species, thus underlining the physiological importance of the peptide hormone and its receptor across a wide range of mammals. We expressed the receptors by stable transfection of BHK cells, both in cell lines with high expression levels of the cloned receptors, as well as in cell lines with lower expression levels, more comparable to endogenous expression of these receptors. High expression levels of cloned GLP-1 receptors markedly increased the potency of GLP-1 and other high affinity ligands, whereas the K(d) values were not affected. For a low affinity ligand like the ago-allosteric modulator Compound 2, expression levels of the human GLP-1 receptor were important for maximal efficacy as well as potency. The two natural metabolites of GLP-1, GLP-1(9-37) and GLP-1(9-36)amide were agonists when tested on a cell line with high expression of the recombinant human GLP-1 receptor, whereas they behaved as (low potent) antagonists on a cell line that expressed the receptor endogenously, as well as cells expressing a moderate level of the recombinant human GLP-1 receptor. The amide form was a more potent agonist than the free acid from. In conclusion, receptor expression level is an important parametre for selecting cell lines with cloned GLP-1 receptors for functional characterization of physiological and pharmaceutical ligands. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. Low-level red laser therapy alters effects of ultraviolet C radiation on Escherichia coli cells

    International Nuclear Information System (INIS)

    Canuto, K.S.; Guimaraes, O.R.; Geller, M.; Sergio, L.P.S.; Paoli, F.; Fonseca, A.S.

    2015-01-01

    Low-level lasers are used at low power densities and doses according to clinical protocols supplied with laser devices or based on professional practice. Although use of these lasers is increasing in many countries, the molecular mechanisms involved in effects of low-level lasers, mainly on DNA, are controversial. In this study, we evaluated the effects of low-level red lasers on survival, filamentation, and morphology of Escherichia coli cells that were exposed to ultraviolet C (UVC) radiation. Exponential and stationary wild-type and uvrA-deficient E. coli cells were exposed to a low-level red laser and in sequence to UVC radiation. Bacterial survival was evaluated to determine the laser protection factor (ratio between the number of viable cells after exposure to the red laser and UVC and the number of viable cells after exposure to UVC). Bacterial filaments were counted to obtain the percentage of filamentation. Area-perimeter ratios were calculated for evaluation of cellular morphology. Experiments were carried out in duplicate and the results are reported as the means of three independent assays. Pre-exposure to a red laser protected wild-type and uvrA-deficient E. coli cells against the lethal effect of UVC radiation, and increased the percentage of filamentation and the area-perimeter ratio, depending on UVC fluence and physiological conditions in the cells. Therapeutic, low-level red laser radiation can induce DNA lesions at a sub-lethal level. Consequences to cells and tissues should be considered when clinical protocols based on this laser are carried out. (author)

  8. Low-level red laser therapy alters effects of ultraviolet C radiation on Escherichia coli cells

    Energy Technology Data Exchange (ETDEWEB)

    Canuto, K.S.; Guimaraes, O.R.; Geller, M. [Centro Universitario Serra dos Orgaos, Teresopolis, RJ (Brazil). Centro de Ciencias da Saude; Sergio, L.P.S. [Instituto de Biologia Roberto Alcantara Gomes, Rio de Janeiro, RJ (Brazil). Departamento de Biofisica e Biometria; Paoli, F. [Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora, MG (Brazil). Departamento de Morfologia; Fonseca, A.S., E-mail: adnfonseca@ig.com.br [Universidade Federal do Estado do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ (Brazil). Departamento de Ciencias Fisiologicas

    2015-10-15

    Low-level lasers are used at low power densities and doses according to clinical protocols supplied with laser devices or based on professional practice. Although use of these lasers is increasing in many countries, the molecular mechanisms involved in effects of low-level lasers, mainly on DNA, are controversial. In this study, we evaluated the effects of low-level red lasers on survival, filamentation, and morphology of Escherichia coli cells that were exposed to ultraviolet C (UVC) radiation. Exponential and stationary wild-type and uvrA-deficient E. coli cells were exposed to a low-level red laser and in sequence to UVC radiation. Bacterial survival was evaluated to determine the laser protection factor (ratio between the number of viable cells after exposure to the red laser and UVC and the number of viable cells after exposure to UVC). Bacterial filaments were counted to obtain the percentage of filamentation. Area-perimeter ratios were calculated for evaluation of cellular morphology. Experiments were carried out in duplicate and the results are reported as the means of three independent assays. Pre-exposure to a red laser protected wild-type and uvrA-deficient E. coli cells against the lethal effect of UVC radiation, and increased the percentage of filamentation and the area-perimeter ratio, depending on UVC fluence and physiological conditions in the cells. Therapeutic, low-level red laser radiation can induce DNA lesions at a sub-lethal level. Consequences to cells and tissues should be considered when clinical protocols based on this laser are carried out. (author)

  9. Low-level red laser therapy alters effects of ultraviolet C radiation on Escherichia coli cells

    Directory of Open Access Journals (Sweden)

    K.S. Canuto

    2015-01-01

    Full Text Available Low-level lasers are used at low power densities and doses according to clinical protocols supplied with laser devices or based on professional practice. Although use of these lasers is increasing in many countries, the molecular mechanisms involved in effects of low-level lasers, mainly on DNA, are controversial. In this study, we evaluated the effects of low-level red lasers on survival, filamentation, and morphology of Escherichia coli cells that were exposed to ultraviolet C (UVC radiation. Exponential and stationary wild-type and uvrA-deficient E. coli cells were exposed to a low-level red laser and in sequence to UVC radiation. Bacterial survival was evaluated to determine the laser protection factor (ratio between the number of viable cells after exposure to the red laser and UVC and the number of viable cells after exposure to UVC. Bacterial filaments were counted to obtain the percentage of filamentation. Area-perimeter ratios were calculated for evaluation of cellular morphology. Experiments were carried out in duplicate and the results are reported as the means of three independent assays. Pre-exposure to a red laser protected wild-type and uvrA-deficient E. coli cells against the lethal effect of UVC radiation, and increased the percentage of filamentation and the area-perimeter ratio, depending on UVC fluence and physiological conditions in the cells. Therapeutic, low-level red laser radiation can induce DNA lesions at a sub-lethal level. Consequences to cells and tissues should be considered when clinical protocols based on this laser are carried out.

  10. Altered intracellular pH regulation in cells with high levels of P-glycoprotein expression.

    Science.gov (United States)

    Young, Gregory; Reuss, Luis; Altenberg, Guillermo A

    2011-01-01

    P-glycoprotein is an ATP-binding-cassette transporter that pumps many structurally unrelated drugs out of cells through an ATP-dependent mechanism. As a result, multidrug-resistant cells that overexpress P-glycoprotein have reduced intracellular steady-state levels of a variety of chemotherapeutic agents. In addition, increased cytosolic pH has been a frequent finding in multidrug-resistant cells that express P-glycoprotein, and it has been proposed that this consequence of P-glycoprotein expression may contribute to the lower intracellular levels of chemotherapeutic agents. In these studies, we measured intracellular pH and the rate of acid extrusion in response to an acid load in two cells with very different levels of P-glycoprotein expression: V79 parental cells and LZ-8 multidrug resistant cells. Compared to the wild-type V79 cells, LZ-8 cells have a lower intracellular pH and a slower recovery of intracellular pH after an acid load. The data also show that LZ-8 cells have reduced ability to extrude acid, probably due to a decrease in Na(+)/H(+) exchanger activity. The alterations in intracellular pH and acid extrusion in LZ-8 cells are reversed by 24-h exposure to the multidrug-resistance modulator verapamil. The lower intracellular pH in LZ-8 indicates that intracellular alkalinization is not necessary for multidrug resistance. The reversal by verapamil of the decreased acid-extrusion suggests that P-glycoprotein can affect other membrane transport mechanism.

  11. Wild-type p53 controls the level of fibronectin expression in breast cancer cells.

    Science.gov (United States)

    You, Daeun; Jung, Seung Pil; Jeong, Yisun; Bae, Soo Youn; Kim, Sangmin

    2017-10-01

    Aberrant fibronectin (FN) expression is associated with poor prognosis, cell adhesion, and cell motility in a variety of cancer cells. In this study, we investigated the relationship between p53 and FN expression in breast cancer cells. Basal FN expression was significantly decreased by treatment with the p53 activator III, RITA, in MCF7 breast cancer cells with wild-type p53. In addition, overexpression of wild-type p53 markedly decreased the level of FN expression in p53-mutant breast cancer cells. To examine the mechanism underlying the relationship between p53 and FN expression, we treated MCF7 breast cancer cells with the tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate). Our results showed that basal FN expression was increased by TPA treatment in a time-dependent manner. In contrast, the level of p53 expression was decreased by TPA treatment. However, the expression of FN and p53 was not altered by TPA in p53-mutant breast cancer cells. Furthermore, the alterations in FN and p53 expression in response to TPA were prevented by a specific MEK inhibitor, UO126. Finally, we demonstrated that TPA triggers degradation of p53 through the proteasomal pathway in MCF7 cells. TPA-induced FN expression was decreased by the proteasome inhibitor MG132. Under the same condition, p53 protein expression, but not mRNA expression, was reversed by MG132. Taken together, our data demonstrate that the level of FN expression is associated with the status and expression of p53 in breast cancer cells.

  12. SCA-1 Expression Level Identifies Quiescent Hematopoietic Stem and Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Mina N.F. Morcos

    2017-06-01

    Full Text Available Blood cell generation depends on continuous cellular output by the sequential hierarchy of hematopoietic stem cell (HSC and progenitor populations that all contain quiescent and actively cycling cells. Hematopoietic stem and progenitor cells (HSPCs express the surface molecule Stem cell antigen 1 (SCA-1/LY6A. Using histone 2B-red fluorescent fusion protein label retention and cell-cycle reporter mice, we demonstrate that high SCA-1 expression (SCA-1hi identifies not only quiescent HSCs but quiescent cells on all hierarchical levels within the lineage−SCA-1+KIT+ (LSK population. Each transplanted SCA-1hi HSPC population also displayed self-renewal potential superior to that of the respective SCA-1lo population. SCA-1 expression is inducible by type I interferon (IFN. We show, however, that quiescence and high self-renewal capacity of cells with brighter SCA-1 expression at steady state were independent of type I IFN signaling. We conclude that SCA-1 expression levels can be used to prospectively isolate functionally heterogeneous HSPC subpopulations.

  13. Regulation of hTERT by BCR-ABL at multiple levels in K562 cells

    International Nuclear Information System (INIS)

    Chai, Juin Hsien; Zhang, Yong; Tan, Wei Han; Chng, Wee Joo; Li, Baojie; Wang, Xueying

    2011-01-01

    The cytogenetic characteristic of Chronic Myeloid Leukemia (CML) is the formation of the Philadelphia chromosome gene product, BCR-ABL. Given that BCR-ABL is the specific target of Gleevec in CML treatment, we investigated the regulation of the catalytic component of telomerase, hTERT, by BCR-ABL at multiple levels in K562 cells. Molecular techniques such as over expression, knockdown, real-time PCR, immunoprecipitation, western blotting, reporter assay, confocal microscopy, telomerase assays and microarray were used to suggest that hTERT expression and activity is modulated by BCR-ABL at multiple levels. Our results suggest that BCR-ABL plays an important role in regulating hTERT in K562 (BCR-ABL positive human leukemia) cells. When Gleevec inhibited the tyrosine kinase activity of BCR-ABL, phosphorylation of hTERT was downregulated, therefore suggesting a positive correlation between BCR-ABL and hTERT. Gleevec treatment inhibited hTERT at mRNA level and significantly reduced telomerase activity (TA) in K562 cells, but not in HL60 or Jurkat cells (BCR-ABL negative cells). We also demonstrated that the transcription factor STAT5a plays a critical role in hTERT gene regulation in K562 cells. Knockdown of STAT5a, but not STAT5b, resulted in a marked downregulation of hTERT mRNA level, TA and hTERT protein level in K562 cells. Furthermore, translocation of hTERT from nucleoli to nucleoplasm was observed in K562 cells induced by Gleevec. Our data reveal that BCR-ABL can regulate TA at multiple levels, including transcription, post-translational level, and proper localization. Thus, suppression of cell growth and induction of apoptosis by Gleevec treatment may be partially due to TA inhibition. Additionally, we have identified STAT5a as critical mediator of the hTERT gene expression in BCR-ABL positive CML cells, suggesting that targeting STAT5a may be a promising therapeutic strategy for BCR-ABL positive CML patients

  14. Salt tolerance at single cell level in giant-celled Characeae.

    Science.gov (United States)

    Beilby, Mary J

    2015-01-01

    Characean plants provide an excellent experimental system for electrophysiology and physiology due to: (i) very large cell size, (ii) position on phylogenetic tree near the origin of land plants and (iii) continuous spectrum from very salt sensitive to very salt tolerant species. A range of experimental techniques is described, some unique to characean plants. Application of these methods provided electrical characteristics of membrane transporters, which dominate the membrane conductance under different outside conditions. With this considerable background knowledge the electrophysiology of salt sensitive and salt tolerant genera can be compared under salt and/or osmotic stress. Both salt tolerant and salt sensitive Characeae show a rise in membrane conductance and simultaneous increase in Na(+) influx upon exposure to saline medium. Salt tolerant Chara longifolia and Lamprothamnium sp. exhibit proton pump stimulation upon both turgor decrease and salinity increase, allowing the membrane PD to remain negative. The turgor is regulated through the inward K(+) rectifier and 2H(+)/Cl(-) symporter. Lamprothamnium plants can survive in hypersaline media up to twice seawater strength and withstand large sudden changes in salinity. Salt sensitive C. australis succumbs to 50-100 mM NaCl in few days. Cells exhibit no pump stimulation upon turgor decrease and at best transient pump stimulation upon salinity increase. Turgor is not regulated. The membrane PD exhibits characteristic noise upon exposure to salinity. Depolarization of membrane PD to excitation threshold sets off trains of action potentials, leading to further loses of K(+) and Cl(-). In final stages of salt damage the H(+)/OH(-) channels are thought to become the dominant transporter, dissipating the proton gradient and bringing the cell PD close to 0. The differences in transporter electrophysiology and their synergy under osmotic and/or saline stress in salt sensitive and salt tolerant characean cells are

  15. Salt tolerance at single cell level in giant-celled Characeae

    Directory of Open Access Journals (Sweden)

    Mary Jane eBeilby

    2015-04-01

    Full Text Available Characean plants provide an excellent experimental system for electrophysiology and physiology due to: (i very large cell size, (ii position on phylogenetic tree near the origin of land plants and (iii continuous spectrum from very salt sensitive to very salt tolerant species. A range of experimental techniques is described, some unique to characean plants. Application of these methods provided electrical characteristics of membrane transporters, which dominate the membrane conductance under different outside conditions. With this considerable background knowledge the electrophysiology of salt sensitive and salt tolerant genera can be compared under salt and/or osmotic stress. Both salt tolerant and salt sensitive Characeae show a rise in membrane conductance and simultaneous increase in Na+ influx upon exposure to saline medium. Salt tolerant Chara longifolia and Lamprothamnium sp. exhibit proton pump stimulation upon both turgor decrease and salinity increase, allowing the membrane PD to remain negative. The turgor is regulated through the inward K+ rectifier and 2H+/Cl- symporter. Lamprothamnium plants can survive in hypersaline media up to twice seawater strength and withstand large sudden changes in salinity. Salt-sensitive Chara australis succumbs to 50 - 100 mM NaCl in few days. Cells exhibit no pump stimulation upon turgor decrease and at best transient pump stimulation upon salinity increase. Turgor is not regulated. The membrane PD exhibits characteristic noise upon exposure to salinity. Depolarization of membrane PD to excitation threshold sets off trains of action potentials, leading to further loses of K+ and Cl-. In final stages of salt damage the H+/OH- channels are thought to become the dominant transporter, dissipating the proton gradient and bringing the cell PD close to 0. The differences in transporter electrophysiology and their synergy under osmotic and/or saline stress in salt sensitive and salt tolerant characean cells

  16. Upregulation of mitochondrial NAD+ levels impairs the clonogenicity of SSEA1+ glioblastoma tumor-initiating cells

    OpenAIRE

    Son, Myung Jin; Ryu, Jae-Sung; Kim, Jae Yun; Kwon, Youjeong; Chung, Kyung-Sook; Mun, Seon Ju; Cho, Yee Sook

    2017-01-01

    Emerging evidence has emphasized the importance of cancer therapies targeting an abnormal metabolic state of tumor-initiating cells (TICs) in which they retain stem cell-like phenotypes and nicotinamide adenine dinucleotide (NAD+) metabolism. However, the functional role of NAD+ metabolism in regulating the characteristics of TICs is not known. In this study, we provide evidence that the mitochondrial NAD+ levels affect the characteristics of glioma-driven SSEA1+ TICs, including clonogenic gr...

  17. Combining Laser Microsurgery and Finite Element Modeling to Assess Cell-Level Epithelial Mechanics

    Science.gov (United States)

    Hutson, M. Shane; Veldhuis, J.; Ma, Xiaoyan; Lynch, Holley E.; Cranston, P. Graham; Brodland, G. Wayne

    2009-01-01

    Abstract Laser microsurgery and finite element modeling are used to determine the cell-level mechanics of the amnioserosa—a morphogenetically crucial epithelium on the dorsal surface of fruit fly embryos (Drosophila melanogaster). In the experiments, a tightly focused laser ablates a subcellular hole (1 μm in diameter) that passes clean through the epithelium. The surrounding cells recoil from the wound site with a large range of initial recoil velocities. These depend on the embryo's developmental stage and the subcellular wound site. The initial recoil (up to 0.1 s) is well reproduced by a base finite element model, which assumes a uniform effective viscosity inside the cells, a constant tension along each cell-cell boundary, and a large, potentially anisotropic, far-field stress—one that far exceeds the stress equivalent of the cell-edge tensions. After 0.1 s, the experimental recoils slow dramatically. This observation can be reproduced by adding viscoelastic rods along cell edges or as a fine prestressed mesh parallel to the apical and basal membranes of the cell. The mesh also reproduces a number of double-wounding experiments in which successive holes are drilled in a single cell. PMID:20006944

  18. Multiweek cell culture project for use in upper-level biology laboratories.

    Science.gov (United States)

    Marion, Rebecca E; Gardner, Grant E; Parks, Lisa D

    2012-06-01

    This article describes a laboratory protocol for a multiweek project piloted in a new upper-level biology laboratory (BIO 426) using cell culture techniques. Human embryonic kidney-293 cells were used, and several culture media and supplements were identified for students to design their own experiments. Treatments included amino acids, EGF, caffeine, epinephrine, heavy metals, and FBS. Students researched primary literature to determine their experimental variables, made their own solutions, and treated their cells over a period of 2 wk. Before this, a sterile technique laboratory was developed to teach students how to work with the cells and minimize contamination. Students designed their experiments, mixed their solutions, seeded their cells, and treated them with their control and experimental media. Students had the choice of manipulating a number of variables, including incubation times, exposure to treatment media, and temperature. At the end of the experiment, students observed the effects of their treatment, harvested and dyed their cells, counted relative cell numbers in control and treatment flasks, and determined the ratio of living to dead cells using a hemocytometer. At the conclusion of the experiment, students presented their findings in a poster presentation. This laboratory can be expanded or adapted to include additional cell lines and treatments. The ability to design and implement their own experiments has been shown to increase student engagement in the biology-related laboratory activities as well as develop the critical thinking skills needed for independent research.

  19. Immune cell distribution and immunoglobulin levels change following sciatic nerve injury in a rat model

    Directory of Open Access Journals (Sweden)

    Wei Yuan

    2016-07-01

    Full Text Available Objective(s: To investigate the systemic and local immune status of two surgical rat models of sciatic nerve injury, a crushed sciatic nerve, and a sciatic nerve transection Materials and Methods:Twenty-four adult male Sprague-Dawley rats were randomly divided into three groups: sham-operation (control group, sciatic nerve crush, and sciatic nerve transaction. Sciatic nerve surgery was performed. The percentage of CD4+ cells and the CD4+/CD8+ratio were determined by flow cytometry. Serum IgM and IgG levels were analyzed by ELISA. T-cells (CD3 and macrophages (CD68 in sciatic nerve tissue sections were identified through immunohistochemistry. Results: Compared to sham-operated controls, in rats that underwent nerve injury, the percentage of CD4+ cells and the CD4+/CD8+ ratio in the peripheral blood were significantly  decreased 7 days after surgery, serum IgM levels were increased 14 days after surgery, and serum IgG levels were increased 21 days after surgery. There were a large number of CD3+ cells and a small number of CD68+ cells in sciatic nerve tissue sections 21 days after surgery, indicating T-cell and macrophage activation and infiltration. Local IgG deposition was also detected at the nerve injury site 21 days after surgery. Conclusion: Rat humoral and cellular immune status changed following sciatic nerve injury, particularly with regard to the cellular immune response at the nerve injury site.

  20. Detection of the Level of Reactive Oxygen Species Induced by Ionizing Radiation in Cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jin Kyu; Chung, Dong Min; Kim, Jin-Hong [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2015-05-15

    By definition, the direct effect is referred to interaction between photon and DNA molecule, whereas the indirect effect is mediated by the reactive oxygen species (ROS) generated by radiolysis and subsequent reaction. It has been reported that ROS produced after exposure to IR can react with cellular materials such as DNA, proteins, carbohydrates and lipids. ROS is free radicals such as the superoxide anion, hydroxyl radicals and the non-radical hydrogen peroxide. Cells generate ROS during aerobic metabolism. Excessive production of ROS can lead to oxidative stress, genetic alteration and even cell death. It has been reported that ROS plays a critical role in radiation-induced cell injury. Thus, it is of great interest to determine the radiation-induced ROS level. Many kinds of methods to detect the level of ROS have been developed so far. There were random changes of fluorescence intensity in the treatment after irradiation. This result meant that this protocol was not appropriate for determination of radiation-induced ROS. On the other hand, the fluorescence intensity was increased in a dose-dependent manner when the cells were treated with the DCFH-DA solution before irradiation. Conclusions can be drawn from the experimental results of this study. In order to properly measure the ROS level in the cells exposed to ionizing radiation, the cells should be treated with the DCFH-DA solution before irradiation.

  1. Performance analysis of three-dimensional-triple-level cell and two-dimensional-multi-level cell NAND flash hybrid solid-state drives

    Science.gov (United States)

    Sakaki, Yukiya; Yamada, Tomoaki; Matsui, Chihiro; Yamaga, Yusuke; Takeuchi, Ken

    2018-04-01

    In order to improve performance of solid-state drives (SSDs), hybrid SSDs have been proposed. Hybrid SSDs consist of more than two types of NAND flash memories or NAND flash memories and storage-class memories (SCMs). However, the cost of hybrid SSDs adopting SCMs is more expensive than that of NAND flash only SSDs because of the high bit cost of SCMs. This paper proposes unique hybrid SSDs with two-dimensional (2D) horizontal multi-level cell (MLC)/three-dimensional (3D) vertical triple-level cell (TLC) NAND flash memories to achieve higher cost-performance. The 2D-MLC/3D-TLC hybrid SSD achieves up to 31% higher performance than the conventional 2D-MLC/2D-TLC hybrid SSD. The factors of different performance between the proposed hybrid SSD and the conventional hybrid SSD are analyzed by changing its block size, read/write/erase latencies, and write unit of 3D-TLC NAND flash memory, by means of a transaction-level modeling simulator.

  2. Alveolar type II cell transplantation restores pulmonary surfactant protein levels in lung fibrosis.

    Science.gov (United States)

    Guillamat-Prats, Raquel; Gay-Jordi, Gemma; Xaubet, Antoni; Peinado, Victor I; Serrano-Mollar, Anna

    2014-07-01

    Alveolar Type II cell transplantation has been proposed as a cell therapy for the treatment of idiopathic pulmonary fibrosis. Its long-term benefits include repair of lung fibrosis, but its success partly depends on the restoration of lung homeostasis. Our aim was to evaluate surfactant protein restoration after alveolar Type II cell transplantation in an experimental model of bleomycin-induced lung fibrosis in rats. Lung fibrosis was induced by intratracheal instillation of bleomycin. Alveolar Type II cells were obtained from healthy animals and transplanted 14 days after bleomycin was administered. Furthermore, one group transplanted with alveolar macrophages and another group treated with surfactant were established to evaluate the specificity of the alveolar Type II cell transplantation. The animals were euthanized at 21 days after bleomycin instillation. Lung fibrosis was confirmed by a histologic study and an evaluation of the hydroxyproline content. Changes in surfactant proteins were evaluated by mRNA expression, Western blot and immunofluorescence studies. The group with alveolar Type II cell transplantation was the only one to show a reduction in the degree of lung fibrosis and a complete recovery to normal levels of surfactant proteins. One of the mechanisms involved in the beneficial effect of alveolar Type II cell transplantation is restoration of lung surfactant protein levels, which is required for proper respiratory function. Copyright © 2014 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.

  3. T cell Bim levels reflect responses to anti–PD-1 cancer therapy

    Science.gov (United States)

    Dronca, Roxana S.; Liu, Xin; Harrington, Susan M.; Chen, Lingling; Cao, Siyu; Kottschade, Lisa A.; McWilliams, Robert R.; Block, Matthew S.; Nevala, Wendy K.; Thompson, Michael A.; Mansfield, Aaron S.; Park, Sean S.; Markovic, Svetomir N.

    2016-01-01

    Immune checkpoint therapy with PD-1 blockade has emerged as an effective therapy for many advanced cancers; however, only a small fraction of patients achieve durable responses. To date, there is no validated blood-based means of predicting the response to PD-1 blockade. We report that Bim is a downstream signaling molecule of the PD-1 pathway, and its detection in T cells is significantly associated with expression of PD-1 and effector T cell markers. High levels of Bim in circulating tumor-reactive (PD-1+CD11ahiCD8+) T cells were prognostic of poor survival in patients with metastatic melanoma who did not receive anti–PD-1 therapy and were also predictive of clinical benefit in patients with metastatic melanoma who were treated with anti–PD-1 therapy. Moreover, this circulating tumor-reactive T cell population significantly decreased after successful anti–PD-1 therapy. Our study supports a crucial role of Bim in both T cell activation and apoptosis as regulated by PD-1 and PD-L1 interactions in effector CD8+ T cells. Measurement of Bim levels in circulating T cells of patients with cancer may provide a less invasive strategy to predict and monitor responses to anti–PD-1 therapy, although future prospective analyses are needed to validate its utility. PMID:27182556

  4. Histamine-induced increases in cyclic AMP levels in bovine adrenal medullary cells.

    OpenAIRE

    Marley, P. D.; Thomson, K. A.; Jachno, K.; Johnston, M. J.

    1991-01-01

    1. The effect of histamine on cellular cyclic AMP levels in cultured bovine adrenal medullary cells has been studied. 2. Histamine (0.3-30 microM) increased cyclic AMP levels transiently, with a maximal response after 5 min, a smaller response after 20 min, and no increase seen after 80 or 180 min. The EC50 at 5 min was approximately 2 microM. Histamine had no effect on cyclic AMP release from the cells over 5 min, but increased it after 90 min. 3. The cyclic AMP response to 5 microM histamin...

  5. Human Lung Mast Cell Products Regulate Airway Smooth Muscle CXCL10 Levels.

    Science.gov (United States)

    Alkhouri, H; Cha, V; Tong, K; Moir, L M; Armour, C L; Hughes, J M

    2014-01-01

    In asthma, the airway smooth muscle (ASM) produces CXCL10 which may attract CXCR3(+) mast/T cells to it. Our aim was to investigate the effects of mast cell products on ASM cell CXCL10 production. ASM cells from people with and without asthma were stimulated with IL-1 β , TNF- α , and/or IFN γ and treated with histamine (1-100  μ M) ± chlorpheniramine (H1R antagonist; 1  μ M) or ranitidine (H2R antagonist; 50  μ M) or tryptase (1 nM) ± leupeptin (serine protease inhibitor; 50  μ M), heat-inactivated tryptase, or vehicle for 4 h or 24 h. Human lung mast cells (MC) were isolated and activated with IgE/anti-IgE and supernatants were collected after 2 h or 24 h. The supernatants were added to ASM cells for 48 h and ASM cell CXCL10 production detected using ELISA (protein) and real-time PCR (mRNA). Histamine reduced IL-1 β /TNF- α -induced CXCL10 protein, but not mRNA, levels independent of H1 and H2 receptor activation, whereas tryptase and MC 2 h supernatants reduced all cytokine-induced CXCL10. Tryptase also reduced CXCL10 levels in a cell-free system. Leupeptin inhibited the effects of tryptase and MC 2 h supernatants. MC 24 h supernatants contained TNF- α and amplified IFN γ -induced ASM cell CXCL10 production. This is the first evidence that MC can regulate ASM cell CXCL10 production and its degradation. Thus MC may regulate airway myositis in asthma.

  6. Multi-Level Communication of Human Retinal Pigment Epithelial Cells via Tunneling Nanotubes

    Science.gov (United States)

    Wittig, Dierk; Wang, Xiang; Walter, Cindy; Gerdes, Hans-Hermann; Funk, Richard H. W.; Roehlecke, Cora

    2012-01-01

    Background Tunneling nanotubes (TNTs) may offer a very specific and effective way of intercellular communication. Here we investigated TNTs in the human retinal pigment epithelial (RPE) cell line ARPE-19. Morphology of TNTs was examined by immunostaining and scanning electron microscopy. To determine the function of TNTs between cells, we studied the TNT-dependent intercellular communication at different levels including electrical and calcium signalling, small molecular diffusion as well as mitochondrial re-localization. Further, intercellular organelles transfer was assayed by FACS analysis. Methodology and Principal Findings Microscopy showed that cultured ARPE-19 cells are frequently connected by TNTs, which are not attached to the substratum. The TNTs were straight connections between cells, had a typical diameter of 50 to 300 nm and a length of up to 120 µm. We observed de novo formation of TNTs by diverging from migrating cells after a short time of interaction. Scanning electron microscopy confirmed characteristic features of TNTs. Fluorescence microscopy revealed that TNTs between ARPE-19 cells contain F-actin but no microtubules. Depolymerisation of F-actin, induced by addition of latrunculin-B, led to disappearance of TNTs. Importantly, these TNTs could function as channels for the diffusion of small molecules such as Lucifer Yellow, but not for large molecules like Dextran Red. Further, organelle exchange between cells via TNTs was observed by microscopy. Using Ca2+ imaging we show the intercellular transmission of calcium signals through TNTs. Mechanical stimulation led to membrane depolarisation, which expand through TNT connections between ARPE-19 cells. We further demonstrate that TNTs can mediate electrical coupling between distant cells. Immunolabelling for Cx43 showed that this gap junction protein is interposed at one end of 44% of TNTs between ARPE-19 cells. Conclusions and Significance Our observations indicate that human RPE cell line ARPE

  7. Activin-A and Bmp4 levels modulate cell type specification during CHIR-induced cardiomyogenesis.

    Directory of Open Access Journals (Sweden)

    Min-Su Kim

    Full Text Available The use of human pluripotent cell progeny for cardiac disease modeling, drug testing and therapeutics requires the ability to efficiently induce pluripotent cells into the cardiomyogenic lineage. Although direct activation of the Activin-A and/or Bmp pathways with growth factors yields context-dependent success, recent studies have shown that induction of Wnt signaling using low molecular weight molecules such as CHIR, which in turn induces the Activin-A and Bmp pathways, is widely effective. To further enhance the reproducibility of CHIR-induced cardiomyogenesis, and to ultimately promote myocyte maturation, we are using exogenous growth factors to optimize cardiomyogenic signaling downstream of CHIR induction. As indicated by RNA-seq, induction with CHIR during Day 1 (Days 0-1 was followed by immediate expression of Nodal ligands and receptors, followed later by Bmp ligands and receptors. Co-induction with CHIR and high levels of the Nodal mimetic Activin-A (50-100 ng/ml during Day 0-1 efficiently induced definitive endoderm, whereas CHIR supplemented with Activin-A at low levels (10 ng/ml consistently improved cardiomyogenic efficiency, even when CHIR alone was ineffective. Moreover, co-induction using CHIR and low levels of Activin-A apparently increased the rate of cardiomyogenesis, as indicated by the initial appearance of rhythmically beating cells by Day 6 instead of Day 8. By contrast, co-induction with CHIR plus low levels (3-10 ng/ml of Bmp4 during Day 0-1 consistently and strongly inhibited cardiomyogenesis. These findings, which demonstrate that cardiomyogenic efficacy is improved by optimizing levels of CHIR-induced growth factors when applied in accord with their sequence of endogenous expression, are consistent with the idea that Nodal (Activin-A levels toggle the entry of cells into the endodermal or mesodermal lineages, while Bmp levels regulate subsequent allocation into mesodermal cell types.

  8. Oxygen levels and the regulation of cell adhesion in the nervous system

    Science.gov (United States)

    Crossin, Kathryn

    2012-01-01

    In this article, I discuss the hallmarks of hypoxia in vitro and in vivo and review work showing that many types of stem cell proliferate more robustly in lowered oxygen. I then discuss recent studies showing that alterations in the levels and the types of cell and substrate adhesion molecules are a notable response to reduced O2 levels in both cultured primary neural stem cells and brain tissues in response to hypoxia in vivo. The ability of O2 levels to regulate adhesion molecule expression is linked to the Wnt signaling pathway, which can control and be controlled by adhesion events. The ability of O2 levels to influence cell adhesion also has far-reaching implications for development, ischemic trauma and neural regeneration, as well as for cancer and other diseases. Finally I discuss the possibility that the fluctuations in O2 levels known to have occurred over evolutionary time could, by influencing adhesion systems, have contributed to early symbiotic events in unicellular organisms and to the emergence of multicellularity. It is not my intention to be exhaustive in these domains, which are far from my own field of study. Rather this article is meant to provoke and stimulate thinking about molecular evolution involving O2 sensing and signaling during eras of geologic and atmospheric change that might inform modern studies on development and disease. PMID:22647940

  9. In vitro differentiation of bone marrow stromal cells into neurons and glial cells and differential protein expression in a two-compartment bone marrow stromal cell/neuron co-culture system.

    Science.gov (United States)

    Qi, Xu; Shao, Ming; Peng, Haisheng; Bi, Zhenggang; Su, Zhiqiang; Li, Hulun

    2010-07-01

    This study was performed to establish a bone marrow stromal cell (BMSC)/neuron two-compartment co-culture model in which differentiation of BMSCs into neurons could occur without direct contact between the two cell types, and to investigate protein expression changes during differentiation of this entirely BMSC-derived population. Cultured BMSCs isolated from Wistar rats were divided into three groups: BMSC culture, BMSC/neuron co-culture and BMSC/neuron two-compartment co-culture. Cells were examined for neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) expression. The electrophysiological behavior of the BMSCs was examined using patch clamping. Proteins that had significantly different expression levels in BMSCs cultured alone and co-cultured with neurons were studied using a protein chip-mass spectroscopy technique. Expression of NSE and GFAP were significantly higher in co-culture cells than in two-compartment co-culture cells, and significantly higher in both co-culture groups than in BMSCs cultured alone. Five proteins showed significant changes in expression during differentiation: TIP39_RAT and CALC_RAT underwent increases, and INSL6_RAT, PNOC_RAT and PCSK1_RAT underwent decreases in expression. We conclude that BMSCs can differentiate into neurons during both contact co-culture with neurons and two-compartment co-culture with neurons. The rate at which BMSCs differentiated into neurons was higher in contact co-culture than in non-contact co-culture.

  10. Elevated levels of cell-free circulating DNA in patients with acute dengue virus infection.

    Directory of Open Access Journals (Sweden)

    Tran Thi Ngoc Ha

    Full Text Available BACKGROUND: Apoptosis is thought to play a role in the pathogenesis of severe dengue and the release of cell-free DNA into the circulatory system in several medical conditions. Therefore, we investigated circulating DNA as a potential biomarker for severe dengue. METHODS AND FINDINGS: A direct fluorometric degradation assay using PicoGreen was performed to quantify cell-free DNA from patient plasma. Circulating DNA levels were significantly higher in patients with dengue virus infection than with other febrile illnesses and healthy controls. Remarkably, the increase of DNA levels correlated with the severity of dengue. Additionally, multivariate logistic regression analysis showed that circulating DNA levels independently correlated with dengue shock syndrome. CONCLUSIONS: Circulating DNA levels were increased in dengue patients and correlated with dengue severity. Additional studies are required to show the benefits of this biomarker in early dengue diagnosis and for the prognosis of shock complication.

  11. Gene-specific correlation of RNA and protein levels in human cells and tissues

    DEFF Research Database (Denmark)

    Edfors, Fredrik; Danielsson, Frida; Hallström, Björn M.

    2016-01-01

    ) conversion factor independent of the tissue type is introduced, thus significantly enhancing the predictability of protein copy numbers from RNA levels. The results show that the RTP ratio varies significantly with a few hundred copies per mRNA molecule for some genes to several hundred thousands of protein...... to measure, at steady-state conditions, absolute protein copy numbers across human tissues and cell lines and compared these levels with the corresponding mRNA levels using transcriptomics. The study shows that the transcript and protein levels do not correlate well unless a gene-specific RNA-to-protein (RTP...... copies per mRNA molecule for others. In conclusion, our data suggest that transcriptome analysis can be used as a tool to predict the protein copy numbers per cell, thus forming an attractive link between the field of genomics and proteomics....

  12. Noncoordinate histone synthesis in heat-shocked Drosophila cells is regulated at multiple levels.

    OpenAIRE

    Farrell-Towt, J; Sanders, M M

    1984-01-01

    Transferring Drosophila tissue culture cells from 25 to 37 degrees C (heat shock) causes histone protein synthesis to become noncoordinate. To determine the level at which this is controlled, the synthesis, degradation, and translation of individual histone mRNAs was studied under both heat shock and control conditions. The increased synthesis of histone H2b protein during heat shock appears to be controlled primarily at the level of translation. During heat shock, H2b mRNA is transcribed at ...

  13. Probing the physiological state of phytoplankton at the single-cell level

    Directory of Open Access Journals (Sweden)

    Frank J. Jochem

    2000-06-01

    Full Text Available Probing the physiological state of phytoplankton at the single-cell level provides valuable insight in ecological studies as well as in environmental monitoring of pollution or UV impacts. This paper reviews the recent progress in assessing the physiological state of phytoplankton with flow cytometry by inherent cell properties such as cell size and chlorophyll autofluorescence, specific fluorescent dyes, and newly developed molecular probes and enzyme substrates. It is reported how nitrogen and iron limitation as well as the effect of copper pollution could be derived from changes in cell inherent properties. Effects of Cu were also recorded by monitoring cell membrane potentials and esterase activity. Photosynthetic capacity of algae was assessed by changes in chlorophyll fluorescence with the electron transport inhibitor DCMU, by a cytometric adaptation of the pump-and-probe approach, and molecular probes for Rubisco. Antibodies were also applied to mark non-terminal stages in the cell DNA replication cycle, to detect non-proliferating cells, to assess DNA damage caused by UV-B radiation and to quantify diatom stickiness. Fluorescein diacetate proved useful to discriminate metabolically active from inactive cells and to reveal strategies of dark survival in algae. The activity of alkaline phosphatase was recorded by a new fluorigenic substrate ELF, and polyclonal antibodies against nitrate reductase (NR provided measurements of the NR abundance. An outlook will show how recent developments in molecular probes might affect the future analysis of marine ecosystems and their communities.

  14. Mammary alveolar cell as evaluation system for casein gene expression involved in glucose level

    Directory of Open Access Journals (Sweden)

    Young Tae Heo

    2017-06-01

    Full Text Available Objective Glucose is an essential fuel in the energy metabolism and synthesis pathways of all mammalian cells. In lactating animals, glucose is the major precursor for lactose and is a substrate for the synthesis of milk proteins and fat in mammary secretory (alveolar epithelial cells. However, clear utilization of glucose in mammary cells during lactogenesis is still unknown, due to the lack of in vitro analyzing models. Therefore, the objective of this study was to test the reliability of the mammary alveolar (MAC-T cell as an in vitro study model for glucose metabolism and lactating system. Methods Undifferentiated MAC-T cells were cultured in three types of Dulbecco’s modified Eagle’s medium with varying levels of glucose (no-glucose: 0 g/L, low-glucose: 1 g/L, and high-glucose: 4.5 g/L for 8 d, after which differentiation to casein secretion was induced. Cell proliferation and expression levels of apoptotic genes, Insulin like growth factor-1 (IGF1 receptor, oxytocin receptor, αS1, αS2, and β casein genes were analyzed at 1, 2, 4, and 8 d after differentiation. Results The proliferation of MAC-T cells with high-glucose treatment was seen to be significantly higher. Expression of apoptotic genes was not affected in any group. However, expression levels of the mammary development related gene (IGF1 receptor and lactation related gene (oxytocin receptor were significantly higher in the low-glucose group. Expressions of αS1-casein, αS2-casein, and β-casein were also higher in the low-glucose treated group as compared to that in the no-glucose and high-glucose groups. Conclusion The results demonstrated that although a high-glucose environment increases cell proliferation in MAC-T cells, a low-glucose treatment to MAC-T cells induces higher expression of casein genes. Our results suggest that the MAC-T cells may be used as an in vitro model to analyze mammary cell development and lactation connected with precise biological effects.

  15. TALK-1 reduces delta-cell endoplasmic reticulum and cytoplasmic calcium levels limiting somatostatin secretion

    Directory of Open Access Journals (Sweden)

    Nicholas C. Vierra

    2018-03-01

    Full Text Available Objective: Single-cell RNA sequencing studies have revealed that the type-2 diabetes associated two-pore domain K+ (K2P channel TALK-1 is abundantly expressed in somatostatin-secreting δ-cells. However, a physiological role for TALK-1 in δ-cells remains unknown. We previously determined that in β-cells, K+ flux through endoplasmic reticulum (ER-localized TALK-1 channels enhances ER Ca2+ leak, modulating Ca2+ handling and insulin secretion. As glucose amplification of islet somatostatin release relies on Ca2+-induced Ca2+ release (CICR from the δ-cell ER, we investigated whether TALK-1 modulates δ-cell Ca2+ handling and somatostatin secretion. Methods: To define the functions of islet δ-cell TALK-1 channels, we generated control and TALK-1 channel-deficient (TALK-1 KO mice expressing fluorescent reporters specifically in δ- and α-cells to facilitate cell type identification. Using immunofluorescence, patch clamp electrophysiology, Ca2+ imaging, and hormone secretion assays, we assessed how TALK-1 channel activity impacts δ- and α-cell function. Results: TALK-1 channels are expressed in both mouse and human δ-cells, where they modulate glucose-stimulated changes in cytosolic Ca2+ and somatostatin secretion. Measurement of cytosolic Ca2+ levels in response to membrane potential depolarization revealed enhanced CICR in TALK-1 KO δ-cells that could be abolished by depleting ER Ca2+ with sarco/endoplasmic reticulum Ca2+ ATPase (SERCA inhibitors. Consistent with elevated somatostatin inhibitory tone, we observed significantly reduced glucagon secretion and α-cell Ca2+ oscillations in TALK-1 KO islets, and found that blockade of α-cell somatostatin signaling with a somatostatin receptor 2 (SSTR2 antagonist restored glucagon secretion in TALK-1 KO islets. Conclusions: These data indicate that TALK-1 reduces δ-cell cytosolic Ca2+ elevations and somatostatin release by limiting δ-cell CICR, modulating the intraislet paracrine signaling

  16. 324 Building radiochemical engineering cells, high-level vault, low-level vault, and associated areas closure plan

    International Nuclear Information System (INIS)

    Barnett, J.M.

    1998-01-01

    The Hanford Site, located adjacent to and north of Richland, Washington, is operated by the US Department of Energy, Richland Operations Office (RL). The 324 Building is located in the 300 Area of the Hanford Site. The 324 Building was constructed in the 1960s to support materials and chemical process research and development activities ranging from laboratory/bench-scale studies to full engineering-scale pilot plant demonstrations. In the mid-1990s, it was determined that dangerous waste and waste residues were being stored for greater than 90 days in the 324 Building Radiochemical Engineering Cells (REC) and in the High-Level Vault/Low-Level Vault (HLV/LLV) tanks. [These areas are not Resource Conservation and Recovery Act of 1976 (RCRA) permitted portions of the 324 Building.] Through the Hanford Federal Facility Agreement and Consent Order (Tri-Party Agreement) Milestone M-89, agreement was reached to close the nonpermitted RCRA unit in the 324 Building. This closure plan, managed under TPA Milestone M-20-55, addresses the identified building areas targeted by the Tri-Party Agreement and provides commitments to achieve the highest degree of compliance practicable, given the special technical difficulties of managing mixed waste that contains high-activity radioactive materials, and the physical limitations of working remotely in the areas within the subject closure unit. This closure plan is divided into nine chapters. Chapter 1.0 provides the introduction, historical perspective, 324 Building history and current mission, and the regulatory basis and strategy for managing the closure unit. Chapters 2.0, 3.0, 4.0, and 5.0 discuss the detailed facility description, process information, waste characteristics, and groundwater monitoring respectively. Chapter 6.0 deals with the closure strategy and performance standard, including the closure activities for the B-Cell, D-Cell, HLV, LLV; piping and miscellaneous associated building areas. Chapter 7.0 addresses the

  17. 324 Building radiochemical engineering cells, high-level vault, low-level vault, and associated areas closure plan

    Energy Technology Data Exchange (ETDEWEB)

    Barnett, J.M.

    1998-03-25

    The Hanford Site, located adjacent to and north of Richland, Washington, is operated by the US Department of Energy, Richland Operations Office (RL). The 324 Building is located in the 300 Area of the Hanford Site. The 324 Building was constructed in the 1960s to support materials and chemical process research and development activities ranging from laboratory/bench-scale studies to full engineering-scale pilot plant demonstrations. In the mid-1990s, it was determined that dangerous waste and waste residues were being stored for greater than 90 days in the 324 Building Radiochemical Engineering Cells (REC) and in the High-Level Vault/Low-Level Vault (HLV/LLV) tanks. [These areas are not Resource Conservation and Recovery Act of 1976 (RCRA) permitted portions of the 324 Building.] Through the Hanford Federal Facility Agreement and Consent Order (Tri-Party Agreement) Milestone M-89, agreement was reached to close the nonpermitted RCRA unit in the 324 Building. This closure plan, managed under TPA Milestone M-20-55, addresses the identified building areas targeted by the Tri-Party Agreement and provides commitments to achieve the highest degree of compliance practicable, given the special technical difficulties of managing mixed waste that contains high-activity radioactive materials, and the physical limitations of working remotely in the areas within the subject closure unit. This closure plan is divided into nine chapters. Chapter 1.0 provides the introduction, historical perspective, 324 Building history and current mission, and the regulatory basis and strategy for managing the closure unit. Chapters 2.0, 3.0, 4.0, and 5.0 discuss the detailed facility description, process information, waste characteristics, and groundwater monitoring respectively. Chapter 6.0 deals with the closure strategy and performance standard, including the closure activities for the B-Cell, D-Cell, HLV, LLV; piping and miscellaneous associated building areas. Chapter 7.0 addresses the

  18. Low Levels of GSTA1 Expression Are Required for Caco-2 Cell Proliferation

    Science.gov (United States)

    Adnan, Humaira; Quach, Holly; MacIntosh, Kimberley; Antenos, Monica; Kirby, Gordon M.

    2012-01-01

    The colonic epithelium continuously regenerates with transitions through various cellular phases including proliferation, differentiation and cell death via apoptosis. Human colonic adenocarcinoma (Caco-2) cells in culture undergo spontaneous differentiation into mature enterocytes in association with progressive increases in expression of glutathione S-transferase alpha-1 (GSTA1). We hypothesize that GSTA1 plays a functional role in controlling proliferation, differentiation and apoptosis in Caco-2 cells. We demonstrate increased GSTA1 levels associated with decreased proliferation and increased expression of differentiation markers alkaline phosphatase, villin, dipeptidyl peptidase-4 and E-cadherin in postconfluent Caco-2 cells. Results of MTS assays, BrdU incorporation and flow cytometry indicate that forced expression of GSTA1 significantly reduces cellular proliferation and siRNA-mediated down-regulation of GSTA1 significantly increases cells in S-phase and associated cell proliferation. Sodium butyrate (NaB) at a concentration of 1 mM reduces Caco-2 cell proliferation, increases differentiation and increases GSTA1 activity 4-fold by 72 hours. In contrast, 10 mM NaB causes significant toxicity in preconfluent cells via apoptosis through caspase-3 activation with reduced GSTA1 activity. However, GSTA1 down-regulation by siRNA does not alter NaB-induced differentiation or apoptosis in Caco-2 cells. While 10 mM NaB causes GSTA1-JNK complex dissociation, phosphorylation of JNK is not altered. These findings suggest that GSTA1 levels may play a role in modulating enterocyte proliferation but do not influence differentiation or apoptosis. PMID:23251616

  19. Acceptable contamination levels in solar grade silicon: From feedstock to solar cell

    International Nuclear Information System (INIS)

    Hofstetter, J.; Lelievre, J.F.; Canizo, C.; Luque, A. del

    2009-01-01

    Ultimately, alternative ways of silicon purification for photovoltaic applications are developed and applied. There is an ongoing debate about what are the acceptable contamination levels within the purified silicon feedstock to specify the material as solar grade silicon. Applying a simple model and making some additional assumptions, we calculate the acceptable contamination levels of different characteristic impurities for each fabrication step of a typical industrial mc-Si solar cell. The acceptable impurity concentrations within the finished solar cell are calculated for SRH recombination exclusively and under low injection conditions. It is assumed that during solar cell fabrication impurity concentrations are only altered by a gettering step. During the crystallization process, impurity segregation at the solid-liquid interface and at extended defects are taken into account. Finally, the initial contamination levels allowed within the feedstock are deduced. The acceptable concentration of iron in the finished solar cell is determined to be 9.7x10 -3 ppma whereas the concentration in the silicon feedstock can be as high as 12.5 ppma. In comparison, the titanium concentration admitted in the solar cell is calculated to be 2.7x10 -4 ppma and the allowed concentration of 2.2x10 -2 ppma in the feedstock is only two orders of magnitude higher. Finally, it is shown theoretically and experimentally that slow cooling rates can lead to a decrease of the interstitial Fe concentration and thus relax the purity requirements in the feedstock.

  20. Microfluidic dielectrophoresis device for trapping, counting and detecting Shewanella oneidensis at the cell level.

    Science.gov (United States)

    Chen, Xiangyu; Liang, Zhiting; Li, Daobo; Xiong, Ying; Xiong, Penghui; Guan, Yong; Hou, Shuangyue; Hu, Yue; Chen, Shan; Liu, Gang; Tian, Yangchao

    2018-01-15

    Shewanella oneidensis, a model organism for electrochemical activity bacteria, has been widely studied at the biofilm level. However, to obtain more information regarding this species, it is essential to develop an approach to trap and detect S. oneidensis at the cell level. In this study, we report a rapid and label-free microfluidic platform for trapping, counting and detecting S. oneidensis cells. A microfluidic chip was integrated with a modified dielectrophoresis (DEP) trapping technique and hole arrays of different hole sizes. By numerical simulation and an elaborate electric field distribution design, S. oneidensis cells were successfully trapped and positioned in the hole arrays. Real time fluorescence imaging was also used to observe the trapping process. With the aid of a homemade image program, the trapped bacteria were accurately counted, and the results demonstrated that the amount of bacteria correlated with the hole sizes. As one of the significant applications of the device, Raman identification and detection of countable S. oneidensis cells was accomplished in two kinds of holes. The microfluidic platform provides a quantitative sample preparation and analysis method at the cell level that could be widely applied in the environmental and energy fields. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Effect of low-level laser irradiation on odontoblast-like cells

    Science.gov (United States)

    Oliveira, C. F.; Hebling, J.; Souza, P. P. C.; Sacono, N. T.; Lessa, F. R.; Lizarelli, R. F. Z.; Costa, C. A. S.

    2008-09-01

    Low-level laser therapy (LLLT), also referred to as therapeutic laser, has been recommended for a wide array of clinical procedures, among which the treatment of dentinal hypersensitivity. However, the mechanism that guides this process remains unknown. Therefore, the objective of this study was to evaluate in vitro the effects of LLL irradiation on cell metabolism (MTT assay), alkaline phosphatase (ALP) expression and total protein synthesis. The expression of genes that encode for collagen type-1 (Col-1) and fibronectin (FN) was analyzed by RT-PCR. For such purposes, odontoblast-like cell line (MDPC-23) was previously cultured in Petri dishes (15000 cells/cm2) and submitted to stress conditions during 12 h. Thereafter, 6 applications with a monochromatic near infrared radiation (GaAlAs) set at predetermined parameters were performed at 12-h intervals. Non-irradiated cells served as a control group. Neither the MTT values nor the total protein levels of the irradiated group differed significantly from those of the control group (Mann-Whitney test; p > 0.05). On the other hand, the irradiated cells showed a decrease in ALP activity (Mann-Whitney test; p 0.05). It may be concluded that, under the tested conditions, the LLLT parameters used in the present study did not influence cell metabolism, but reduced slightly the expression of some specific proteins.

  2. Optimal memory configuration analysis in tri-hybrid solid-state drives with storage class memory and multi-level cell/triple-level cell NAND flash memory

    Science.gov (United States)

    Matsui, Chihiro; Yamada, Tomoaki; Sugiyama, Yusuke; Yamaga, Yusuke; Takeuchi, Ken

    2017-04-01

    This paper analyzes the best mix of memories in a tri-hybrid solid-state drive (SSD) with storage class memory (SCM) and multi-level cell (MLC)/triple-level cell (TLC) NAND flash memory. SCM is fast but its cost is high. Although MLC NAND flash memory is slow, it is more cost effective than SCM. For further cost efficiency, TLC NAND flash memory is denser and less expensive than MLC NAND flash. Performance of tri-hybrid SSD is evaluated in various memory configurations. Moreover, the optimum memory configuration is changed according to the application characteristics. If 10% cost increase is allowed compared to the MLC NAND flash only SSD, SCM/MLC NAND flash hybrid SSD provides the best performance with hot/random workload, whereas SCM/MLC/TLC NAND flash tri-hybrid SSD achieves the best for hot/sequential and cold/random workloads. In addition, it is possible to add long latency but low-cost SCM to the tri-hybrid SSD. As a result, tri-hybrid SSD with slow SCM achieves the best performance.

  3. Visualization and measurement of ATP levels in living cells replicating hepatitis C virus genome RNA.

    Directory of Open Access Journals (Sweden)

    Tomomi Ando

    Full Text Available Adenosine 5'-triphosphate (ATP is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV, a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Förster resonance energy transfer (FRET-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ∼5 mM at possible replicating sites and ∼1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ∼2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome.

  4. Manganese superoxide dismutase level in blood cells of patients with breast cancer

    International Nuclear Information System (INIS)

    Adzic, M.; Niciforovic, A.; Filipovic, D.; Vucic, V.; Neskovic-Konstantinovic, Z.; Radojcic, M.B. . E-mail address of corresponding author: marija@vin.bg.ac.yu; Radojcic, M.B.)

    2005-01-01

    The intracellular MnSOD levels were determined in peripheral blood cells obtained from two age groups of patients with breast cancer (BC): 30-45 year old patients (premenopausal, n=7, clinical stage 1 to 3) and 46-60 year old patients (peri- and postmenopausal women, n=12, clinical stage 3 or 4), at diagnosis, prior to any clinical treatment. The respective healthy women groups were used as controls. Blood cells were also irradiated in vitro with 2- and 9- Gy of gamma-ray radiation from 60 Co source. The MnSOD levels were determined by the specific immunostaining and quantified by the laser densitometry. The MANOVA analysis and Tukey post-hoc test indicated significantly higher MnSOD levels in both groups of BC patients in relation to the respective controls ( F=25.166, p<0.001). The data indicated that the increased initial MnSOD levels in peripheral blood cells may be related to the presence of BC i.e. they may reflect the system response to the presence of malignant tumour. In addition to that, in vitro radiation challenge of blood cells indicate that MnSOD overexpression may be a good indicator for selection of BC patients that would express increased resistance to oxidative stress imposed by the clinical treatment. (author)

  5. Cytochrome P450 levels are altered in patients with esophageal squamous-cell carcinoma

    DEFF Research Database (Denmark)

    Bergheim, I.; Wolfgarten, E.; Bollschweiler, E.

    2007-01-01

    AIM: To investigate the role of cytochrome P450 (CYP) in the carcinogenesis of squamous-cell carcinoma (SCC) in human esophagus by determining expression patterns and protein levels of representative CYPs in esophageal tissue of patients with SCC and controls. METHODS: mRNA expression of CYP2E1...

  6. Low levels of aflatoxin B1, ricin and milk enhance recombinant protein production in mammalian cells

    Science.gov (United States)

    Changing the optimal tissue culture medium by adding low levels of environmental stress such as 1 µM of the fungal toxin aflatoxin B1 (AFB1), 1 ng of the castor bean protein toxin ricin in transduced mammalian cells or 1% reconstituted milk enhances transcription and increases production of the foll...

  7. T cell responsiveness correlates differentially with antibody isotype levels in clinical and asymptomatic filariasis

    NARCIS (Netherlands)

    Yazdanbakhsh, M.; Paxton, W. A.; Kruize, Y. C.; Sartono, E.; Kurniawan, A.; van het Wout, A.; Selkirk, M. E.; Partono, F.; Maizels, R. M.

    1993-01-01

    To establish the relationships among T and B cell responses, active infection, and clinical manifestations in lymphatic filariasis, filarial-specific lymphocyte proliferation, IgG antibody isotypes, and IgE levels were determined in an exposed population: 31 asymptomatic amicrofilaremics, 43

  8. Effect of academic stress on serum cortisol level and CD4 cell count ...

    African Journals Online (AJOL)

    To assess the effect of stress on serum cortisol level and CD4 cell count in young male postgraduate students at Igbinedion University, a cross sectional laboratory based analysis survey was adopted for this study. A total of 104 male volunteer postgraduate students (age 22 + 7.0 years, body mass index 26 + 0.5 kg/m2) ...

  9. Protein C and antithrombin levels in patients with sickle cell anemia ...

    African Journals Online (AJOL)

    Background: Alterations in the components of hemostasis, namely platelet function, the procoagulant, anticoagulant, and the fibrinolytic systems, are observed in sickle cell anemia (SCA) and are in favor of a procoagulant phenotype. Therefore, study of protein C and antithrombin (AT) levels in patients with SCA in steady ...

  10. Microarray analysis of genes associated with cell surface NIS protein levels in breast cancer

    Directory of Open Access Journals (Sweden)

    Richardson Andrea L

    2011-10-01

    Full Text Available Abstract Background Na+/I- symporter (NIS-mediated iodide uptake allows radioiodine therapy for thyroid cancer. NIS is also expressed in breast tumors, raising potential for radionuclide therapy of breast cancer. However, NIS expression in most breast cancers is low and may not be sufficient for radionuclide therapy. We aimed to identify biomarkers associated with NIS expression such that mechanisms underlying NIS modulation in human breast tumors may be elucidated. Methods Published oligonucleotide microarray data within the National Center for Biotechnology Information Gene Expression Omnibus database were analyzed to identify gene expression tightly correlated with NIS mRNA level among human breast tumors. NIS immunostaining was performed in a tissue microarray composed of 28 human breast tumors which had corresponding oligonucleotide microarray data available for each tumor such that gene expression associated with cell surface NIS protein level could be identified. Results and Discussion NIS mRNA levels do not vary among breast tumors or when compared to normal breast tissues when detected by Affymetrix oligonucleotide microarray platforms. Cell surface NIS protein levels are much more variable than their corresponding NIS mRNA levels. Despite a limited number of breast tumors examined, our analysis identified cysteinyl-tRNA synthetase as a biomarker that is highly associated with cell surface NIS protein levels in the ER-positive breast cancer subtype. Conclusions Further investigation on genes associated with cell surface NIS protein levels within each breast cancer molecular subtype may lead to novel targets for selectively increasing NIS expression/function in a subset of breast cancers patients.

  11. Human and great ape red blood cells differ in plasmalogen levels and composition

    Directory of Open Access Journals (Sweden)

    Ely John J

    2011-06-01

    Full Text Available Abstract Background Plasmalogens are ether phospholipids required for normal mammalian developmental, physiological, and cognitive functions. They have been proposed to act as membrane antioxidants and reservoirs of polyunsaturated fatty acids as well as influence intracellular signaling and membrane dynamics. Plasmalogens are particularly enriched in cells and tissues of the human nervous, immune, and cardiovascular systems. Humans with severely reduced plasmalogen levels have reduced life spans, abnormal neurological development, skeletal dysplasia, impaired respiration, and cataracts. Plasmalogen deficiency is also found in the brain tissue of individuals with Alzheimer disease. Results In a human and great ape cohort, we measured the red blood cell (RBC levels of the most abundant types of plasmalogens. Total RBC plasmalogen levels were lower in humans than bonobos, chimpanzees, and gorillas, but higher than orangutans. There were especially pronounced cross-species differences in the levels of plasmalogens with a C16:0 moiety at the sn-1 position. Humans on Western or vegan diets had comparable total RBC plasmalogen levels, but the latter group showed moderately higher levels of plasmalogens with a C18:1 moiety at the sn-1 position. We did not find robust sex-specific differences in human or chimpanzee RBC plasmalogen levels or composition. Furthermore, human and great ape skin fibroblasts showed only modest differences in peroxisomal plasmalogen biosynthetic activity. Human and chimpanzee microarray data indicated that genes involved in plasmalogen biosynthesis show cross-species differential expression in multiple tissues. Conclusion We propose that the observed differences in human and great ape RBC plasmalogens are primarily caused by their rates of biosynthesis and/or turnover. Gene expression data raise the possibility that other human and great ape cells and tissues differ in plasmalogen levels. Based on the phenotypes of humans and

  12. Human and great ape red blood cells differ in plasmalogen levels and composition.

    Science.gov (United States)

    Moser, Ann B; Steinberg, Steven J; Watkins, Paul A; Moser, Hugo W; Ramaswamy, Krishna; Siegmund, Kimberly D; Lee, D Rick; Ely, John J; Ryder, Oliver A; Hacia, Joseph G

    2011-06-17

    Plasmalogens are ether phospholipids required for normal mammalian developmental, physiological, and cognitive functions. They have been proposed to act as membrane antioxidants and reservoirs of polyunsaturated fatty acids as well as influence intracellular signaling and membrane dynamics. Plasmalogens are particularly enriched in cells and tissues of the human nervous, immune, and cardiovascular systems. Humans with severely reduced plasmalogen levels have reduced life spans, abnormal neurological development, skeletal dysplasia, impaired respiration, and cataracts. Plasmalogen deficiency is also found in the brain tissue of individuals with Alzheimer disease. In a human and great ape cohort, we measured the red blood cell (RBC) levels of the most abundant types of plasmalogens. Total RBC plasmalogen levels were lower in humans than bonobos, chimpanzees, and gorillas, but higher than orangutans. There were especially pronounced cross-species differences in the levels of plasmalogens with a C16:0 moiety at the sn-1 position. Humans on Western or vegan diets had comparable total RBC plasmalogen levels, but the latter group showed moderately higher levels of plasmalogens with a C18:1 moiety at the sn-1 position. We did not find robust sex-specific differences in human or chimpanzee RBC plasmalogen levels or composition. Furthermore, human and great ape skin fibroblasts showed only modest differences in peroxisomal plasmalogen biosynthetic activity. Human and chimpanzee microarray data indicated that genes involved in plasmalogen biosynthesis show cross-species differential expression in multiple tissues. We propose that the observed differences in human and great ape RBC plasmalogens are primarily caused by their rates of biosynthesis and/or turnover. Gene expression data raise the possibility that other human and great ape cells and tissues differ in plasmalogen levels. Based on the phenotypes of humans and rodents with plasmalogen disorders, we propose that cross

  13. Elevated levels of 14-3-3 proteins, serotonin, gamma enolase and pyruvate kinase identified in clinical samples from patients diagnosed with colorectal cancer

    Czech Academy of Sciences Publication Activity Database

    Dowling, P.; Hughes, D. J.; Larkin, A.M.; Meiller, J.; Henry, M.; Meleady, P.; Lynch, V.; Pardini, B.; Naccarati, A.; Levý, M.; Vodička, Pavel; Neary, P.; Clynes, M.

    2015-01-01

    Roč. 441, feb. (2015), s. 133-141 ISSN 0009-8981 Institutional support: RVO:68378041 Keywords : biomarkers * colorectal cancer * proteomics * mass spectrometry * 14-3-3 proteins * pyruvate kinase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.799, year: 2015

  14. Nanoparticle uptake and their co-localization with cell compartments - a confocal Raman microscopy study at single cell level

    Energy Technology Data Exchange (ETDEWEB)

    Estrela-Lopis, I; Donath, E [Institute of Medical Physics and Biophysics, Leipzig University, Haertelstrasse 16, 04107 Leipzig (Germany); Romero, G; Rojas, E; Moya, S E, E-mail: Irina.Estrela-Lopis@medizin.uni-leipzig.de [CIC biomaGUNE, Paseo Miramon 182 Edificio Empresarial C, 20009 San Sebastian, Gipuzkoa (Spain)

    2011-07-06

    Confocal Raman Microscopy, a non-invasive, non-destructive and label-free technique, was employed to study the uptake and localization of nanoparticles (NPs) in the Hepatocarcinoma human cell line HepG2 at the level of single cells. Cells were exposed to carbon nanotubes (CNTs) the surface of which was engineered with polyelectrolytes and lipid layers, aluminium oxide and cerium dioxide nanoparticles. Raman spectra deconvolution was applied to obtain the spatial distributions of NPs together with lipids/proteins in cells. The colocalization of the NPs with different intracellular environments, lipid bodies, protein and DNA, was inferred. Lipid coated CNTs associated preferentially with lipid rich regions, whereas polyelectrolyte coated CNTs were excluded from lipid rich regions. Al{sub 2}O{sub 3} NPs were found in the cytoplasm. CeO{sub 2} NPs were readily taken up and have been observed all over the cell. Raman z-scans proved the intracellular distribution of the respective NPs.

  15. Differential expression of the neural cell adhesion molecule NCAM 140 in human pituitary tumors

    OpenAIRE

    Aletsee-Ufrecht, M. C.; Langley, O. K.; Gratzl, O.; Gratzl, Manfred

    1990-01-01

    We have analyzed the expression of the intracellular marker protein neuron specific enolase (NSE), synaptophysin (SYN) and of the cell surface marker NCAM (neural cell adhesion molecule) in both normal human hypophysis and in pituitary adenomas in order to explore their potential use as diagnostic tools. All adenomas (4 prolactinomas, 3 growth hormone (GH) producing adenomas and 4 inactive adenomas) showed SYN and NSE immunoreactivity on tissue sections and this was confirmed by immunoblots. ...

  16. Trans fatty acids increase nitric oxide levels and pancreatic beta-cell necrosis in rats

    Directory of Open Access Journals (Sweden)

    Kusmiyati Tjahjono DK

    2013-04-01

    Full Text Available Background The prevalence of diabetes in Indonesia is increasing due to various factors, including life style changes such as trans fatty acid (TFA intake. High TFA intake is known to be related to blood lipid profile changes resulting in cardiovascular disorders. This study was to identify the effect of TFA on nitric oxide (NO production and on necrosis of pancreatic beta cells. Methods A study of randomized pre-test post–test design with control group. Thirty Sprague Dawley rats were divided into 3 groups, i.e. group K (control, group P1 receiving a diet with 5% TFA, and P2 receiving 10% TFA. The intervention was performed for 8 weeks. NO level and pancreatic beta-cell necrosis were analyzed using Pearson’s chi square test. Results After 4 weeks of treatment there was no change in NO levels in group K, but increased NO in P2 (2.6-3.8 ìM. At 8 weeks after treatment, NO levels in groups P1 and P2 increased to 2.6-3.4 ìM and 4.2-14.3 ìM, respectively, while in group K only 2 rats had increased NO levels of 2.8-2.9 ìM. With Pearson’s chi-square test, there was a signifant difference in the proportions of necrotic pancreatic beta cells after 4 weeks and 8 weeks (p=0.000. No necrosis of beta cells was found in group K, mild necrosis in group P1 (1-25% and moderate necrosis in group P2 (26-50%. Conclusion TFA consumption significantly increases NO levels in Sprague Dawley rats and also results in moderate grades of necrosis of pancreatic beta cells.

  17. Trans fatty acids increase nitric oxide levels and pancreatic beta-cell necrosis in rats

    Directory of Open Access Journals (Sweden)

    Kusmiyati Tjahjono DK

    2015-12-01

    Full Text Available BACKGROUND The prevalence of diabetes in Indonesia is increasing due to various factors, including life style changes such as trans fatty acid (TFA intake. High TFA intake is known to be related to blood lipid profile changes resulting in cardiovascular disorders. This study was to identify the effect of TFA on nitric oxide (NO production and on necrosis of pancreatic beta cells. METHODS A study of randomized pre-test post–test design with control group. Thirty Sprague Dawley rats were divided into 3 groups, i.e. group K (control, group P1 receiving a diet with 5% TFA, and P2 receiving 10% TFA. The intervention was performed for 8 weeks. NO level and pancreatic beta-cell necrosis were analyzed using Pearson’s chi square test. RESULTS After 4 weeks of treatment there was no change in NO levels in group K, but increased NO in P2 (2.6-3.8 ìM. At 8 weeks after treatment, NO levels in groups P1 and P2 increased to 2.6-3.4 ìM and 4.2-14.3 ìM, respectively, while in group K only 2 rats had increased NO levels of 2.8-2.9 ìM. With Pearson’s chi-square test, there was a signifant difference in the proportions of necrotic pancreatic beta cells after 4 weeks and 8 weeks (p= 0.000. No necrosis of beta cells was found in group K, mild necrosis in group P1 (1-25% and moderate necrosis in group P2 (26-50%. CONCLUSION TFA consumption significantly increases NO levels in Sprague Dawley rats and also results in moderate grades of necrosis of pancreatic beta cells

  18. Oxidized low density lipoprotein increases RANKL level in human vascular cells. Involvement of oxidative stress

    Energy Technology Data Exchange (ETDEWEB)

    Mazière, Cécile, E-mail: maziere.cecile@chu-amiens.fr [Biochemistry Laboratory, South Hospital University, René Laennec Avenue, Amiens 80000 (France); Salle, Valéry [Internal Medicine, North Hospital University, Place Victor Pauchet, Amiens 80000 (France); INSERM U1088 (EA 4292), SFR CAP-Santé (FED 4231), University of Picardie – Jules Verne (France); Gomila, Cathy; Mazière, Jean-Claude [Biochemistry Laboratory, South Hospital University, René Laennec Avenue, Amiens 80000 (France)

    2013-10-18

    Highlights: •Oxidized LDL enhances RANKL level in human smooth muscle cells. •The effect of OxLDL is mediated by the transcription factor NFAT. •UVA, H{sub 2}O{sub 2} and buthionine sulfoximine also increase RANKL level. •All these effects are observed in human fibroblasts and endothelial cells. -- Abstract: Receptor Activator of NFκB Ligand (RANKL) and its decoy receptor osteoprotegerin (OPG) have been shown to play a role not only in bone remodeling but also in inflammation, arterial calcification and atherosclerotic plaque rupture. In human smooth muscle cells, Cu{sup 2+}-oxidized LDL (CuLDL) 10–50 μg/ml increased reactive oxygen species (ROS) and RANKL level in a dose-dependent manner, whereas OPG level was not affected. The lipid extract of CuLDL reproduced the effects of the whole particle. Vivit, an inhibitor of the transcription factor NFAT, reduced the CuLDL-induced increase in RANKL, whereas PKA and NFκB inhibitors were ineffective. LDL oxidized by myeloperoxidase (MPO-LDL), or other pro-oxidant conditions such as ultraviolet A (UVA) irradiation, incubation with H{sub 2}O{sub 2} or with buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis{sub ,} also induced an oxidative stress and enhanced RANKL level. The increase in RANKL in pro-oxidant conditions was also observed in fibroblasts and endothelial cells. Since RANKL is involved in myocardial inflammation, vascular calcification and plaque rupture, this study highlights a new mechanism whereby OxLDL might, by generation of an oxidative stress, exert a deleterious effect on different cell types of the arterial wall.

  19. Low-level lasers on microRNA and uncoupling protein 2 mRNA levels in human breast cancer cells

    Science.gov (United States)

    Canuto, K. S.; Teixeira, A. F.; Rodrigues, J. A.; Paoli, F.; Nogueira, E. M.; Mencalha, A. L.; Fonseca, A. S.

    2017-06-01

    MicroRNA is short non-coding RNA and is a mediator of post-transcriptional regulation of gene expression. In addition, uncoupling proteins (UCPs) regulate thermogenesis, metabolic and energy balance, and decrease reactive oxygen species production. Both microRNA and UCP2 expression can be altered in cancer cells. At low power, laser wavelength, frequency, fluence and emission mode deternube photobiological responses, which are the basis of low-level laser therapy. There are few studies on miRNA and UCP mRNA levels after low-level laser exposure on cancer cells. In this work, we evaluate the micrRNA (mir-106b and mir-15a) and UCP2 mRNA levels in human breast cancer cells exposed to low-level lasers. MDA-MB-231 human breast cancer cells were exposed to low-level red and infrared lasers, total RNA was extracted for cDNA synthesis and mRNA levels by real time quantitative polymerase chain reaction were evaluated. Data show that mir-106b and mir-15a relative levels are not altered, but UCP2 mRNA relative levels are increased in MDA-MB-231 human breast cancer cells exposed to low-level red and infrared lasers at fluences used in therapeutic protocols.

  20. The Level of Circulating Endothelial Progenitor Cell Is Associated with Cerebral Vasoreactivity: A Pilot Study

    Directory of Open Access Journals (Sweden)

    Chih-Ping Chung

    2015-01-01

    Full Text Available Endothelial progenitor cell is known to be able to repair injured vessels. We assessed the hypothesis that endothelial progenitor cell also modulates cerebral endothelial function in healthy status. We used transcranial color-coded sonography to measure middle cerebral arterial vasoreactivity to CO2 (breath-holding index in healthy subjects and observed its relationship with the number of circulating CD34CD133+ cells. To detect significant correlations between each characteristic and breath-holding index of middle cerebral artery, we used univariate and multivariate regression analyses. 22 young healthy subjects were included in the present study (6 men, 16 women; mean age: 28.45 ± 3.98 years, range: 22–34 years. The mean breath-holding index and CD45lowCD34+CD133+ cells number were 0.95 ± 0.48% and 0.52 ± 0.26, respectively. The level of CD34CD133+ cells was independently associated with middle cerebral artery’s vasoreactivity (r=0.439,P=0.04. Our results suggest that endothelial progenitor cell also modulates healthy cerebral vessels’ endothelial function. This ability of endothelial progenitor cell could be potentially applied therapeutically and for prevention in conditions with cerebral endothelial dysfunction and cerebral ischemia.

  1. Acanthamoeba castellanii metabolites increase the intracellular calcium level and cause cytotoxicity in wish cells.

    Science.gov (United States)

    Mattana, A; Bennardini, F; Usai, S; Fiori, P L; Franconi, F; Cappuccinelli, P

    1997-08-01

    Previous studies have shown that trophozoites of the pathogenic free-living amoeba Acanthamoeba castellanii rapidly lyse a variety of cells in vitro. However, the role played by cytolitic molecules that may participate in Acanthamoebal cytopathogenicity has yet to be completely elucidated. The aim of this work was to study whether soluble molecules released by A. castellanii trophozoites could induce cytopathic effect in human epithelial cells in vitro. The results obtained indicate that A. castellanii trophozoites constitutively elaborate and release soluble factors that immediately elicit a cytosolic free-calcium increase in target cells. This phenomenon is induced by low molecular weight amoebic metabolites and depends on a transmembrane influx of extracellular calcium. Morphological changes, cytoskeletal damage, cell death and cytolysis followed the elevation of cytosolic free-calcium levels. Calcium ions are very important for cell homeostasis, in fact, they control the functions of a variety of cellular responses, including secretion, cell proliferation and apoptosis. Our results suggest that the substained elevation of the cytosolic free-calcium in response to A. castellanii metabolites might play a fundamental role in target cell damage during Acanthamoeba infections.

  2. Sodium Chloride Increases Aβ Levels by Suppressing Aβ Clearance in Cultured Cells.

    Science.gov (United States)

    Cheng, Xiao-Juan; Gao, Yuan; Zhao, Yu-Wu; Cheng, Xiao-Dong

    2015-01-01

    Recent studies suggest that high-salt diet is associated with cognitive decline in human and mouse. The fact that genetic factors account for less than 50% cases of sporadic Alzheimer's disease (AD) highlights the important contribution of environmental factors, such as high-salt diet, in AD pathogenesis. However, whether and how high-salt diet fits the "amyloid cascade" hypothesis remains unexplored. Here, we show sodium chloride (NaCl) could increase Aβ levels in the medium of HEK293 cells overexpressing amyloid precursor protein (APP) or C99 fragment. NaCl treatment dose not affect APP level, gamma secretase level or activity. Instead, NaCl treatment suppresses the capacity of cells to clear Aβ and reduces Apolipoprotein E (ApoE) level. Finally, NaCl treated THP-1 or BV2 cells are inefficient in clearing Aβ when co-cultured with rat primary neurons. Our study suggests that high-salt diet may increase AD risk by directly modulating Aβ levels.

  3. Metabolic syndrome and its components with neuron-specific enolase: a cross-sectional study in large health check-up population in China.

    Science.gov (United States)

    Wang, Shu-Yi; Zha, Xiao-Juan; Zhu, Xin-Ying; Li, Wen-Bo; Ma, Jun; Wu, Ze-Wei; Wu, Huan; Jiang, Ming-Fei; Wen, Yu-Feng

    2018-04-10

    This study was aimed at investigating the relationship between neuron-specific enolase (NSE) and components of metabolic syndrome (MS). Cross-sectional study. Chinese health check-up population. 40 684 health check-up people were enrolled in this study from year 2014 to 2016. OR and coefficient for MS. The percentage of abnormal NSE and MS was 26.85% and 8.85%, respectively. There were significant differences in sex, body mass index, drinking habit, triglycerides (TGs), high-density lipoprotein cholesterol (HDL-C), blood pressure and MS between low-NSE and high-NSE groups. In logistic regression analysis, elevated NSE was present in MS, higher body mass index, hypertriglyceridaemia, hypertension and low-HDL groups. Stepwise linear analysis showed a negative correlation between NSE and fasting blood glucose (FBG) (<6.0 mmol/L), and a positive correlation between NSE and TGs (<20 mmol/L), systolic blood pressure (75-200 mm Hg), HDL-C (0.75-2.50 mmol/L), diastolic blood pressure (<70 mm Hg) and FBG (6.00-20.00 mmol/L). Furthermore, MS was positively correlated with NSE within the range of 2.00-7.50 ng/mL, but had a negative correlation with NSE within the range of 7.50-23.00 ng/mL. There are associations between NSE with MS and its components. The result suggests that NSE may be a potential predictor of MS. Further research could be conducted in discussing the potential mechanism involved. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  4. Low levels of aflatoxin B1, ricin, and milk enhance recombinant protein production in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Reuven Rasooly

    Full Text Available Gene expression in transduced mammalian cells correlates with virus titer, but high doses of vector for gene therapy leads to toxicity in humans and in animals. Changing the optimal tissue culture medium by adding low levels of environmental stressors, such as 1 µM of the fungal toxin aflatoxin B1 (AFB1, 1 ng of the castor bean protein toxin ricin, or 1% reconstituted milk, enhances transcription and increases production of proteins in transduced mammalian cells as demonstrated by production of the following three recombinant proteins: firefly luciferase, β-galactosidase, and green fluorescent protein (GFP. Higher concentrations of the stress-producing substances damage the cells beyond recovery, resulting in inhibited gene expression and cell death. We also evaluated the effect of the stressor substances on the enhanced infectivity of virus. The presented findings extend methods for large-scale transient recombinant protein production in mammalian cells and suggest that it may be possible to reduce the cytotoxicity of the adenovirus by reducing the virus titer without adversely affecting gene expression levels.

  5. Low levels of aflatoxin B1, ricin, and milk enhance recombinant protein production in mammalian cells.

    Science.gov (United States)

    Rasooly, Reuven; Hernlem, Bradley; Friedman, Mendel

    2013-01-01

    Gene expression in transduced mammalian cells correlates with virus titer, but high doses of vector for gene therapy leads to toxicity in humans and in animals. Changing the optimal tissue culture medium by adding low levels of environmental stressors, such as 1 µM of the fungal toxin aflatoxin B1 (AFB1), 1 ng of the castor bean protein toxin ricin, or 1% reconstituted milk, enhances transcription and increases production of proteins in transduced mammalian cells as demonstrated by production of the following three recombinant proteins: firefly luciferase, β-galactosidase, and green fluorescent protein (GFP). Higher concentrations of the stress-producing substances damage the cells beyond recovery, resulting in inhibited gene expression and cell death. We also evaluated the effect of the stressor substances on the enhanced infectivity of virus. The presented findings extend methods for large-scale transient recombinant protein production in mammalian cells and suggest that it may be possible to reduce the cytotoxicity of the adenovirus by reducing the virus titer without adversely affecting gene expression levels.

  6. A Bayesian framework for cell-level protein network analysis for multivariate proteomics image data

    Science.gov (United States)

    Kovacheva, Violet N.; Sirinukunwattana, Korsuk; Rajpoot, Nasir M.

    2014-03-01

    The recent development of multivariate imaging techniques, such as the Toponome Imaging System (TIS), has facilitated the analysis of multiple co-localisation of proteins. This could hold the key to understanding complex phenomena such as protein-protein interaction in cancer. In this paper, we propose a Bayesian framework for cell level network analysis allowing the identification of several protein pairs having significantly higher co-expression levels in cancerous tissue samples when compared to normal colon tissue. It involves segmenting the DAPI-labeled image into cells and determining the cell phenotypes according to their protein-protein dependence profile. The cells are phenotyped using Gaussian Bayesian hierarchical clustering (GBHC) after feature selection is performed. The phenotypes are then analysed using Difference in Sums of Weighted cO-dependence Profiles (DiSWOP), which detects differences in the co-expression patterns of protein pairs. We demonstrate that the pairs highlighted by the proposed framework have high concordance with recent results using a different phenotyping method. This demonstrates that the results are independent of the clustering method used. In addition, the highlighted protein pairs are further analysed via protein interaction pathway databases and by considering the localization of high protein-protein dependence within individual samples. This suggests that the proposed approach could identify potentially functional protein complexes active in cancer progression and cell differentiation.

  7. Biological effects of low-level laser irradiation on umbilical cord mesenchymal stem cells

    Science.gov (United States)

    Chen, Hongli; Wang, Hong; Li, Yingxin; Liu, Weichao; Wang, Chao; Chen, Zhuying

    2016-04-01

    Low-level laser irradiation (LLLI) can enhance stem cell (SC) activity by increasing migration and proliferation. This study investigated the effects of LLLI on proliferation, enzymatic activity, and growth factor production in human umbilical cord mesenchymal SCs (hUC-MSCs) as well as the underlying mechanisms. hUC-MSCs were assigned to a control group (non-irradiation group) and three LLLI treatment groups (635 nm group, 808 nm group, and 635/808 nm group). Laser power density and energy density of 20 mW/cm2 and 12 J/cm2, respectively, were used for each experiment. The proliferation rate was higher in the 635 nm as compared to the other groups. LLLI at 808 nm did not induce cell proliferation. ROS levels in cells exposed to 635, 808, and 635/808 nm radiation were increased by 52.81%, 26.89%, and 21.15%, respectively, relative to the control group. CAT, tGPx, and SOD activity was increased. LLLI at 808 nm increased the levels of IL-1, IL-6, and NFκB but not VEGF. LLLI improved hUC-MSCs function and increased antioxidant activity. Dual-wavelength LLLI had more potent effects on hUC-MSCs than single-wavelength treatment. LLLI has potential applications in the preconditioning of hUC-MSCs in vitro prior to transplantation, which could improve the regenerative capacity of cells.

  8. Biological effects of low-level laser irradiation on umbilical cord mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Hongli; Wang, Hong; Li, Yingxin, E-mail: yingxinli2005@126.com; Liu, Weichao; Chen, Zhuying [Key Laboratory of Laser Medicine of Tianjin, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300192 (China); Wang, Chao [Biomedical Engineering and Technology College, Tianjin Medical University, Tianjin, 300070 (China)

    2016-04-15

    Low-level laser irradiation (LLLI) can enhance stem cell (SC) activity by increasing migration and proliferation. This study investigated the effects of LLLI on proliferation, enzymatic activity, and growth factor production in human umbilical cord mesenchymal SCs (hUC-MSCs) as well as the underlying mechanisms. hUC-MSCs were assigned to a control group (non-irradiation group) and three LLLI treatment groups (635 nm group, 808 nm group, and 635/808 nm group). Laser power density and energy density of 20 mW/cm{sup 2} and 12 J/cm{sup 2}, respectively, were used for each experiment. The proliferation rate was higher in the 635 nm as compared to the other groups. LLLI at 808 nm did not induce cell proliferation. ROS levels in cells exposed to 635, 808, and 635/808 nm radiation were increased by 52.81%, 26.89%, and 21.15%, respectively, relative to the control group. CAT, tGPx, and SOD activity was increased. LLLI at 808 nm increased the levels of IL-1, IL-6, and NFκB but not VEGF. LLLI improved hUC-MSCs function and increased antioxidant activity. Dual-wavelength LLLI had more potent effects on hUC-MSCs than single-wavelength treatment. LLLI has potential applications in the preconditioning of hUC-MSCs in vitro prior to transplantation, which could improve the regenerative capacity of cells.

  9. Reduced Levels of Proteasome Products in a Mouse Striatal Cell Model of Huntington's Disease.

    Directory of Open Access Journals (Sweden)

    Sayani Dasgupta

    Full Text Available Huntington's disease is the result of a long polyglutamine tract in the gene encoding huntingtin protein, which in turn causes a large number of cellular changes and ultimately results in neurodegeneration of striatal neurons. Although many theories have been proposed, the precise mechanism by which the polyglutamine expansion causes cellular changes is not certain. Some evidence supports the hypothesis that the long polyglutamine tract inhibits the proteasome, a multiprotein complex involved in protein degradation. However, other studies report normal proteasome function in cells expressing long polyglutamine tracts. The controversy may be due to the methods used to examine proteasome activity in each of the previous studies. In the present study, we measured proteasome function by examining levels of endogenous peptides that are products of proteasome cleavage. Peptide levels were compared among mouse striatal cell lines expressing either 7 glutamines (STHdhQ7/Q7 or 111 glutamines in the huntingtin protein, either heterozygous (STHdhQ7/Q111 or homozygous (STHdhQ111/Q111. Both of the cell lines expressing huntingtin with 111 glutamines showed a large reduction in nearly all of the peptides detected in the cells, relative to levels of these peptides in cells homozygous for 7 glutamines. Treatment of STHdhQ7/Q7 cells with proteasome inhibitors epoxomicin or bortezomib also caused a large reduction in most of these peptides, suggesting that they are products of proteasome-mediated cleavage of cellular proteins. Taken together, these results support the hypothesis that proteasome function is impaired by the expression of huntingtin protein containing long polyglutamine tracts.

  10. High-Cholesterol Diet Disrupts the Levels of Hormones Derived from Anterior Pituitary Basophilic Cells.

    Science.gov (United States)

    Yang, J; Zhang, X; Liu, Z; Yuan, Z; Song, Y; Shao, S; Zhou, X; Yan, H; Guan, Q; Gao, L; Zhang, H; Zhao, J

    2016-03-01

    Emerging evidence shows that elevated cholesterol levels are detrimental to health. However, it is unclear whether there is an association between cholesterol and the pituitary. We investigated the effects of a high-cholesterol diet on pituitary hormones using in vivo animal studies and an epidemiological study. In the animal experiments, rats were fed a high-cholesterol or control diet for 28 weeks. In rats fed the high-cholesterol diet, serum levels of thyroid-stimulating hormone (TSH; also known as thyrotrophin), luteinising hormone (LH) and follicle-stimulating hormone (FSH) produced by the basophilic cells of the anterior pituitary were elevated in a time-dependent manner. Among these hormones, TSH was the first to undergo a significant change, whereas adrenocorticotrophic hormone (ACTH), another hormone produced by basophilic cells, was not changed significantly. As the duration of cholesterol feeding increased, cholesterol deposition increased gradually in the pituitary. Histologically, basophilic cells, and especially thyrotrophs and gonadotrophs, showed an obvious increase in cell area, as well as a potential increase in their proportion of total pituitary cells. Expression of the β-subunit of TSH, FSH and LH, which controls hormone specificity and activity, exhibited a corresponding increase. In the epidemiological study, we found a similar elevation of serum TSH, LH and FSH and a decrease in ACTH in patients with hypercholesterolaemia. Significant positive correlations existed between serum total cholesterol and TSH, FSH or LH, even after adjusting for confounding factors. Taken together, the results of the present study suggest that the high-cholesterol diet affected the levels of hormones derived from anterior pituitary basophilic cells. This phenomenon might contribute to the pituitary functional disturbances described in hypercholesterolaemia. © 2016 British Society for Neuroendocrinology.

  11. Effect of vitamin E levels on the cell-mediated immunity of broilers vaccinated against coccidiosis

    Directory of Open Access Journals (Sweden)

    ICM da Silva

    2011-03-01

    Full Text Available Studies on the relationships between animal nutrition and immunity have sought reliable methodologies to measure responses. Cell-mediated immune response is similarly studied in humans. The cutaneous basophil hypersensitivity test (CBH is one of the methods to measure that response and consists in the infiltration of inflammatory cells, particularly of lymphocytes and basophils, as result of the application of substances capable of inducing cell proliferation in determined sites, such as wings, wattle, and interdigital space in birds. CBH is considered a simple and fast method and can be applied in birds of different ages. In immunocompetence studies with poultry, phytohemagglutinin-P (PHA-P is a commonly used substance, despite the variability of the response related to the method of application (intradermal injection and the antigens used. In the present experiment, PHA-P was used to observe the cell-mediated immune response of 216 chicks fed three dietary levels of vitamin E from 1 to 36 days of age. All birds were immunologically challenged by vaccination against coccidiosis at three days of age and against Newcastle Disease (NCD at 14 and 30 days of age. At 36 days of age, birds were submitted to the CBH test according to the methodology of Corrier & DeLoach (1990. Birds fed 65mg/kg of vitamin E presented lasting cell reaction (p<0.08, which indicates that this vitamin E level improved cell immune response of birds due to its antioxidant and immunomodulating properties. The use of this vitamin E level can be considered by nutritionists under practical conditions, aiming to improve broiler immunity.

  12. Calmodulin antagonists effect on Ca(2+ level in the mitochondria and cytoplasm of myometrium cells

    Directory of Open Access Journals (Sweden)

    S. G. Shlykov

    2015-10-01

    Full Text Available It is known that Са2+-dependent regulation of this cation exchange in mitochondria is carried out with participation of calmodulin. We had shown in a previous work using two experimental models: isolated mitochondria and intact myometrium cells, that calmodulin antagonists reduce the level of mitochondrial membrane polarization. The aim of this work was to investigate the influence of calmodulin antagonists on the level of ionized Са in mitochondria and cytoplasm of uterine smooth muscle cells using spectrofluorometry and confocal microscopy. It was shown that myometrium mitochondria, in the presence of АТР and MgCl2 in the incubation medium, accumulate Са ions in the matrix. Incubation of mitochondria in the presence of СССР inhibited cation accumulation, but did not cease it. Calmodulin antagonist such as trifluoperazine (100 µМ considerably increased the level of ionized Са in the mitochondrial matrix. Preliminary incubation of mitochondria with 100 µМ Са2+, before adding trifluoperazine to the incubation medium, partly prevented influence of the latter on the cation level in the matrix. Incubation of myometrium cells (primary culture with another calmodulin antagonist calmidazolium (10 µМ was accompanied by depolarization of mitochondrial membrane and an increase in the concentration of ionized Са in cytoplasm. Thus, using two models, namely, isolated mitochondria and intact myometrium cells, it has been shown that calmodulin antagonists cause depolarization of mitochondrial membranes and an increase of the ionized Са concentration in both the mitochondrial matrix and the cell cytoplasm.

  13. Application of gelatin zymography for evaluating low levels of contaminating neutrophils in red blood cell samples.

    Science.gov (United States)

    Achilli, Cesare; Ciana, Annarita; Balduini, Cesare; Risso, Angela; Minetti, Giampaolo

    2011-02-15

    Supposedly "homogeneous" red blood cell (RBC) samples are commonly obtained by "washing" whole blood free of plasma, platelets, and white cells with physiological solutions, a procedure that does not result, however, in sufficient removal of polymorphonuclear neutrophils (PMNs), leading to possible artifactual results. Pure RBC samples can be obtained only by leukodepletion procedures. Proposed here is a version of gelatin zymography adapted to detect matrix metalloproteinase 9 (MMP-9), selectively expressed by PMNs, in heterogeneous mixtures of RBCs and PMNs that can reveal contamination at levels as low as 1 PMN/10⁶ RBCs. Copyright © 2010 Elsevier Inc. All rights reserved.

  14. Keeping the intracellular vitamin C at a physiologically relevant level in endothelial cell culture

    DEFF Research Database (Denmark)

    Frikke-Schmidt, Henriette Rønne; Lykkesfeldt, Jens

    2010-01-01

    It is generally accepted that the addition of vitamin C to cell culture medium improves cell growth. However, once added, the vitamin C concentration declines rapidly. This situation differs from the in vivo environment where the endothelium is constantly supplied with ascorbate from the blood....... With a focus on intracellular vitamin C, we simulated constant supply of ascorbate by the hourly addition of freshly prepared medium containing 75 lM ascorbate and subsequently compared it with more practical regimens using combinations of ascorbate and 2-phosphoascorbate. We found that a single supplement...... of ascorbate and 2-phosphoascorbate adequately maintains intracellular vitamin C at physiological levels for up to 72 h....

  15. Establishment and characterization of a platinum- and paclitaxel-resistant high grade serous ovarian carcinoma cell line.

    Science.gov (United States)

    Teng, Pang-Ning; Bateman, Nicholas W; Wang, Guisong; Litzi, Tracy; Blanton, Brian E; Hood, Brian L; Conrads, Kelly A; Ao, Wei; Oliver, Kate E; Darcy, Kathleen M; McGuire, William P; Paz, Keren; Sidransky, David; Hamilton, Chad A; Maxwell, G Larry; Conrads, Thomas P

    2017-07-01

    High grade serous ovarian cancer (HGSOC) patients have a high recurrence rate after surgery and adjuvant chemotherapy due to inherent or acquired drug resistance. Cell lines derived from HGSOC tumors that are resistant to chemotherapeutic agents represent useful pre-clinical models for drug discovery. Here, we describe establishment of a human ovarian carcinoma cell line, which we term WHIRC01, from a patient-derived mouse xenograft established from a chemorefractory HGSOC patient who did not respond to carboplatin and paclitaxel therapy. This newly derived cell line is platinum- and paclitaxel-resistant with cisplatin, carboplatin, and paclitaxel half-maximal lethal doses of 15, 130, and 20 µM, respectively. Molecular characterization of this cell line was performed using targeted DNA exome sequencing, transcriptomics (RNA-seq), and mass spectrometry-based proteomic analyses. Results from exomic sequencing revealed mutations in TP53 consistent with HGSOC. Transcriptomic and proteomic analyses of WHIRC01 showed high level of alpha-enolase and vimentin, which are associated with cell migration and epithelial-mesenchymal transition. WHIRC01 represents a chemorefractory human HGSOC cell line model with a comprehensive molecular profile to aid future investigations of drug resistance mechanisms and screening of chemotherapeutic agents.

  16. Low level of 5-Hydroxymethylcytosine predicts poor prognosis in non-small cell lung cancer

    Science.gov (United States)

    LIAO, YUNFEI; GU, JIE; WU, YONGBING; LONG, XIANG; GE, DI; XU, JIANJUN; DING, JIANYONG

    2016-01-01

    The loss of 5-hydroxymethylcytosine (5-hmC) has previously been demonstrated to be implicated in the initiation and progression of various tumors. However, its role in non-small cell lung cancer (NSCLC) remains unknown. The present study aimed to determine the level of 5-hmC in NSCLC and their adjacent normal lung tissues by immunohistochemistry and dot-blot analysis; then the relationship between 5-hmC level and the clinicopathological features of NSCLC and the prognostic significance of 5-hmC level in NSCLC patients were analyzed. By employing the dot-blot analysis, a significant reduction of 5-hmC level in NSCLC tissues compared with the adjacent normal tissues was detected, which were further verified by the immunohistochemistry results on tissue microarrays. Further analyses demonstrated that 65.38% (136/208) presented with low 5-hmC level, and low 5-hmC level was significantly associated with lymph node metastasis (PhmC levels were significantly lower than patients with high 5-hmC levels (PhmC level was identified as independent prognostic factor in patients' overall survival. In conclusion, downregulation of 5-hmC may serve as a useful biomarker for NSCLC prognosis evaluation. PMID:27313688

  17. Low level of 5-Hydroxymethylcytosine predicts poor prognosis in non-small cell lung cancer.

    Science.gov (United States)

    Liao, Yunfei; Gu, Jie; Wu, Yongbing; Long, Xiang; Ge, D I; Xu, Jianjun; Ding, Jianyong

    2016-06-01

    The loss of 5-hydroxymethylcytosine (5-hmC) has previously been demonstrated to be implicated in the initiation and progression of various tumors. However, its role in non-small cell lung cancer (NSCLC) remains unknown. The present study aimed to determine the level of 5-hmC in NSCLC and their adjacent normal lung tissues by immunohistochemistry and dot-blot analysis; then the relationship between 5-hmC level and the clinicopathological features of NSCLC and the prognostic significance of 5-hmC level in NSCLC patients were analyzed. By employing the dot-blot analysis, a significant reduction of 5-hmC level in NSCLC tissues compared with the adjacent normal tissues was detected, which were further verified by the immunohistochemistry results on tissue microarrays. Further analyses demonstrated that 65.38% (136/208) presented with low 5-hmC level, and low 5-hmC level was significantly associated with lymph node metastasis (PhmC levels were significantly lower than patients with high 5-hmC levels (PhmC level was identified as independent prognostic factor in patients' overall survival. In conclusion, downregulation of 5-hmC may serve as a useful biomarker for NSCLC prognosis evaluation.

  18. Changes in peripheral blood level of regulatory T cells in patients with malignant melanoma during treatment with dendritic cell vaccination and low-dose IL-2

    DEFF Research Database (Denmark)

    Bjoern, J; Brimnes, M K; Andersen, M H

    2011-01-01

    In this study, changes in peripheral blood regulatory T cell (Treg) levels were evaluated in 46 progressive patients with melanoma treated with a dendritic cell-based vaccine and concomitant low-dose IFN-α and IL-2. The regulatory subset of CD4 T cells, characterized by CD25(high) , was prospecti......In this study, changes in peripheral blood regulatory T cell (Treg) levels were evaluated in 46 progressive patients with melanoma treated with a dendritic cell-based vaccine and concomitant low-dose IFN-α and IL-2. The regulatory subset of CD4 T cells, characterized by CD25(high...

  19. Interleukin-18 alters protein expressions of neurodegenerative diseases-linked proteins in human SH-SY5Y neuron-like cells

    Directory of Open Access Journals (Sweden)

    Elina M Sutinen

    2014-08-01

    Full Text Available Chronic inflammation and oxidative stress (OS are present in Alzheimer´s disease (AD brains in addition to neuronal loss, Amyloid-β (Aβ plaques and hyperphosphorylated tau-protein neurofibrillary tangles. Previously we showed that levels of the pro-inflammatory cytokine, interleukin-18 (IL-18, are elevated in post-mortem AD brains. IL-18 can modulate the tau kinases, Cdk5 and GSK3β, as well as Aβ-production. IL-18 levels are also increased in AD risk diseases, including type-2 diabetes and obesity. Here, we explored other IL-18 regulated proteins in neuron-like SH-SY5Y cells. Differentiated SH-SY5Y cells, incubated with IL-18 for 24, 48 or 72h, were analyzed by two-dimensional gel electrophoresis (2D-DIGE. Specific altered protein spots were chosen and identified with mass spectrometry and verified by western immunoblotting. IL-18 had time-dependent effects on the SH-SY5Y proteome, modulating numerous protein levels/modifications. We concentrated on those related to OS (DDAH2, peroxiredoxins 2, 3 and 6, DJ-1, BLVRA, Aβ-degradation (MMP14, TIMP2, Aβ-aggregation (Septin-2 and modifications of axon growth and guidance associated, collapsing response mediator protein 2 (CRMP2. IL-18 significantly increased antioxidative enzymes, indicative of OS, and altered levels of glycolytic α- and γ-enolase and multifunctional 14-3-3γ and -ε, commonly affected in neurodegenerative diseases. MMP14, TIMP2, α-enolase and 14-3-3ε, indirectly involved in Aβ metabolism, as well as Septin-2 showed changes that increase Aβ levels. Increased 14-3-3γ may contribute to GSK3β driven tau hyperphosphorylation and CRMP2 Thr514 and Ser522 phosphorylation with the Thr555-site, a target for Rho kinase, showing time-dependent changes. IL-18 also increased caspase-1 levels and vacuolization of the cells. Although our SH-SY5Y cells were not aged, as neurons in AD, our work suggests that heightened or prolonged IL-18 levels can drive protein changes of known

  20. T-cell independent reconstitution of the immunoglobulin levels in nu/nu mice

    International Nuclear Information System (INIS)

    Mannhardt, W.; Schulte-Wissermann, H.; Gardilcic, S.; Leon, F. de

    1982-01-01

    Nude mice were transplanted under the renal capsule either with allogeneic or human thymus that were long-term precultured or pretreated in vitro with Carrageenan for three days. None of the thymus tissue transplants showed lymphatic repopulation 9 wk after transplantation. Histological investigation of the peripheral lymphatic tissue did not reveal any change in the thymus-dependent area. On the other hand, plasma cells and germinal centers could be found in significantly increased numbers. In addition, a normalization of the serum immunoglobulin concentrations could be found, as no specific antibodies against thymus-dependent antigens were present after immunization and T-cell function did not improve. Similar results were obtained 9 wk after injection of irradiated thymocyte suspensions or of peritoneal macrophages from immunocompetent donors. It is concluded that thymus epithelial cells could act via macrophages on the polyclonal maturation and differentiation of B cells without involvement of T cells. This would be in agreement with the experience in some patients with severe combined immunodeficiency (SCID) in which reconstitution of the immunoglobulin levels is observed after transplantation of cultured thymus tissue before T-cell reconstitution can be demonstrated. (Auth.)

  1. Relationship of visfatin level to pancreatic endocrine hormone level, HOMA-IR index, and HOMA β-cell index in overweight women who performed hydraulic resistance exercise

    OpenAIRE

    Ha, Chang Ho; Swearingin, Brenda; Jeon, Yong Kyun

    2015-01-01

    [Purpose] This study aimed to examine the correlation of visfatin level to pancreatic endocrine hormone level, homeostasis model assessment of insulin resistance (HOMA-IR) index, and HOMA β-cell index in hydraulic resistance exercise. Furthermore, it investigated the relationship between visfatin level and other variables affected by exercise in overweight women. [Subjects and Methods] The exercise group trained for 12 weeks, 70 minutes/day, 5 days/week. Visfatin level, pancreatic endocrine h...

  2. Mast cell lines HMC-1 and LAD2 in comparison with mature human skin mast cells--drastically reduced levels of tryptase and chymase in mast cell lines.

    Science.gov (United States)

    Guhl, Sven; Babina, Magda; Neou, Angelos; Zuberbier, Torsten; Artuc, Metin

    2010-09-01

    To circumvent the costly isolation procedure associated with tissue mast cells (MC), two human MC lines, i.e. HMC-1 and LAD2, are frequently employed, but their relation to mature MC is unknown. Here, we quantitatively assessed their expression of MC markers in direct comparison to skin MC (sMC). sMC expressed all lineage markers at highest and HMC-1 cells at lowest levels. LAD2 cells expressed comparable high-affinity IgE receptor alpha (FcepsilonRIalpha) and FcepsilonRIgamma but less FcepsilonRIbeta than sMC and displayed slightly reduced, but robust FcepsilonRI-mediated histamine release. Only minor differences were found for total histamine content and c-Kit expression. Huge, and to this level unexpected, differences were found for MC tryptase and chymase, with sMC > LAD2 > HMC-1. Taken together, HMC-1 cells represent very immature malignantly transformed MC, whereas LAD2 cells can be considered intermediately differentiated. Because of the minute levels of MC proteases, MC lines can serve as surrogates of tissue MC to a limited degree only.

  3. Reduced levels of SCD1 accentuate palmitate-induced stress in insulin-producing β-cells

    Directory of Open Access Journals (Sweden)

    Hovsepyan Meri

    2010-09-01

    Full Text Available Abstract Background Stearoyl-CoA desaturase 1 (SCD1 is an ER resident enzyme introducing a double-bond in saturated fatty acids. Global knockout of SCD1 in mouse increases fatty acid oxidation and insulin sensitivity which makes the animal resistant to diet-induced obesity. Inhibition of SCD1 has therefore been proposed as a potential therapy of the metabolic syndrome. Much of the work has focused on insulin target tissue and very little is known about how reduced levels of SCD1 would affect the insulin-producing β-cell, however. The aim of the present study was therefore to investigate how reduced levels of SCD1 affect the β-cell. Results Insulin-secreting MIN6 cells with reduced levels of SCD1 were established by siRNA mediated knockdown. When fatty acid oxidation was measured, no difference between cells with reduced levels of SCD1 and mock-transfected cells were found. Also, reducing levels of SCD1 did not affect insulin secretion in response to glucose. To investigate how SCD1 knockdown affected cellular mechanisms, differentially regulated proteins were identified by a proteomic approach. Cells with reduced levels of SCD1 had higher levels of ER chaperones and components of the proteasome. The higher amounts did not protect the β-cell from palmitate-induced ER stress and apoptosis. Instead, rise in levels of p-eIF2α and CHOP after palmitate exposure was 2-fold higher in cells with reduced levels of SCD1 compared to mock-transfected cells. Accordingly, apoptosis rose to higher levels after exposure to palmitate in cells with reduced levels of SCD1 compared to mock-transfected cells. Conclusions In conclusion, reduced levels of SCD1 augment palmitate-induced ER stress and apoptosis in the β-cell, which is an important caveat when considering targeting this enzyme as a treatment of the metabolic syndrome.

  4. [Level of Th17 cell and CD4(+);CD25(+); Foxp3(+); regulatory T cell in peripheral blood mononuclear cells of primary nephrotic syndrome in children].

    Science.gov (United States)

    Wang, Li; Li, Qiu; Wang, Li-jia; Li, Xin

    2010-08-01

    To investigate the levels and functions of Th17 cells and CD4(+); CD25(+); Foxp3(+); regulate cells(Treg) and explore their role in pathogenesis of primary nephrotic syndrome (PNS) in children. Children with PNS were divided into simple type nephritic syndrome group (SNS) (n = 20), and nephritic type nephritic syndrome group (NNS) (n = 15). 20 healthy subjects were selected as control group. The circulating frequencies of Th17 cells and Treg were measured by FCM. Real-time PCR were used to analyze the mRNA expressions of RORC, IL-23p19 and Foxp3 in peripheral blood mononuclear cells. The serum of IL-1ß, IL-6, TGF-ß1 were measured by ELISA. Circulating frequencies of Th17 cells, the mRNA levels of RORC, IL-23p19 and the serum of IL-1ß, IL-6 were higher in SNS and NNS groups than control group (P 0.05). Imbalance of Th17 and Treg cells might contribute to the pathogenesis of PNS in children and have associated with clinical presentation, pathological type, glucocorticoid sensitivity and prognosis of the disease.

  5. Differential cellular responses by oncogenic levels of c-Myc expression in long-term confluent retinal pigment epithelial cells.

    Science.gov (United States)

    Wang, Yiping; Cheng, Xiangdong; Samma, Muhammad Kaleem; Kung, Sam K P; Lee, Clement M; Chiu, Sung Kay

    2017-11-29

    c-Myc is a highly pleiotropic transcription factor known to control cell cycle progression, apoptosis, and cellular transformation. Normally, ectopic expression of c-Myc is associated with promoting cell proliferation or triggering cell death via activating p53. However, it is not clear how the levels of c-Myc lead to different cellular responses. Here, we generated a series of stable RPE cell clones expressing c-Myc at different levels, and found that consistent low level of c-Myc induced cellular senescence by activating AP4 in post-confluent RPE cells, while the cells underwent cell death at high level of c-Myc. In addition, high level of c-Myc could override the effect of AP4 on cellular senescence. Further knockdown of AP4 abrogated senescence-like phenotype in cells expressing low level of c-Myc, and accelerated cell death in cells with medium level of c-Myc, indicating that AP4 was required for cellular senescence induced by low level of c-Myc.

  6. Tumor-promoting phorbol ester transiently down-modulates the p53 level and blocks the cell cycle

    DEFF Research Database (Denmark)

    Skouv, J.; Jensen, P O; Forchhammer, J

    1994-01-01

    a decrease in the p53 mRNA level in the cell lines. Normal diploid as well as various tumor cell lines were tested. Two tumor cell lines, HeLa and A549, both containing the wild-type p53 gene, but very different levels of p53 protein, were studied in detail. In both cell lines, the level of p53 m......RNA was minimal after 9 h of exposure to PMA. After approximately 120 h, the p53 mRNA level was similar to the pretreatment level. PMA induced a similar transient decrease in the level of p53 protein in the A549 cell line. The decrease in the p53 mRNA level could not be explained by changes in the transcriptional...

  7. Single versus Serial Measurements of Neuron-Specific Enolase and Prediction of Poor Neurological Outcome in Persistently Unconscious Patients after Out-Of-Hospital Cardiac Arrest - A TTM-Trial Substudy

    DEFF Research Database (Denmark)

    Wiberg, Sebastian; Hassager, Christian; Stammet, Pascal

    2017-01-01

    of the biomarker neuron-specific enolase (NSE) in combination with other predictors of outcome in patients admitted after out-of-hospital cardiac arrest (OHCA). This study sought to investigate the ability of NSE to predict poor outcome in patients remaining unconscious at day three after OHCA. In addition......, this study sought to investigate if serial NSE measurements add incremental prognostic information compared to a single NSE measurement at 48 hours in this population. METHODS: This study is a post-hoc sub-study of the TTM trial, randomizing OHCA patients to a course of TTM at either 33°C or 36°C. Patients...

  8. Relationship between plasma cholesterol levels and cholesterol esterification in isolated human mononuclear cells

    International Nuclear Information System (INIS)

    Dallongeville, J.; Davignon, J.; Lussier-Cacan, S.

    1990-01-01

    The authors studied the relationship between plasma lipoprotein concentrations and cholesterol esterification in freshly isolated human mononuclear cells from 27 normolipidemic and 32 hyperlipidemic individuals. Cells were either incubated for 5 hours with radiolabeled oleate immediately after isolation or were preincubated for 18 hours in the presence of exogenous cholesterol, and then incubated with [ 14 C]sodium-oleate-albumin complex. In the absence of exogenous cholesterol, control and hypercholesterolemic subjects had similarly low values of intracellular cholesterol esterification. In the presence of exogenous cholesterol, both hypertriglyceridemic and hypercholesterolemic subjects had higher cholesterol esterification than controls. There was a significant correlation between the rate of cholesterol esterification and plasma total cholesterol. These results suggest that plasma cholesterol levels may regulate mononuclear cell intra-cellular cholesterol esterification in humans

  9. HLA-G Level on Monocytoid Dendritic Cells Correlates with Regulatory T Cell Foxp3 Expression in Liver Transplant Tolerance

    Science.gov (United States)

    Castellaneta, Antonino; Mazariegos, George V; Nayyar, Navdeep; Zeevi, Adriana; Thomson, Angus W

    2011-01-01

    Background Human leukocyte antigen (HLA)-G is a non-classical HLA class I molecule expressed as membrane-bound and soluble isoforms. Interaction of HLA-G with its receptor, immunoglobulin (Ig)-like transcript (ILT) 4 on dendritic cells (DC) down-regulates their T cell stimulatory ability. Methods We examined expression of HLA-G, ILT4, other immune regulatory molecules (inducible costimulator ligand and glucocorticoid-induced tumor necrosis factor-related receptor ligand), and the activation marker CMRF44 on circulating monocytoid (m) and plasmacytoid (p)DC by monoclonal antibody staining and flow cytometry. Three groups of stable liver transplant recipients,-operationally tolerant (TOL), prospective immunosuppressive drug weaning (PW) and maintenance immunosuppression (MI) were studied, together with healthy controls (HC). Serum HLA-G levels were measured by enzyme-linked immunosorbent assay. Results In TOL patients, mDC but not pDC expressed higher HLA-G than in MI patients or HC. In TOL patients, the incidence of CD4+CD25hiCD127− regulatory T cells (Treg) and the intensity of Treg forkhead box p3 (Foxp3) expression were significantly higher than in the MI group. HLA-G expression on circulating mDC correlated significantly with that of Foxp3 in the TOL group. There was no correlation between immunosuppressive drug (tacrolimus) dose or trough level and HLA-G expression or Treg frequency or Foxp3 expression. The incidence of patients with circulating HLA-G levels >100ng/ml was highest in the TOL group, although statistical significance was not achieved. Conclusions Higher HLA-G expression on circulating mDC in TOL recipients compared with MI or HC, suggests a possible role of HLA-G in immune regulation possibly mediated by enhanced host Treg Foxp3 expression. PMID:21423069

  10. Protection against UVA-induced photooxidative damage in mammalian cell lines expressing increased levels of metallothionein

    International Nuclear Information System (INIS)

    Dudek, E.J.; Roth, R.M.

    1990-01-01

    Metallothionein (MT) is an endogenous low molecular weight protein that is inducible in a variety of eukaryotic cells and has the ability to selectivity bind heavy metal ions such as zinc and the cadmium. Although the exact physiological role of MT is still not understood, there is strong evidence that MT is involved in providing cellular resistance against the damaging effects of heavy metals and in the regulation of intracellular zinc and copper. Recently, it has been demonstrated that MT can scavenge radiation-induced reactive oxygen intermediates in vitro, specifically hydroxyl and superoxide radicals, and because of these observations it has been suggested that MT may provide protection against radiation-induced oxidative stress in vivo. Cell lines expressing increased levels of MT have demonstrated resistance to ionizing radiation, to ultraviolet radiation, and also to various DNA damaging agents including melphalan and cis-diaminedichloroplatinum. It is therefore important to gain some insight into the relationship between cellular MT content and cellular resistance to radiation and other DNA damaging agents. In this study we investigated the role of MT in providing protection against monochromatic 365-nm UVA radiation, which is known to generate intracellular reactive oxygen species that are involved in both DNA damage and cell killing. For this purpose, we used zinc acetate, a potent inducer of MT, to elevate MT levels in V79 Chinese hamster fibroblasts prior to UVA exposure and determined cell survival for uninduced and induced cultures. In order to eliminate any zinc effects other than MT induction, we also isolated and characterized cadmium chloride-resistant clones of V79 cells that have increased steady-state levels of both MT mRNA and protein, and we examined their survival characteristics against 365-nm radiation in the absence of zinc acetate. 14 refs., 3 figs

  11. Low level of 5-Hydroxymethylcytosine predicts poor prognosis in non-small cell lung cancer

    OpenAIRE

    LIAO, YUNFEI; GU, JIE; WU, YONGBING; LONG, XIANG; GE, DI; XU, JIANJUN; DING, JIANYONG

    2016-01-01

    The loss of 5-hydroxymethylcytosine (5-hmC) has previously been demonstrated to be implicated in the initiation and progression of various tumors. However, its role in non-small cell lung cancer (NSCLC) remains unknown. The present study aimed to determine the level of 5-hmC in NSCLC and their adjacent normal lung tissues by immunohistochemistry and dot-blot analysis; then the relationship between 5-hmC level and the clinicopathological features of NSCLC and the prognostic significance of 5-h...

  12. Lifetime Estimation of Electrolytic Capacitors in Fuel Cell Power Converter at Various Confidence Levels

    DEFF Research Database (Denmark)

    Zhou, Dao; Wang, Huai; Blaabjerg, Frede

    2016-01-01

    based lifetime expectancy of the individual capacitor and the capacitor bank is estimated in a fuel cell backup power converter operating in both standby mode and operation mode. The lifetime prediction of the capacitor banks at different confidence levels is also obtained.......DC capacitors in power electronic converters are a major constraint on improvement of the power density and the reliability. In this paper, according to the degradation data of tested capacitors, the lifetime model of the component is analyzed at various confidence levels. Then, the mission profile...

  13. Serum B cell-activating factor (BAFF) level in connective tissue disease associated interstitial lung disease.

    Science.gov (United States)

    Hamada, Tsutomu; Samukawa, Takuya; Kumamoto, Tomohiro; Hatanaka, Kazuhito; Tsukuya, Go; Yamamoto, Masuki; Machida, Kentaro; Watanabe, Masaki; Mizuno, Keiko; Higashimoto, Ikkou; Inoue, Yoshikazu; Inoue, Hiromasa

    2015-09-30

    Interstitial lung diseases (ILDs) are common in patients with connective tissue diseases (CTDs). Although the diagnosis of an underlying CTD in ILD (CTD-ILD) affects both prognosis and treatment, it is sometimes difficult to distinguish CTD-ILD from chronic fibrosing interstitial pneumonia (CFIP). B cell-activating factor belonging to the tumour necrosis factor family (BAFF) plays a crucial role in B cell development, survival, and antibody production. We examined serum levels of BAFF, surfactant protein D (SP-D), and Krebs von den Lungen-6 (KL-6) in 33 patients with CTD-ILD, 16 patients with undifferentiated CTD-ILD, 19 patients with CFIP, and 26 healthy volunteers. And we analysed the relationship between serum BAFF levels and pulmonary function, as well as the expression of BAFF in the lung tissue of patients with CTD-ILD. Serum levels of BAFF were significantly higher in CTD-ILD patients compared to healthy subjects and CFIP patients. However, there were no significant differences in serum levels of SP-D and KL-6. Furthermore, serum BAFF levels in CTD-ILD patients were inversely correlated with pulmonary function. BAFF was strongly expressed in the lungs of CTD-ILD patients, but weakly in normal lungs. This is the first study to demonstrate that serum BAFF levels were significantly higher in CTD-ILD patients compared to healthy subjects and CFIP patients. Furthermore, serum BAFF levels were correlated with pulmonary function. We consider that serum BAFF levels in patients with CTD-ILD reflect the presence of ILDs disease activity and severity. These finding suggest that BAFF may be a useful marker for distinguishing CTD-ILD from CFIP.

  14. Clinical management of small-cell carcinoma of the urinary tract: a 10-year single-center's experience.

    Science.gov (United States)

    Carranza, Omar E; Castañón, Eduardo; Abella, Luis E; Zudaire, María E; Castillo, Ainhoa; Arévalo, Estefanía; Fusco, Juan P; Zudaire, Juan J; Carías, Rafael; Cambeiro, Mauricio; Martínez-Monge, Rafael; Gil-Bazo, Ignacio

    2013-06-01

    Small-cell carcinoma (SCC) comprises 1% of primary bladder tumors and approximately 2% of prostate neoplasms. Metastatic disease at diagnosis is common, and survival outcomes are extremely poor. There is controversy about the ideal clinical management of these patients. The neuron-specific enolase (NSE) serum levels have never been studied in patients with small-cell carcinoma of the urinary tract (SCCUT). We report the clinical outcome of 12 consecutive SCCUT patients treated during the past 10 years. We also study the NSE levels at diagnosis and during treatment. Patients with limited disease (LD) experienced a non-significant longer progression-free survival (PFS) and overall survival (OS) compared with extensive disease (ED) subjects. Patients with bladder SCC showed a significantly higher median PFS compared with prostate SCCUT patients (22 vs. 6 months; P = .034), although that difference did not impact on a significant longer OS. NSE levels decreased during chemotherapy administration in all patients with ED and baseline high levels. Our patients showed a poor prognosis as described in previous studies. A better outcome for patients with bladder SCC compared with prostate SCC could be suggested. Serum NSE levels should be further evaluated to prove its potential use in early diagnosis and treatment monitoring during chemotherapy. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. The glomerular parietal epithelial cell's responses are influenced by SM22 alpha levels.

    Science.gov (United States)

    Naito, Shokichi; Pippin, Jeffrey W; Shankland, Stuart J

    2014-11-06

    Studies have shown in several diseases initially affecting podocytes, that the neighboring glomerular parietal epithelial cells (PECs) are secondarily involved. The PEC response might be reparative under certain circumstances, yet injurious under others. The factors governing these are not well understood. We have shown that SM22α, an actin-binding protein considered a marker of smooth muscle differentiation, is upregulated in podocytes and PECs in several models of podocyte disease. However, the impact of SM22α levels on PECs is not known. Experimental glomerular disease, characterized by primary podocyte injury, was induced in aged-matched SM22α+/+ and SM22α-/-mice by intraperitoneal injection of sheep anti-rabbit glomeruli antibody. Immunostaining methods were employed on days 7 and 14 of disease. The number of PEC transition cells, defined as cells co-expressing a PEC protein (PAX2) and podocyte protein (Synaptopodin) was higher in diseased SM22α-/-mice compared with SM22α+/+mice. WT1 staining along Bowman's capsule is higher in diseased SM22α-/-mice. This was accompanied by increased PEC proliferation (measured by ki-67 staining), and an increase in immunostaining for the progenitor marker NCAM, in a subpopulation of PECs in diseased SM22α-/-mice. In addition, immunostaining for vimentin and alpha smooth muscle actin, markers of epithelial-to-mesenchymal transition (EMT), was lower in diseased SM22α-/-mice compared to diseased SM22α+/+mice. SM22α levels may impact how PECs respond following a primary podocyte injury in experimental glomerular disease. Absent/lower levels favor an increase in PEC transition cells and PECs expressing a progenitor marker, and a lower EMT rate compared to SM22α+/+mice, where SM22 levels are markedly increased in PECs.

  16. Photovoltaic reciprocity and quasi-Fermi level splitting in nanostructure-based solar cells (Conference Presentation)

    Science.gov (United States)

    Aeberhard, Urs

    2017-04-01

    The photovoltaic reciprocity theory relates the electroluminescence spectrum of a solar cell under applied bias to the external photovoltaic quantum efficiency of the device as measured at short circuit conditions [1]. So far, the theory has been verified for a wide range of devices and material systems and forms the basis of a growing number of luminesecence imaging techniques used in the characterization of photovoltaic materials, cells and modules [2-5]. However, there are also some examples where the theory fails, such as in the case of amorphous silicon. In our contribution, we critically assess the assumptions made in the derivation of the theory and compare its predictions with rigorous formal relations as well as numerical computations in the framework of a comprehensive quantum-kinetic theory of photovoltaics [6] as applied to ultra-thin absorber architectures [7]. One of the main applications of the photovoltaic reciprocity relation is the determination of quasi-Fermi level splittings (QFLS) in solar cells from the measurement of luminescence. In nanostructure-based photovoltaic architectures, the determination of QFLS is challenging, but instrumental to assess the performance potential of the concepts. Here, we use our quasi-Fermi level-free theory to investigate existence and size of QFLS in quantum well and quantum dot solar cells. [1] Uwe Rau. Reciprocity relation between photovoltaic quantum efficiency and electrolumines- cent emission of solar cells. Phys. Rev. B, 76(8):085303, 2007. [2] Thomas Kirchartz and Uwe Rau. Electroluminescence analysis of high efficiency cu(in,ga)se2 solar cells. J. Appl. Phys., 102(10), 2007. [3] Thomas Kirchartz, Uwe Rau, Martin Hermle, Andreas W. Bett, Anke Helbig, and Jrgen H. Werner. Internal voltages in GaInP-GaInAs-Ge multijunction solar cells determined by electro- luminescence measurements. Appl. Phys. Lett., 92(12), 2008. [4] Thomas Kirchartz, Anke Helbig, Wilfried Reetz, Michael Reuter, Jürgen H. Werner, and

  17. Influence of oxygen levels on chondrogenesis of porcine mesenchymal stem cells cultured in polycaprolactone scaffolds.

    Science.gov (United States)

    Rodenas-Rochina, Joaquin; Kelly, Daniel J; Gómez Ribelles, Jose Luis; Lebourg, Myriam

    2017-06-01

    Chondrogenesis of mesenchymal stem cells (MSCs) is known to be regulated by a number of environmental factors, including local oxygen levels. The hypothesis of this study is that the response of MSCs to hypoxia is dependent on the physical and chemical characteristics of the substrate used. The objective of this study was to explore how different modifications to polycaprolactone (PCL) scaffolds influenced the response of MSCs to hypoxia. PCL, PCL-hyaluronic acid (HA), and PCL-Bioglass ® (BG) scaffolds were seeded with MSCs derived from bone marrow and cultured for 35 days under normoxic or low oxygen conditions, and the resulting biochemical properties of the MSC laden construct were assessed. Low oxygen tension has a positive effect over cell proliferation and macromolecules biosynthesis. Furthermore, hypoxia enhanced the distribution of collagen and glycosaminoglycans (GAGs) deposition through the scaffold. On the other hand, MSCs displayed certain material dependent responses to hypoxia. Low oxygen tension had a positive effect on cell proliferation in BG and HA scaffolds, but only a positive effect on GAGs synthesis in PCL and HA scaffolds. In conclusion, hypoxia increased cell viability and expression of chondrogenic markers but the cell response was modulated by the type of scaffold used. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1684-1691, 2017. © 2017 Wiley Periodicals, Inc.

  18. Neutrophil granulocytes uniquely express, among human blood cells, high levels of Methionine-sulfoxide-reductase enzymes.

    Science.gov (United States)

    Achilli, Cesare; Ciana, Annarita; Rossi, Antonio; Balduini, Cesare; Minetti, Giampaolo

    2008-01-01

    L-Methionine (Met), in its free form or when inserted in proteins, is sensitive to oxidation of its thioether group by reactive oxygen species from exogenous or endogenous sources. Two stable diastereomers of Met sulfoxide [Met-(O)] may be formed [Met-S-(O) and Met-R-(O)], but these can be reduced by two classes of Methionine-sulfoxide-reductase (Msr) enzymes: MsrA, which reduces the S, and MsrB, which reduces the R sulfoxide. In this study, we have examined the levels of expression of Msr in human blood cells by enzymatic activity assay, Western blotting, and RT-PCR of purified populations of polymorphonuclear neutrophils and eosinophils, mononuclear cells, platelets, and erythrocytes. Our data indicate that of the blood cells analyzed, neutrophils expressed the highest activity, which was mainly of MsrB type. During degranulation of activated neutrophils, Msr activity was not released but remained confined within the cell, indicating a non-granular localization. Immunoprecipitation and RT-PCR studies indicated the almost complete lack of mitochondrial forms of Msrs in granulocytes. It is thus likely that Msrs are important as antioxidant/repair systems for neutrophils, cells with enormous capacity for the generation of reactive oxidants and hence, susceptible to oxidative damage.

  19. Impacts of Bone Marrow Stem Cells on Caspase-3 Levels after Spinal Cord Injury in Mice.

    Science.gov (United States)

    Gashmardi, Noushin; Hosseini, Seyed Ebrahim; Mehrabani, Davood; Edalatmanesh, Mohammad Amin; Khodabandeh, Zahra

    2017-11-01

    Spinal cord injury (SCI) is a drastic disability that leads to spinal cord impairment. This study sought to determine the effects of bone marrow stem cells (BMSCs) on caspase-3 levels after acute SCI in mice. Forty-two mice were randomly divided into 3 groups: control (2 subcategories), subjected to no intervention; sham (3 subcategories), subjected to acute SCI; and experimental (2 subcategories), subjected to SCI and cell transplantation. In the experimental group, 2×10 5 BMSCs were injected intravenously 1 day after SCI. The mesenchymal property of the cells was assessed. The animals in the 3 groups were sacrificed 1, 21, and 35 days after the induction of injury and caspase-3 levels were evaluated using a caspase-3 assay kit. The obtained values were analyzed with ANOVA and Tukey tests using GraphPad and SPSS. Based on the assessments, the transplanted cells were spindle-shaped and were negative for the hematopoietic markers of CD34 and CD45 and positive for the expression of the mesenchymal marker of CD90 and osteogenic induction. The caspase-3 levels showed a significant increase in the sham and experimental groups in comparison to the control group. One day after SCI, the caspase-3 level was significantly higher in the sham group (1.157±0.117) than in the other groups (P<0.000). Twenty-one days after SCI, the caspase-3 level was significantly lower in the experimental group than in the sham group (0.4±0.095 vs. 0.793±0.076; P˂0.000). Thirty-five days following SCI, the caspase-3 level was lower in the experimental group than in the sham group (0.223±0.027 vs. 0.643±0.058; P˂0.000). We conclude that BMSC transplantation was able to downregulate the caspase-3 level after acute SCI, underscoring the role of caspase-3 as a marker for the assessment of treatment efficacy in acute SCI.

  20. Impacts of Bone Marrow Stem Cells on Caspase-3 Levels after Spinal Cord Injury in Mice

    Directory of Open Access Journals (Sweden)

    Noushin Gashmardi

    2017-11-01

    Full Text Available Spinal cord injury (SCI is a drastic disability that leads to spinal cord impairment. This study sought to determine the effects of bone marrow stem cells (BMSCs on caspase-3 levels after acute SCI in mice. Forty-two mice were randomly divided into 3 groups: control (2 subcategories, subjected to no intervention; sham (3 subcategories, subjected to acute SCI; and experimental (2 subcategories, subjected to SCI and cell transplantation. In the experimental group, 2×105 BMSCs were injected intravenously 1 day after SCI. The mesenchymal property of the cells was assessed. The animals in the 3 groups were sacrificed 1, 21, and 35 days after the induction of injury and caspase-3 levels were evaluated using a caspase-3 assay kit. The obtained values were analyzed with ANOVA and Tukey tests using GraphPad and SPSS. Based on the assessments, the transplanted cells were spindle-shaped and were negative for the hematopoietic markers of CD34 and CD45 and positive for the expression of the mesenchymal marker of CD90 and osteogenic induction. The caspase-3 levels showed a significant increase in the sham and experimental groups in comparison to the control group. One day after SCI, the caspase-3 level was significantly higher in the sham group (1.157±0.117 than in the other groups (P<0.000. Twenty-one days after SCI, the caspase-3 level was significantly lower in the experimental group than in the sham group (0.4±0.095 vs. 0.793±0.076; P˂0.000. Thirty-five days following SCI, the caspase-3 level was lower in the experimental group than in the sham group (0.223±0.027 vs. 0.643±0.058; P˂0.000. We conclude that BMSC transplantation was able to downregulate the caspase-3 level after acute SCI, underscoring the role of caspase-3 as a marker for the assessment of treatment efficacy in acute SCI.

  1. MG132, a proteasome inhibitor, induces human pulmonary fibroblast cell death via increasing ROS levels and GSH depletion.

    Science.gov (United States)

    Park, Woo Hyun; Kim, Suhn Hee

    2012-04-01

    MG132 as a proteasome inhibitor can induce apoptotic cell death in lung cancer cells. However, little is known about the toxicological cellular effects of MG132 on normal primary lung cells. Here, we investigated the effects of N-acetyl cysteine (NAC) and vitamin C (well known antioxidants) or L-buthionine sulfoximine (BSO; an inhibitor of GSH synthesis) on MG132-treated human pulmonary fibroblast (HPF) cells in relation to cell death, reactive oxygen species (ROS) and glutathione (GSH). MG132 induced growth inhibition and death in HPF cells, accompanied by the loss of mitochondrial membrane potential (MMP; ∆ψm). MG132 increased ROS levels and GSH-depleted cell numbers in HPF cells. Both antioxidants, NAC and vitamin C, prevented growth inhibition, death and MMP (∆ψm) loss in MG132-treated HPF cells and also attenuated ROS levels in these cells. BSO showed a strong increase in ROS levels in MG132-treated HPF cells and slightly enhanced the growth inhibition, cell death, MMP (∆ψm) loss and GSH depletion. In addition, NAC decreased anonymous ubiquitinated protein levels in MG132-treated HPF cells. Furthermore, superoxide dismutase (SOD) 2, catalase (CTX) and GSH peroxidase (GPX) siRNAs enhanced HPF cell death by MG132, which was not correlated with ROS and GSH level changes. In conclusion, MG132 induced the growth inhibition and death of HPF cells, which were accompanied by increasing ROS levels and GSH depletion. Both NAC and vitamin C attenuated HPF cell death by MG132, whereas BSO slightly enhanced the death.

  2. Do serum and red blood cell folate levels indicate iron response in hemodialysis patients?

    Science.gov (United States)

    Mitsopoulos, Efstathios; Zanos, Stavros; Ginikopoulou, Eudoxia; Tsiatsiou, Maria; Giannakou, Anastasia; Pavlitou, Aikaterini; Sakellariou, Georgios

    2006-01-01

    The relationship among iron status, ferritin, and folate levels, and the possible contribution of folate measurement in the prediction of iron response in hemodialysis patients, have not been assessed. In addition to serum ferritin and transferrin saturation (TSAT), serum and red blood cell (RBC) folate levels were evaluated as indices for intravenous iron therapy responsiveness in 60 hemodialysis patients. Patients were classified as iron responders or nonresponders depending on whether they exhibited a rise in hemoglobin above 1 g/dl after administration of 1 g of iron sucrose. An inverse relation between serum ferritin concentration and RBC folate levels was found in iron responders (n=26, r=-0.62, p<0.001) but not in nonresponders (n=34, r=0.07, p=nonsignificant). Only serum and RBC folate levels could predict iron response in patients with ferritin levels above 150 microg/l (n=25), with a sensitivity of 83.3% and a specificity of 94.7%. Our findings suggest that RBC folate concentration is inversely related with ferritin levels in iron-responsive but not in non-responsive hemodialysis patients. Serum and RBC folate concentration seems to predict response to iron administration better than serum ferritin or TSAT in patients with ferritin levels above 150 microg/l; therefore, in these patients, it might be used to guide iron management.

  3. Efficient handling of high-level radioactive cell waste in a vitrification facility analytical laboratory

    International Nuclear Information System (INIS)

    Roberts, D.W.; Collins, K.J.

    1998-01-01

    The Savannah River Site''s (SRS) Defense Waste Processing Facility (DWPF) near Aiken, South Carolina, is the world''s largest and the United State''s first high level waste vitrification facility. For the past 1.5 years, DWPF has been vitrifying high level radioactive liquid waste left over from the Cold War. The vitrification process involves the stabilization of high level radioactive liquid waste into borosilicate glass. The glass is contained in stainless steel canisters. DWPF has filled more than 200 canisters 3.05 meters (10 feet) long and 0.61 meters (2 foot) diameter. Since operations began at DWPF in March of 1996, high level radioactive solid waste continues to be generated due to operating the facility''s analytical laboratory. The waste is referred to as cell waste and is routinely removed from the analytical laboratories. Through facility design, engineering controls, and administrative controls, DWPF has established efficient methods of handling the high level waste generated in its laboratory facility. These methods have resulted in the prevention of undue radiation exposure, wasted man-hours, expenses due to waste disposal, and the spread of contamination. This level of efficiency was not reached overnight, but it involved the collaboration of Radiological Control Operations and Laboratory personnel working together to devise methods that best benefited the facility. This paper discusses the methods that have been incorporated at DWPF for the handling of cell waste. The objective of this paper is to provide insight to good radiological and safety practices that were incorporated to handle high level radioactive waste in a laboratory setting

  4. Increased viability of odontoblast-like cells subjected to low-level laser irradiation

    Science.gov (United States)

    Oliveira, C. F.; Basso, F. G.; Lins, E. C.; Kurachi, C.; Hebling, J.; Bagnato, V. S.; de Souza Costa, C. A.

    2010-07-01

    Studies have shown that the increase of cell metabolism depends on the low level laser therapy (LLLT) parameters used to irradiate the cells. However, the optimal laser dose to up-regulate pulp cell activity remains unknown. Consequently, the aim of this study was to evaluate the metabolic response of odontoblast-like cells (MDPC-23) exposed to different LLLT doses. Cells at 20000 cells/cm2 were seeded in 24-well plates using plain culture medium (DMEM) and were incubated in a humidified incubator with 5% CO2 at 37°C. After 24 h, the culture medium was replaced by fresh DMEM supplemented with 5% (stress by nutritional deficit) or 10% fetal bovine serum (FBS). The cells were exposed to different laser doses from a near infrared diode laser prototype designed to provide a uniform irradiation of the wells. The experimental groups were: G1: 1.5 J/cm2 + 5% FBS; G2: 1.5 J/cm2 + 10% FBS; G3: 5 J/cm2 + 5% FBS; G4: 5 J/cm2 + 10% FBS; G5: 19 J/cm2 + 5% FBS; G6: 19 J/cm2 + 10% FBS. LLLT was performed in 3 consecutive irradiation cycles with a 24-hour interval. Non-irradiated cells cultured in DMEM supplemented with either 5 or 10% FBS served as control groups. The analysis of the metabolic response was performed by the MTT assay 3 h after the last irradiation. G1 presented an increase in SDH enzyme activity and differed significantly (Mann-Whitney test, p < 0.05) from the other groups. Analysis by scanning electron microscopy showed normal cell morphology in all groups. Under the tested conditions, LLLT stimulated the metabolic activity of MDPC-23 cultured in DMEM supplemented with 5% FBS and exposed to a laser dose of 1.5 J/cm2. These findings are relevant for further studies on the action of near infrared lasers on cells with odontoblast phenotype.

  5. Changes in metabolic proteins in ex vivo rat retina during glutamate-induced neural progenitor cell induction.

    Science.gov (United States)

    Tokuda, Kazuhiro; Kuramitsu, Yasuhiro; Baron, Byron; Kitagawa, Takao; Tokuda, Nobuko; Kobayashi, Masaaki; Kimura, Kazuhiro; Sonoda, Koh-Hei; Nakamura, Kazuyuki

    2016-08-01

    Understanding how energy metabolism and related proteins influence neural progenitor cells in adult tissues is critical for developing new strategies in clinical tissue regeneration therapy. We have recently reported that a subtoxic concentration of glutamate-induced neural progenitor cells in the mature ex vivo rat retina. We herein explore changes in the metabolic pathways during the process. We firstly observed an increase in lactate and lactate dehydrogenase concentration in the glutamate-treated retina. We then investigated the levels of glycolytic enzymes and confirmed significant upregulation of pyruvate kinase M type (PKM), especially PKM2, enolase, phosphoglycerate mutase 1 (PGAM1), and inosine-5'-monophosphate dehydrogenase (IMPDH1) in the glutamate-treated retina compared to the untreated retina. An analysis of the subcellular localization of PKM2 revealed nuclear translocation in the treated retina, which has been reported to regulate cell cycle proliferation and glycolytic enzymes. Our findings indicate that the mature rat retina undergoes an increase in aerobic glycolysis. PKM2, both in the cytoplasm and in the nucleus, may thus play an important role during neural progenitor cell induction, as it does in other proliferating cells.

  6. CEA, SCC and NSE levels in exhaled breath condensate--possible markers for early detection of lung cancer.

    Science.gov (United States)

    Zou, Yingchang; Wang, Lin; Zhao, Cong; Hu, Yanjie; Xu, Shan; Ying, Kejing; Wang, Ping; Chen, Xing

    2013-12-01

    Lung cancer (LC) is the leading cause of cancer-related death. The sensitive and non-invasive diagnostic tools in the early stage are still poor. We present a pilot study on the early diagnosis of LC by detecting markers in exhaled breath condensate (EBC). EBC samples were collected from 105 patients with LC and 56 healthy controls. We applied chemiluminescence immunoassay to detect CEA (carcinoembryonic antigen), SCC (squamous cell carcinoma) antigen and NSE (neuron specific enolase) in EBC and serum. Concentrations of markers were compared between independent groups and subgroups. A significantly higher concentration level of each marker was found in patients with LC than healthy controls. The areas under curve of receiver operating characteristic (ROC) curves were 0.800, 0.771, 0.659, 0.679, 0.636 and 0.626 for EBC-CEA, serum-CEA, EBC-SCC, serum-SCC, EBC-NSE and serum-NSE, respectively. Markers in EBC had a higher positive rate (PR) and were more specific to histologic types than markers in serum. In addition, multivariate analysis was performed to evaluate the association of presenting markers with the stages of non-small cell lung cancer (NSCLC). EBC-CEA showed the best predictive characteristic (p tumor markers in EBC may have a better diagnostic performance for LC than those in serum. With further investigation on the combination of markers in EBC, detection of EBC could probably be a novel and non-invasive method to detect NSCLC earlier.

  7. Fatigue, serum cytokine levels, and blood cell counts during radiotherapy of patients with breast cancer

    International Nuclear Information System (INIS)

    Geinitz, Hans; Zimmermann, Frank B.; Stoll, Peter; Thamm, Reinhard; Kaffenberger, Walter; Ansorg, Kai; Keller, Monika; Busch, Raymonde; Beuningen, Dirk van; Molls, Michael

    2001-01-01

    Purpose: To assess the level of fatigue during the course of adjuvant radiotherapy (RT) of breast cancer patients and its relation to anxiety, depression, serum cytokines, and blood count levels. Methods and Materials: Forty-one patients who received adjuvant RT after breast-conserving surgery were prospectively studied. All patients underwent RT without concomitant chemotherapy. Patients rated their fatigue with two standardized self-assessment instruments, the Fatigue Assessment Questionnaire and a visual analog scale on fatigue intensity, before RT, during weeks 1-5 of RT, and 2 months after RT completion. In addition, the anxiety and depression levels were assessed with the Hospital Anxiety and Depression Scale. A differential blood cell count and the serum levels of the cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor-α were determined in parallel to the fatigue assessments. Results: Fatigue intensity as assessed with the visual analog scale increased (p<0.001) until treatment week 4 and remained elevated until week 5. Two months after RT, the values had fallen to the pretreatment levels. Fatigue measured with the Fatigue Assessment Questionnaire did not increase significantly during treatment, but the subscores on physical (p=0.035) and cognitive (p=0.015) fatigue were elevated during treatment weeks 4 and 5. Affective fatigue did not change significantly. Anxiety, as rated with the Hospital Anxiety and Depression Scale, declined during RT (p=0.002), but the Hospital Anxiety and Depression Scale depression score did not change significantly. IL-1β, IL-6, and tumor necrosis factor-α levels did not change during therapy and did not correlate with fatigue. Peripheral blood cell levels declined significantly during therapy and were still low 2 months after treatment. Until treatment week 5, lymphocytes were reduced to almost 50% of their initial values. Hemoglobin levels did not correlate with fatigue. Conclusions: We observed an increase in

  8. DECREASED LEVEL OF CORD BLOOD CIRCULATING ENDOTHELIAL COLONY-FORMING CELLS IN PREECLAMPSIA

    Science.gov (United States)

    Muñoz-Hernandez, Rocio; Miranda, Maria L.; Stiefel, Pablo; Lin, Ruei-Zeng; Praena-Fernández, Juan M.; Dominguez-Simeon, Maria J.; Villar, Jose; Moreno-Luna, Rafael; Melero-Martin, Juan M.

    2014-01-01

    Preeclampsia is a pregnancy-related disorder associated with increased cardiovascular risk for the offspring. Endothelial colony-forming cells (ECFCs) are a subset of circulating endothelial progenitor cells that participate in the formation of vasculature during development. However, the effect of preeclampsia on fetal levels of ECFCs is largely unknown. In this study, we sought to determine whether cord blood ECFC abundance and function are altered in preeclampsia. We conducted a prospective cohort study that included women with normal (n=35) and preeclamptic (n=15) pregnancies. We measured ECFC levels in the umbilical cord blood of neonates and characterized ECFC phenotype, cloning-forming ability, proliferation and migration towards VEGF-A and FGF-2, in vitro formation of capillary-like structures, and in vivo vasculogenic ability in immunodeficient mice. We found that the level of cord blood ECFCs was statistically lower in preeclampsia than in control pregnancies (P = .04), a reduction that was independent of other obstetric factors. In addition, cord blood ECFCs from preeclamptic pregnancies required more time to emerge in culture than control ECFCs. However, once derived in culture, ECFC function was deemed normal and highly similar between preeclampsia and control, including the ability to form vascular networks in vivo. This study demonstrates that preeclampsia affects ECFC abundance in neonates. A reduced level of ECFCs during preeclamptic pregnancies may contribute to an increased risk of developing future cardiovascular events. PMID:24752434

  9. DNA damage in blood cells exposed to low-level lasers.

    Science.gov (United States)

    Sergio, Luiz Philippe da Silva; Silva, Ana Paula Almeida da; Amorim, Philipi Freitas; Campos, Vera Maria Araújo; Magalhães, Luis Alexandre Gonçalves; de Paoli, Flavia; de Souza da Fonseca, Adenilson

    2015-04-01

    In regenerative medicine, there are increasing applications of low-level lasers in therapeutic protocols for treatment of diseases in soft and in bone tissues. However, there are doubts about effects on DNA, and an adequate dosimetry could improve the safety of clinical applications of these lasers. This work aimed to evaluate DNA damage in peripheral blood cells of Wistar rats induced by low-level red and infrared lasers at different fluences, powers, and emission modes according to therapeutic protocols. Peripheral blood samples were exposed to lasers and DNA damage was accessed by comet assay. In other experiments, DNA damage was accessed in blood cells by modified comet assay using formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III enzymes. Data show that exposure to low-level red and infrared lasers induce DNA damage depending on fluence, power and emission mode, which are targeted by Fpg and endonuclease III. Oxidative DNA damage should be considered for therapeutic efficacy and patient safety in clinical applications based on low-level red and infrared lasers. © 2015 Wiley Periodicals, Inc.

  10. Study on serum TNF-α level, B-cell count and T-cell subsets distribution in peripheral blood in patients with rheumatoid arthritis

    International Nuclear Information System (INIS)

    Shi Buqing

    2006-01-01

    Objective: To study the changes of serum TNF-α levels, B-cell count and T-cell subsets distribution in peripheral blood in patients with rheumatoid arthritis. Methods: Serum TNF-α levels (with RIA), B cell as well as T cell subsets distribution type (with monoclonal antibody technique) were examined in 37 patients with rheumatoid arthritis and 30 controls. Results Serum TNF-α levels and B lymphocytes count were significantly higher in the patients than those in controls (P 3 , CD 4 and CD 4 /CD 8 were obviously lower (P<0.01). Conclusion: Rheumatoid arthritis is an autoimmune disease with abnormal immunoregulation. (authors)

  11. Radiosensitizing Effects of Ectopic miR-101 on Non-Small-Cell Lung Cancer Cells Depend on the Endogenous miR-101 Level

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Susie; Wang Hongyan; Ng, Wooi Loon; Curran, Walter J. [Department of Radiation Oncology, School of Medicine and the Winship Cancer Institute, Emory University, Atlanta, GA (United States); Wang Ya, E-mail: ywang94@emory.edu [Department of Radiation Oncology, School of Medicine and the Winship Cancer Institute, Emory University, Atlanta, GA (United States)

    2011-12-01

    Purpose: Previously, we showed that ectopic miR-101 could sensitize human tumor cells to radiation by targeting ATM and DNA-PK catalytic subunit (DNA-PKcs) to inhibit DNA repair, as the endogenous miR-101 levels are low in tumors in general. However, the heterogeneity of human cancers may result in an exception. The purpose of this study was to test the hypothesis that a few tumor cell lines with a high level of endogenous miR-101 would prove less response to ectopic miR-101. Methods and Materials: Fourteeen non-small-cell lung cancer (NSCLC) cell lines and one immortalized non-malignant lung epithelial cell line (NL20) were used for comparing endogenous miR-101 levels by real-time reverse transcription-polymerase chain reaction. Based on the different miR-101 levels, four cell lines with different miR-101 levels were chosen for transfection with a green fluorescent protein-lentiviral plasmid encoding miR-101. The target protein levels were measured by using Western blotting. The radiosensitizing effects of ectopic miR-101 on these NSCLC cell lines were determined by a clonogenic assay and xenograft mouse model. Results: The endogenous miR-101 level was similar or lower in 13 NSCLC cell lines but was 11-fold higher in one cell line (H157) than in NL20 cells. Although ectopic miR-101 efficiently decreased the ATM and DNA-PKcs levels and increased the radiosensitization level in H1299, H1975, and A549 cells, it did not change the levels of the miR-101 targets or radiosensitivity in H157 cells. Similar results were observed in xenograft mice. Conclusions: A small number of NSCLC cell lines could have a high level of endogenous miR-101. The ectopic miR-101 was able to radiosensitize most NSCLC cells, except for the NSCLC cell lines that had a much higher endogenous miR-101 level. These results suggest that when we choose one miRNA as a therapeutic tool, the endogenous level of the miRNA in each tumor should be considered.

  12. Radiosensitizing Effects of Ectopic miR-101 on Non–Small-Cell Lung Cancer Cells Depend on the Endogenous miR-101 Level

    International Nuclear Information System (INIS)

    Chen, Susie; Wang Hongyan; Ng, Wooi Loon; Curran, Walter J.; Wang Ya

    2011-01-01

    Purpose: Previously, we showed that ectopic miR-101 could sensitize human tumor cells to radiation by targeting ATM and DNA-PK catalytic subunit (DNA-PKcs) to inhibit DNA repair, as the endogenous miR-101 levels are low in tumors in general. However, the heterogeneity of human cancers may result in an exception. The purpose of this study was to test the hypothesis that a few tumor cell lines with a high level of endogenous miR-101 would prove less response to ectopic miR-101. Methods and Materials: Fourteeen non–small-cell lung cancer (NSCLC) cell lines and one immortalized non-malignant lung epithelial cell line (NL20) were used for comparing endogenous miR-101 levels by real-time reverse transcription–polymerase chain reaction. Based on the different miR-101 levels, four cell lines with different miR-101 levels were chosen for transfection with a green fluorescent protein–lentiviral plasmid encoding miR-101. The target protein levels were measured by using Western blotting. The radiosensitizing effects of ectopic miR-101 on these NSCLC cell lines were determined by a clonogenic assay and xenograft mouse model. Results: The endogenous miR-101 level was similar or lower in 13 NSCLC cell lines but was 11-fold higher in one cell line (H157) than in NL20 cells. Although ectopic miR-101 efficiently decreased the ATM and DNA-PKcs levels and increased the radiosensitization level in H1299, H1975, and A549 cells, it did not change the levels of the miR-101 targets or radiosensitivity in H157 cells. Similar results were observed in xenograft mice. Conclusions: A small number of NSCLC cell lines could have a high level of endogenous miR-101. The ectopic miR-101 was able to radiosensitize most NSCLC cells, except for the NSCLC cell lines that had a much higher endogenous miR-101 level. These results suggest that when we choose one miRNA as a therapeutic tool, the endogenous level of the miRNA in each tumor should be considered.

  13. Bystander normal human fibroblasts reduce damage response in radiation targeted cancer cells through intercellular ROS level modulation

    Energy Technology Data Exchange (ETDEWEB)

    Widel, Maria, E-mail: maria.widel@polsl.pl [Biosystem Group, Department of Automatics, Electronics and Informatics, Silesian University of Technology, 16 Akademicka Street, 44-100 Gliwice (Poland); Przybyszewski, Waldemar M., E-mail: wmp@io.gliwice.pl [Center for Translational Research and Molecular Biology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Branch Gliwice, 15 Wybrzeze Armii Krajowej, 44-101 Gliwice (Poland); Cieslar-Pobuda, Artur [Biosystem Group, Department of Automatics, Electronics and Informatics, Silesian University of Technology, 16 Akademicka Street, 44-100 Gliwice (Poland); Saenko, Yuri V. [Department of Pharmacology and Biochemistry, Center of Nanotechnology and Materials, Ulyanovsk State University (Russian Federation); Rzeszowska-Wolny, Joanna [Biosystem Group, Department of Automatics, Electronics and Informatics, Silesian University of Technology, 16 Akademicka Street, 44-100 Gliwice (Poland)

    2012-03-01

    The radiation-induced bystander effect is a well-established phenomenon which results in damage in non-irradiated cells in response to signaling from irradiated cells. Since communication between irradiated and bystander cells could be reciprocal, we examined the mutual bystander response between irradiated cells and co-cultured with them non-irradiated recipients. Using a transwell culture system, irradiated human melanoma (Me45) cells were co-cultured with non-irradiated Me45 cells or normal human dermal fibroblasts (NHDF) and vice versa. The frequency of micronuclei and of apoptosis, ROS level, and mitochondrial membrane potential were used as the endpoints. Irradiated Me45 and NHDF cells induced conventional bystander effects detected as modest increases of the frequency of micronuclei and apoptosis in both recipient neighbors; the increase of apoptosis was especially high in NHDF cells co-cultured with irradiated Me45 cells. However, the frequencies of micronuclei and apoptosis in irradiated Me45 cells co-cultured with NHDF cells were significantly reduced in comparison with those cultured alone. This protective effect was not observed when irradiated melanomas were co-cultured with non-irradiated cells of the same line, or when irradiated NHDF fibroblasts were co-cultured with bystander melanomas. The increase of micronuclei and apoptosis in irradiated Me45 cells was paralleled by an increase in the level of intracellular reactive oxygen species (ROS), which was reduced significantly when they were co-cultured for 24 h with NHDF cells. A small but significant elevation of ROS level in NHDF cells shortly after irradiation was also reduced by co-culture with non-irradiated NHDF cells. We propose that in response to signals from irradiated cells, non-irradiated NHDF cells trigger rescue signals, whose nature remains to be elucidated, which modify the redox status in irradiated cells. This inverse bystander effect may potentially have implications in clinical

  14. HDAC inhibition induces increased choline uptake and elevated phosphocholine levels in MCF7 breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Christopher S Ward

    Full Text Available Histone deacetylase (HDAC inhibitors have emerged as effective antineoplastic agents in the clinic. Studies from our lab and others have reported that magnetic resonance spectroscopy (MRS-detectable phosphocholine (PC is elevated following SAHA treatment, providing a potential noninvasive biomarker of response. Typically, elevated PC is associated with cancer while a decrease in PC accompanies response to antineoplastic treatment. The goal of this study was therefore to elucidate the underlying biochemical mechanism by which HDAC inhibition leads to elevated PC. We investigated the effect of SAHA on MCF-7 breast cancer cells using (13C MRS to monitor [1,2-(13C] choline uptake and phosphorylation to PC. We found that PC synthesis was significantly higher in treated cells, representing 154±19% of control. This was within standard deviation of the increase in total PC levels detected by (31P MRS (129±7% of control. Furthermore, cellular choline kinase activity was elevated (177±31%, while cytidylyltransferase activity was unchanged. Expression of the intermediate-affinity choline transporter SLC44A1 and choline kinase α increased (144% and 161%, respectively relative to control, as determined by mRNA microarray analysis with protein-level confirmation by Western blotting. Taken together, our findings indicate that the increase in PC levels following SAHA treatment results from its elevated synthesis. Additionally, the concentration of glycerophosphocholine (GPC increased significantly with treatment to 210±45%. This is likely due to the upregulated expression of several phospholipase A2 (PLA2 isoforms, resulting in increased PLA2 activity (162±18% in SAHA-treated cells. Importantly, the levels of total choline (tCho-containing metabolites, comprised of choline, PC and GPC, are readily detectable clinically using (1H MRS. Our findings thus provide an important step in validating clinically translatable non-invasive imaging methods for follow

  15. S100 protein expression in human melanoma cells: Comparison of levels of expression among different cell lines and individual cells in different phases of the cell cycle

    Energy Technology Data Exchange (ETDEWEB)

    Marks, A.; O' Hanlon, D.; Dunn, R. (Univ. of Toronto, Ontario (Canada)); Petsche, D.; Baumal, R. (Hospital for Sick Children, Toronto, Ontario (Canada)); Kwong, P.C.; Stead, R. (McMaster Univ., Hamilton, Ontario (Canada)); Liao, S.K. (McMaster Univ., Hamilton, Ontario (Canada) Biotherapeutics, Inc., Franklin, TN (United States))

    1990-03-01

    The synthesis of S100 protein in cultured human melanoma cells was examined using metabolic labeling with ({sup 35}S)methionine, immunoprecipitation with anti-S100 protein antiserum, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Six of seven cell lines derived from melanomas synthesized relatively large amounts of S100 protein, whereas three cell lines derived from normal melanocytes synthesized lesser amounts. Synthesis of S100 protein was not detected in 10 human cell lines of non-neuroectodermal origin. Analysis of poly(A{sup +}) RNA form one melanoma cell line by Northern blot hybridization with a probe specific for the {beta} subunit of rat S100 protein revealed a single mRNA species of 1.0 kb coding for the human protein. Flow cytometric analysis of individual cells of two melanoma cell lines and the rat glioma cell line C6 indicated that G0/G1 cells were heterogeneous with respect to S100 protein expression, while almost all the cells in S+G2+M expressed S100 protein. These results suggest that expression of S100 protein in G0/G1 could be a prerequisite for progression of the cells through the cell cycle.

  16. Constructing disease-specific gene networks using pair-wise relevance metric: application to colon cancer identifies interleukin 8, desmin and enolase 1 as the central elements.

    Science.gov (United States)

    Jiang, Wei; Li, Xia; Rao, Shaoqi; Wang, Lihong; Du, Lei; Li, Chuanxing; Wu, Chao; Wang, Hongzhi; Wang, Yadong; Yang, Baofeng

    2008-08-10

    With the advance of large-scale omics technologies, it is now feasible to reversely engineer the underlying genetic networks that describe the complex interplays of molecular elements that lead to complex diseases. Current networking approaches are mainly focusing on building genetic networks at large without probing the interaction mechanisms specific to a physiological or disease condition. The aim of this study was thus to develop such a novel networking approach based on the relevance concept, which is ideal to reveal integrative effects of multiple genes in the underlying genetic circuit for complex diseases. The approach started with identification of multiple disease pathways, called a gene forest, in which the genes extracted from the decision forest constructed by supervised learning of the genome-wide transcriptional profiles for patients and normal samples. Based on the newly identified disease mechanisms, a novel pair-wise relevance metric, adjusted frequency value, was used to define the degree of genetic relationship between two molecular determinants. We applied the proposed method to analyze a publicly available microarray dataset for colon cancer. The results demonstrated that the colon cancer-specific gene network captured the most important genetic interactions in several cellular processes, such as proliferation, apoptosis, differentiation, mitogenesis and immunity, which are known to be pivotal for tumourigenesis. Further analysis of the topological architecture of the network identified three known hub cancer genes [interleukin 8 (IL8) (p approximately 0), desmin (DES) (p = 2.71 x 10(-6)) and enolase 1 (ENO1) (p = 4.19 x 10(-5))], while two novel hub genes [RNA binding motif protein 9 (RBM9) (p = 1.50 x 10(-4)) and ribosomal protein L30 (RPL30) (p = 1.50 x 10(-4))] may define new central elements in the gene network specific to colon cancer. Gene Ontology (GO) based analysis of the colon cancer-specific gene network and the sub-network that

  17. Constructing disease-specific gene networks using pair-wise relevance metric: Application to colon cancer identifies interleukin 8, desmin and enolase 1 as the central elements

    Directory of Open Access Journals (Sweden)

    Jiang Wei

    2008-08-01

    Full Text Available Abstract Background With the advance of large-scale omics technologies, it is now feasible to reversely engineer the underlying genetic networks that describe the complex interplays of molecular elements that lead to complex diseases. Current networking approaches are mainly focusing on building genetic networks at large without probing the interaction mechanisms specific to a physiological or disease condition. The aim of this study was thus to develop such a novel networking approach based on the relevance concept, which is ideal to reveal integrative effects of multiple genes in the underlying genetic circuit for complex diseases. Results The approach started with identification of multiple disease pathways, called a gene forest, in which the genes extracted from the decision forest constructed by supervised learning of the genome-wide transcriptional profiles for patients and normal samples. Based on the newly identified disease mechanisms, a novel pair-wise relevance metric, adjusted frequency value, was used to define the degree of genetic relationship between two molecular determinants. We applied the proposed method to analyze a publicly available microarray dataset for colon cancer. The results demonstrated that the colon cancer-specific gene network captured the most important genetic interactions in several cellular processes, such as proliferation, apoptosis, differentiation, mitogenesis and immunity, which are known to be pivotal for tumourigenesis. Further analysis of the topological architecture of the network identified three known hub cancer genes [interleukin 8 (IL8 (p ≈ 0, desmin (DES (p = 2.71 × 10-6 and enolase 1 (ENO1 (p = 4.19 × 10-5], while two novel hub genes [RNA binding motif protein 9 (RBM9 (p = 1.50 × 10-4 and ribosomal protein L30 (RPL30 (p = 1.50 × 10-4] may define new central elements in the gene network specific to colon cancer. Gene Ontology (GO based analysis of the colon cancer-specific gene network and

  18. Deep level study of cadmium sulfide/cadmium telluride solar cells

    Science.gov (United States)

    Komin, Valery Vitalyevich

    The goal of this project is to develop a reliable approach to characterize defects in CdTe/CdS solar cells, taking into account the requirements dictated by the polycrystallinity of the device materials, to characterize solar cells prepared in different processing conditions, to develop an understanding on how processing variations correlate with changes in the overall device performance and stability, and to develop recommendations on the optimization of processing conditions based on this analysis. Polycrystalline CdTe/CdS solar cells were studied using double boxcar deep level transient spectroscopy (DLTS) and correlation DLTS measurements to investigate the effect of the post-growth CdCl2 heat treatment, and the effect of the HgTe:Cu-doped graphite and Ni2P-based back contacts. The limitations of each deep-level characterization technique dictated by the polycrystalline nature of the CdTe/CdS heterostructure, were taken in consideration. Dark C-V, dark and illuminated J-V measurements were performed in order to monitor changes in the solar cell parameters during the deep level studies. To avoid issues associated with the metastability of some defects, all experiments were performed in the dark. Twelve traps were detected in the temperature range from 90K to 360K. Preliminary solar cell simulations using AMPS-1D were used to help understand the role of these traps in the current transport mechanism and the impact of their presence on the overall device performance. A donor-like defect with activation energy EA = 0.140 eV was identified as a chlorine-related DX2-state of (VCd2--ClTe+) complex. Its presence in the CdTe layer was associated with significant degradation of overall solar cell performance. An acceptor-like defect with activation energy EA = 0.350 was identified as a defect Cu Cd- or complex (Cui+-2Cu Cd-)-. Recommendations for processing condition optimization are made based on the results of this study.

  19. Function of Metallothionein-3 in Neuronal Cells: Do Metal Ions Alter Expression Levels of MT3?

    Science.gov (United States)

    Bousleiman, Jamie; Pinsky, Alexa; Ki, Sohee; Su, Angela; Morozova, Irina; Kalachikov, Sergey; Wiqas, Amen; Silver, Rae; Sever, Mary; Austin, Rachel Narehood

    2017-01-01

    A study of factors proposed to affect metallothionein-3 (MT3) function was carried out to elucidate the opaque role MT3 plays in human metalloneurochemistry. Gene expression of Mt2 and Mt3 was examined in tissues extracted from the dentate gyrus of mouse brains and in human neuronal cell cultures. The whole-genome gene expression analysis identified significant variations in the mRNA levels of genes associated with zinc homeostasis, including Mt2 and Mt3. Mt3 was found to be the most differentially expressed gene in the identified groups, pointing to the existence of a factor, not yet identified, that differentially controls Mt3 expression. To examine the expression of the human metallothioneins in neurons, mRNA levels of MT3 and MT2 were compared in BE(2)C and SH-SY5Y cell cultures treated with lead, zinc, cobalt, and lithium. MT2 was highly upregulated by Zn2+ in both cell cultures, while MT3 was not affected, and no other metal had an effect on either MT2 or MT3. PMID:28587098

  20. Kaempferol increases levels of coenzyme Q in kidney cells and serves as a biosynthetic ring precursor.

    Science.gov (United States)

    Fernández-Del-Río, Lucía; Nag, Anish; Gutiérrez Casado, Elena; Ariza, Julia; Awad, Agape M; Joseph, Akil I; Kwon, Ohyun; Verdin, Eric; de Cabo, Rafael; Schneider, Claus; Torres, Jorge Z; Burón, María I; Clarke, Catherine F; Villalba, José M

    2017-09-01

    Coenzyme Q (Q) is a lipid-soluble antioxidant essential in cellular physiology. Patients with Q deficiencies, with few exceptions, seldom respond to treatment. Current therapies rely on dietary supplementation with Q 10 , but due to its highly lipophilic nature, Q 10 is difficult to absorb by tissues and cells. Plant polyphenols, present in the human diet, are redox active and modulate numerous cellular pathways. In the present study, we tested whether treatment with polyphenols affected the content or biosynthesis of Q. Mouse kidney proximal tubule epithelial (Tkpts) cells and human embryonic kidney cells 293 (HEK 293) were treated with several types of polyphenols, and kaempferol produced the largest increase in Q levels. Experiments with stable isotope 13 C-labeled kaempferol demonstrated a previously unrecognized role of kaempferol as an aromatic ring precursor in Q biosynthesis. Investigations of the structure-function relationship of related flavonols showed the importance of two hydroxyl groups, located at C3 of the C ring and C4' of the B ring, both present in kaempferol, as important determinants of kaempferol as a Q biosynthetic precursor. Concurrently, through a mechanism not related to the enhancement of Q biosynthesis, kaempferol also augmented mitochondrial localization of Sirt3. The role of kaempferol as a precursor that increases Q levels, combined with its ability to upregulate Sirt3, identify kaempferol as a potential candidate in the design of interventions aimed on increasing endogenous Q biosynthesis, particularly in kidney. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Circulating cell-free DNA levels increase variably following chorionic villus sampling.

    Science.gov (United States)

    Vora, Neeta L; Johnson, Kirby L; Peter, Inga; Tighiouart, Hocine; Ralston, Steven J; Craigo, Sabrina D; Bianchi, Diana W

    2010-04-01

    Cell-free fetal DNA (cffDNA) in maternal plasma results from degradation of fetal and/or placental cells. Our objective was to determine if chorionic villus sampling (CVS) causes increased release of fetal and/or maternal DNA. Fifty-two pregnant women were recruited prior to CVS, performed for clinical indications, at 10 5/7 to 13 2/7 weeks. Maternal blood was collected before and within 15 min after CVS. cffDNA was extracted from plasma. Real-time polymerase chain reaction (PCR) amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the Y chromosome sequence DYS1 were used as measures of total and fetal DNA, respectively. All samples were analyzed in triplicate without knowledge of fetal gender. Sensitivity of DYS1 detection in male fetuses was 100% (n = 30); specificity in female fetuses was 100% (n = 22). While a majority of women had > 50% post-procedure increases in both fetal and total DNA, some showed post-procedure decreases. However, overall median proportional increases were not statistically significant. Gestational age (GA), placental location, and individual CVS operator did not correlate with changes in DNA levels. While there were no statistically significant overall changes in DNA levels after CVS, as-yet undiscovered variables may influence the extent of post-procedure release of cell-free DNA in the circulation of pregnant women. Copyright (c) 2010 John Wiley & Sons, Ltd.

  2. Cystatins in non-small cell lung cancer: tissue levels, localization and relation to prognosis.

    Science.gov (United States)

    Werle, Bernd; Schanzenbächer, Ulrike; Lah, Tamara Turensek; Ebert, Eileen; Jülke, Britta; Ebert, Werner; Fiehn, Werner; Kayser, Klaus; Spiess, Eberhard; Abrahamson, Magnus; Kos, Janko

    2006-10-01

    Cystatins regulate tumour-associated cysteine proteases, however, their role in tumour progression is not clear yet. To assess their relevance in the progression of non-small cell lung cancer (NSCLC) the protein level, cysteine protease activity (CPI) and localization of type I (stefins A and B) and type II (C, E/M and F) cystatins were defined in tumours and control lung counterparts from 165 patients. The medians of CPI activity, stefins A and B were significantly greater in tumour than in lung tissue (2.1-fold, 1.7-fold, 1.2-fold, respectively, all pcystatin C and cystatin E/M were lower in tumour tissue (0.9-fold, p=0.06; 0.6-fold, pcystatin F were below the detection limit. Immunohistochemical analysis revealed the presence of all cystatins in tumour cells and infiltrated inflammatory cells such as macrophages and neutrophils. In univariate survival analysis patients with high levels of stefin A, stefin B and CPI activity exhibited a better survival probability (p=0.05, p=0.05, pcystatins C and E/M provided no prognostic information. In multivariate analysis the most powerful predictor of survival was the pTNM stage (pcystatins, are up-regulated in lung tumours and thus able to counteract harmful tumour-associated proteolytic activity. As biological markers they may add independent prognostic information for better assessment of low- and high-risk patients with NSCLC.

  3. Effects of low-level laser therapy on stem cells from human exfoliated deciduous teeth

    Directory of Open Access Journals (Sweden)

    Ana Paula FERNANDES

    Full Text Available ABSTRACT Low-Level Laser Therapy stimulates the proliferation of a variety of types of cells. However, very little is known about its effect on stem cells from human exfoliated deciduous teeth (SHED. Objective This study aimed to evaluate the influence of different laser therapy energy densities on SHED viability and proliferation. Material and Methods SHED were irradiated according to the groups: I (1.2 J/cm2 - 0.5 mW – 10 s, II (2.5 J/cm2 – 10 mW – 10 s, III (3.7 J/cm2 – 15 mW – 10 s, IV (5.0 J/cm2 – 20 mW – 10 s, V (6.2 J/cm2 – 25 mW – 10 s, and VI (not irradiated – control group. Cell viability was assessed 6 and 24 h after irradiation measuring the mitochondrial activity and using the Crystal Violet assay. Cell proliferation was assessed after 24, 48, and 72 h of irradiation by SRB assay. Results MTT assay demonstrated differences from 6 to 24 hours after irradiation. After 24 h, groups I and IV showed higher absorbance values than those of control group. Crystal Violet assay showed statistically differences in the absorbance rate from 6 to 24 h after irradiation for groups III and VI. At 24 h after irradiation, Group III absorbance rate was greater than that of groups I, II, and IV. Group VI absorbance rate was greater than that of groups I and IV. SRB assay showed that the group I had higher rates than those of groups II, III, V, and VI, at 24 h after irradiation. After 48 h, group I exhibited the greatest cell proliferation rate followed by groups III, V, and VI. After 72 h, group III exhibited the lowest cell proliferation rate than those of groups II, IV, and V. Conclusions The Low-Level Laser Therapy energy densities used in this study did not cause loss of cell viability and stimulated SHED proliferation within the parameters described in this study.

  4. Dengue viral RNA levels in peripheral blood mononuclear cells are associated with disease severity and preexisting dengue immune status.

    Directory of Open Access Journals (Sweden)

    Anon Srikiatkhachorn

    Full Text Available Infection with dengue viruses (DENV causes a wide range of manifestations from asymptomatic infection to a febrile illness called dengue fever (DF, to dengue hemorrhagic fever (DHF. The in vivo targets of DENV and the relation between the viral burden in these cells and disease severity are not known.The levels of positive and negative strand viral RNA in peripheral blood monocytes, T/NK cells, and B cells and in plasma of DF and DHF cases were measured by quantitative RT-PCR.Positive strand viral RNA was detected in monocytes, T/NK cells and B cells with the highest amounts found in B cells. Viral RNA levels in CD14+ cells and plasma were significantly higher in DHF compared to DF, and in cases with a secondary infection compared to those undergoing a primary infection. The distribution of viral RNA among cell subpopulations was similar in DF and DHF cases. Small amounts of negative strand RNA were found in a few cases only. The severity of plasma leakage correlated with viral RNA levels in plasma and in CD14+ cells.B cells were the principal cells containing DENV RNA in peripheral blood, but overall there was little active DENV RNA replication detectable in peripheral blood mononuclear cells (PBMC. Secondary infection and DHF were associated with higher viral burden in PBMC populations, especially CD14+ monocytes, suggesting that viral infection of these cells may be involved in disease pathogenesis.

  5. Fluorescent reporter signals, EGFP and DsRed, encoded in HIV-1 facilitate the detection of productively infected cells and cell-associated viral replication levels

    Directory of Open Access Journals (Sweden)

    Kazutaka eTerahara

    2012-01-01

    Full Text Available Flow cytometric analysis is a reliable and convenient method for investigating molecules at the single cell level. Previously, recombinant human immunodeficiency virus type 1 (HIV-1 strains were constructed that express a fluorescent reporter, either enhanced green fluorescent protein or DsRed, which allow the monitoring of HIV-1-infected cells by flow cytometry. The present study further investigated the potential of these recombinant viruses in terms of whether the HIV-1 fluorescent reporters would be helpful in evaluating viral replication based on fluorescence intensity. When primary CD4+ T cells were infected with recombinant viruses, the fluorescent reporter intensity measured by flow cytometry was associated with the level of CD4 downmodulation and Gag p24 expression in infected cells. Interestingly, some HIV-1-infected cells, in which CD4 was only moderately downmodulated, were reporter-positive but Gag p24-negative. Furthermore, when the activation status of primary CD4+ T cells was modulated by T cell receptor-mediated stimulation, we confirmed the preferential viral production upon strong stimulation and showed that the intensity of the fluorescent reporter within a proportion of HIV-1-infected cells was correlated with the viral replication level. These findings indicate that a fluorescent reporter encoded within HIV-1 is useful for the sensitive detection of productively-infected cells at different stages of infection and for evaluating cell-associated viral replication at the single cell level.

  6. Dynamic expression of the translational machinery during Bacillus subtilis life cycle at a single cell level.

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    Alex Rosenberg

    Full Text Available The ability of bacteria to responsively regulate the expression of translation components is crucial for rapid adaptation to fluctuating environments. Utilizing Bacillus subtilis (B. subtilis as a model organism, we followed the dynamics of the translational machinery at a single cell resolution during growth and differentiation. By comprehensive monitoring the activity of the major rrn promoters and ribosomal protein production, we revealed diverse dynamics between cells grown in rich and poor medium, with the most prominent dissimilarities exhibited during deep stationary phase. Further, the variability pattern of translational activity varied among the cells, being affected by nutrient availability. We have monitored for the first time translational dynamics during the developmental process of sporulation within the two distinct cellular compartments of forespore and mother-cell. Our study uncovers a transient forespore specific increase in expression of translational components. Finally, the contribution of each rrn promoter throughout the bacterium life cycle was found to be relatively constant, implying that differential expression is not the main purpose for the existence of multiple rrn genes. Instead, we propose that coordination of the rrn operons serves as a strategy to rapidly fine tune translational activities in a synchronized fashion to achieve an optimal translation level for a given condition.

  7. Development of a unit cell model for interim performance assessment of vitrified low level waste disposal

    International Nuclear Information System (INIS)

    Kline, N.W.

    1995-09-01

    The unit cell modeling approach has been developed and used in analysis of some design options for a vitrified low level waste disposal facility. The unit cell modeling approach is likely to be useful in interim performance assessment for the facility. The present unit cell model will probably need to be refitted in terms of some model parameters for the latter purpose. Two present disposal facility concepts differ in the length of a capillary barrier proposed to limit effective recharge through the top of the facility. Results of the study summarized herein suggest design of a capillary barrier which can reduce a recharge rate of 0.1 cm/yr by one or two orders of magnitude seems feasible for both concepts. A benchmark comparison of the unit cell model against a full facility model shows comparable predictive accuracy in less than one percent of the computer time. Results suggest that model parameters include capillary barrier performance, inter-canister spacing, rate of moisture withdrawal due to glass corrosion, contaminant inventory, and the well interceptor factor. It is also important that variations of waste form hydraulic parameters suggest that transport through the waste form is dominated by diffusion

  8. Caffeic Acid Phenethyl Ester Regulates PPAR’s Levels in Stem Cells-Derived Adipocytes

    Directory of Open Access Journals (Sweden)

    Luca Vanella

    2016-01-01

    Full Text Available Hypertrophic obesity inhibits activation of peroxisome proliferators-activated receptor gamma (PPARγ, considered the key mediator of the fully differentiated and insulin sensitive adipocyte phenotype. We examined the effects of Caffeic Acid Phenethyl Ester (Cape, isolated from propolis, a honeybee hive product, on Adipose Stem Cells (ASCs differentiation to the adipocyte lineage. Finally we tested the effects of Cape on insulin-resistant adipocytes. Quantification of Oil Red O-stained cells showed that lipid droplets decreased following Cape treatment as well as radical oxygen species formation. Additionally, exposure of ASC to high glucose levels decreased adiponectin and increased proinflammatory cytokines mRNA levels, which were reversed by Cape-mediated increase of insulin sensitivity. Cape treatment resulted in decreased triglycerides synthesis and increased beta-oxidation. Exposure of ASCs to Lipopolysaccharide (LPS induced a reduction of PPARγ, an increase of IL-6 levels associated with a well-known stimulation of lipolysis; Cape partially attenuated the LPS-mediated effects. These observations reveal the main role of PPARγ in the adipocyte function and during ASC differentiation. As there is now substantial interest in functional food and nutraceutical products, the observed therapeutic value of Cape in insulin-resistance related diseases should be taken into consideration.

  9. Interaction of Leptospira interrogans with Human Proteolytic Systems Enhances Dissemination through Endothelial Cells and Protease Levels

    Science.gov (United States)

    Vieira, Monica L.; Alvarez-Flores, Miryam P.; Kirchgatter, Karin; Romero, Eliete C.; Alves, Ivy J.; de Morais, Zenaide M.; Vasconcellos, Silvio A.; Chudzinski-Tavassi, Ana M.

    2013-01-01

    We have recently reported the ability of Leptospira to capture plasminogen (PLG) and generate plasmin (PLA) bound on the microbial surface in the presence of exogenous activators. In this work, we examined the effects of leptospiral PLG binding for active penetration through the endothelial cell barrier and activation. The results indicate that leptospires with PLG association or PLA activation have enhanced migration activity through human umbilical vein endothelial cell (HUVEC) monolayers compared with untreated bacteria. Leptospira cells coated with PLG were capable of stimulating the expression of PLG activators by HUVECs. Moreover, leptospires endowed with PLG or PLA promoted transcriptional upregulation matrix metalloprotease 9 (MMP-9). Serum samples from patients with confirmed leptospirosis showed higher levels of PLG activators and total MMP-9 than serum samples from normal (healthy) subjects. The highest level of PLG activators and total MMP-9 was detected with microscopic agglutination test (MAT)-negative serum samples, suggesting that this proteolytic activity stimulation occurs at the early stage of the disease. Furthermore, a gelatin zymography profile obtained for MMPs with serum samples from patients with leptospirosis appears to be specific to leptospiral infection because serum samples from patients with unrelated infectious diseases produced no similar degradation bands. Altogether, the data suggest that the Leptospira-associated PLG or PLA might represent a mechanism that contributes to bacterial penetration of endothelial cells through an activation cascade of events that enhances the proteolytic capability of the organism. To our knowledge, this is the first proteolytic activity associated with leptospiral pathogenesis described to date. PMID:23478319

  10. A low-level diode laser therapy reduces the lipopolysaccharide (LPS)-induced periodontal ligament cell inflammation

    International Nuclear Information System (INIS)

    Huang, T H; Chen, C C; Liu, S L; Lu, Y C; Kao, C T

    2014-01-01

    The purpose of this study was to investigate the cytologic effects of inflammatory periodontal ligament cells in vitro after low-level laser therapy. Human periodontal ligament cells were cultured, exposed to lipopolysaccharide and subjected to low-level laser treatment of 5 J cm −2 or 10 J cm −2 using a 920 nm diode laser. A periodontal ligament cell attachment was observed under a microscope, and the cell viability was quantified by a mitochondrial colorimetric assay. Lipopolysaccharide-treated periodontal ligament cells were irradiated with the low-level laser, and the expression levels of several inflammatory markers, iNOS, TNF-α and IL-1, and pErk kinase, were analyzed by reverse transcription polymerase chain reaction and western blot. The data were collected and analyzed by one-way analysis of variance; p < 0.05 indicated a statistically significant difference. The low-level laser treatment of periodontal ligament cells increased their ability to attach and survive. After irradiation, the expression levels of iNOS, TNF-α and IL-1 in lipopolysaccharide-exposed periodontal ligament cells decreased over time (p < 0.05). In periodontal ligament cells, low-level diode laser treatment increased the cells’ proliferative ability and decreased the expression of the examined inflammatory mediators. (letters)

  11. Unit cell modeling in support of interim performance assessment for low level tank waste disposal

    Energy Technology Data Exchange (ETDEWEB)

    Kline, N.W., Westinghouse Hanford

    1996-08-01

    A unit cell model is used to simulate the base analysis case and related sensitivity cases for the interim performance assessment of low level tank waste disposal. Simulation case results are summarized in terms of fractional contaminant release rates to the vadose zone and to the water table at the unconfined aquifer. Results suggest that the crushed glass water conditioning layer at the top of the facility and the chemical retardation pad at the bottom of the facility can be important components of the facility. Results also suggest that the release rates to the water table are dominated by the release rate from the waste form.

  12. Effects of low level radiation upon the hematopoietic stem cell: Implications for leukemogenesis

    International Nuclear Information System (INIS)

    Cronkite, E.P.; Bond, V.P.; Carsten, A.L.; Miller, M.E.; Bullis, J.E.; Inoue, T.; Yokohama City Univ.

    1987-01-01

    These studies have addressed firstly the effect of single small doses of X-rays upon murine hematopoietic stem cells to obtain a better estimate of the D q . It is small, of the order of 20 rad. Secondly, a dose fractionation schedule that does not kill or perturb the kinetcs of hemopoietic cell proliferation was sought in order to investigate the leukemogenic potential of low level radiation upon an unperturbed hemopoietic system. Doses used by others in past radiation leukemogenesis studies clearly perturb hemopoiesis and kill a detectable fraction of stem cells. The studies reported herein show that 1.25 rad every day decrease the CFU-S content of bone marrow by the time 80 rads are accumulated. Higher daily doses as used in published studies on radiation leukemogenesis produce greater effects. Studies on the effect of 0.5, 1.0, 2.0, and 3.0 rad 3 times per week are under way. Two rad 3 times per week produced a modest decrease in CFU-S content of bone marrow after an accumulation of 68 rad. With 3.0 rad 3 times per week an accumulation of 102 rad produced a significant decrease in CFU-S content of bone marrow. Dose fractionation at 0.5 and 1.0 rad 3 times per week has not produced a CFU-S depression after accumulation of 17 and 34 rad. Radiation leukemogenesis studies published to date have utilized single doses and chronic exposure schedules that probably have significantly perturbed the kinetcs of hematopoietic stem cells. Whether radiation will produce leukemia in animal models with dose schedules that do not perturb kinetics of hematopoietic stem cells remains to be seen. (orig.)

  13. Effects of low-level radiation upon the haematopoietic stem cell. Implications for leukaemogenesis

    International Nuclear Information System (INIS)

    Cronkite, E.P.; Bond, V.P.; Carsten, A.L.; Miller, M.E.; Bullis, J.E.

    1983-01-01

    These studies address first the effect of single small doses of X-rays upon murine haematopoietic stem cells to obtain a better estimate of the Dsub(q). It is small, of the order of 20 rad. Second, a dose fractionation schedule that does not kill or perturb the kinetics of haematopoietic cell proliferation was sought to investigate the leukaemogenic potential of low-level radiation upon an unperturbed haematopoietic system. Doses used by others in past radiation leukaemogenesis studies clearly perturb haematopoiesis and kill a detectable fraction of stem cells. The studies reported in the paper show that 1.25 rad every day decreases the CFU-S content of bone marrow by the time 80 rad are accumulated. Higher daily doses as used in published studies on radiation leukaemogenesis produce greater effects. Studies of the effect of 0.5, 1.0, 2.0 and 3.0 rad three times per week are under way. Two rad three times per week produce a modest decrease in CFU-S content of bone marrow after an accumulation of 68 rad. With 3.0 rad three times per week, an accumulation of 102 rad produces a significant decrease in CFU-S content of bone marrow. Dose fractionation at 0.5 and 1.0 rad three times per week has not produced a CFU-S depression after accumulation of 17 and 34 rad. Radiation leukaemogenesis studies published to date have used single doses and chronic exposure schedules that probably have significantly perturbed the kinetics of haematopoietic stem cells. Whether radiation will produce leukaemia in animal models with dose schedules that do not perturb the kinetics of haematopoietic stem cells remains to be seen. (author)

  14. High level expression, purification and activation of human dipeptidyl peptidase I from mammalian cells.

    Science.gov (United States)

    Yang, Wei; Xia, Wenjuan; Mao, Jingjing; Xu, Daqi; Chen, Jianhe; Feng, Shan; Wang, Jianhua; Li, Hua; Theisen, Claus Friis; Petersen, Jørn Meidahl; Thórólfsson, Matthías; Rasmussen, Hanne Benedicte; Junker, Flemming; Boel, Esper; Su, Jing

    2011-03-01

    Dipeptidyl peptidase I (DPPI) plays a crucial role in maturation of many regulatory peptides and has been suggested as a pharmaceutical target in several inflammatory diseases. It is also a useful processing enzyme for the generation of authentic protein products by catalyzing the removal of N-terminal fusion peptides. We used a robust transient transfection system in human embryonic kidney 293 cells to exploit expression and activation of DPPI from chicken, rat and man for the development of an industrial production process. The expression of human and rat DPPI was significantly higher in the human HEK293 cell line than that obtained with avian DPPI. A CHO K1SV stable cell line was selected as the optimal stable host system for production of human DPPI yielding expression levels higher than 1.5 g/L. The secreted pro-DPPI underwent auto-maturation during defined buffer conditions during the purification steps. Active human DPPI was purified with a three-step purification strategy employing: Butyl Sepharose 4 Fast Flow, Sephadex G-25 Medium and Q Sepharose Fast Flow chromatography. The final yield of active enzyme was approximately 1 g/L cell culture. The enzyme exhibited exopeptidase activity against both a dipeptide-p-nitroanilide substrate and N-terminally extended MEAE-hGH (Met-Glu-Ala-Glu-human growth hormone). In conclusion, an efficient production process for recombinant human DPPI has been developed including a highly efficient and stable CHO cell system and an efficient purification procedure, which is simple and easy to scale for industrial purposes. The present data facilitates not only industrial applications of DPPI as a processing enzyme, but also provides active enzyme useful in the identification of small molecule inhibitors. Copyright © 2010 Elsevier Inc. All rights reserved.

  15. Synergism between arsenite and proteasome inhibitor MG132 over cell death in myeloid leukaemic cells U937 and the induction of low levels of intracellular superoxide anion

    Energy Technology Data Exchange (ETDEWEB)

    Lombardo, Tomás [Laboratorio de Immunotoxicologia (LaITO), IDEHU-CONICET, Hospital de Clínicas, José de San Martín, Universidad de Buenos Aires (UBA), Buenos Aires (Argentina); Cavaliere, Victoria; Costantino, Susana N. [Laboratorio de Inmunología Tumoral (LIT), IDEHU-CONICET, Facultad de Farmacia y Bioquímica, UBA, Buenos Aires (Argentina); Kornblihtt, Laura [Servicio de Hematología, Hospital de Clínicas, José de San Martín (UBA), Buenos Aires (Argentina); Alvarez, Elida M. [Laboratorio de Inmunología Tumoral (LIT), IDEHU-CONICET, Facultad de Farmacia y Bioquímica, UBA, Buenos Aires (Argentina); Blanco, Guillermo A., E-mail: gblanco@ffyb.uba.ar [Laboratorio de Immunotoxicologia (LaITO), IDEHU-CONICET, Hospital de Clínicas, José de San Martín, Universidad de Buenos Aires (UBA), Buenos Aires (Argentina)

    2012-02-01

    Increased oxygen species production has often been cited as a mechanism determining synergism on cell death and growth inhibition effects of arsenic-combined drugs. However the net effect of drug combination may not be easily anticipated solely from available knowledge of drug-induced death mechanisms. We evaluated the combined effect of sodium arsenite with the proteasome inhibitor MG132, and the anti-leukaemic agent CAPE, on growth-inhibition and cell death effect in acute myeloid leukaemic cells U937 and Burkitt's lymphoma-derived Raji cells, by the Chou–Talalay method. In addition we explored the association of cytotoxic effect of drugs with changes in intracellular superoxide anion (O{sub 2}{sup −}) levels. Our results showed that combined arsenite + MG132 produced low levels of O{sub 2}{sup −} at 6 h and 24 h after exposure and were synergic on cell death induction in U937 cells over the whole dose range, although the combination was antagonistic on growth inhibition effect. Exposure to a constant non-cytotoxic dose of 80 μM hydrogen peroxide together with arsenite + MG132 changed synergism on cell death to antagonism at all effect levels while increasing O{sub 2}{sup −} levels. Arsenite + hydrogen peroxide also resulted in antagonism with increased O{sub 2}{sup −} levels in U937 cells. In Raji cells, arsenite + MG132 also produced low levels of O{sub 2}{sup −} at 6 h and 24 h but resulted in antagonism on cell death and growth inhibition. By contrast, the combination arsenite + CAPE showed high levels of O{sub 2}{sup −} production at 6 h and 24 h post exposure but resulted in antagonism over cell death and growth inhibition effects in U937 and Raji cells. We conclude that synergism between arsenite and MG132 in U937 cells is negatively associated to O{sub 2}{sup −} levels at early time points after exposure. -- Highlights: ► Arsenic combined cytotoxic and anti-proliferative effects by Chou–Talalay method. ► Cytotoxic effect

  16. Salivary Levels of ErbB2 and CEA in Oral Squamous Cell Carcinoma Patients.

    Science.gov (United States)

    Honarmand, Marieh Honarmand; Farhad-Mollashahi, Leila; Nakhaee, Alireza; Nehi, Masoume

    2016-01-01

    Oral squamous cell carcinoma (OSCC) is one of the ten most common causes of cancer death worldwide. Assessment of molecular changes can help detect and control lesions. The aim of this study was evaluation of salivary levels of ErbB2 and CEA in OSCC patients. In this case-control study, 27 OSCC patients and 26 healthy controls participated. After obtaining consent and filling out a questionnaire, unstimulated saliva samples were collected from people in the morning for measurement of the two markers using ELISA. Data were analyzed using Mann Whitney U test in SPSS 19 software with Ptumor marker in patients with OSCC must still be regarded as controversial and needs further studies to clarify any significance for early detection or screening. In contrast the salivary level of CEA may find application for early detection of patients.

  17. Variation of DNA damage levels in peripheral blood mononuclear cells isolated in different laboratories

    DEFF Research Database (Denmark)

    Godschalk, Roger W L; Ersson, Clara; Stępnik, Maciej

    2014-01-01

    collected in the same way and processed using the same blood isolation procedure. The inter-laboratory variation was the prominent contributor to the overall variation. The inter-laboratory coefficient of variation decreased for both DNA strand breaks (from 68 to 26%) and FPG sensitive sites (from 57 to 12......This study investigated the levels of DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG) sensitive sites, as assessed by the comet assay, in peripheral blood mononuclear cells (PBMC) from healthy women from five different countries in Europe. The laboratory in each country (referred......%) by standardisation of the primary comet assay endpoint with calibration curve samples. The level of DNA strand breaks in the samples from two of the centres (0.56-0.61 lesions/10(6) bp) was significantly higher compared with the other three centres (0.41-0.45 lesions/10(6) bp). In contrast, there was no difference...

  18. Genes adopt non-optimal codon usage to generate cell cycle-dependent oscillations in protein levels

    DEFF Research Database (Denmark)

    Frenkel-Morgenstern, Milana; Danon, Tamar; Christian, Thomas

    2012-01-01

    The cell cycle is a temporal program that regulates DNA synthesis and cell division. When we compared the codon usage of cell cycle-regulated genes with that of other genes, we discovered that there is a significant preference for non-optimal codons. Moreover, genes encoding proteins that cycle...... at the protein level exhibit non-optimal codon preferences. Remarkably, cell cycle-regulated genes expressed in different phases display different codon preferences. Here, we show empirically that transfer RNA (tRNA) expression is indeed highest in the G2 phase of the cell cycle, consistent with the non...... that non-optimal (wobbly) matching codons influence protein synthesis during the cell cycle. We describe a new mathematical model that shows how codon usage can give rise to cell-cycle regulation. In summary, our data indicate that cells exploit wobbling to generate cell cycle-dependent dynamics...

  19. Cells have sex chromosomes and circadian clocks: Implications for organismal level functions.

    Science.gov (United States)

    Silver, Rae

    2018-04-01

    A great number of stakeholders have a keen interest in issues surrounding sex differences. These participants in the discourse often use the same evidence to draw opposite conclusions, with implications for individuals and society as a whole. One part of the maelstrom and associated emotionality derives from confounds between the concepts of "sex" vs. "gender", even among professionals. Here, the oft-repeated point is made that evidence for gender differences can't be derived from the animal research, once the generally accepted conception of gender as a process unique to humans, is acknowledged. Nevertheless, considered at a more general level, the developmental and epigenetic mechanisms that give rise to differences in behavior among individuals and groups is exquisitely explored in animal studies but relatively poorly in research on humans. The focus on animal research here, starts with the fact that virtually each cell of the body has sex chromosomes (XX and XY), along with the intracellular genetic and cytoplasmic mechanisms associated with circadian (circa-about, dies-day) timing. The consequences of these sex×circadian interactions for physiology and behavior at cellular and higher levels of organization are considered in systems where compelling evidence is available. These include sex differences in the circadian timing system, the hypothalamic-pituitary-adrenal (HPA) axis, and in metabolism. The evidence highlights sex differences in cells throughout the body and thus has implications for higher level processes and systems such as sleep/wake patterns. In a more general sense, they point to mechanisms that could give rise to gender differences. In summary, the viewpoint presented here is that the circadian timing system can be used very elegantly to explore the contributions of genetic and hormonal sex differences on biological systems at many levels. Copyright © 2017. Published by Elsevier Inc.

  20. The level of circulating endothelial progenitor cells may be associated with the occurrence and recurrence of chronic subdural hematoma

    Directory of Open Access Journals (Sweden)

    Yan Song

    2013-01-01

    Full Text Available OBJECTIVES: The onset of chronic subdural hematoma may be associated with direct or indirect minor injuries to the head or a poorly repaired vascular injury. Endothelial progenitor cells happen to be one of the key factors involved in hemostasis and vascular repair. This study was designed to observe the levels of endothelial progenitor cells, white blood cells, platelets, and other indicators in the peripheral blood of patients diagnosed with chronic subdural hematoma to determine the possible relationship between the endothelial progenitor cells and the occurrence, development, and outcomes of chronic subdural hematoma. METHOD: We enrolled 30 patients with diagnosed chronic subdural hematoma by computer tomography scanning and operating procedure at Tianjin Medical University General Hospital from July 2009 to July 2011. Meanwhile, we collected 30 cases of peripheral blood samples from healthy volunteers over the age of 50. Approximately 2 ml of blood was taken from veins of the elbow to test the peripheral blood routine and coagulation function. The content of endothelial progenitor cells in peripheral blood mononuclear cells was determined by flow cytometry. RESULTS: The level of endothelial progenitor cells in peripheral blood was significantly lower in preoperational patients with chronic subdural hematomas than in controls. There were no significant differences between the two groups regarding the blood routine and coagulation function. However, the levels of circulating endothelial progenitor cells were significantly different between the recurrent group and the non-recurrent group. CONCLUSIONS: The level of circulating endothelial progenitor cells in chronic subdural hematoma patients was significantly lower than the level in healthy controls. Meanwhile, the level of endothelial progenitor cells in recurrent patients was significantly lower than the level in patients without recurrence. Endothelial progenitor cells may be related to the

  1. [A preliminary study on the autophagy level of human periodontal ligament cells regulated by nicotine].

    Science.gov (United States)

    Yang, Du; Shuai, Yuan; Zhifei, Zhou; Lizheng, Wu; Lulu, Wang; Xing'an, Wu; Xiaojing, Wang

    2017-04-01

    To explore the effect of nicotine on the autophagy level of human periodontal ligament cells (hPDLCs). Periodontal tissues collected from premolars for orthodontic treatment reasons were used to culture hPDLCs. Western blot analysis was performed to test the most optimal time and concentration of nicotine on the autophagy level of the hPDLCs. Transmission electron microscope and immunofluorescence observation were carried out to detect the form of autophagosomes and expression of autophagy related protein LC3 in hPDLCs under this optimal condition. Protein expression of LC3Ⅱ was up regulated with the 12 h nicotine stimulating. Besides that, the up regulation of the protein expression of LC3Ⅱ was concentration dependent and nicotine with a concentration of 1×10⁻⁵ mol·L⁻¹ was the most optimal condition. Transmission electron microscope and immunofluorescence observations indicated that nicotine would activate the autophagy level of hPDLCs by increasing the number of autophagosomes and up regulating the expression of autophagy related protein LC3. Nicotine could increase autophagy level of hPDLCs, thus affecting the occurrence and development of smoking related periodontitis.

  2. Acute coronary syndrome patients with depression have low blood cell membrane omega-3 fatty acid levels.

    Science.gov (United States)

    Amin, Alpesh A; Menon, Rishi A; Reid, Kimberly J; Harris, William S; Spertus, John A

    2008-10-01

    To determine the extent to which levels of membrane eicosapentaenoic (EPA)+docosahexaenoic acids (DHA) (the omega-3 index) were associated with depression in patients with acute coronary syndrome (ACS). Depression is associated with worse cardiovascular (CV) outcomes in patients with ACS. Reduced levels of blood cell membrane omega-3 (n-3) fatty acids (FAs), an emerging risk factor for both CV disease and depression, may help to explain the link between depression and adverse CV outcomes. We measured membrane FA composition in 759 patients with confirmed ACS. The analysis included not only EPA and DHA but also the n-6 FAs linoleic and arachidonic acids (LA and AA). Depressive symptoms were measured with the Patient Health Questionnaire-9 (PHQ). Multivariable linear regression was used to adjust for demographic and clinical characteristics. There was a significant inverse relationship between the n-3 index and depressive symptoms (PHQ) in the fully adjusted model (p = .034). For every 4.54% point rise in the n-3 index, there was a 1-point decline in depressive symptoms. In contrast to the n-3 FAs, membrane levels of the n-6 FAs LA and AA were not different between depressed and nondepressed ACS patients. We found an inverse relationship between the n-3 index and the prevalence of depressive symptoms in patients with ACS. Therefore, this study supports the hypothesis that reduced n-3 FA tissue levels are a common and potentially modifiable link between depression and adverse CV outcomes.

  3. Epalrestat increases intracellular glutathione levels in Schwann cells through transcription regulation

    Directory of Open Access Journals (Sweden)

    Keisuke Sato

    2014-01-01

    Full Text Available Epalrestat (EPS, approved in Japan, is the only aldose reductase inhibitor that is currently available for the treatment of diabetic neuropathy. Here we report that EPS at near-plasma concentration increases the intracellular levels of glutathione (GSH, which is important for protection against oxidative injury, through transcription regulation. Treatment of Schwann cells with EPS caused a dramatic increase in intracellular GSH levels. EPS increased the mRNA levels of γ-glutamylcysteine synthetase (γ-GCS, the enzyme catalyzing the first and rate-limiting step in de novo GSH synthesis. Nuclear factor erythroid 2-related factor 2 (Nrf2 is a key transcription factor that plays a central role in regulating the expression of γ-GCS. ELISA revealed that EPS increased nuclear Nrf2 levels. Knockdown of Nrf2 by siRNA suppressed the EPS-induced GSH biosynthesis. Furthermore, pretreatment with EPS reduced the cytotoxicity induced by H2O2, tert-butylhydroperoxide, 2,2'-azobis (2-amidinopropane dihydrochloride, and menadione, indicating that EPS plays a role in protecting against oxidative stress. This is the first study to show that EPS induces GSH biosynthesis via the activation of Nrf2. We suggest that EPS has new beneficial properties that may prevent the development and progression of disorders caused by oxidative stress.

  4. Rhodanine dyes for dye-sensitized solar cells : spectroscopy, energy levels and photovoltaic performance.

    Science.gov (United States)

    Marinado, Tannia; Hagberg, Daniel P; Hedlund, Maria; Edvinsson, Tomas; Johansson, Erik M J; Boschloo, Gerrit; Rensmo, Håkan; Brinck, Tore; Sun, Licheng; Hagfeldt, Anders

    2009-01-07

    Three new sensitizers for photoelectrochemical solar cells were synthesized consisting of a triphenylamine donor, a rhodanine-3-acetic acid acceptor and a polyene connection. The conjugation length was systematically increased, which resulted in two effects: first, it led to a red-shift of the optical absorption of the dyes, resulting in an improved spectral overlap with the solar spectrum. Secondly, the oxidation potential decreased systematically. The excited state levels were, however, calculated to be nearly stationary. The experimental trends were in excellent agreement with density functional theory (DFT) computations. The photovoltaic performance of this set of dyes as sensitizers in mesoporous TiO2 solar cells was investigated using electrolytes containing the iodide/triiodide redox couple. The dye with the best absorption characteristics showed the poorest solar cell efficiency, due to losses by recombination of electrons in TiO2 with triiodide. Addition of 4-tert butylpyridine to the electrolyte led to a strongly reduced photocurrent for all dyes due to a reduced electron injection efficiency, caused by a 0.15 V negative shift of the TiO2 conduction band potential.

  5. The significance of serum leptin level in patients with early stage nonsmall cell lung cancer.

    Science.gov (United States)

    Karatas, Fatih; Yalcin, Bulent; Sahin, Suleyman; Akbulut, Hakan; Utkan, Gungor; Demirkazik, Ahmet; Icli, Fikri

    2017-01-01

    The serum leptin level (SLL) has been shown to increase in patients with nonsmall cell lung cancer (NSCLC). However, available data regarding the relation between SLL and tumor subtypes, survival, cachexia, and tumor respectability in NSCLC are still under debate. The aim of this study is to evaluate SLL in NSCLC patients with and without cachexia. A total of 71 patients with early stage NSCLC were enrolled in this prospective study. SLL was measured by enzyme-linked immunosorbent assay. The relationship between SLL and clinicopathological factors including histopathological subtypes, weight loss, overall survival, and tumor resectability were evaluated. Of the 71 patients, 57 (81%) were male with a mean age of 63.3 ± 8.2 years. The rates of histological subtypes of NSCLC were as follows: Squamous cell carcinoma 60.5%, adenocarcinoma 32%, and others 7.2%. Mean SLL was 12.9 ± 38.4 pmol/mL. There was no distinctive difference between SLL, weight loss, and survival. However, when stratifying the groups according to the lung cancer histological subtypes, mean SLL was significantly higher in patients with adenocarcinoma than those with squamous cell subtype (26.9 ± 6.2 pmol/mL vs. 5.1 ± 9.1 pmol/mL, P = 0.004). SLL might be beneficial as a useful biomarker in preclinical setting of NSCLC to guide detecting the lung cancer subtypes as well as monitoring the patients.

  6. Low-level (gallium-aluminum-arsenide) laser irradiation of Par-C10 cells and acinar cells of rat parotid gland.

    Science.gov (United States)

    Onizawa, Katsuhiro; Muramatsu, Takashi; Matsuki, Miwako; Ohta, Kazumasa; Matsuzaka, Kenichi; Oda, Yutaka; Shimono, Masaki

    2009-03-01

    We investigated cell response, including cell proliferation and expression of heat stress protein and bcl-2, to clarify the influence of low-level [gallium-aluminum-arsenide (Ga-Al-As) diode] laser irradiation on Par-C10 cells derived from the acinar cells of rat parotid glands. Furthermore, we also investigated amylase release and cell death from irradiation in acinar cells from rat parotid glands. The number of Par-C10 cells in the laser-irradiated groups was higher than that in the non-irradiated group at days 5 and 7, and the difference was statistically significant (P < 0.01). Greater expression of heat shock protein (HSP)25 and bcl-2 was seen on days 1 and 3 in the irradiated group. Assay of the released amylase showed no significant difference statistically between the irradiated group and the non-irradiated group. Trypan blue exclusion assay revealed that there was no difference in the ratio of dead to live cells between the irradiated and the non-irradiated groups. These results suggest that low-level laser irradiation promotes cell proliferation and expression of anti-apoptosis proteins in Par-C10 cells, but it does not significantly affect amylase secretion and does not induce rapid cell death in isolated acinar cells from rat parotid glands.

  7. DESTINY: A Comprehensive Tool with 3D and Multi-Level Cell Memory Modeling Capability

    Directory of Open Access Journals (Sweden)

    Sparsh Mittal

    2017-09-01

    Full Text Available To enable the design of large capacity memory structures, novel memory technologies such as non-volatile memory (NVM and novel fabrication approaches, e.g., 3D stacking and multi-level cell (MLC design have been explored. The existing modeling tools, however, cover only a few memory technologies, technology nodes and fabrication approaches. We present DESTINY, a tool for modeling 2D/3D memories designed using SRAM, resistive RAM (ReRAM, spin transfer torque RAM (STT-RAM, phase change RAM (PCM and embedded DRAM (eDRAM and 2D memories designed using spin orbit torque RAM (SOT-RAM, domain wall memory (DWM and Flash memory. In addition to single-level cell (SLC designs for all of these memories, DESTINY also supports modeling MLC designs for NVMs. We have extensively validated DESTINY against commercial and research prototypes of these memories. DESTINY is very useful for performing design-space exploration across several dimensions, such as optimizing for a target (e.g., latency, area or energy-delay product for a given memory technology, choosing the suitable memory technology or fabrication method (i.e., 2D v/s 3D for a given optimization target, etc. We believe that DESTINY will boost studies of next-generation memory architectures used in systems ranging from mobile devices to extreme-scale supercomputers. The latest source-code of DESTINY is available from the following git repository: https://bitbucket.org/sparshmittal/destinyv2.

  8. The phenotypic equilibrium of cancer cells: From average-level stability to path-wise convergence.

    Science.gov (United States)

    Niu, Yuanling; Wang, Yue; Zhou, Da

    2015-12-07

    The phenotypic equilibrium, i.e. heterogeneous population of cancer cells tending to a fixed equilibrium of phenotypic proportions, has received much attention in cancer biology very recently. In the previous literature, some theoretical models were used to predict the experimental phenomena of the phenotypic equilibrium, which were often explained by different concepts of stabilities of the models. Here we present a stochastic multi-phenotype branching model by integrating conventional cellular hierarchy with phenotypic plasticity mechanisms of cancer cells. Based on our model, it is shown that: (i) our model can serve as a framework to unify the previous models for the phenotypic equilibrium, and then harmonizes the different kinds of average-level stabilities proposed in these models; and (ii) path-wise convergence of our model provides a deeper understanding to the phenotypic equilibrium from stochastic point of view. That is, the emergence of the phenotypic equilibrium is rooted in the stochastic nature of (almost) every sample path, the average-level stability just follows from it by averaging stochastic samples. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Plasma Riboflavin Level is Associated with Risk, Relapse, and Survival of Esophageal Squamous Cell Carcinoma.

    Science.gov (United States)

    Li, Shan-Shan; Xu, Yi-Wei; Wu, Jian-Yi; Tan, Hua-Zhen; Wu, Zhi-Yong; Xue, Yu-Jie; Zhang, Jian-Jun; Li, En-Min; Xu, Li-Yan

    2017-01-01

    Riboflavin is an essential micronutrient for normal cellular activity, and deficiency may result in disease, such as cancer. We performed a case-control study to explore the association of riboflavin levels with risk and prognosis of esophageal squamous cell carcinoma (ESCC). Plasma riboflavin levels, as measured by enzyme-linked immunosorbent assay (ELISA), in ESCC patients were significantly lower than in those of healthy controls (7.04 ± 6.34 ng/ml vs. 9.32 ± 12.40 ng/ml; P riboflavin level and risk of ESCC (odds ratio (OR) = 0.97, 95% confidence interval (CI) = 0.95-0.99, P =  0.02). The 5-year relapse-free and overall survival rates were significantly lower when riboflavin levels were ≤0.8 ng/ml than >0.8 ng/ml (relapse-free survival rate: 29.4% vs. 54.8%; overall survival rate: 28.6% vs. 55.6%). Plasma riboflavin level was an independent protective factor for both relapse-free (hazard ratio (HR) = 0.325, 95% CI = 0.161-0.657, P = 0.002) and overall survival of ESCC patients (HR = 0.382, 95% CI = 0.190-0.768, P = 0.007). In conclusion, plasma riboflavin levels are significantly related to risk and prognosis of ESCC patients, suggesting that moderate supplementation of riboflavin will decrease risk and prevent recurrence of ESCC and also improve prognosis of ESCC patients.

  10. Inhibition of hyaluronan synthesis reduces versican and fibronectin levels in trabecular meshwork cells.

    Directory of Open Access Journals (Sweden)

    Kate E Keller

    Full Text Available Hyaluronan (HA is a major component of the extracellular matrix (ECM and is synthesized by three HA synthases (HAS. Similarities between the HAS2 knockout mouse and the hdf mutant mouse, which has a mutation in the versican gene, suggest that HA and versican expression may be linked. In this study, the relationship between HA synthesis and levels of versican, fibronectin and several other ECM components in trabecular meshwork cells from the anterior segment of the eye was investigated. HA synthesis was inhibited using 4-methylumbelliferone (4MU, or reduced by RNAi silencing of each individual HAS gene. Quantitative RT-PCR and immunoblotting demonstrated a reduction in mRNA and protein levels of versican and fibronectin. Hyaluronidase treatment also reduced versican and fibronectin levels. These effects could not be reversed by addition of excess glucose or glucosamine or exogenous HA to the culture medium. CD44, tenascin C and fibrillin-1 mRNA levels were reduced by 4MU treatment, but SPARC and CSPG6 mRNA levels were unaffected. Immunostaining of trabecular meshwork tissue after exposure to 4MU showed an altered localization pattern of HA-binding protein, versican and fibronectin. Reduction of versican by RNAi silencing did not affect HA concentration as assessed by ELISA. Together, these data imply that HA concentration affects synthesis of certain ECM components. Since precise regulation of the trabecular meshwork ECM composition and organization is required to maintain the aqueous humor outflow resistance and intraocular pressure homeostasis in the eye, coordinated coupling of HA levels and several of its ECM binding partners should facilitate this process.

  11. Live-Cell Imaging Tool Optimization To Study Gene Expression Levels and Dynamics in Single Cells of Bacillus cereus

    NARCIS (Netherlands)

    Eijlander, Robyn T.; Kuipers, Oscar P.

    Single-cell methods are a powerful application in microbial research to study the molecular mechanism underlying phenotypic heterogeneity and cell-to-cell variability. Here, we describe the optimization and application of single-cell time-lapse fluorescence microscopy for the food spoilage bacterium

  12. Effects of low-level radiation upon the hematopoietic steam cell: implications for leukemogenesis

    International Nuclear Information System (INIS)

    Cronkite, E.P.; Bond, V.P.; Carsten, A.L.; Miller, M.E.; Bullis, J.E.

    1983-01-01

    These studies have addressed firstly the effect of single small doses of x-ray upon murine hematopoietic stem cells to obtain a better estimate of the D/sub q/. It is small, of the order of 20 rads. Secondly, a dose fractionation schedule tht does not kill or perturb the kinetics of hemopoietic cell proliferation was sought in order to investigate the leukemogenic potential of low level radiation upon an unperturbed hemopoietic system. The studies reported herein show tht 1.25 rads every other day decrease the CFU-S content of bone marrow by the time 40 rads are accumulated. Studies on the effect of 0.5, 1.0, 2.0, and 3.0 rads 3 times per week are under way. Two rads 3 times per week produced a modest decrease in CFU-S content of bone marrow after an accumulation of 68 rads. With 3.0 rads 3 times per week an accumulation of 102 rads produces a significant decrease in CFU-S content of bone marrow. Dose fractionation at 0.5 and 1.0 rad 3 times per week has not produced a CFU-S depression after accumulation of 17 and 34 rads. Radiation leukemogenesis studies published to date have utilized single doses and chronic exposure schedules that probably have significantly perturbed the kinetics of hematopoietic stem cells. Whether radiation will produce leukemia in animal models with dose schedules that do not perturb kinetics of hematopoietic stem cells remains to be seen

  13. Intestinal epithelial cell caveolin 1 regulates fatty acid and lipoprotein cholesterol plasma levels.

    Science.gov (United States)

    Otis, Jessica P; Shen, Meng-Chieh; Quinlivan, Vanessa; Anderson, Jennifer L; Farber, Steven A

    2017-03-01

    Caveolae and their structural protein caveolin 1 (CAV1) have roles in cellular lipid processing and systemic lipid metabolism. Global deletion of CAV1 in mice results in insulin resistance and increases in atherogenic plasma lipids and cholesterol, but protects from diet-induced obesity and atherosclerosis. Despite the fundamental role of the intestinal epithelia in the regulation of dietary lipid processing and metabolism, the contributions of CAV1 to lipid metabolism in this tissue have never been directly investigated. In this study the cellular dynamics of intestinal Cav1 were visualized in zebrafish and the metabolic contributions of CAV1 were determined with mice lacking CAV1 in intestinal epithelial cells (CAV1 IEC-KO ). Live imaging of Cav1-GFP and fluorescently labeled caveolae cargos shows localization to the basolateral and lateral enterocyte plasma membrane (PM), suggesting Cav1 mediates transport between enterocytes and the submucosa. CAV1 IEC-KO mice are protected from the elevation in circulating fasted low-density lipoprotein (LDL) cholesterol associated with a high-fat diet (HFD), but have increased postprandial LDL cholesterol, total free fatty acids (FFAs), palmitoleic acid, and palmitic acid. The increase in circulating FAs in HFD CAV1 IEC-KO mice is mirrored by decreased hepatic FAs, suggesting a non-cell-autonomous role for intestinal epithelial cell CAV1 in promoting hepatic FA storage. In conclusion, CAV1 regulates circulating LDL cholesterol and several FA species via the basolateral PM of enterocytes. These results point to intestinal epithelial cell CAV1 as a potential therapeutic target to lower circulating FFAs and LDL cholesterol, as high levels are associated with development of type II diabetes and cardiovascular disease. © 2017. Published by The Company of Biologists Ltd.

  14. Functionalized silica nanoparticles: a platform for fluorescence imaging at the cell and small animal levels.

    Science.gov (United States)

    Wang, Kemin; He, Xiaoxiao; Yang, XiaoHai; Shi, Hui

    2013-07-16

    Going in vivo, including living cells and the whole body, is very important for gaining a better understanding of the mystery of life and requires specialized imaging techniques. The diversity, composition, and temporal-spatial variation of life activities from cells to the whole body require the analysis techniques to be fast-response, noninvasive, highly sensitive, and stable, in situ and in real-time. Functionalized nanoparticle-based fluorescence imaging techniques have the potential to meet such needs through real-time and noninvasive visualization of biological events in vivo. Functionalized silica nanoparticles (SiNPs) doped with fluorescent dyes appear to be an ideal and flexible platform for developing fluorescence imaging techniques used in living cells and the whole body. We can select and incorporate different dyes inside the silica matrix either noncovalently or covalently. These form the functionalized hybrid SiNPs, which support multiplex labeling and ratiometric sensing in living systems. Since the silica matrix protects dyes from outside quenching and degrading factors, this enhances the photostability and biocompatibility of the SiNP-based probes. This makes them ideal for real-time and long-time tracking. One nanoparticle can encapsulate large numbers of dye molecules, which amplifies their optical signal and temporal-spatial resolution response. Integrating fluorescent dye-doped SiNPs with targeting ligands using various surface modification techniques can greatly improve selective recognition. Along with the endocytosis, functionalized SiNPs can be efficiently internalized into cells for noninvasive localization, assessment, and monitoring. These unique characteristics of functionalized SiNPs substantially support their applications in fluorescence imaging in vivo. In this Account, we summarize our efforts to develop functionalized dye-doped SiNPs for fluorescence imaging at the cell and small animal levels. We first discuss how to design and

  15. Direct measurement of actual levels of nitric oxide (NO in cell culture conditions using soluble NO donors

    Directory of Open Access Journals (Sweden)

    Weilue He

    2016-10-01

    Full Text Available Applying soluble nitric oxide (NO donors is the most widely used method to expose cells of interest to exogenous NO. Because of the complex equilibria that exist between components in culture media, the donor compound and NO itself, it is very challenging to predict the dose and duration of NO cells actually experience. To determine the actual level of NO experienced by cells exposed to soluble NO donors, we developed the CellNO Trap, a device that allows continuous, real-time monitoring of the level of NO adherent cells produce and/or experience in culture without the need to alter cell culturing procedures. Herein, we directly measured the level of NO that cells grown in the CellNO Trap experienced when soluble NO donors were added to solutions in culture wells and we characterized environmental conditions that effected the level of NO in in vitro culture conditions. Specifically, the dose and duration of NO generated by the soluble donors S-nitroso-N-acetylpenicillamine (SNAP, S-nitrosoglutathione (GSNO, S-nitrosocysteine (CysNO and the diazeniumdiolate diethyltriamine (DETA/NO were investigated in both phosphate buffered saline (PBS and cell culture media. Other factors that were studied that potentially affect the ultimate NO level achieved with these donors included pH, presence of transition metals (ion species, redox level, presence of free thiol and relative volume of media. Then murine smooth muscle cell (MOVAS with different NO donors but with the same effective concentration of available NO were examined and it was demonstrated that the cell proliferation ratio observed does not correlate with the half-lives of NO donors characterized in PBS, but does correlate well with the real-time NO profiles measured under the actual culture conditions. This data demonstrates the dynamic characteristic of the NO and NO donor in different biological systems and clearly illustrates the importance of tracking individual NO profiles under the actual

  16. Fermentation temperature modulates phosphatidylethanolamine and phosphatidylinositol levels in the cell membrane of Saccharomyces cerevisiae.

    Science.gov (United States)

    Henderson, Clark M; Zeno, Wade F; Lerno, Larry A; Longo, Marjorie L; Block, David E

    2013-09-01

    During alcoholic fermentation, Saccharomyces cerevisiae is exposed to a host of environmental and physiological stresses. Extremes of fermentation temperature have previously been demonstrated to induce fermentation arrest under growth conditions that would otherwise result in complete sugar utilization at "normal" temperatures and nutrient levels. Fermentations were carried out at 15°C, 25°C, and 35°C in a defined high-sugar medium using three Saccharomyces cerevisiae strains with diverse fermentation characteristics. The lipid composition of these strains was analyzed at two fermentation stages, when ethanol levels were low early in stationary phase and in late stationary phase at high ethanol concentrations. Several lipids exhibited dramatic differences in membrane concentration in a temperature-dependent manner. Principal component analysis (PCA) was used as a tool to elucidate correlations between specific lipid species and fermentation temperature for each yeast strain. Fermentations carried out at 35°C exhibited very high concentrations of several phosphatidylinositol species, whereas at 15°C these yeast strains exhibited higher levels of phosphatidylethanolamine and phosphatidylcholine species with medium-chain fatty acids. Furthermore, membrane concentrations of ergosterol were highest in the yeast strain that experienced stuck fermentations at all three temperatures. Fluorescence anisotropy measurements of yeast cell membrane fluidity during fermentation were carried out using the lipophilic fluorophore diphenylhexatriene. These measurements demonstrate that the changes in the lipid composition of these yeast strains across the range of fermentation temperatures used in this study did not significantly affect cell membrane fluidity. However, the results from this study indicate that fermenting S. cerevisiae modulates its membrane lipid composition in a temperature-dependent manner.

  17. A systems-level approach for metabolic engineering of yeast cell factories.

    Science.gov (United States)

    Kim, Il-Kwon; Roldão, António; Siewers, Verena; Nielsen, Jens

    2012-03-01

    The generation of novel yeast cell factories for production of high-value industrial biotechnological products relies on three metabolic engineering principles: design, construction, and analysis. In the last two decades, strong efforts have been put on developing faster and more efficient strategies and/or technologies for each one of these principles. For design and construction, three major strategies are described in this review: (1) rational metabolic engineering; (2) inverse metabolic engineering; and (3) evolutionary strategies. Independent of the selected strategy, the process of designing yeast strains involves five decision points: (1) choice of product, (2) choice of chassis, (3) identification of target genes, (4) regulating the expression level of target genes, and (5) network balancing of the target genes. At the construction level, several molecular biology tools have been developed through the concept of synthetic biology and applied for the generation of novel, engineered yeast strains. For comprehensive and quantitative analysis of constructed strains, systems biology tools are commonly used and using a multi-omics approach. Key information about the biological system can be revealed, for example, identification of genetic regulatory mechanisms and competitive pathways, thereby assisting the in silico design of metabolic engineering strategies for improving strain performance. Examples on how systems and synthetic biology brought yeast metabolic engineering closer to industrial biotechnology are described in this review, and these examples should demonstrate the potential of a systems-level approach for fast and efficient generation of yeast cell factories. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  18. Transcript levels of several epigenome regulatory genes in bovine somatic donor cells are not correlated with their cloning efficiency.

    Science.gov (United States)

    Zhou, Wenli; Sadeghieh, Sanaz; Abruzzese, Ronald; Uppada, Subhadra; Meredith, Justin; Ohlrichs, Charletta; Broek, Diane; Polejaeva, Irina

    2009-09-01

    Among many factors that potentially affect somatic cell nuclear transfer (SCNT) embryo development is the donor cell itself. Cloning potentials of somatic donor cells vary greatly, possibly because the cells have different capacities to be reprogrammed by ooplasma. It is therefore intriguing to identify factors that regulate the reprogrammability of somatic donor cells. Gene expression analysis is a widely used tool to investigate underlying mechanisms of various phenotypes. In this study, we conducted a retrospective analysis investigating whether donor cell lines with distinct cloning efficiencies express different levels of genes involved in epigenetic reprogramming including histone deacetylase-1 (HDAC1), -2 (HDAC2); DNA methyltransferase-1 (DNMT1), -3a (DNMT3a),-3b (DNMT3b), and the bovine homolog of yeast sucrose nonfermenting-2 (SNF2L), a SWI/SNF family of ATPases. Cell samples from 12 bovine donor cell lines were collected at the time of nuclear transfer experiments and expression levels of the genes were measured using quantitative polymerase chain reaction (PCR). Our results show that there are no significant differences in expression levels of these genes between donor cell lines of high and low cloning efficiency defined as live calving rates, although inverse correlations are observed between in vitro embryo developmental rates and expression levels of HDAC2 and SNF2L. We also show that selection of stable reference genes is important for relative quantification, and different batches of cells can have different gene expression patterns. In summary, we demonstrate that expression levels of these epigenome regulatory genes in bovine donor cells are not correlated with cloning potential. The experimental design and data analysis method reported here can be applied to study any genes expressed in donor cells.

  19. Autistic Children Exhibit Decreased Levels of Essential Fatty Acids in Red Blood Cells

    Directory of Open Access Journals (Sweden)

    Sarah A. Brigandi

    2015-05-01

    Full Text Available Omega-6 (n-6 and omega-3 (n-3 polyunsaturated fatty acids (PUFA are essential nutrients for brain development and function. However, whether or not the levels of these fatty acids are altered in individuals with autism remains debatable. In this study, we compared the fatty acid contents between 121 autistic patients and 110 non-autistic, non-developmentally delayed controls, aged 3–17. Analysis of the fatty acid composition of red blood cell (RBC membrane phospholipids showed that the percentage of total PUFA was lower in autistic patients than in controls; levels of n-6 arachidonic acid (AA and n-3 docosahexaenoic acid (DHA were particularly decreased (p < 0.001. In addition, plasma levels of the pro-inflammatory AA metabolite prostaglandin E2 (PGE2 were higher in a subset of the autistic participants (n = 20 compared to controls. Our study demonstrates an alteration in the PUFA profile and increased production of a PUFA-derived metabolite in autistic patients, supporting the hypothesis that abnormal lipid metabolism is implicated in autism.

  20. Detection of TTV in peripheral blood cells from patients with altered ALT and AST levels.

    Science.gov (United States)

    de Oliveira, Jaqueline Carvalho; Nasser, Thiago Franco; Oda, Julie Massayo Maeda; Aoki, Mateus Nóbrega; Carneiro, Juliana Laino do Val; Barbosa, Décio Sabbatini; Reiche, Edna Maria Vissoci; Watanabe, Maria Angelica Ehara

    2008-04-01

    This work analyzes the prevalence of TTV DNA in peripheral blood cells from patients with hepatic alterations and healthy blood donors and measures levels of sodium, potassium, urea, creatinine, phosphatase alkaline, total and direct bilirubin, gamma glutamyl transferase (GGT), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in certain randomly selected patients. DNA samples from 111 individuals were evaluated. They were divided into two groups, "A" (study) and "B" (control), including 54 patients with liver enzyme alterations (ALT/AST) presenting non-B-non-C hepatitis and 57 blood donors, respectively. TTV DNA was determined by nested PCR. Certain products of the second-round PCR were sequenced. Serum biochemical assay was performed and disclosed TTV in 31.48% (17/54) of patients in group A and 5.26% (3/57) in the control group B. TTV prevalence was significantly higher in patients with liver disease than in healthy donors. In group A, sodium, potassium, urea, creatinine, phosphatase alkaline, total and direct bilirubin, gamma glutamyl transferase (GGT), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were analyzed in certain randomly selected patients and no significant difference in biochemical levels (p>0.05) was found when TTV infected and noninfected individuals were compared. Knowledge related to TTV has rapidly increased, but many fundamental aspects remain unclear. This led us to question the role of TTV and doubt remains as to whether or not it is just a commensal virus. Further studies are necessary to confirm and extend these findings.

  1. White blood cell counts, insulin resistance, vitamin D levels and sarcopenia in Korean elderly men.

    Science.gov (United States)

    Kim, Sang-Hwan; Kwon, Hyun Seok; Hwang, Hee-Jin

    2017-05-01

    Sarcopenia is a major determinant of frailty, disability and mortality in the elderly. Whether low-grade inflammation, insulin resistance and vitamin D are independently associated with sarcopenia remains unclear. In our study, sarcopenia was defined as an appendicular skeletal muscle mass divided by height squared (ASM/Ht 2 ) that was sarcopenia in Korean elderly men aged more than 65 years was 11.2%. ASM/Ht 2 were positively associated with vitamin D levels, but negatively associated with white blood cell counts and HOMA-IR by multiple regression analysis. After adjustment for covariables, sarcopenia was associated with the highest quartile of WBC counts (OR = 2.93, 95% CI = 1.21-7.14) and the highest quartile of serum vitamin D levels (OR = 0.38, 95% CI = 0.15-0.95). In conclusion, the study findings suggest that higher WBC counts and lower vitamin D levels are independently associated with the presence of sarcopenia in community-dwelling elderly men. They also provide a basis for further studies of the complex immune-endocrine network in sarcopenia.

  2. Vitamin d levels affect outcome in pediatric hematopoietic stem cell transplantation.

    Science.gov (United States)

    Hansson, Magnus E A; Norlin, Anna-Carin; Omazic, Brigitta; Wikström, Ann-Charlotte; Bergman, Peter; Winiarski, Jacek; Remberger, Mats; Sundin, Mikael

    2014-10-01

    The importance of vitamin D in immunologic processes has recently emerged, but whether it has any impact on the course of allogeneic hematopoietic stem cell transplantation (HSCT) has not been determined. Reports indicate that HSCT recipients, particularly children, often suffer from vitamin D deficiency. This study investigated the role of vitamin D in 123 children undergoing HSCT from 2004 to 2011. Vitamin D (ie, serum calcidiol) was analyzed in collected cryostored samples. Patients were grouped according to pre-HSCT calcidiol level: insufficient (disease occurred more frequently in the vitamin D sufficient group (47% versus 30%, P = .05), whereas no difference was demonstrated for chronic graft-versus-host disease. The neutrophil granulocytes rose significantly faster in the vitamin D sufficient group. No difference in lymphocyte counts, immunoglobulin levels, or infectious disease burden during the first year post-HSCT were observed. Among children with malignancies, overall survival was significantly better in the sufficient group (87% versus 50%, P = .01). In addition, rejection (0% versus 11%, P = .06) and relapse (4% versus 33%, P = .03) rates were lower in patients with sufficient vitamin D levels. To conclude, vitamin D may have an important impact on the outcome of pediatric HSCT, particularly in patients with malignant disease. Further studies investigating whether vitamin D acts as an immunomodulator or is merely a surrogate marker of patient health or nutritional status are warranted. Copyright © 2014 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

  3. Inverse association of leptin levels with renal cell carcinoma: results from a case-control study.

    Science.gov (United States)

    Spyridopoulos, Themistoklis N; Petridou, Eleni Th; Dessypris, Nick; Terzidis, Agapios; Skalkidou, Alkistis; Deliveliotis, Charalambos; Chrousos, George P

    2009-01-01

    Leptin is primarily produced in adipose tissue and appears to play a modulatory role between metabolism and immunity. Given that obesity, a state of chronic inflammation, is an established risk factor for Renal Cell Carcinoma (RCC), we investigated the association between plasma leptin levels and RCC risk. This case-control study included 70 patients with newly diagnosed, histologically confirmed RCC and 280 age-, gender- and district of residence-matched controls. Anthropometric data, socio-demographic variables, medical history, lifestyle habits and dietary data were derived from a personal interview. Serum leptin and adiponectin levels were determined using standard commercial kits. Adjusted odds ratios for RCC risk were derived through multiple logistic regression analyses. Leptin levels were inversely associated with RCC risk (OR: 0.53, CI: 0.28- 0.99, p = 0.05), even after controlling for potential confounding factors, such as Body Mass Index (BMI), recent weight change, history of diabetes mellitus and other obesity related hormones, notably adiponectin. The precise mechanism linking obesity with RCC remains unclear; however, the inverse association of leptin with RCC might be attributed, at least in part, to hormonal cross-talk with complex neuron-endocrine and immune circuits. These findings, if confirmed in prospective and interventional studies, might further elucidate the underlying mechanisms.

  4. SCA-1 Expression Level Identifies Quiescent Hematopoietic Stem and Progenitor Cells

    NARCIS (Netherlands)

    Morcos, Mina N.F.; Schoedel, Kristina B.; Hoppe, Anja; Behrendt, Rayk; Basak, Onur; Clevers, Hans C.; Roers, Axel; Gerbaulet, Alexander

    2017-01-01

    Blood cell generation depends on continuous cellular output by the sequential hierarchy of hematopoietic stem cell (HSC) and progenitor populations that all contain quiescent and actively cycling cells. Hematopoietic stem and progenitor cells (HSPCs) express the surface molecule Stem cell antigen 1

  5. Levels and function of regulatory T cells in patients with polymorphic light eruption: relation to photohardening.

    Science.gov (United States)

    Schweintzger, N; Gruber-Wackernagel, A; Reginato, E; Bambach, I; Quehenberger, F; Byrne, S N; Wolf, P

    2015-08-01

    We hypothesized that regulatory T cells (Tregs) are involved in the immunological abnormalities seen in patients with polymorphic light eruption (PLE). To investigate the number and suppressive function of peripheral Tregs in patients with PLE compared with healthy controls. Blood sampling was done in 30 patients with PLE [seeking or not seeking 311-nm ultraviolet (UV)B photohardening] as well as 19 healthy controls at two time points: TP1, March to June (before phototherapy); and TP2, May to August (after phototherapy). We compared the number of CD4(+) CD25(high) CD127(-) FoxP3(+) Tregs by flow cytometry and their function by assessing FoxP3 mRNA levels and effector T cell/Treg suppression assays. Tregs isolated from healthy controls significantly suppressed the proliferation of effector T cells at TP1 by 68% (P = 0·0156). In contrast, Tregs from patients with PLE entirely lacked the capacity to suppress effector T-cell proliferation at that time point. The medical photohardening seen in 23 patients with PLE resulted in a significant increase in the median percentage of circulating Tregs [both as a proportion of all lymphocytes; 65 6% increase (P = 0·0049), and as a proportion of CD4(+) T cells; 32.5% increase (P = 0·0049)]. This was accompanied by an increase in the expression of FoxP3 mRNA (P = 0·0083) and relative immunosuppressive function of Tregs (P = 0·083) comparing the two time points in representative subsets of patients with healthy controls tested. Seven patients with PLE not receiving 311-nm UVB also exhibited an increase in the number of Tregs but this was not statistically significant. No significant differences in Treg numbers were observed in healthy subjects between the two time points. An impaired Treg function is likely to play a role in PLE pathogenesis. A UV-induced increase in the number of Tregs (either naturally or therapeutically) may be a compensatory mechanism by which the immune system counteracts the susceptibility to

  6. Identifying long-term memory B-cells in vaccinated children despite waning antibody levels specific for Bordetella pertussis proteins

    NARCIS (Netherlands)

    Hendrikx, Lotte H.; Ozturk, Kemal; de Rond, Lia G. H.; Veenhoven, Reinier H.; Sanders, Elisabeth A. M.; Berbers, Guy A. M.; Buisman, Anne-Marie

    2011-01-01

    Whooping cough is a respiratory disease caused by Bordetella pertussis. Since the 1950s in developed countries pertussis vaccinations are included in the national immunization program. However, antibody levels rapidly wane after both whole cell and acellular pertussis vaccination. Therefore

  7. Nonlethal Levels of Zeaxanthin Inhibit Cell Migration, Invasion, and Secretion of MMP-2 via NF-κB Pathway in Cultured Human Uveal Melanoma Cells

    Directory of Open Access Journals (Sweden)

    Ming-Chao Bi

    2016-01-01

    Full Text Available Zeaxanthin at nonlethal dosages (3–10 μM significantly inhibited the cell migration of cultured uveal melanoma cells (C918 cell line as determined by wound healing assay and Boyden chamber assay. Matrigel invasion assay showed that cell invasion of uveal melanoma cells could be significantly inhibited by zeaxanthin. Secretion of MMP-2 by melanoma cells was significantly inhibited by zeaxanthin in a dose-dependent manner as measured by ELISA kit. Zeaxanthin also significantly inhibited the NF-κB levels in nuclear extracts of the UM cells, which is the upstream of the MMP-2 secretion. These results suggest that zeaxanthin might be a potentially therapeutic approach in the prevention of metastasis in uveal melanoma.

  8. Insulin induces an increase in cytosolic glucose levels in 3T3-L1 cells with inhibited glycogen synthase activation.

    Science.gov (United States)

    Chowdhury, Helena H; Kreft, Marko; Jensen, Jørgen; Zorec, Robert

    2014-10-02

    Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence resonance energy transfer-based glucose nanosensors has made it possible to measure intracellular glucose more accurately. Here, we used the fluorescent indicator protein (FLIPglu-600µ) to monitor cytosolic glucose dynamics in mouse 3T3-L1 cells in which glucose utilization for glycogen synthesis was inhibited. The results show that cells exhibit a low resting cytosolic glucose concentration. However, in cells with inhibited glycogen synthase activation, insulin induced a robust increase in cytosolic free glucose. The insulin-induced increase in cytosolic glucose in these cells is due to an imbalance between the glucose transported into the cytosol and the use of glucose in the cytosol. In untreated cells with sensitive glycogen synthase activation, insulin stimulation did not result in a change in the cytosolic glucose level. This is the first report of dynamic measurements of cytosolic glucose levels in cells devoid of the glycogen synthesis pathway.

  9. Does fetal gender affect cytotrophoblast cell activity in the human term placenta? Correlation with maternal hCG levels.

    Science.gov (United States)

    Gol, Mert; Tuna, Burçin; Dogan, Erbil; Gulekli, Bülent; Bagci, Mustafa; Altunyurt, Sabahattin; Saygili, Ugur

    2004-08-01

    Pregnant women with female fetuses have higher maternal serum human chorionic gonadotropin (hCG) levels than pregnant women with male fetuses. Ki-67, a cell proliferation and activity marker, is confined mostly in the nuclei of villous cytotrophoblasts of the human placenta. In this study, we examined the effect of fetal gender on the cytotrophoblast cell activity in human term placenta, with special regard to maternal serum and cord blood hCG levels. Thirty-four uncomplicated, singleton, term pregnancies (17 male and 17 female fetuses) were recruited in the study. hCG was measured in maternal peripheral serum and umbilical cord blood. Placental samples were collected in each patient during the cesarean section. Cytotrophoblast cell activity was measured by using immunohistochemistry for Ki-67 antigen. Ki-67 staining index values of the cytotrophoblasts were compared between the female and male placentas. Maternal serum and cord blood hCG levels were higher in pregnant women with female fetuses than in those carrying male fetuses. There was no sex difference in Ki-67 immunostaining rates of the cytotrophoblast cells. There was no correlation between maternal serum and cord blood hCG levels and Ki-67 staining index values of the cytotrophoblast cells. The difference in maternal serum and cord blood hCG levels in correlation with the fetal gender is not associated with cytotrophoblast cell activity in the human term placenta. The gender of the fetus does not seem to affect the regulation of cytotrophoblast cell proliferation.

  10. Single-cell level based approach to investigate acetate metabolism during batch industrial fermentation

    DEFF Research Database (Denmark)

    Nierychlo, Marta; Larsen, Poul; Eriksen, Niels T.

    accumulation causes the decrease of productivity as it represents a waste of carbon source that would otherwise be converted to biomass and product. As the acetate accumulation problem is of the utmost importance in batch fermentation processes, various strategies have been developed to explain, understand...... during glucose fermentation. Batch fermentations were performed in order to examine consecutive stages of acetate metabolism during the fermentation process (production, co-consumption with glucose, consumption as single substrate). Uptake of glucose and acetate at single-cell level was observed...... and control the overflow metabolism phenomenon in E. coli. Even though acetate formation by E. coli have been studied for more than three decades, the literature published presents the results based on the average measurement of the whole population. The averaged data can mask the distribution of the activity...

  11. Single-cell level based approach to investigate bacterial metabolism during batch industrial fermentation

    DEFF Research Database (Denmark)

    Nierychlo, Marta; Larsen, Poul; Eriksen, Niels T.

    , and performance of Escherichia coli. An insight into glucose and acetate fate on the level of individual cell can provide the type of information which are valuable for the understanding of bacterial metabolism in fermentation process and can shed more light on the differentiation of isogenic fermenting...... can exhibit different phenotypes under specific environmental conditions that show significant differences in physiological parameters from the population average. However, studies concerning segregation of populations into metabolically diversified subpopulations are scarce. Acetate is a product...... of Escherichia coli overflow metabolism when the bacteria are grown under aerobic conditions and glucose is present in excessive concentrations. Acetate accumulation is of the utmost importance in batch fermentation processes as it is an undesirable byproduct that negatively affects growth, physiology...

  12. Perovskite Solar Cells: From the Atomic Level to Film Quality and Device Performance.

    Science.gov (United States)

    Saliba, Michael; Correa-Baena, Juan-Pablo; Grätzel, Michael; Hagfeldt, Anders; Abate, Antonio

    2018-03-01

    Organic-inorganic perovskites have made tremendous progress in recent years due to exceptional material properties such as high panchromatic absorption, charge carrier diffusion lengths, and a sharp optical band edge. The combination of high-quality semiconductor performance with low-cost deposition techniques seems to be a match made in heaven, creating great excitement far beyond academic ivory towers. This is particularly true for perovskite solar cells (PSCs) that have shown unprecedented gains in efficiency and stability over a time span of just five years. Now there are serious efforts for commercialization with the hope that PSCs can make a major impact in generating inexpensive, sustainable solar electricity. In this Review, we will focus on perovskite material properties as well as on devices from the atomic to the thin film level to highlight the remaining challenges and to anticipate the future developments of PSCs. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Transcending the biomarker mindset: deciphering disease mechanisms at the single cell level.

    Science.gov (United States)

    Danna, Erika A; Nolan, Garry P

    2006-02-01

    The application of proteomics to disease research promises to enhance the understanding and treatment of many human maladies through the identification of molecular profiles associated with each disease. However, although much is made of the utility of molecular signatures as markers of disease state, insufficient emphasis is often placed on the simultaneous need for biological mechanism inquiry. Focused and detailed analyses of disease-associated signaling networks have the potential to be more mechanistically informative than large-scale proteomic profiling approaches, providing insight into the cellular processes involved in pathogenesis, disease progression and therapeutic resistance; while still providing diagnostic or clinical management direction. Phospho-specific flow cytometry provides a method for the analysis of pathological signaling networks, enabling the investigation of disease mechanisms at the single-cell level.

  14. Melanogenesis stimulation in B16-F10 melanoma cells induces cell cycle alterations, increased ROS levels and a differential expression of proteins as revealed by proteomic analysis

    Energy Technology Data Exchange (ETDEWEB)

    Cunha, Elizabeth S.; Kawahara, Rebeca [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil); Kadowaki, Marina K. [Universidade Estadual do Oeste do Parana, Cascavel, PR (Brazil); Amstalden, Hudson G.; Noleto, Guilhermina R.; Cadena, Silvia Maria S.C.; Winnischofer, Sheila M.B. [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil); Martinez, Glaucia R., E-mail: grmartinez@ufpr.br [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil)

    2012-09-10

    Considering that stimulation of melanogenesis may lead to alterations of cellular responses, besides melanin production, our main goal was to study the cellular effects of melanogenesis stimulation of B16-F10 melanoma cells. Our results show increased levels of the reactive oxygen species after 15 h of melanogenesis stimulation. Following 48 h of melanogenesis stimulation, proliferation was inhibited (by induction of cell cycle arrest in the G1 phase) and the expression levels of p21 mRNA were increased. In addition, melanogenesis stimulation did not induce cellular senescence. Proteomic analysis demonstrated the involvement of proteins from other pathways besides those related to the cell cycle, including protein disulfide isomerase A3, heat-shock protein 70, and fructose biphosphate aldolase A (all up-regulated), and lactate dehydrogenase (down-regulated). In RT-qPCR experiments, the levels of pyruvate kinase M2 mRNA dropped, whereas the levels of ATP synthase (beta-F1) mRNA increased. These data indicate that melanogenesis stimulation of B16-F10 cells leads to alterations in metabolism and cell cycle progression that may contribute to an induction of cell quiescence, which may provide a mechanism of resistance against cellular injury promoted by melanin synthesis. -- Highlights: Black-Right-Pointing-Pointer Melanogenesis stimulation by L-tyrosine+NH{sub 4}Cl in B16-F10 melanoma cells increases ROS levels. Black-Right-Pointing-Pointer Melanogenesis inhibits cell proliferation, and induced cell cycle arrest in the G1 phase. Black-Right-Pointing-Pointer Proteomic analysis showed alterations in proteins of the cell cycle and glucose metabolism. Black-Right-Pointing-Pointer RT-qPCR analysis confirmed alterations of metabolic targets after melanogenesis stimulation.

  15. Peripheral blood mononuclear cells HIV DNA levels impact intermittently on neurocognition.

    Directory of Open Access Journals (Sweden)

    Lucette A Cysique

    Full Text Available To determine the contribution of peripheral blood mononuclear cells' (PBMCs HIV DNA levels to HIV-associated dementia (HAD and non-demented HIV-associated neurocognitive disorders (HAND in chronically HIV-infected adults with long-term viral suppression on combined antiretroviral treatment (cART.Eighty adults with chronic HIV infection on cART (>97% with plasma and CSF HIV RNA <50 copies/mL were enrolled into a prospective observational cohort and underwent assessments of neurocognition and pre-morbid cognitive ability at two visits 18 months apart. HIV DNA in PBMCs was measured by real-time PCR at the same time-points.At baseline, 46% had non-demented HAND; 7.5% had HAD. Neurocognitive decline occurred in 14% and was more likely in those with HAD (p<.03. Low pre-morbid cognitive ability was uniquely associated with HAD (p<.05. Log10 HIV DNA copies were stable between study visits (2.26 vs. 2.22 per 106 PBMC. Baseline HIV DNA levels were higher in those with lower pre-morbid cognitive ability (p<.04, and higher in those with no ART treatment during HIV infection 1st year (p = .03. Baseline HIV DNA was not associated with overall neurocognition. However, % ln HIV DNA change was associated with decline in semantic fluency in unadjusted and adjusted analyses (p = .01-.03, and motor-coordination (p = .02-.12 to a lesser extent.PBMC HIV DNA plays a role in HAD pathogenesis, and this is moderated by pre-morbid cognitive ability in the context of long-term viral suppression. While the HIV DNA levels in PBMC are not associated with current non-demented HAND, increasing HIV DNA levels were associated with a decline in neurocognitive functions associated with HAND progression.

  16. The influence of 60Co gamma rays to cell reproduction (An experiment using low dose levels on vero and primary monkey kidney cells)

    International Nuclear Information System (INIS)

    Danusupadmo, C.J. Sugiarto

    1985-01-01

    Vero and primary monkey kidney cells in culture were gamma irradiated with doses of 0, 0.4 and 0.8 Gy at a dose-rate of 1.30-1.45x10 3 Gy/hour. At harvest time 3 days post irradiation, 0.4 Gy proved to be able to lower the number of vero cells in such a degree that it became significantly different from the control, whereas 0.8 Gy could not suppress the number of primary cells to a level that differed significantly from its control. At harvest time of 7 days post irradiation, 0.4 Gy was found effective in lowering both vero and primary cells so that the number of the harvested cells were significantly different from the controls. At harvest time of 3 days post irradiation, 0.8 Gy caused both cell types reached levels that were not significantly different from 0.4 Gy, but at 7 days post irradiation the number of vero cells was very significantly different from that of 0.4 Gy, while the number of primary cells remained equal to that of 0.4 Gy. This phenomenon showed that irradiation could cause greater injurious effect at more advanced post irradiation times, while the more proliferative vero cells proved to be more susceptible to irradiation than primary cells, but at the same time more potential in performing repair. (author)

  17. Real-time Recording of Cytosolic Calcium Levels in Arabidopsis thaliana Cell Cultures during Parabolic Flights

    Science.gov (United States)

    Neef, Maren; Ecke, Margret; Hampp, Rüdiger

    2015-07-01

    In plants, like in other organisms, calcium (Ca2+) is an important second messenger which participates in the conversion of environmental signals into molecular responses. There is increasing evidence, that sensing of changes in gravitation or reorientation of tissues is an example for such signaling cascades in which Ca2+ is involved. In order to determine g-dependent changes in the cytosolic calcium (Ca^{2+}_{ {cyt}}) concentration of plant cells, semisolid transgenic callus cell cultures of Arabidopsis thaliana (A.t.), expressing the calcium sensor YC3.6 (cameleon), were exposed to g-forces between 1.8 g and μ g during parabolic flights. Using such cells, intracellular calcium transients can be monitored by FRET in vivo and in real-time. Interestingly we observed a slight decrease of the Ca^{2+}_{ {cyt}} level during the hypergravity phases of a parabola but a significant increase of the Ca^{2+}_{ {cyt}} concentration during microgravity. Application of known Ca2+ inhibitors and antagonists yielded the following effects: nifedipine (Ca2+ channel blocker) showed no effect, whereas LaCl3, GdCl3 (both inhibitors of uptake at the plasma membrane), DPI (inhibitor of NADP oxidase), and DMSO (solvent) diminished the gravity-alteration-related Ca^{2+}_{ {cyt}} response. EGTA (binding of Ca2+) and eosin yellow (inhibitor of a plasma membrane-located Ca2+ pump) suppressed the respective Ca^{2+}_{ {cyt}} changes entirely. We thus conclude that the significant increase in Ca^{2+}_{ {cyt}} under microgravity is largely due to extracellular Ca2+ sources.

  18. Auxins differentially regulate root system architecture and cell cycle protein levels in maize seedlings.

    Science.gov (United States)

    Martínez-de la Cruz, Enrique; García-Ramírez, Elpidio; Vázquez-Ramos, Jorge M; Reyes de la Cruz, Homero; López-Bucio, José

    2015-03-15

    Maize (Zea mays) root system architecture has a complex organization, with adventitious and lateral roots determining its overall absorptive capacity. To generate basic information about the earlier stages of root development, we compared the post-embryonic growth of maize seedlings germinated in water-embedded cotton beds with that of plants obtained from embryonic axes cultivated in liquid medium. In addition, the effect of four different auxins, namely indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-D) on root architecture and levels of the heat shock protein HSP101 and the cell cycle proteins CKS1, CYCA1 and CDKA1 were analyzed. Our data show that during the first days after germination, maize seedlings develop several root types with a simultaneous and/or continuous growth. The post-embryonic root development started with the formation of the primary root (PR) and seminal scutellar roots (SSR) and then continued with the formation of adventitious crown roots (CR), brace roots (BR) and lateral roots (LR). Auxins affected root architecture in a dose-response fashion; whereas NAA and IBA mostly stimulated crown root formation, 2,4-D showed a strong repressing effect on growth. The levels of HSP101, CKS1, CYCA1 and CDKA in root and leaf tissues were differentially affected by auxins and interestingly, HSP101 registered an auxin-inducible and root specific expression pattern. Taken together, our results show the timing of early branching patterns of maize and indicate that auxins regulate root development likely through modulation of the HSP101 and cell cycle proteins. Copyright © 2014 Elsevier GmbH. All rights reserved.

  19. Elevated lead levels and adverse effects on natural killer cells in children from an electronic waste recycling area.

    Science.gov (United States)

    Zhang, Yu; Huo, Xia; Cao, Junjun; Yang, Tian; Xu, Long; Xu, Xijin

    2016-06-01

    Lead (Pb) has been proved to exert immunotoxicity to influence immune homeostasis in humans. To monitor the internal Pb level and evaluate its effect on natural killer (NK) cells and cytokine/chemokine concentrations, we recruited 285 preschool children from Guiyu, one of the largest electronic waste (e-waste) destinations and recycling areas in the world, and known to have high concentrations of Pb in the air, soil, water, sediment and plants. A total of 126 preschool children were selected from Haojiang as a reference group. Results showed that children in Guiyu, the exposed area, had higher blood Pb levels and lower percentages of NK cells than children from the reference area. A significantly negative association was found between the percentage of NK cells and increasing Pb levels. Moreover, children in Guiyu area had higher platelet counts and IL-1β concentrations, and lower levels of IL-2, IL-27, MIP-1α and MIP-1β were observed in the exposed children. These changes might not be conducive to the development and differentiation of NK cells. Taken together, the elevated Pb levels result in the lower percentages of NK cells, but also alter the levels of platelets, IL-1β and IL-27, which might be unconducive to the activity and function of NK cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Global levels of histone modifications in peripheral blood mononuclear cells of subjects with exposure to nickel.

    Science.gov (United States)

    Arita, Adriana; Niu, Jingping; Qu, Qingshan; Zhao, Najuan; Ruan, Ye; Nadas, Arthur; Chervona, Yana; Wu, Fen; Sun, Hong; Hayes, Richard B; Costa, Max

    2012-02-01

    Occupational exposure to nickel (Ni) is associated with an increased risk for lung and nasal cancers. Ni compounds exhibit weak mutagenic activity, cause gene amplification, and disrupt cellular epigenetic homeostasis. However, the Ni-induced changes in global histone modification levels have only been tested in vitro. This study was conducted in a Chinese population to determine whether occupational exposure to Ni is associated with alterations of global histone modification levels and to evaluate the inter- and intraindividual variance of global histone modification levels. Forty-five subjects with occupational exposure to Ni and 75 referents were recruited. Urinary Ni and global H3K4 trimethylation, H3K9 acetylation, and H3K9 dimethylation levels were measured in peripheral blood mononuclear cells (PBMCs) of subjects. H3K4me3 was elevated in Ni-exposed subjects (0.25% ± 0.11%) compared with referents (0.15% ± 0.04%; p = 0.0004), and H3K9me2 was decreased (Ni-exposed subjects, 0.11% ± 0.05%; referents, 0.15% ± 0.04%; p = 0.003). H3K4me3 was positively (r = 0.4, p = 0.0008) and H3K9ac was negatively (r = 0.1, p = 0.01) associated with urinary Ni. Interindividual variances of H3K4me3, H3K9ac, and H3K9me2 were larger compared with intraindividual variance in both exposure test groups, resulting in reliability coefficients (an estimate of consistency of a set of measurements) of 0.60, 0.67, and 0.79 for H3K4me3, H3K9ac, and H3K9me2, respectively, for Ni-exposed subjects and of 0.75, 0.74, and 0.97, respectively, for referent subjects. The results of this study indicate that occupational exposure to Ni is associated with alterations of global histone modification levels and that measurements of global levels of histone modifications are relatively stable over time in human PBMCs.

  1. Identifying long-term memory B-cells in vaccinated children despite waning antibody levels specific for Bordetella pertussis proteins.

    Science.gov (United States)

    Hendrikx, Lotte H; Oztürk, Kemal; de Rond, Lia G H; Veenhoven, Reinier H; Sanders, Elisabeth A M; Berbers, Guy A M; Buisman, Anne-Marie

    2011-02-04

    Whooping cough is a respiratory disease caused by Bordetella pertussis. Since the 1950s in developed countries pertussis vaccinations are included in the national immunization program. However, antibody levels rapidly wane after both whole cell and acellular pertussis vaccination. Therefore protection against pertussis may depend largely on long-term B- and T-cell immunities. We investigated long-term pertussis-specific memory B-cell responses in children who were primed at infant age with the Dutch wP-vaccine (ISRCTN65428640). Purified B-cells were characterized by FACS-analysis and after polyclonal stimulation memory B-cells were detected by ELISPOT-assays specific for pertussis toxin, filamentous haemagglutinin, pertactin and tetanus. In addition, plasma IgG levels directed to the same antigens were measured by a fluorescent bead-based multiplex immunoassay. Two and 3 years after wP priming as well as 2 and 5 years after the aP booster at the age of 4, low plasma IgG levels to the pertussis proteins were found. At the same time, however pertussis protein-specific memory B-cells could be detected and their number increased with age. The number of tetanus-specific memory B-cells was similar in all age groups, whereas IgG-tetanus levels were high 2 years after tetanus booster compared to pre- and 5 years post-booster levels. This study shows the presence of long-term pertussis protein-specific memory B-cells in children despite waning antibody levels after vaccination, which suggests that memory B-cells in addition to antibodies may contribute to protection against pertussis. Copyright © 2010 Elsevier Ltd. All rights reserved.

  2. Modulation of DNA methylation levels sensitizes doxorubicin-resistant breast adenocarcinoma cells to radiation-induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Luzhna, Lidia [Department of Biological Sciences, University of Lethbridge, AB, Canada T1K 3M4 (Canada); Kovalchuk, Olga, E-mail: olga.kovalchuk@uleth.ca [Department of Biological Sciences, University of Lethbridge, AB, Canada T1K 3M4 (Canada)

    2010-02-05

    Chemoresistant tumors often fail to respond to other cytotoxic treatments such as radiation therapy. The mechanisms of chemo- and radiotherapy cross resistance are not fully understood and are believed to be epigenetic in nature. We hypothesize that MCF-7 cells and their doxorubicin-resistant variant MCF-7/DOX cells may exhibit different responses to ionizing radiation due to their dissimilar epigenetic status. Similar to previous studies, we found that MCF-7/DOX cells harbor much lower levels of global DNA methylation than MCF-7 cells. Furthermore, we found that MCF-7/DOX cells had lower background apoptosis levels and were less responsive to radiation than MCF-7 cells. Decreased radiation responsiveness correlated to significant global DNA hypomethylation in MCF-7/DOX cells. Here, for the first time, we show that the radiation resistance of MCF-7/DOX cells can be reversed by an epigenetic treatment - the application of methyl-donor SAM. SAM-mediated reversal of DNA methylation led to elevated radiation sensitivity in MCF-7/DOX cells. Contrarily, application of SAM on the radiation sensitive and higher methylated MCF-7 cells resulted in a decrease in their radiation responsiveness. This data suggests that a fine balance of DNA methylation is needed to insure proper radiation and drug responsiveness.

  3. CD4+ T-cell counts and plasma HIV-1 RNA levels beyond 5 years of highly active antiretroviral therapy.

    Science.gov (United States)

    Li, Xiuhong; Margolick, Joseph B; Jamieson, Beth D; Rinaldo, Charles R; Phair, John P; Jacobson, Lisa P

    2011-08-15

    The heterogeneity of CD4 T-cell counts and HIV-1 RNA at 5-12 years after the initiation of highly active antiretroviral therapy (HAART) remains largely uncharacterized. In the Multicenter AIDS Cohort Study, 614 men who initiated HAART contributed data 5-12 years subsequently. Multivariate regression was used to evaluate the predictors of CD4 counts and HIV-1 RNA levels. At 5 to 12 years post-HAART, the median CD4 T-cell count was 586 (interquartile range, 421-791) cells per microliter and 78% of the HIV-1 RNA measurements were undetectable. Higher CD4 T-cell counts 5-12 years post HAART were predicted by higher CD4 T-cell counts and higher total lymphocyte count pre HAART, lack of hepatitis B or C virus coinfections, and greater CD4 T-cell change and suppressed HIV-1 RNA in the first 5 years after starting HAART. Men who were 50 years and older with 351-500 CD4 cells per microliter at HAART initiation had adjusted mean CD4 T-cell count of 643 cells per microliter at 10-12 years post HAART, which was similar to the adjusted mean CD4 T-cell count (670 cells/μL, P = 0.45) in this period for younger men starting HAART with lower CD4 T-cell counts. HIV-1 RNA suppression in the first 5 years post HAART predicted subsequent viral suppression. Immunological and virological responses in the first 5 years post HAART predicted subsequent CD4 T-cell counts and HIV-1 RNA levels. The association between age and subsequent CD4 T-cell count supports incorporating age in the guidelines for use of HAART.

  4. Low-level laser therapy vs. pulsed electromagnetic field on neonatal rat calvarial osteoblast-like cells.

    Science.gov (United States)

    Emes, Yusuf; Akça, Kivanç; Aybar, Buket; Yalçın, Serhat; Çavuşoğlu, Yeliz; Baysal, Uğur; Işsever, Halim; Atalay, Belir; Vural, Pervin; Ergüven, Mine; Çehreli, Murat Cavit; Bilir, Ayhan

    2013-05-01

    To compare the effects of pulsed electromagnetic field (PEMF) and low-level laser therapy (LLLT) on osteoblast cells in a cell culture model. Fifty thousand neonatal rat calvarial osteoblast-like cells per milliliter were seeded and 0.06 mT PEMF, 0.2 mT PEMF, and LLLT at 808 nm were applied for 24 and 96 h on the cells. To evaluate cellular proliferation and differentiation, specimens were examined for DNA synthesis, alkaline phosphatase (ALP) activity, cell numbers, and viability of the cells. Morphological appearances of the cells were observed using scanning electron microcopy after 24 and 96 h of incubation. At 24 and 96 h, the control group had a higher cell proliferation than 0.06 and 0.2 mT PEMF groups (p=0.001). At 96 h, 0.2 mT PEMF group had higher cell proliferation rate than 0.06 mT PEMF and LLLT groups (p=0.001). The cell count and cell viability in 0.2 mT PEMF group were higher than the 0.06-mT PEMF and LLLT groups, although these differences were not statistically significant at 96 h (p>0.05). At 24 and 96 h, cell viability in the control group was higher than the test groups. Alkaline phosphatase levels of the groups were comparable in both time intervals (p>0.05). 0.2 mT PEMF application on osteoblast-like cells led to cell proliferation and differentiation better than 0.06 mT PEMF and LLLT at 808 nm, although a remarkable effect of both PEMF and LLLT could not be detected. The ALP activity of 0.2 and 0.06 mT PEMF and LLLT were comparable.

  5. Influence of βS-globin haplotypes and hydroxyurea on tumor necrosis factor-alpha levels in sickle cell anemia.

    Science.gov (United States)

    Laurentino, Marília Rocha; Maia, Pedro Aurio; Barbosa, Maritza Cavalcante; Bandeira, Izabel Cristina Justino; Rocha, Lilianne Brito da Silva; Gonçalves, Romelia Pinheiro

    2014-03-01

    Sickle cell anemia is a chronic inflammatory disease characterized by an increased production of proinflammatory cytokines including tumor necrosis factor-alpha. Hydroxyurea, by decreasing the polymerization of hemoglobin, reduces inflammatory states. The effect of the genetic polymorphisms of sickle cell patients on tumor necrosis factor-alpha levels remains unknown. The aim of this study was to investigate the association of tumor necrosis factor-alpha levels with β-globin haplotypes and the use of hydroxyurea. A cross-sectional study was performed of 67 patients with sickle cell anemia diagnosed at steady-state in a referral hospital in Fortaleza, Ceará, Brazil. A group of 26 healthy individuals was used as control. βS-haplotype analysis was performed by restriction fragment length polymorphism-polymerase chain reaction. The tumor necrosis factor-alpha levels were measured by the enzyme-linked immunosorbent assay test. Laboratory data (complete blood count and fetal hemoglobin) and information regarding the use of hydroxyurea were obtained from medical records. Statistical analysis was performed using R software with the Kruskal-Wallis and Mann-Whitney tests. Statistical significance was established for p-values sickle cell anemia had significantly higher tumor necrosis factor-alpha levels than controls (p-values sickle cell anemia patients who were receiving hydroxyurea treatment than those who were not (p-value = 0.1249). Sickle cell anemia patients with Bantu/n genotype had significantly higher levels than patients with the Bantu/Benin genotype (p-value = 0.0021). In summary, βS-globin haplotypes, but not hydroxyurea therapy, have a role in modulating tumor necrosis factor-alpha levels in sickle cell anemia adults at steady-state. Many previous studies have investigated prognosis and inflammatory states in sickle cell anemia patients, but the discovery that tumor necrosis factor-alpha levels vary according to the genetic polymorphism of the patient is a

  6. MK-801 treatment affects glycolysis in oligodendrocytes more than in astrocytes and neuronal cells: insights for schizophrenia

    Directory of Open Access Journals (Sweden)

    Paul C Guest

    2015-05-01

    Full Text Available As a multifactorial disease, the underlying causes of schizophrenia require analysis by multiplex methods such as proteomics to allow identification of whole protein networks. Previous post-mortem proteomic studies on brain tissues from schizophrenia patients have demonstrated changes in activation of glycolytic and energy metabolism pathways. However, it is not known whether these changes occur in neurons or in glial cells. To address this question, we treated neuronal, astrocyte and oligodendrocyte cell lines with the NMDA receptor antagonist MK-801 and measured the levels of six glycolytic enzymes by Western blot analysis. MK-801 acts on the glutamatergic system and has been proposed as a pharmacological means of modeling schizophrenia. Treatment with MK-801 resulted in significant changes in the levels of glycolytic enzymes in all cell types. Most of the differences were found in oligodendrocytes, which had altered levels of hexokinase 1 (HK1, enolase 2 (ENO2, phosphoglycerate kinase (PGK and phosphoglycerate mutase 1 (PGAM1 after acute MK-801 treatment (8 hours, and HK1, ENO2, PGK and triosphosphate isomerase (TPI following long term treatment (72 hours. Addition of the antipsychotic clozapine to the cultures resulted in counter-regulatory effects to the MK-801 treatment by normalizing the levels of ENO2 and PGK in both the acute and long term cultures. In astrocytes, MK-801 affected only aldolase C (ALDOC under both acute conditions and HK1 and ALDOC following long term treatment, and TPI was the only enzyme affected under long term conditions in the neuronal cells. In conclusion, MK-801 affects glycolysis in oligodendrocytes to a larger extent than neuronal cells and this may be modulated by antipsychotic treatment. Although cell culture studies do not necessarily reflect the in vivo pathophysiology and drug effects within the brain, these results suggest that neurons, astrocytes and oligodendrocytes are affected differently in

  7. Response of Listeria monocytogenes to disinfection stress at the single-cell and population levels as monitored by intracellular pH measurements and viable-cell counts

    DEFF Research Database (Denmark)

    Kastbjerg, Vicky Gaedt; Nielsen, Dennis S.; Arneborg, Nils

    2009-01-01

    of the bacterium. In situ analyses of Listeria monocytogenes single cells were performed during exposure to different concentrations of the disinfectant Incimaxx DES to study a possible population subdivision. Bacterial survival was quantified with plate counting and disinfection stress at the single-cell level...... by measuring intracellular pH (pHi) over time by fluorescence ratio imaging microscopy. pHi values were initially 7 to 7.5 and decreased in both attached and planktonic L. monocytogenes cells during exposure to sublethal and lethal concentrations of Incimaxx DES. The response of the bacterial population...... that a population of L. monocytogenes cells, whether planktonic or attached, is homogenous with respect to sensitivity to an acidic disinfectant studied on the single-cell level. Hence a major subpopulation more tolerant to disinfectants, and hence more persistent, does not appear to be present....

  8. ULK1 regulates melanin levels in MNT-1 cells independently of mTORC1.

    Directory of Open Access Journals (Sweden)

    Eyal Kalie

    Full Text Available Melanosomes are lysosome-related organelles that serve as specialized sites of melanin synthesis and storage in melanocytes. The progression of melanosomes through the different stages of their formation requires trafficking of specific proteins and membrane constituents in a sequential manner, which is likely to deploy ubiquitous cellular machinery along with melanocyte-specific proteins. Recent evidence revealed a connection between melanogenesis and the autophagy machinery, suggesting a novel role for members of the latter in melanocytes. Here we focused on ULK1, a key autophagy protein which is negatively regulated by mTORC1, to assess its potential role in melanogenesis in MNT-1 cells. We found that ULK1 depletion causes an increase in melanin levels, suggesting an inhibitory function for this protein in melanogenesis. Furthermore, this increase was accompanied by increased transcription of MITF (microphthalmia-associated transcription factor and tyrosinase and by elevated protein levels of tyrosinase, the rate-limiting factor in melanin biogenesis. We also provide evidence to show that ULK1 function in this context is independent of the canonical ULK1 autophagy partners, ATG13 and FIP200. Furthermore we show that regulation of melanogenesis by ULK1 is independent of mTORC1 inhibition. Our data thus provide intriguing insights regarding the involvement of the key regulatory autophagy machinery in melanogenesis.

  9. Dissecting miRNA gene repression on single cell level with an advanced fluorescent reporter system

    Science.gov (United States)

    Lemus-Diaz, Nicolas; Böker, Kai O.; Rodriguez-Polo, Ignacio; Mitter, Michael; Preis, Jasmin; Arlt, Maximilian; Gruber, Jens

    2017-01-01

    Despite major advances on miRNA profiling and target predictions, functional readouts for endogenous miRNAs are limited and frequently lead to contradicting conclusions. Numerous approaches including functional high-throughput and miRISC complex evaluations suggest that the functional miRNAome differs from the predictions based on quantitative sRNA profiling. To resolve the apparent contradiction of expression versus function, we generated and applied a fluorescence reporter gene assay enabling single cell analysis. This approach integrates and adapts a mathematical model for miRNA-driven gene repression. This model predicts three distinct miRNA-groups with unique repression activities (low, mid and high) governed not just by expression levels but also by miRNA/target-binding capability. Here, we demonstrate the feasibility of the system by applying controlled concentrations of synthetic siRNAs and in parallel, altering target-binding capability on corresponding reporter-constructs. Furthermore, we compared miRNA-profiles with the modeled predictions of 29 individual candidates. We demonstrate that expression levels only partially reflect the miRNA function, fitting to the model-projected groups of different activities. Furthermore, we demonstrate that subcellular localization of miRNAs impacts functionality. Our results imply that miRNA profiling alone cannot define their repression activity. The gene regulatory function is a dynamic and complex process beyond a minimalistic conception of “highly expressed equals high repression”. PMID:28338079

  10. Obesity suppresses circulating level and function of endothelial progenitor cells and heart function

    Directory of Open Access Journals (Sweden)

    Tsai Tzu-Hsien

    2012-07-01

    Full Text Available Abstract Background and aim This study tested the hypothesis that obesity suppresses circulating number as well as the function of endothelial progenitor cells (EPCs and left ventricular ejection fraction (LVEF. Methods High fat diet (45 Kcal% fat was given to 8-week-old C57BL/6 J mice (n = 8 for 20 weeks to induce obesity (group 1. Another age-matched group (n = 8 were fed with control diet for 20 weeks as controls (group 2. The animals were sacrificed at the end of 20 weeks after obesity induction. Results By the end of study period, the heart weight, body weight, abdominal fat weight, serum levels of total cholesterol and fasting blood sugar were remarkably higher in group 1 than in group 2 (all p Conclusions Obesity diminished circulating EPC level, impaired the recovery of damaged endothelium, suppressed EPC angiogenesis ability and LVEF, and increased LV remodeling.

  11. Predictive and prognostic value of preoperative serum tumor markers is EGFR mutation-specific in resectable non-small-cell lung cancer

    Science.gov (United States)

    Jiang, Richeng; Wang, Xinyue; Li, Kai

    2016-01-01

    Background The predictive and prognostic value of carcinoembryonic antigen (CEA), cytokeratin-19 fragments (Cyfra21-1), squamous cell carcinoma antigen (SCCA) and neuron-specific enolase (NSE) has been investigated in non-small-cell lung cancer (NSCLC) patients. However, few studies have directly focused on the association between these markers and epidermal growth factor receptor (EGFR) mutation status or mutation subtypes. Patients and methods We retrospectively analyzed 1016 patients with stage I-IIIA NSCLC who underwent complete resection between 2008 and 2012. Correlations between serum tumor marker levels and EGFR mutations and survival parameters were analyzed and prognostic factors were identified. Results Cyfra21-1 levels (P = 0.032 for disease-free survival [DFS]; P CEA levels (P CEA (P = 0.005) and clinical stage were predictive factors of DFS, while elevated CEA (P = 0.005) and Cyfra21-1 (P = 0.027) were independent prognostic factors. Conclusion Cyfra21-1 and CEA exhibit different predictive and prognostic values between EGFR-mutated and wild-type adenocarcinomas, as well as between EGFR mutation subtypes. The prognostic impact of preoperative serum tumor markers should be evaluated together with EGFR mutation status. PMID:27072585

  12. Correlation Between Th1, Th2 Cells and Levels of Serum MMP-2, MMP-9 in Children with Asthma

    Directory of Open Access Journals (Sweden)

    Xuan WANG

    2015-12-01

    Full Text Available Abstract Objective: To explore the correlation between Th1 and Th2 cells and the levels of serum matrix metalloproteinase-2 (MMP-2 and MMP-9 in children with asthma. Methods: A total of 89 children with asthma were divided into acute group (n=48 and chronic group (n=41 according to the course of disease, and 40 healthy children at the same term were collected as control group. The ratios of Th1 and Th2 cells as well as levels of MMP-2 and MMP-9 were compared in three groups, and the correlation between Th1 and Th2 cells and levels of MMP-2, MMP-9 was analyzed in acute group and chronic group. Results: When compared with control group, the ratios of Th1 and Th2 cells went down in both acute group and chronic group (P<0.01, while the levels of serum MMP-2 and MMP-9 up (P<0.01. The levels of serum MMP-2 and MMP-9 in acute group were dramatically higher than those in chronic group, and there was statistical significance (P<0.01. Pearson correlation analysis revealed that there was no significant correlation between Th1 and Th2 cells and MMP-2 level (r=0.148, P=0.314, r=0.299, P=0.058; r=0.183, P=0.214, r=0.289, P=0.067, whereas both Th1 and Th2 cells were negatively correlated with MMP-9 level in acute group and chronic group (r=-0.489, P=0.000, r=-0.324, P=0.039; r=-0.352, P=0.014, r=-0.357, P=0.022. Conclusion: Aberrant secretion of Th cells can not only damage the immune function of children with asthma, but also decrease the level of serum MMP-9, consequently affecting the collagen degradation and airway remodeling.

  13. Angiotensin II potentiates prostaglandin stimulation of cyclic AMP levels in intact bovine adrenal medulla cells but not adenylate cyclase in permeabilized cells.

    Science.gov (United States)

    Boarder, M R; Plevin, R; Marriott, D B

    1988-10-25

    The level of cyclic AMP in primary cultures of bovine adrenal medulla cells is elevated by prostaglandin E1. Angiotensin II is commonly reported to act on receptors linked to phosphoinositide metabolism or to inhibition of adenylate cyclase. We have investigated the effect of angiotensin II on prostaglandin E1-stimulated cyclic AMP levels in these primary cultures. Rather than reducing cyclic AMP levels, we have found that angiotensin II powerfully potentiates prostaglandin E1-stimulated cyclic AMP accumulation in intact cells, both in the presence and absence of phosphodiesterase inhibitors. The 50% maximal response was similar to that for stimulation of phosphoinositide breakdown by angiotensin II in these cultures. The potentiation of stimulated cyclic AMP levels was seen, although to a smaller maximum, with the protein kinase C (Ca2+/phospholipid-dependent enzyme) activating phorbol ester tetradecanoyl phorbolacetate and with the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol; pretreatment (24 h) with active phorbol ester, which would be expected to diminish protein kinase C levels, attenuated the angiotensin II potentiation of cyclic AMP. Using digitonin-permeabilized cells we showed that adenylate cyclase activity was stimulated by prostaglandin E1 with the same dose-response relationship as was cyclic AMP accumulation in intact cells, but the permeabilized cells showed no response to angiotensin II. The results are discussed with respect to the hypothesis that the angiotensin II influence on cyclic AMP levels is mediated, in part, by diacylglycerol stimulation of protein kinase C.

  14. Curcumin elevates sirtuin level but does not postpone in vitro senescence of human cells building the vasculature

    Science.gov (United States)

    Grabowska, Wioleta; Suszek, Małgorzata; Wnuk, Maciej; Lewinska, Anna; Wasiak, Emilia; Sikora, Ewa; Bielak-Zmijewska, Anna

    2016-01-01

    It is believed that curcumin, a component of the turmeric that belongs to hormetins, possesses anti-aging propensity. This property of curcumin can be partially explained by its influence on the level of sirtuins. Previously, we have shown that relatively high (2.5-10 μM) doses of curcumin induce senescence of cancer cells and cells building the vasculature. In the present study we examined whether curcumin at low doses (0.1 and 1 μM) is able to delay cell senescence and upregulate the level of sirtuins in human cells building the vasculature, namely vascular smooth muscle (VSMC) and endothelial (EC) cells. To this end we used cells senescing in a replicative and premature manner. We showed that low doses of curcumin in case of VSMC neither postponed the replicative senescence nor protected from premature senescence induced by doxorubicin. Moreover, curcumin slightly accelerated replicative senescence of EC. Despite some fluctuations, a clear increasing tendency in the level of sirtuins was observed in curcumin-treated young, senescing or already senescent cells. Sirtuin activation could be caused by the activation of AMPK resulting from superoxide elevation and ATP reduction. Our results show that curcumin at low doses can increase the level of sirtuins without delaying senescence of VSMC. PMID:27034011

  15. High Intracellular Seed Train BiP Levels Correlate with Poor Production Culture Performance in CHO Cells.

    Science.gov (United States)

    Tung, Meg; Tang, Danming; Wang, Szu-Han; Zhan, Dejin; Kiplinger, Karen; Pan, Shu; Jing, Yifeng; Shen, Amy; Ahyow, Patrick; Snedecor, Brad; Gawlitzek, Martin; Misaghi, Shahram

    2018-04-10

    Consistent cell culture performance is a prerequisite to ensure product quality consistency and achieve productivity goals for the manufacture of recombinant protein therapeutics, including monoclonal antibodies. Here we report a peculiar observation where high levels of intracellular BiP in seed train cultures are consistently predictive of poor cell culture performance in the subsequent inoculum and production cultures for a monoclonal antibody produced in CHO cells. Our investigations suggest that in this cell line the high intracellular BiP levels in the seed train are triggered by a slightly lower culture pH, which interferes with proper antibody folding and secretion. While the seed train culture does not display any obvious signs of the problem at slightly lower culture pH, inoculum trains and production cultures sourced from these low pH seed trains display significantly lower cell growth and cell size. High intracellular BiP levels may interfere with UPR signaling, thereby hampering a proper and timely UPR response in the production media. Studies of other problematic cell lines have shown a similar correlation between intracellular BiP accumulation and poor production performance. We believe intracellular BiP levels in seed train should hence be low in order to increase the success rate in production. This article is protected by copyright. All rights reserved.

  16. Characteristics of action of nitrosoalkylureas on cell level in winter wheat

    Directory of Open Access Journals (Sweden)

    M. M. Nazarenko

    2016-05-01

    Full Text Available Here we report the cytogenetic characteristics of variability of mutation induction in new wheat varieties and some relationships between the means of the cytogenetic characteristics and different doses and types of mutagens. Analysis of chromosomal aberrations caused by mutagenic action of any kind of mutagen by the meto-anaphase method is one of the best known and most precise methods which we can use for determining the fact of mutagenic action on plants, and identifying the nature of the mutagenic factor. The strategy of investigation combined the identification of genotypes with a specific low-sensitivity to mutagenic factors using cytological analysis screening of mutagen treated wheat populations with the approach of comparing different varieties by breeding methods to reveal their connections and differences, specific sensitivity to mutagenic effects on the cell level. Dry seeds of 8 varieties of winter wheat were treated by nitrosomethilurea at concentrations 0.0125%, 0.0250%, and nitrosoethilurea 0.010%, 0.025%, which are normal levels for mutagenic selection of winter wheat mutation. The frequency and spectra of chromosomal aberrations were investigated. The overall correlations between the frequency of chromosomal aberrations and the value of a concentration were at the level 0.7–0.8. It was established that the fragments-bridges ratio is a reliable parameter for identifyingthe nature of mutagen. We identified genotypes and groups of genotypes which have a resistance to different types of mutagenic action. The resistance of a group is dependant on the breeding method of the variety initially obtained. It expressed as a decrease in the frequency of aberrations. Varieties obtained through chemical mutagenesis were less sensitive to the same chemical mutagens. Higher rates of chromosomal aberrations were typical for varieties obtained through field hybridization without any mutagen treatment or when the initial material was changed by

  17. Cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser.

    Science.gov (United States)

    Alexsandra da Silva Neto Trajano, Larissa; da Silva, Camila Luna; de Carvalho, Simone Nunes; Cortez, Erika; Mencalha, André Luiz; de Souza da Fonseca, Adenilson; Stumbo, Ana Carolina

    2016-07-01

    Low-level infrared laser is considered safe and effective for treatment of muscle injuries. However, the mechanism involved on beneficial effects of laser therapy are not understood. The aim was to evaluate cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser at therapeutic fluences. C2C12 myoblast cultures at different (2 and 10 %) fetal bovine serum (FBS) concentrations were exposed to low-level infrared laser (808 nm, 100 mW) at different fluences (10, 35, and 70 J/cm(2)) and evaluated after 24, 48, and 72 h. Cell viability was evaluated by WST-1 assay; reactive oxygen species (ROS), apoptosis, and necrosis were evaluated by flow cytometry. Cell viability was decreased atthe lowest FBS concentration. Laser exposure increased the cell viability in myoblast cultures at 2 % FBS after 48 and 72 h, but no significant increase in ROS was observed. Apoptosis was decreased at the higher fluence and necrosis was increased at lower fluence in myoblast cultures after 24 h of laser exposure at 2 % FBS. No laser-induced alterations were obtained at 10 % FBS. Results show that level of reactive oxygen species is not altered, at least to those evaluated in this study, but low-level infrared laser exposure affects cell viability, apoptosis, and necrosis in myoblast cultures depending on laser fluence and physiologic conditions of cells.

  18. Response of single bacterial cells to stress gives rise to complex history dependence at the population level

    Science.gov (United States)

    Mathis, Roland; Ackermann, Martin

    2016-01-01

    Most bacteria live in ever-changing environments where periods of stress are common. One fundamental question is whether individual bacterial cells have an increased tolerance to stress if they recently have been exposed to lower levels of the same stressor. To address this question, we worked with the bacterium Caulobacter crescentus and asked whether exposure to a moderate concentration of sodium chloride would affect survival during later exposure to a higher concentration. We found that the effects measured at the population level depended in a surprising and complex way on the time interval between the two exposure events: The effect of the first exposure on survival of the second exposure was positive for some time intervals but negative for others. We hypothesized that the complex pattern of history dependence at the population level was a consequence of the responses of individual cells to sodium chloride that we observed: (i) exposure to moderate concentrations of sodium chloride caused delays in cell division and led to cell-cycle synchronization, and (ii) whether a bacterium would survive subsequent exposure to higher concentrations was dependent on the cell-cycle state. Using computational modeling, we demonstrated that indeed the combination of these two effects could explain the complex patterns of history dependence observed at the population level. Our insight into how the behavior of single cells scales up to processes at the population level provides a perspective on how organisms operate in dynamic environments with fluctuating stress exposure. PMID:26960998

  19. Effect of copper intrauterine device vs. injectable contraceptive on serum hormone levels and cell mitotic activity in endometrium

    Directory of Open Access Journals (Sweden)

    Ebtesam Moustafa Kamal

    2013-09-01

    Conclusion: Either copper intrauterine device or injectable contraceptive usage for more than 9 months results in significant decrease in endometrial proliferative or cell mitotic activity. While copper IUD has no effect on serum estradiol or progesterone levels, DMPA usage increased serum progesterone level with no effect on serum estradiol.

  20. The effects of low level laser therapy on both HIV-1 infected and uninfected TZM-bl cells

    CSIR Research Space (South Africa)

    Lugongolo, Masixole Y

    2017-01-01

    Full Text Available that would completely eradicate the virus in infected individuals, the quest for new therapies continues. Low level laser therapy (LLLT) involves the exposure of cells to low levels of red or infrared light. LLLT has been widely used in different medical...

  1. High normal fasting glucose level in obese youth: a marker for insulin resistance and beta cell dysregulation.

    LENUS (Irish Health Repository)

    O'Malley, G

    2010-06-01

    A high but normal fasting plasma glucose level in adults is a risk factor for future development of type 2 diabetes mellitus and cardiovascular disease. We investigated whether normal fasting plasma glucose levels (<5.60 mmol\\/l) are associated with decreases in insulin sensitivity and beta cell function, as well as an adverse cardiovascular profile in obese youth.

  2. High level of chromosomal instability in circulating tumor cells of ROS1-rearranged non-small-cell lung cancer

    OpenAIRE

    Pailler, E.; Auger, N.; Lindsay, C. R.; Vielh, P.; Islas-Morris-Hernandez, A.; Borget, I.; Ngo-Camus, M.; Planchard, D.; Soria, J.-C.; Besse, B.; Farace, F.

    2015-01-01

    Background Genetic aberrations affecting the c-ros oncogene 1 (ROS1) tyrosine kinase gene have been reported in a small subset of patients with non-small-cell lung cancer (NSCLC). We evaluated whether ROS1-chromosomal rearrangements could be detected in circulating tumor cells (CTCs) and examined tumor heterogeneity of CTCs and tumor biopsies in ROS1-rearranged NSCLC patients. Patients and methods Using isolation by size of epithelial tumor cells (ISET) filtration and filter-adapted-fluoresce...

  3. Low-level laser therapy: Effects on human face aged skin and cell viability of HeLa cells exposed to UV radiation

    Directory of Open Access Journals (Sweden)

    Mezghani Sana

    2015-01-01

    Full Text Available Chronic and excessive exposure to UV radiation leads to photoaging and photocarcinogenesis. Adequate protection of the skin against the deleterious effects of UV irradiation is essential. Low-level laser therapy (LLLT is a light source in the red to near-infrared range that has been accepted in a variety of medical applications. In this study, we explored the effect of LLLT in human face aged skin and the cell viability of HeLa cells exposed to UV radiation. We found that LLLT significantly reduced visible wrinkles and the loss of firmness of facial skin in aging subjects. Additionally, treatment of cultured HeLa cells with LLLT prior to or post UVA or UVB exposure significantly protected cells from UV-mediated cell death. All results showed the beneficial effects of LLLT on relieving signs of skin aging and its prevention and protection of the cell viability against UV-induced damage.

  4. Use of INAA to study the determination of Se, Th, Zn, Co and Fe levels of yeast cells

    International Nuclear Information System (INIS)

    Czauderna, M.; Turska, M.; Sitowska, B.

    1996-01-01

    Differences in the effects of seleno-cystine (CySe) 2 , glutathione (GSH), Se(IV) [as SeO 2 ] and Se(VI) [as (NH 4 ) 2 SeO 4 ] on Th(IV) [as Th(CO 3 ) 2 ] uptake by the cells, Saccharomyces cerevisiae, have been studied. The Th, Se, Zn, Co and Fe levels of the yeast cells were measured by instrumental neutron activation analysis. Results obtained show that the addition of Th alone to the culture medium resulting in the Th content of the cells and the Th level of the yeast slightly decreased during the incubation. The addition of Th in combination with GSH produced a higher decrease of the Th content in comparison with the single Th dosage. During the initial 48 h of the incubation the presence of Th and Se(VI) in the medium produced a decrease of the Th level of the cells in comparison with the addition of Th alone. (CySe) 2 or SeO 2 does not produce a regular change of the Th level of the cells. Th uptake by the yeast influenced the retention of Se in the cells. In fact, the Se levels of the cells were always significantly higher when the yeast was incubated in the medium containing Th and SeO 2 or Se(VI). The enhance in the Se level of the cells rises increasing concentrations of SeO 2 in the culture medium. Th decreased the Se content of the yeast when the cells were incubated in the medium containing (CySe) 2 and Th. GSH supply in combination with Th and SeO 2 produced a very significant enhancement of the Se abundance in the cells in comparison with the single addition of SeO 2 . Se-compounds and/or Th dosages affected the Zn, Co and Fe contents of the cells. The Fe level of the yeast is below the quantitative detection limit of Fe when the cells were incubated in the medium containing Th. (Author)

  5. Decreased drug accumulation and increased tolerance to DNA damage in tumor cells with a low level of cisplatin resistance.

    Science.gov (United States)

    Lanzi, C; Perego, P; Supino, R; Romanelli, S; Pensa, T; Carenini, N; Viano, I; Colangelo, D; Leone, R; Apostoli, P; Cassinelli, G; Gambetta, R A; Zunino, F

    1998-04-15

    In an attempt to examine the cellular changes associated with cisplatin resistance, we selected a cisplatin-resistant (A43 1/Pt) human cervix squamous cell carcinoma cell line following continuous in vitro drug exposure. The resistant subline was characterized by a 2.5-fold degree of resistance. In particular, we investigated the expression of cellular defence systems and other cellular factors probably involved in dealing with cisplatin-induced DNA damage. Resistant cells exhibited decreased platinum accumulation and reduced levels of DNA-bound platinum and interstrand cross-link frequency after short-term drug exposure. Analysis of the effect of cisplatin on cell cycle progression revealed a cisplatin-induced G2M arrest in sensitive and resistant cells. Interestingly, a slowdown in S-phase transit was found in A431/Pt cells. A comparison of the ability of sensitive and resistant cells to repair drug-induced DNA damage suggested that resistant cells were able to tolerate higher levels of cisplatin-induced DNA damage than their parental counterparts. Analysis of the expression of proteins involved in DNA mismatch repair showed a decreased level of MSH2 in resistant cells. Since MSH2 seems to be involved in recognition of drug-induced DNA damage, this change may account for the increased tolerance to DNA damage observed in the resistant subline. In conclusion, the involvement of accumulation defects and the increased tolerance to cisplatin-induced DNA damage in these cisplatin-resistant cells support the notion that multiple changes contribute to confer a low level of cisplatin resistance.

  6. Determination of gamma radiation lethal dose (LD{sub 50}) and resveratrol cytotoxicity level in tumor cells line

    Energy Technology Data Exchange (ETDEWEB)

    Magalhaes, Vanessa D.; Rogero, Sizue O.; Rogero, Jose R. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Cruz, Aurea S. [Instituto Adolfo Lutz (IAL-SP) Secao de Culturas Celulares, SP (Brazil)

    2011-07-01

    Cancer is a disease with high incidence and it is considered a worldwide public health problem. Resveratrol is a polyphenol occurring naturally in a wide variety of plants according to response of ultraviolet radiation (UV) exposition or according to mechanical stress resulting of pathogens or chemical and physical agents. This polyphenol possesses a pharmacological activity of carcinogenesis inhibition in multiple levels. It also protects cells by scavenging the free radicals which are considered toxic products. These free radicals are formed of natural process of cell aging and also by incidence of ionizing radiation in the organism. Thus, resveratrol is considered as a cell radioprotector. On the other hand, in some elevated concentrations resveratrol may be considered as a radiosensitizing. The aim of this work was the determination of radiation lethal dose (LD{sub 50}) and also verifies the cytotoxicity level of resveratrol in tumor cells line: muco epidermoid pulmonary carcinoma cells (NCI-H292) and rhabdomyosarcoma cells (RD). The cytotoxicity test was performed by neutral red uptake assay. The results of resveratrol IC{sub 50%} in NCI-H292 cells was 192{mu}M and in RD cells was 128{mu}M; and RD cells gamma radiation LD{sub 50} was 435Gy. (author)

  7. High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Whittaker, J.; Okamoto, A.K.; Thys, R.; Bell, G.I.; Steiner, D.F.; Hofmann, C.A.

    1987-08-01

    In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10/sup 6/ receptors per cell. The cell line with the highest /sup 125/I-insulin binding (NIH 3T3 HIR3.5) had 6 x 10/sup 6/ receptors with a K/sub d/ of 10/sup -9/ M. This level was not dependent on exposure to metals but could be increased further to 2 x 10/sup 7/ receptors per cell by addition of sodium butyrate to the culture medium. The ..cap alpha.. and ..beta.. subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the ..cap alpha.. and ..beta.. subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis

  8. High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells

    International Nuclear Information System (INIS)

    Whittaker, J.; Okamoto, A.K.; Thys, R.; Bell, G.I.; Steiner, D.F.; Hofmann, C.A.

    1987-01-01

    In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10 6 receptors per cell. The cell line with the highest 125 I-insulin binding (NIH 3T3 HIR3.5) had 6 x 10 6 receptors with a K/sub d/ of 10 -9 M. This level was not dependent on exposure to metals but could be increased further to 2 x 10 7 receptors per cell by addition of sodium butyrate to the culture medium. The α and β subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the α and β subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis

  9. Changes in cytokine and biomarker blood levels in patients with colorectal cancer during dendritic cell-based vaccination

    DEFF Research Database (Denmark)

    Burgdorf, Stefan K; Claesson, Mogens Helweg; Nielsen, Hans J

    2009-01-01

    Introduction. Immunotherapy based on dendritic cell vaccination has exciting perspectives for treatment of cancer. In order to clarify immunological mechanisms during vaccination it is essential with intensive monitoring of the responses. This may lead to optimization of treatment and prediction...... of responding patients. The aim of this study was to evaluate cytokine and biomarker responses in patients with colorectal cancer treated with a cancer vaccine based on dendritic cells pulsed with an allogeneic melanoma cell lysate. Material and methods. Plasma and serum samples were collected prior...... disease showed increasing levels of plasma GM-CSF, TNF-alpha, IFN-gamma, IL-2, and IL-5. Patients with progressive disease showed significant increase in CEA and TIMP-1 levels, while patients with stable disease showed relatively unaltered levels. Conclusion. The increased levels of key pro...

  10. Oleate ameliorates palmitate-induced reduction of NAMPT activity and NAD levels in primary human hepatocytes and hepatocarcinoma cells.

    Science.gov (United States)

    Penke, Melanie; Schuster, Susanne; Gorski, Theresa; Gebhardt, Rolf; Kiess, Wieland; Garten, Antje

    2017-10-03

    Nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide adenine dinucleotide (NAD) levels are crucial for liver function. The saturated fatty acid palmitate and the unsaturated fatty acid oleate are the main free fatty acids in adipose tissue and human diet. We asked how these fatty acids affect cell survival, NAMPT and NAD levels in HepG2 cells and primary human hepatocytes. HepG2 cells were stimulated with palmitate (0.5mM), oleate (1mM) or a combination of both (0.5mM/1mM) as well as nicotinamide mononucleotide (NMN) (0.5 mM) or the specific NAMPT inhibitor FK866 (10nM). Cell survival was measured by WST-1 assay and Annexin V/propidium iodide staining. NAD levels were determined by NAD/NADH Assay or HPLC. Protein and mRNA levels were analysed by Western blot analyses and qPCR, respectively. NAMPT enzyme activity was measured using radiolabelled 14 C-nicotinamide. Lipids were stained by Oil red O staining. Palmitate significantly reduced cell survival and induced apoptosis at physiological doses. NAMPT activity and NAD levels significantly declined after 48h of palmitate. In addition, NAMPT mRNA expression was enhanced which was associated with increased NAMPT release into the supernatant, while intracellular NAMPT protein levels remained stable. Oleate alone did not influence cell viability and NAMPT activity but ameliorated the negative impact of palmitate on cell survival, NAMPT activity and NAD levels, as well as the increased NAMPT mRNA expression and secretion. NMN was able to normalize intracellular NAD levels but did not ameliorate cell viability after co-stimulation with palmitate. FK866, a specific NAMPT inhibitor did not influence lipid accumulation after oleate-treatment. Palmitate targets NAMPT activity with a consequent cellular depletion of NAD. Oleate protects from palmitate-induced apoptosis and variation of NAMPT and NAD levels. Palmitate-induced cell stress leads to an increase of NAMPT mRNA and accumulation in the supernatant. However

  11. Circulating concentrations of stem-cell-mobilizing cytokines are associated with levels of osteoprogenitor cells and aortic calcification severity

    International Nuclear Information System (INIS)

    Pal, S.N.; Clancy, P.; Golledge, J.

    2011-01-01

    The background of this study was to investigate the association between aortic calcification, concentrations of stem-cell-mobilizing cytokines and osteocalcin-positive mononuclear cells in a mouse model and patients with peripheral artery disease. We estimated the concentration of the stem-cell-mobilizing cytokines stromal cell-derived factor α (SDF-1α), granulocyte colony stimulating factor and stem cell factor in a mouse model of aortic calcification developed in osteoprotegerin-deficient (OPG -/- ) mice, as well as in patient plasma samples. Calcification was estimated by a colorimetric assay of extracts of harvested mice aortas and by computed tomographic angiogram images in patients. The cytokine concentrations were assessed for association with the severity of calcification and the percentage of osteocalcin-positive mononuclear cells (OCN + MNC) using non-parametric analysis. The serum concentration of stromal SDF-1α and granulocyte-colony stimulating factor (G-CSF) were significantly greater in OPG -/- compared to control mice. The percentage of circulating OCN + MNC was correlated to the concentration of SDF-1α in OPG -/- mice. These cytokines also correlated with the severity of calcification in OPG -/- mice. Patients with more severe calcification had a higher plasma concentration of the cytokines than those with less marked aortic calcification. The concentrations also correlated with circulating OCN + MNC and aortic calcification volumes. The association between stem cell cytokines, OCN + MNC and calcification suggests a possible role of bone-derived osteoprogenitor cells in the pathogenesis of vascular calcification. (author)

  12. Efficient Strategies for Predictive Cell-Level Control of Lithium-Ion Batteries

    Science.gov (United States)

    Xavier, Marcelo A.

    This dissertation introduces a set of state-space based model predictive control (MPC) algorithms tailored to a non-zero feedthrough term to account for the ohmic resistance that is inherent to the battery dynamics. MPC is herein applied to the problem of regulating cell-level measures of performance for lithium-ion batteries; the control methodologies are used first to compute a fast charging profile that respects input, output, and state constraints, i.e., input current, terminal voltage, and state of charge for an equivalent circuit model of the battery cell, and extended later to a linearized physics-based reduced-order model. The novelty of this work can summarized as follows: (1) the MPC variants are employed to a physics based reduce-order model in order to make use of the available set of internal electrochemical variables and mitigate internal mechanisms of cell degradation. (e.g., lithium plating); (2) we developed a dual-mode MPC closed-loop paradigm that suits the battery control problem with the objective of reducing computational effort by solving simpler optimization routines and guaranteeing stability; and finally (3) we developed a completely new approach of the use of a predictive control strategy where MPC is employed as a "smart sensor" for power estimation. Results are presented that show the comparative performance of the MPC algorithms for both EMC and PBROM These results highlight that dual-mode MPC can deliver optimal input current profiles by using a shorter horizon while still guaranteeing stability. Additionally, rigorous mathematical developments are presented for the development of the MPC algorithms. The use of MPC as a "smart sensor" presents it self as an appealing method for power estimation, since MPC permits a fully dynamic input profile that is able to achieve performance right at the proper constraint boundaries. Therefore, MPC is expected to produce accurate power limits for each computed sample time when compared to the

  13. Lamprey Prohibitin2 Arrest G2/M Phase Transition of HeLa Cells through Down-regulating Expression and Phosphorylation Level of Cell Cycle Proteins.

    Science.gov (United States)

    Shi, Ying; Guo, Sicheng; Wang, Ying; Liu, Xin; Li, Qingwei; Li, Tiesong

    2018-03-02

    Prohibitin 2(PHB2) is a member of the SFPH trans-membrane family proteins. It is a highly conserved and functionally diverse protein that plays an important role in preserving the structure and function of the mitochondria. In this study, the lamprey PHB2 gene was expressed in HeLa cells to investigate its effect on cell proliferation. The effect of Lm-PHB2 on the proliferation of HeLa cells was determined by treating the cells with pure Lm-PHB2 protein followed by MTT assay. Using the synchronization method with APC-BrdU and PI double staining revealed rLm-PHB2 treatment induced the decrease of both S phase and G0/G1 phase and then increase of G2/M phase. Similarly, cells transfected with pEGFP-N1-Lm-PHB2 also exhibited remarkable reduction in proliferation. Western blot and quantitative real-time PCR(qRT-PCR) assays suggested that Lm-PHB2 caused cell cycle arrest in HeLa cells through inhibition of CDC25C and CCNB1 expression. According to our western blot analysis, Lm-PHB2 was also found to reduce the expression level of Wee1 and PLK1 and the phosphorylation level of CCNB1, CDC25C and CDK1 in HeLa cells. Lamprey prohibitin 2 could arrest G2/M phase transition of HeLa cells through down-regulating expression and phosphorylation level of cell cycle proteins.

  14. Expression profiling indicating low selenium-sensitive microRNA levels linked to cell cycle and cell stress response pathways in the CaCo-2 cell line.

    Science.gov (United States)

    McCann, Mark J; Rotjanapun, Kunjana; Hesketh, John E; Roy, Nicole C

    2017-05-01

    Se is an essential micronutrient for human health, and fluctuations in Se levels and the potential cellular dysfunction associated with it may increase the risk for disease. Although Se has been shown to influence several biological pathways important in health, little is known about the effect of Se on the expression of microRNA (miRNA) molecules regulating these pathways. To explore the potential role of Se-sensitive miRNA in regulating pathways linked with colon cancer, we profiled the expression of 800 miRNA in the CaCo-2 human adenocarcinoma cell line in response to a low-Se (72 h at <40 nm) environment using nCounter direct quantification. These data were then examined using a range of in silico databases to identify experimentally validated miRNA-mRNA interactions and the biological pathways involved. We identified ten Se-sensitive miRNA (hsa-miR-93-5p, hsa-miR-106a-5p, hsa-miR-205-5p, hsa-miR-200c-3p, hsa-miR-99b-5p, hsa-miR-302d-3p, hsa-miR-373-3p, hsa-miR-483-3p, hsa-miR-512-5p and hsa-miR-4454), which regulate 3588 mRNA in key pathways such as the cell cycle, the cellular response to stress, and the canonical Wnt/β-catenin, p53 and ERK/MAPK signalling pathways. Our data show that the effects of low Se on biological pathways may, in part, be due to these ten Se-sensitive miRNA. Dysregulation of the cell cycle and of the stress response pathways due to low Se may influence key genes involved in carcinogenesis.

  15. Coffee Polyphenols Change the Expression of STAT5B and ATF-2 Modifying Cyclin D1 Levels in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Carlota Oleaga

    2012-01-01

    Full Text Available Background. Epidemiological studies suggest that coffee consumption reduces the risk of cancer, but the molecular mechanisms of its chemopreventive effects remain unknown. Objective. To identify differentially expressed genes upon incubation of HT29 colon cancer cells with instant caffeinated coffee (ICC or caffeic acid (CA using whole-genome microarrays. Results. ICC incubation of HT29 cells caused the overexpression of 57 genes and the underexpression of 161, while CA incubation induced the overexpression of 12 genes and the underexpression of 32. Using Venn-Diagrams, we built a list of five overexpressed genes and twelve underexpressed genes in common between the two experimental conditions. This list was used to generate a biological association network in which STAT5B and ATF-2 appeared as highly interconnected nodes. STAT5B overexpression was confirmed at the mRNA and protein levels. For ATF-2, the changes in mRNA levels were confirmed for both ICC and CA, whereas the decrease in protein levels was only observed in CA-treated cells. The levels of cyclin D1, a target gene for both STAT5B and ATF-2, were downregulated by CA in colon cancer cells and by ICC and CA in breast cancer cells. Conclusions. Coffee polyphenols are able to affect cyclin D1 expression in cancer cells through the modulation of STAT5B and ATF-2.

  16. Natural killer cells from psoriasis vulgaris patients have reduced levels of cytotoxicity associated degranulation and cytokine production.

    Science.gov (United States)

    Dunphy, S E; Sweeney, C M; Kelly, G; Tobin, A M; Kirby, B; Gardiner, C M

    2017-04-01

    Psoriasis vulgaris is a chronic inflammatory disease of the skin with a strong genetic component and immune system involvement. Although some evidence suggests that Natural Killer (NK) cells may play a part in psoriasis, their role is relatively unstudied and results are controversial. In this current study, NK cells from psoriasis patients exhibited reduced degranulation and produced lower levels of the pro-inflammatory cytokines IFN-γ and TNF-α. Further investigation found that NK cells from psoriasis patients and healthy controls expressed similar levels of activation markers, NK cell receptors and apoptosis-inducing molecules. In addition, comparable levels of several cytokines important in NK cell biology were found in the serum of psoriasis patients and healthy controls. Genotyping analysis revealed that HLA-C2, which provides a ligand for killer-cell immunoglobulin-like receptors (KIR) expressed by NK cells, was strongly associated with psoriasis susceptibility. However, no link between the KIR genes themselves and disease was found. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Cyclic AMP-dependent regulation of tyrosine hydroxylase mRNA and immunofluorescence levels in rat retinal precursor cells.

    Science.gov (United States)

    Voisin, Pierre; Bernard, Marianne

    2013-05-01

    Stimulation of tyrosine hydroxylase (TH) gene transcription by cyclic AMP (cAMP) has been clearly established in adrenal medula cells and neural-crest-derived cell lines but information on this mechanism is still lacking in dopaminergic neurons. Because they are easily amenable to in vitro experiments, dopaminergic amacrine cells of the retina might constitute a valuable model system to study this mechanism. We have used real-time reverse transcription with the polymerase chain reaction to quantify TH mRNA levels in the rat retina during post-natal development and in retinal precursor cells obtained from neonatal rats and cultured for 3 days in serum-free medium. Whereas the TH mRNA concentration remains consistantly low in control cultures, treatment with cAMP-increasing agents (forskolin, membrane depolarization, phosphodiesterase inhibitors) is sufficient to raise it to the level observed in adult retina (15-fold increase). Treatment of the cultured cells can be delayed by up to 2 days with identical results at the TH mRNA level, thus ruling out a survival-promoting effect of cAMP. TH immunofluorescence has confirmed cAMP-dependent regulation of TH expression at the protein level and indicates that the frequency of TH-positive cells in the cultures is similar to that observed in the adult retina. Selective phosphodiesterase inhibitors suggest that PDE4 is the major subtype involved in the dopaminergic amacrine cell response. Our data clearly establish the cAMP-dependent regulation of TH mRNA and immunofluorescence levels in retinal precursor cells. The possible role of this regulation mechanism in the developmental activation of TH gene expression is discussed.

  18. RACK1 downregulates levels of the pro-apoptotic protein Fem1b in apoptosis-resistant colon cancer cells.

    Science.gov (United States)

    Subauste, M Cecilia; Ventura-Holman, Tereza; Du, Liqin; Subauste, Jose S; Chan, Shing-Leng; Yu, Victor C; Maher, Joseph F

    2009-12-01

    Evasion of apoptosis plays an important role in colon cancer progression. Following loss of the Apc tumor suppressor gene in mice, the gene encoding Fem1b is upregulated early in neoplastic intestinal epithelium. Fem1b is a pro-apoptotic protein that interacts with Fas, TNFR1 and Apaf-1, and increased expression of Fem1b induces apoptosis of cancer cells. Fem1b is a homolog of FEM-1, a protein in Caenorhabditis elegans that is negatively regulated by ubiquitination and proteasomal degradation. To study Fem1b regulation in colon cancer progression, we used apoptotis-sensitive SW480 cells, derived from a primary colon cancer, and their isogenic, apoptosis-resistant counterparts SW620 cells, derived from a subsequent metastatic lesion in the same patient. Treatment with proteasome inhibitor increased Fem1b protein levels in SW620 cells, but not in SW480 cells. In SW620 cells we found that endogenous Fem1b co-immunoprecipitates in complexes with RACK1, a protein known to mediate ubiquitination and proteasomal degradation of other pro-apoptotic proteins and to be upregulated in colon cancer. Full-length Fem1b, or the N-terminal region of Fem1b, associated with RACK1 when co-expressed in HEK293T cells, and RACK1 stimulated ubiquitination of Fem1b. RACK1 overexpression in SW620 cells led to downregulation of Fem1b protein levels. Conversely, downregulation of RACK1 led to upregulation of Fem1b protein levels, associated with induction of apoptosis, and this apoptosis was inhibited by blocking Fem1b protein upregulation. In conclusion, RACK1 downregulates levels of the pro-apoptotic protein Fem1b in metastatic, apoptosis-resistant colon cancer cells, which may promote apoptosis-resistance during progression of colon cancer.

  19. Resolution of Conflicting Signals at the Single-Cell Level in the Regulation of Cyanobacterial Photosynthesis and Nitrogen Fixation

    Science.gov (United States)

    Mohr, Wiebke; Vagner, Tomas; Kuypers, Marcel M. M.; Ackermann, Martin; LaRoche, Julie

    2013-01-01

    Unicellular, diazotrophic cyanobacteria temporally separate dinitrogen (N2) fixation and photosynthesis to prevent inactivation of the nitrogenase by oxygen. This temporal segregation is regulated by a circadian clock with oscillating activities of N2 fixation in the dark and photosynthesis in the light. On the population level, this separation is not always complete, since the two processes can overlap during transitions from dark to light. How do single cells avoid inactivation of nitrogenase during these periods? One possibility is that phenotypic heterogeneity in populations leads to segregation of the two processes. Here, we measured N2 fixation and photosynthesis of individual cells using nanometer-scale secondary ion mass spectrometry (nanoSIMS) to assess both processes in a culture of the unicellular, diazotrophic cyanobacterium Crocosphaera watsonii during a dark-light and a continuous light phase. We compared single-cell rates with bulk rates and gene expression profiles. During the regular dark and light phases, C. watsonii exhibited the temporal segregation of N2 fixation and photosynthesis commonly observed. However, N2 fixation and photosynthesis were concurrently measurable at the population level during the subjective dark phase in which cells were kept in the light rather than returned to the expected dark phase. At the single-cell level, though, cells discriminated against either one of the two processes. Cells that showed high levels of photosynthesis had low nitrogen fixing activities, and vice versa. These results suggest that, under ambiguous environmental signals, single cells discriminate against either photosynthesis or nitrogen fixation, and thereby might reduce costs associated with running incompatible processes in the same cell. PMID:23805199

  20. Human induced pluripotent cells resemble embryonic stem cells demonstrating enhanced levels of DNA repair and efficacy of nonhomologous end-joining

    Energy Technology Data Exchange (ETDEWEB)

    Fan Jinshui; Robert, Carine [Department of Radiation Oncology, University of Maryland School of Medicine, 655 West Baltimore Street, BRB 7-023A, Baltimore, MD 21201 (United States); Jang, Yoon-Young; Liu Hua; Sharkis, Saul; Baylin, Stephen Bruce [Johns Hopkins University School of Medicine, Department of Oncology, Baltimore, MD 21231-1000 (United States); Rassool, Feyruz Virgilia, E-mail: frassool@som.umaryland.edu [Department of Radiation Oncology, University of Maryland School of Medicine, 655 West Baltimore Street, BRB 7-023A, Baltimore, MD 21201 (United States)

    2011-08-01

    Highlights: {yields} iPSC and hESC demonstrate a similar cell cycle profile, with increased S phase cells and decreased G0/G1. {yields} iPSC and hESC increased ROS and decreased DSBs, compared with differentiated parental cells. {yields} iPSC and hESC demonstrate elevated DSB repair activity, including nonhomologous end-joining, compared with differentiated parental cells. {yields} iPSC however show a partial apoptotic response to DNA damage, compared to hESC. {yields} DNA damage responses may constitute important markers for the efficacy of iPSC reprogramming. - Abstract: To maintain the integrity of the organism, embryonic stem cells (ESC) need to maintain their genomic integrity in response to DNA damage. DNA double strand breaks (DSBs) are one of the most lethal forms of DNA damage and can have disastrous consequences if not repaired correctly, leading to cell death, genomic instability and cancer. How human ESC (hESC) maintain genomic integrity in response to agents that cause DSBs is relatively unclear. Adult somatic cells can be induced to 'dedifferentiate' into induced pluripotent stem cells (iPSC) and reprogram into cells of all three germ layers. Whether iPSC have reprogrammed the DNA damage response is a critical question in regenerative medicine. Here, we show that hESC demonstrate high levels of endogenous reactive oxygen species (ROS) which can contribute to DNA damage and may arise from high levels of metabolic activity. To potentially counter genomic instability caused by DNA damage, we find that hESC employ two strategies: First, these cells have enhanced levels of DNA repair proteins, including those involved in repair of DSBs, and they demonstrate elevated nonhomologous end-joining (NHEJ) activity and repair efficacy, one of the main pathways for repairing DSBs. Second, they are hypersensitive to DNA damaging agents, as evidenced by a high level of apoptosis upon irradiation. Importantly, iPSC, unlike the parent cells they are derived

  1. Effect of DHA supplementation on oxylipin levels in plasma and immune cell stimulated blood.

    Science.gov (United States)

    Schuchardt, Jan Philipp; Ostermann, Annika I; Stork, Lisa; Fritzsch, Sabrina; Kohrs, Heike; Greupner, Theresa; Hahn, Andreas; Schebb, Nils Helge

    2017-06-01

    EPA and DHA cause different physiological effects, which are in many cases mediated via their oxidative metabolites (oxylipins). However, metabolism studies investigating the effect of either EPA or DHA on comprehensive oxylipin patterns are lacking. The short and long term (1, 3, 6, and 12 week) effect of 1076mg/d DHA (free of EPA) on free (unesterified) oxylipin concentrations in plasma and lipopolysacharid (LPS) stimulated blood of 12 healthy men (mean age 25.1 ± 1.5 years) was investigated. After DHA supplementation, plasma levels of all DHA-oxylipins (HDHAs, EpDPEs, DiHDPEs) significantly increased (up to 600%) in a time-dependent fashion. Oxylipins of EPA and arachidonic acid (AA) were also affected. Whereas a slight increase in several EPA-derived hydroxy-FAs (including the RvE1 precursor 18-HEPE) and dihydroxy-FAs was observed after DHA supplementation, a trend to a slight decline in AA-derived oxylipin levels was found. In LPS stimulated blood, it is shown that DHA supplementation significantly reduces the ability of immune cells to form AA-derived COX (TXB2 and PGB2) and 12-LOX (12-HETE) eicosanoids. While no increase in EPA COX metabolites was found, n-3 PUFA 12-LOX metabolites of EPA (12-HEPE) and DHA (14-HDHA) were highly induced. We demonstrated that DHA supplementation causes a time-dependent shift in the entire oxylipin profile suggesting a cross-linked metabolism of PUFAs and subsequent formation of oxygenated lipid mediators. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Saliva CA125 and TPS levels in patients with oral squamous cell carcinoma.

    Science.gov (United States)

    Geng, Xue-fei; Du, Meng; Han, Jing-xiu; Zhang, Min; Tang, Xiao-fei; Xing, Ru-dong

    2013-01-01

    To determine the levels of carbohydrate antigen 125 (CA125) and tissue polypeptide-specific antigen (TPS) in saliva of patients with oral squamous cell carcinoma (OSCC) and patients with nonneoplastic disease of the oral cavity, and to investigate their diagnostic value and their relationship with pathological grade and clinical stage. A total of 97 hospitalized patients with OSCC, 36 patients with nonneoplastic disease of the oral cavity and 50 healthy individuals were included in this investigation. Mixed saliva was collected from these patients and the healthy controls before treatment. Saliva samples were analyzed by enzyme-linked immunosorbent assay (ELISA). The saliva CA125 and TPS concentrations were significantly higher in patients with OSCC than in patients with nonneoplastic disease and healthy controls, but not significantly different between patients with nonneoplastic disease and controls. Neither the saliva CA125 nor the TPS level was correlated with pathological grade and clinical stage. The sensitivity, specificity and accuracy of saliva CA125 and TPS for the diagnosis of OSCC were 80.0%, 66.0%, 75.7%, and 82.1%, 74.0%, 79.3%, respectively. When CA125 and TPS were analyzed independently, there was no significant difference in sensitivity, specificity and accuracy between the two markers. When CA125 and TPS were analyzed in combination, there was no significant difference in sensitivity, specificity and accuracy between independent detection and combined detection. The saliva CA125 and TPS concentrations were elevated in patients with OSCC. CA125 and TPS may prove to be useful tumor markers in OSCC.

  3. Energy level and thickness control on PEDOT:PSS layer for efficient planar heterojunction perovskite cells

    Science.gov (United States)

    Wang, Chunhua; Zhang, Chujun; Tong, Sichao; Xia, Huayan; Wang, Lijuan; Xie, Haipeng; Gao, Yongli; Yang, Junliang

    2018-01-01

    Efficient planar heterojunction perovskite solar cells (PHJ-PSCs) with an architecture of ITO/PEDOT:PSS/CH3NH3PbI3/PCBM/Al were fabricated by controlling the energy level and thickness of the PEDOT:PSS layer, where the PEDOT:PSS precursor was diluted with deionized water (H2O) and isopropyl alcohol (IPA), i.e. W-PEDOT:PSS and I-PEDOT:PSS. The performance parameters of the PHJ-PSCs showed soaring enhancement after employing W-PEDOT:PSS or I-PEDOT:PSS instead of pristine PEDOT:PSS (P-PEDOT:PSS), resulting in an increase of the power conversion efficiency (PCE) of W-PEDOT:PSS-based PHJ-PSCs to 15.60% from 11.95% for P-PEDOT:PSS-based PHJ-PSCs. The performance improvement results from two aspects. On the one hand, as compared to P-PEDOT:PSS, the occupied molecular orbital energy (HOMO) level of dilute PEDOT:PSS showed an impressive decrease and can well match the valence band of CH3NH3PbI3 film, resulting in less energy loss and a significant improvement in the open-circuit voltage (V oc). On the other hand, the dilute PEDOT:PSS could produce a thinner film as compared with the P-PEDOT:PSS, which also played an important role in the performance of the PHJ-PSCs. Furthermore, the electrochemical impedance spectroscopy (EIS) results indicated that the interface between perovskite and PEDOT:PSS was greatly improved by employing W-PEDOT:PSS or I-PEDOT:PSS, leading to an obvious decrease in the series resistance (R s) and an increase in the recombination resistance (R rec). The research demonstrated that diluting PEDOT:PSS with a common solvent, such as H2O and IPA, is a feasible low-temperature way of achieving efficient PHJ-PSCs.

  4. Smoking decreases the level of circulating CD34+ progenitor cells in young healthy women - a pilot study

    Directory of Open Access Journals (Sweden)

    Baumann Gert

    2010-05-01

    Full Text Available Abstract Background Decreased levels of circulating bone marrow-derived progenitor cells have been associated with risk factors and cardiovascular diseases. Smoking is the most important modifiable risk factor for atherosclerosis in young women. The aim of this pilot study was to assess in healthy premenopausal women without other risk factors for cardiovascular disease the influence of nicotine abuse on the number of circulating progenitor cells in relation to endothelial function. Methods The number of endothelial progenitor cells, measured as colony-forming units in a cell-culture assay (EPC-CFU and the number of circulating CD34 + and CD34 + /CD133 + cells, measured by flow cytometry, was estimated in 32 women at the menstrual phase of the menstrual cycle. In addition, flow-mediated dilation (FMD was assessed as a marker for vascular function. In a subgroup of these women (n = 20, progenitor cells were also investigated at the mid-follicular and luteal phases of the menstrual cycle. Results Compared to non-smokers, the abundance of circulating CD34 + cells was significantly lower in smoking women in the menstrual, mid-luteal, and mid-follicular phases of the menstrual cycle. The number of CD34 + progenitor cells was revealed to have significant positive correlation with FMD in young healthy women, whereas CD34 + /CD133 + progenitor cells and EPC-CFU showed no significant correlation. Conclusion The number of CD34 + progenitor cells positively correlates with FMD in young healthy women and is decreased by smoking.

  5. How the charge-neutrality level of interface states controls energy level alignment in cathode contacts of organic bulk-heterojunction solar cells.

    Science.gov (United States)

    Guerrero, Antonio; Marchesi, Luís F; Boix, Pablo P; Ruiz-Raga, Sonia; Ripolles-Sanchis, Teresa; Garcia-Belmonte, Germà; Bisquert, Juan

    2012-04-24

    Electronic equilibration at the metal-organic interface, leading to equalization of the Fermi levels, is a key process in organic optoelectronic devices. How the energy levels are set across the interface determines carrier extraction at the contact and also limits the achievable open-circuit voltage under illumination. Here, we report an extensive investigation of the cathode energy equilibration of organic bulk-heterojunction solar cells. We show that the potential to balance the mismatch between the cathode metal and the organic layer Fermi levels is divided into two contributions: spatially extended band bending in the organic bulk and voltage drop at the interface dipole layer caused by a net charge transfer. We scan the operation of the cathode under a varied set of conditions, using metals of different work functions in the range of ∼2 eV, different fullerene acceptors, and several cathode interlayers. The measurements allow us to locate the charge-neutrality level within the interface density of sates and calculate the corresponding dipole layer strength. The dipole layer withstands a large part of the total Fermi level mismatch when the polymer:fullerene blend ratio approaches ∼1:1, producing the practical alignment between the metal Fermi level and the charge-neutrality level. Origin of the interface states is linked with fullerene reduced molecules covering the metal contact. The dipole contribution, and consequently the band bending, is highly sensitive to the nature and amount of fullerene molecules forming the interface density of states. Our analysis provides a detailed picture of the evolution of the potentials in the bulk and the interface of the solar cell when forward voltage is applied or when photogeneration takes place.

  6. Cell-free DNA levels and correlation to stage and outcome following treatment of locally advanced rectal cancer.

    Science.gov (United States)

    Boysen, Anders Kindberg; Wettergren, Yvonne; Sorensen, Boe Sandahl; Taflin, Helena; Gustavson, Bengt; Spindler, Karen-Lise Garm

    2017-11-01

    Accurate staging of rectal cancer remains essential for optimal patient selection for combined modality treatment, including radiotherapy, chemotherapy and surgery. We aimed at examining the correlation of cell free DNA with the pathologic stage and subsequent risk of recurrence for patients with locally advanced rectal cancer undergoing preoperative chemoradiation. We examined 75 patients with locally advanced rectal cancer receiving preoperative chemoradiation. Blood samples for translational use were drawn prior to rectal surgery. The level of cell free DNA was quantified by digital droplet PCR and expressed as copy number of beta 2 microglobulin. We found a median level of cell free DNA in the AJCC stages I-III of 3100, 8300, and 10,700 copies/mL respectively. For patients with 12 sampled lymph nodes or above, the median level of cell free DNA were 2400 copies/mL and 4400 copies/mL (p = 0.04) for node negative and node positive disease respectively. The median follow-up was 39 months and 11 recurrences were detected (15%). The median level for patients with recurrent disease was 13,000 copies/mL compared to 5200 copies/mL for non-recurrent patients (p = 0.08). We have demonstrated a correlation between the level of total cell free DNA and the pathologic stage and nodal involvement. Furthermore, we have found a trend towards a correlation with the risk of recurrence following resection of localized rectal cancer.

  7. Cytokine levels correlate with immune cell infiltration after anti-VEGF therapy in preclinical mouse models of breast cancer.

    Directory of Open Access Journals (Sweden)

    Christina L Roland

    2009-11-01

    Full Text Available The effect of blocking VEGF activity in solid tumors extends beyond inhibition of angiogenesis. However, no studies have compared the effectiveness of mechanistically different anti-VEGF inhibitors with respect to changes in tumor growth and alterations in the tumor microenvironment. In this study we use three distinct breast cancer models, a MDA-MB-231 xenograft model, a 4T1 syngenic model, and a transgenic model using MMTV-PyMT mice, to explore the effects of various anti-VEGF therapies on tumor vasculature, immune cell infiltration, and cytokine levels. Tumor vasculature and immune cell infiltration were evaluated using immunohistochemistry. Cytokine levels were evaluated using ELISA and electrochemiluminescence. We found that blocking the activation of VEGF receptor resulted in changes in intra-tumoral cytokine levels, specifically IL-1beta, IL-6 and CXCL1. Modulation of the level these cytokines is important for controlling immune cell infiltration and ultimately tumor growth. Furthermore, we demonstrate that selective inhibition of VEGF binding to VEGFR2 with r84 is more effective at controlling tumor growth and inhibiting the infiltration of suppressive immune cells (MDSC, Treg, macrophages while increasing the mature dendritic cell fraction than other anti-VEGF strategies. In addition, we found that changes in serum IL-1beta and IL-6 levels correlated with response to therapy, identifying two possible biomarkers for assessing the effectiveness of anti-VEGF therapy in breast cancer patients.

  8. Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels

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    M. Ryan Smith

    2016-08-01

    Full Text Available Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP, decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231 breast adenocarcinoma cells up to 6 days after an initial 24 h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10 µM of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC protein levels, although other protein levels were

  9. A framework to quantify karyotype variation associated with CHO cell line instability at a single-cell level.

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    Baik, Jong Youn; Lee, Kelvin H

    2017-05-01

    Chinese hamster ovary (CHO) cells, the major mammalian host cells for biomanufacturing of therapeutic proteins, have been extensively investigated to enhance productivity and product quality. However, cell line instability resulting in unexpected changes in productivity or product quality continues to be a challenge. Based on previous reports about causes and characteristics of production instability, we hypothesized that chromosomal rearrangements due to genomic instability are associated with production instability and that these events can be characterized. We developed a production instability model using secreted alkaline phosphatase (SEAP)-expressing CHO cells (CHO-SEAP) as well as a framework to quantify chromosomal rearrangements by karyotyping. In the absence of methotrexate (MTX), CHO-SEAP cells exhibited a slightly increased growth rate, a significantly decreased specific productivity, and changes in the chromosomal rearrangement ratio of seven chromosomes. In contrast, when MTX was re-introduced, the growth rate and SEAP productivity reversed to the initial values, demonstrating the reversibility of production instability in CHO-SEAP cells. Fluorescence in situ hybridization analysis identified that the SEAP genes were incorporated in the chromosomal rearrangement (insertion) part of the der(Z9) chromosome. Karyotype analysis indicated that the insertion ratio of the der(Z9) chromosome decreased in the CHO-SEAP cells grown without MTX, demonstrating a correlation between chromosomal rearrangement and production instability. Our results support a mechanism for production instability, wherein a randomly generated chromosomal rearrangement (or genotype) results in cells with a growth advantage that is also associated with non (or low)-producing traits. As a result, the non-producing cells grow faster and thereby outgrow the producing population. Biotechnol. Bioeng. 2017;114: 1045-1053. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  10. Inverse associations between obesity indicators and thymic T-cell production levels in aging atomic-bomb survivors.

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    Kengo Yoshida

    Full Text Available Reduction of the naive T-cell population represents a deteriorating state in the immune system that occurs with advancing age. In animal model studies, obesity compromises the T-cell immune system as a result of enhanced adipogenesis in primary lymphoid organs and systemic inflammation. In this study, to test the hypothesis that obesity may contribute to the aging of human T-cell immunity, a thousand atomic-bomb survivors were examined for obesity status and ability to produce naive T cells, i.e., T-cell receptor excision circle (TREC numbers in CD4 and CD8 T cells. The number of TRECs showed a strong positive correlation with naive T cell numbers, and lower TREC numbers were associated with higher age. We found that the TREC number was inversely associated with levels of obesity indicators (BMI, hemoglobin A1c and serum CRP levels. Development of type-2 diabetes and fatty liver was also associated with lower TREC numbers. This population study suggests that obesity with enhanced inflammation is involved in aging of the human T-cell immune system. Given the fact that obesity increases the risk of numerous age-related diseases, attenuated immune competence is a possible mechanistic link between obesity and disease development among the elderly.

  11. Inverse associations between obesity indicators and thymic T-cell production levels in aging atomic-bomb survivors.

    Science.gov (United States)

    Yoshida, Kengo; Nakashima, Eiji; Kubo, Yoshiko; Yamaoka, Mika; Kajimura, Junko; Kyoizumi, Seishi; Hayashi, Tomonori; Ohishi, Waka; Kusunoki, Yoichiro

    2014-01-01

    Reduction of the naive T-cell population represents a deteriorating state in the immune system that occurs with advancing age. In animal model studies, obesity compromises the T-cell immune system as a result of enhanced adipogenesis in primary lymphoid organs and systemic inflammation. In this study, to test the hypothesis that obesity may contribute to the aging of human T-cell immunity, a thousand atomic-bomb survivors were examined for obesity status and ability to produce naive T cells, i.e., T-cell receptor excision circle (TREC) numbers in CD4 and CD8 T cells. The number of TRECs showed a strong positive correlation with naive T cell numbers, and lower TREC numbers were associated with higher age. We found that the TREC number was inversely associated with levels of obesity indicators (BMI, hemoglobin A1c) and serum CRP levels. Development of type-2 diabetes and fatty liver was also associated with lower TREC numbers. This population study suggests that obesity with enhanced inflammation is involved in aging of the human T-cell immune system. Given the fact that obesity increases the risk of numerous age-related diseases, attenuated immune competence is a possible mechanistic link between obesity and disease development among the elderly.

  12. Proteomic profiling of rabbit embryonic stem cells derived from parthenotes and fertilized embryos.

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    Payungsuk Intawicha

    Full Text Available Rabbit embryonic stem (rES cells can be derived from various sources of embryos. However, understanding of the gene expression profile, which distincts embryonic stem (ES cells from other cell types, is still extremely limited. In this study, we compared the protein profiles of three independent lines of rabbit cells, i.e., fibroblasts, fertilized embryo-derived stem (f-rES cells, and parthenote-derived ES (p-rES cells. Proteomic analyses were performed using two-dimensional gel electrophoresis (2-DE and mass spectrometry. Collectively, the expression levels of 100 out of 284 protein spots differed significantly among these three cell types (p<0.05. Of those differentially expressed spots, 91% were identified in the protein database and represented 63 distinct proteins. Proteins with known identities are mainly localized in the cytoplasmic compartments (48%, nucleus (14%, and cytoskeletal machineries (13%. These proteins were majorly involved in biological functions of energy and metabolic pathways (25%, cell growth and maintenance (25%, signal transduction (14%, and protein metabolisms (10%. When protein expression levels among cell types were compared, six proteins associated with a variety of cellular activities, including structural constituents of the cytoskeleton (tubulins, structural molecule (KRT8, catalytic molecules (α-enolase, receptor complex scaffold (14-3-3 protein sigma, microfilament motor proteins (Myosin-9, and heat shock protein (HSP60, were found highly expressed in p-rES cells. Two proteins related to HSP activity and structural constituent of cytoskeleton in f-rES cells, and one structural molecule activity protein in fibroblasts showed significantly higher expression levels (p<0.05. Marker protein expressions in f-rES and p-rES cells were further confirmed by Western blotting and immunocytochemical staining. This study demonstrated unique proteomic profiles of the three rabbit cell types and revealed some novel proteins

  13. Organ-level quorum sensing directs regeneration in hair stem cell populations

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    Chen, Chih-Chiang; Wang, Lei; Plikus, Maksim V.; Jiang, Ting Xin; Murray, Philip J.; Ramos, Raul; Guerrero-Juarez, Christian F.; Hughes, Michael W; Lee, Oscar K.; Shi, Songtao; Widelitz, Randall B.; Lander, Arthur D.; Chuong, Cheng Ming

    2015-01-01

    SUMMARY Coordinated organ behavior is crucial for an effective response to environmental stimuli. By studying regeneration of hair follicles in response to patterned hair removal, we demonstrate that organ-level quorum sensing allows coordinated responses to skin injury. Removing hair at different densities leads to a regeneration of up to 5 times more neighboring, unplucked resting hairs, indicating activation of a collective decision-making process. Through data modeling, the range of the quorum signal was estimated to be on the order of 1 mm, greater than expected for a diffusible molecular cue. Molecular and genetic analysis uncovered a two-step mechanism, where release of CCL2 from injured hairs leads to recruitment of TNF-α secreting macrophages, which accumulate and signal to both plucked and unplucked follicles. By coupling immune response with regeneration, this mechanism allows skin to respond predictively to distress, disregarding mild injury, while meeting stronger injury with full-scale cooperative activation of stem cells. PMID:25860610

  14. Educational level of patients with germ cell tumor radiated in childhood

    International Nuclear Information System (INIS)

    Nakamizo, Akira; Inamura, Takanori; Inoha, Satoshi; Nishio, Shunji; Ikezaki, Kiyonobu; Fukui, Masashi

    2001-01-01

    In order to estimate the influence of radiotherapy on the intellectual development of children with brain tumor, we investigated the educational level of 21 patients with germ cell tumor who had undergone radiotherapy. They were divided into three groups in accordance with their age at the time of radiation; under school age group (under 6 years of age), elementary school age group (from 7 to 12 years of age), and junior high and high school age group (from 13 to 18 years of age). There were 2 cases in the under school age group, one of them graduated from high school and the other is presently a junior high school student. There were 5 cases in the elementary school age group. Three of these graduated from university, 1 is presently a university student and 1 is a high school student. There were 14 cases in the junior high and high school age group. Two of these are university students, 7 graduated from high school, 1 is presently a junior high school student, and 4 died because of tumor progression. The mean period of hospitalization of the patients who have been admitted to university was 63.0 days, and that of patients who have not been admitted university was 135 days. There is a statistical difference (p<0.05). It could be concluded that the period of hospitalization rather than radiotherapy seemed to influence the educational status of children with brain tumor. (author)

  15. Mast cell mediator tryptase levels after inhalation or intravenous administration of high doses pharmaceutically prepared heroin

    NARCIS (Netherlands)

    Rook, E. J.; van Zanten, A. P.; van den Brink, W.; van Ree, J. M.; Beijnen, J. H.

    2006-01-01

    BACKGROUND: Opioids like morphine and heroin induce mast cell degranulation in vitro. The release of mast cell mediators like histamine and tryptase may lead to allergic symptoms. In this study it was investigated whether mast cell mediator release also occurs in vivo in addicted patients who

  16. Ethanol increases tumor necrosis factor-alpha receptor-1 (TNF-R1) levels in hepatic, intestinal, and cardiac cells.

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    Rodriguez, Diego A; Moncada, Claudio; Núñez, Marco T; Lavandero, Sergio; Ponnappa, Biddanda C; Israel, Yedy

    2004-05-01

    Chronic ethanol consumption leads to cell injury in virtually every tissue. Tumor necrosis factor-alpha (TNF-alpha) constitutes a major factor in the development of alcohol-induced liver injury. In alcohol-dependent subjects, elevated levels of plasma TNF-alpha are strongly predictive of mortality. Binding of TNF-alpha to TNF-alpha receptor-1 (TNF-R1) activates death domain pathways, leading to necrosis and apoptosis in most tissues, and it also increases the expression of intercellular adhesion molecules (i.e., ICAM-1), which promote inflammation. We determined whether ethanol exposure leads to increases in cellular TNF-R1. We incubated HepG2 human hepatoma cells and H4-II-E-C3 rat hepatoma cells with 25, 50, and 100 mM ethanol for various intervals of time up to 48 h. Human colonic adenocarcinoma cells (Caco-2 cells) and neonatal rat primary cardiomyocytes were also incubated with different concentrations of ethanol. Levels of TNF-R1 were measured either by a sandwich enzyme-linked immunosorbent assay (ELISA) method or by determining the extracellular transmembrane domain of TNF-R1 by an intact-cell ELISA method. Ethanol exposure for 48 h increased TNF-R1 levels in human hepatoma cells in a dose-dependent manner. Levels increased significantly by 164% at 50 mM and by 240% at 100 mM ethanol. Effects were time dependent and did not reach a plateau at 48 h. Similar increases in TNF-R1 were also observed in rat hepatoma cells (90% at 50 mM and 230% at 100 mM ethanol). Under similar conditions, Caco-2 cells showed a significant 80% increase in TNF-R1 levels at 200 mM ethanol, a concentration found in intestine. Neonatal rat primary cardiomyocytes showed TNF-R1 increases of 36% at 50 mM and 44% at 100 mM ethanol. These results indicate that exposure of different cell types to pharmacologic concentrations of ethanol increases TNF-R1 levels and may augment TNF-alpha-mediated cell injury in different tissues.

  17. Serum glial cell line-derived neurotrophic factor levels and postoperative cognitive dysfunction after surgery for rheumatic heart disease.

    Science.gov (United States)

    Duan, Xiaoxia; Zhu, Tao; Chen, Chan; Zhang, Guanpeng; Zhang, Junhui; Wang, Lin; Zhang, Luye; Wang, Maohua; Wang, Xiaobin

    2018-03-01

    Postoperative cognitive dysfunction is an important complication of cardiac surgery with poor outcomes. Serum glial cell line-derived neurotrophic factor levels are decreased in patients with Alzheimer's disease, but the association between glial cell line-derived neurotrophic factor levels and postoperative cognitive dysfunction is poorly understood. The present study aimed to investigate the prognostic value of postoperative serum glial cell line-derived neurotrophic factor levels to predict postoperative cognitive dysfunction in patients with rheumatic heart disease undergoing heart valve replacement. This was a prospective observational study of 80 patients undergoing elective heart valve replacement surgery from June 2015 to June 2016 at the Affiliated Hospital of Southeast Medical University. Cognitive functions were assessed 1 day before and 7 days after surgery. Serum glial cell line-derived neurotrophic factor levels were measured by an enzyme-linked immunosorbent assay before (T1) and 1 (T2), 2 (T3), and 7 (T4) days after surgery. Perioperative parameters were evaluated to assess the relationship between glial cell line-derived neurotrophic factors and postoperative cognitive dysfunction. Postoperative cognitive dysfunction was identified in 38 patients (47.5%) 7 days after surgery. Average glial cell line-derived neurotrophic factor levels at 2 and 7 days after surgery in the postoperative cognitive dysfunction group were lower than in the nonpostoperative cognitive dysfunction group at the same time points (P derived neurotrophic factor (T1-T3) and Δglial cell line-derived neurotrophic factor (T1-T4) were identified as good predictors of postoperative cognitive dysfunction with threshold for postoperative cognitive dysfunction detection of 49.10 and 60.90, respectively. The perioperative glial cell line-derived neurotrophic factor levels in patients with postoperative cognitive dysfunction were lower than in patients without postoperative

  18. Serum levels of leptin in Nigerian patients with sickle cell anaemia

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    Aina Ayoola O

    2011-05-01

    Full Text Available Abstract Background Several studies have shown that the pathophysiology of homozygous sickle cell anaemia (SCA results in a myriad of metabolic, nutritional, haematological and clinical effects that interact with other co-morbid factors to determine the quality of life and life expectancy of afflicted patients. Because of its critical roles in nutrition and metabolism, inflammation, haematopoiesis and cellular immunity, this study determined the plasma levels of leptin in steady and unsteady states of HbSS in Nigerian patients. Methods A total of 51 SCA patients aged 5 - 35 years with 34 (61.8% being females who were either on admission or visiting four medical centres in Lagos, Nigeria together with 22 non-SCD controls aged 5 -30 years comprising 12 (54.5% females were enrolled after obtaining their informed consent and ethical approval. Patients were further stratified into steady and unsteady cases of SCA based on clinical presentations, while blood samples collected by venipuncture from each of the study participants were analyzed haematologically for full blood count and HbF level and microscopically for malaria, while plasma leptin was assayed using ELISA method. Body composition defined by weight, fat mass and body mass index (BMI was determined using standard methods. Data obtained for cases and controls were analyzed statistically. Results Twenty - one patients had unsteady HbSS and elicited greater and significant (P P. falciparum parasitaemia (4613.7 vs. 749.6 - 1078.4 parasites/uL, pyrexia rate (58.3 vs. 25.8% when compared with steady state patients or non-SCD controls. Compared to the control, significant decreases in plasma leptin before and after controlling for body fat that was worsened by crisis were observed among the SCD patients. Unlike the non-SCD controls, leptin correlated non-significantly (P > 0.05 with all body composition indices measured in the patients except for fat mass in unsteady cases. Multivariate regression

  19. Deciphering Mineral Homeostasis in Barley Seed Transfer Cells at Transcriptional Level.

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    Behrooz Darbani

    Full Text Available In addition to the micronutrient inadequacy of staple crops for optimal human nutrition, a global downtrend in crop-quality has emerged from intensive breeding for yield. This trend will be aggravated by elevated levels of the greenhouse gas carbon dioxide. Therefore, crop biofortification is inevitable to ensure a sustainable supply of minerals to the large part of human population who is dietary dependent on staple crops. This requires a thorough understanding of plant-mineral interactions due to the complexity of mineral homeostasis. Employing RNA sequencing, we here communicate transfer cell specific effects of excess iron and zinc during grain filling in our model crop plant barley. Responding to alterations in mineral contents, we found a long range of different genes and transcripts. Among them, it is worth to highlight the auxin and ethylene signaling factors Arfs, Abcbs, Cand1, Hps4, Hac1, Ecr1, and Ctr1, diurnal fluctuation components Sdg2, Imb1, Lip1, and PhyC, retroelements, sulfur homeostasis components Amp1, Hmt3, Eil3, and Vip1, mineral trafficking components Med16, Cnnm4, Aha2, Clpc1, and Pcbps, and vacuole organization factors Ymr155W, RabG3F, Vps4, and Cbl3. Our analysis introduces new interactors and signifies a broad spectrum of regulatory levels from chromatin remodeling to intracellular protein sorting mechanisms active in the plant mineral homeostasis. The results highlight the importance of storage proteins in metal ion toxicity-resistance and chelation. Interestingly, the protein sorting and recycling factors Exoc7, Cdc1, Sec23A, and Rab11A contributed to the response as well as the polar distributors of metal-transporters ensuring the directional flow of minerals. Alternative isoform switching was found important for plant adaptation and occurred among transcripts coding for identical proteins as well as transcripts coding for protein isoforms. We also identified differences in the alternative-isoform preference between

  20. Deciphering Mineral Homeostasis in Barley Seed Transfer Cells at Transcriptional Level.

    Science.gov (United States)

    Darbani, Behrooz; Noeparvar, Shahin; Borg, Søren

    2015-01-01

    In addition to the micronutrient inadequacy of staple crops for optimal human nutrition, a global downtrend in crop-quality has emerged from intensive breeding for yield. This trend will be aggravated by elevated levels of the greenhouse gas carbon dioxide. Therefore, crop biofortification is inevitable to ensure a sustainable supply of minerals to the large part of human population who is dietary dependent on staple crops. This requires a thorough understanding of plant-mineral interactions due to the complexity of mineral homeostasis. Employing RNA sequencing, we here communicate transfer cell specific effects of excess iron and zinc during grain filling in our model crop plant barley. Responding to alterations in mineral contents, we found a long range of different genes and transcripts. Among them, it is worth to highlight the auxin and ethylene signaling factors Arfs, Abcbs, Cand1, Hps4, Hac1, Ecr1, and Ctr1, diurnal fluctuation components Sdg2, Imb1, Lip1, and PhyC, retroelements, sulfur homeostasis components Amp1, Hmt3, Eil3, and Vip1, mineral trafficking components Med16, Cnnm4, Aha2, Clpc1, and Pcbps, and vacuole organization factors Ymr155W, RabG3F, Vps4, and Cbl3. Our analysis introduces new interactors and signifies a broad spectrum of regulatory levels from chromatin remodeling to intracellular protein sorting mechanisms active in the plant mineral homeostasis. The results highlight the importance of storage proteins in metal ion toxicity-resistance and chelation. Interestingly, the protein sorting and recycling factors Exoc7, Cdc1, Sec23A, and Rab11A contributed to the response as well as the polar distributors of metal-transporters ensuring the directional flow of minerals. Alternative isoform switching was found important for plant adaptation and occurred among transcripts coding for identical proteins as well as transcripts coding for protein isoforms. We also identified differences in the alternative-isoform preference between the treatments