WorldWideScience

Sample records for cellular arginase activity

  1. Protein energy malnutrition increases arginase activity in monocytes and macrophages.

    Science.gov (United States)

    Corware, Karina; Yardley, Vanessa; Mack, Christopher; Schuster, Steffen; Al-Hassi, Hafid; Herath, Shanthi; Bergin, Philip; Modolell, Manuel; Munder, Markus; Müller, Ingrid; Kropf, Pascale

    2014-01-01

    Protein energy malnutrition is commonly associated with immune dysfunctions and is a major factor in susceptibility to infectious diseases. In this study, we evaluated the impact of protein energy malnutrition on the capacity of monocytes and macrophages to upregulate arginase, an enzyme associated with immunosuppression and increased pathogen replication. Our results show that monocytes and macrophages are significantly increased in the bone marrow and blood of mice fed on a protein low diet. No alteration in the capacity of bone marrow derived macrophages isolated from malnourished mice to phagocytose particles, to produce the microbicidal molecule nitric oxide and to kill intracellular Leishmania parasites was detected. However, macrophages and monocytes from malnourished mice express significantly more arginase both in vitro and in vivo. Using an experimental model of visceral leishmaniasis, we show that following protein energy malnutrition, the increased parasite burden measured in the spleen of these mice coincided with increased arginase activity and that macrophages provide a more permissive environment for parasite growth. Taken together, these results identify a novel mechanism in protein energy malnutrition that might contributes to increased susceptibility to infectious diseases by upregulating arginase activity in myeloid cells.

  2. Obesity-induced vascular inflammation involves elevated arginase activity.

    Science.gov (United States)

    Yao, Lin; Bhatta, Anil; Xu, Zhimin; Chen, Jijun; Toque, Haroldo A; Chen, Yongjun; Xu, Yimin; Bagi, Zsolt; Lucas, Rudolf; Huo, Yuqing; Caldwell, Ruth B; Caldwell, R William

    2017-11-01

    Obesity-induced vascular dysfunction involves pathological remodeling of the visceral adipose tissue (VAT) and increased inflammation. Our previous studies showed that arginase 1 (A1) in endothelial cells (ECs) is critically involved in obesity-induced vascular dysfunction. We tested the hypothesis that EC-A1 activity also drives obesity-related VAT remodeling and inflammation. Our studies utilized wild-type and EC-A1 knockout (KO) mice made obese by high-fat/high-sucrose (HFHS) diet. HFHS diet induced increases in body weight, fasting blood glucose, and VAT expansion. This was accompanied by increased arginase activity and A1 expression in vascular ECs and increased expression of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interleukin-10 (IL-10), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) mRNA and protein in both VAT and ECs. HFHS also markedly increased circulating inflammatory monocytes and VAT infiltration by inflammatory macrophages, while reducing reparative macrophages. Additionally, adipocyte size and fibrosis increased and capillary density decreased in VAT. These effects of HFHS, except for weight gain and hyperglycemia, were prevented or reduced in mice lacking EC-A1 or treated with the arginase inhibitor 2-( S )-amino-6-boronohexanoic acid (ABH). In mouse aortic ECs, exposure to high glucose (25 mM) and Na palmitate (200 μM) reduced nitric oxide production and increased A1, TNF-α, VCAM-1, ICAM-1, and MCP-1 mRNA, and monocyte adhesion. Knockout of EC-A1 or ABH prevented these effects. HFHS diet-induced VAT inflammation is mediated by EC-A1 expression/activity. Limiting arginase activity is a possible therapeutic means of controlling obesity-induced vascular and VAT inflammation.

  3. Arginase in Retinopathy

    Science.gov (United States)

    Narayanan, S. Priya; Rojas, Modesto; Suwanpradid, Jutamas; Toque, Haroldo A.; Caldwell, R. William; Caldwell, Ruth B.

    2013-01-01

    Ischemic retinopathies, such as diabetic retinopathy (DR), retinopathy of prematurity and retinal vein occlusion are a major cause of blindness in developed nations worldwide. Each of these conditions is associated with early neurovascular dysfunction. However, conventional therapies target clinically significant macula edema or neovascularization, which occur much later. Intraocular injections of anti-VEGF show promise in reducing retinal edema, but the effects are usually transient and the need for repeated injections increases the risk of intraocular infection. Laser photocoagulation can control pathological neovascularization, but may impair vision and in some patients the retinopathy continues to progress. Moreover, neither treatment targets early stage disease or promotes repair. This review examines the potential role of the ureahydrolase enzyme arginase as a therapeutic target for the treatment of ischemic retinopathy. Arginase metabolizes L-arginine to form proline, polyamines and glutamate. Excessive arginase activity reduces the L-arginine supply for nitric oxide synthase (NOS), causing it to become uncoupled and produce superoxide and less NO. Superoxide and NO react and form the toxic oxidant peroxynitrite. The catabolic products of polyamine oxidation and glutamate can induce more oxidative stress and DNA damage, both of which can cause cellular injury. Studies indicate that neurovascular injury during retinopathy is associated with increased arginase expression/activity, decreased NO, polyamine oxidation, formation of superoxide and peroxynitrite and dysfunction and injury of both vascular and neural cells. Furthermore, data indicate that the cytosolic isoform arginase I (AI) is involved in hyperglycemia-induced dysfunction and injury of vascular endothelial cells whereas the mitochondrial isoform arginase II (AII) is involved in neurovascular dysfunction and death following hyperoxia exposure. Thus, we postulate that activation of the arginase

  4. Arginase activity of Leishmania isolated from patients with cutaneous leishmaniasis.

    Science.gov (United States)

    Badirzadeh, A; Taheri, T; Abedi-Astaneh, F; Taslimi, Y; Abdossamadi, Z; Montakhab-Yeganeh, H; Aghashahi, M; Niyyati, M; Rafati, S

    2017-09-01

    Cutaneous leishmaniasis (CL) is one of the most important vector-borne parasitic diseases, highly endemic in Iran, and its prevalence is increasing all over the country. Arginase (ARG) activity in isolated Leishmania parasites from CL patients is yet to be explored. This study aimed to compare the ARG activity of isolated Leishmania promastigotes from CL patients with a standard strain of Leishmania major and its influences on the disease pathogenesis. We recruited 16 confirmed CL patients from Qom Province, in central Iran; after detection of Leishmania species using PCR-RFLP, we assessed the levels of ARG in the isolated promastigotes and determined the parasites' growth rate. Only L. major was identified from CL patients. The level of ARG activity in the isolated Leishmania promastigotes from CL patients was significantly higher than that obtained from the standard strain of L. major. No significant correlations between ARG activity and lesion size, number or duration were observed; in contrast, a significant negative correlation was seen between ARG level and Leishmania' growth rate. The obtained results suggest that increased ARG expression and activity in the isolated Leishmania promastigotes might contribute to the higher parasite infectivity and play a major role in the pathogenicity of the CL. © 2017 John Wiley & Sons Ltd.

  5. Expression and activity of arginase isoenzymes during normal and diabetes-impaired skin repair.

    Science.gov (United States)

    Kämpfer, Heiko; Pfeilschifter, Josef; Frank, Stefan

    2003-12-01

    Within the past years, an important role for nitric oxide (NO) in skin repair has been well defined. As NO is synthesized from L-arginine by NO synthases (NOS), the availability of L-arginine might be one rate-limiting factor of NO production at the wound site. Upon injury, arginase-1 and -2 mRNA, protein, and activity were strongly induced reaching a maximum between day 3 and day 7 postwounding. Immunohistochemistry colocalized both arginases and the inducible NOS (iNOS) at epithelial sites at the margins of the wound. Notably, diabetes-impaired skin repair in leptin-deficient mice (diabetes/diabetes, db/db; and obese/obese, ob/ob) was characterized by an abnormally elevated arginase activity in wound tissue in the absence of an expression of iNOS. Expression analyses demonstrated that arginase-1 contributed to increased arginase activities in impaired repair. Interestingly, an improved healing of chronic wound situations in leptin-supplemented ob/ob mice was strongly associated with an adjustment of the dysregulated expression of L-arginine-converting enzymes: an attenuated iNOS expression was upregulated early in repair and an augmented arginase-1 expression and activity was downregulated in the presence of markedly elevated numbers of macrophages during late repair. These data suggest a coordinated consumption of L-arginine by the NOS and arginase enzymatic pathways at the wound site as a prerequisite for a balanced NO (via iNOS) and polyamine (via arginases) synthesis that drives a normal skin repair.

  6. NOX2-Induced Activation of Arginase and Diabetes-Induced Retinal Endothelial Cell Senescence

    Directory of Open Access Journals (Sweden)

    Modesto Rojas

    2017-06-01

    Full Text Available Increases in reactive oxygen species (ROS and decreases in nitric oxide (NO have been linked to vascular dysfunction during diabetic retinopathy (DR. Diabetes can reduce NO by increasing ROS and by increasing activity of arginase, which competes with nitric oxide synthase (NOS for their commons substrate l-arginine. Increased ROS and decreased NO can cause premature endothelial cell (EC senescence leading to defective vascular repair. We have previously demonstrated the involvement of NADPH oxidase 2 (NOX2-derived ROS, decreased NO and overactive arginase in DR. Here, we investigated their impact on diabetes-induced EC senescence. Studies using diabetic mice and retinal ECs treated with high glucose or H2O2 showed that increases in ROS formation, elevated arginase expression and activity, and decreased NO formation led to premature EC senescence. NOX2 blockade or arginase inhibition prevented these effects. EC senescence was also increased by inhibition of NOS activity and this was prevented by treatment with a NO donor. These results indicate that diabetes/high glucose-induced activation of arginase and decreases in NO bioavailability accelerate EC senescence. NOX2-generated ROS contribute importantly to this process. Blockade of NOX2 or arginase represents a strategy to prevent diabetes-induced premature EC senescence by preserving NO bioavailability.

  7. Neuroblastoma arginase activity creates an immunosuppressive microenvironment that impairs autologous and engineered immunity

    Science.gov (United States)

    Mussai, Francis; Egan, Sharon; Hunter, Stuart; Webber, Hannah; Fisher, Jonathan; Wheat, Rachel; McConville, Carmel; Sbirkov, Yordan; Wheeler, Kate; Bendle, Gavin; Petrie, Kevin; Anderson, John; Chesler, Louis; De Santo, Carmela

    2015-01-01

    Neuroblastoma is the most common extra cranial solid tumour of childhood, and survival remains poor for patients with advanced disease. Novel immune therapies are currently in development, but clinical outcomes have not matched preclinical results. Here, we describe key mechanisms in which neuroblastoma inhibits the immune response. We show that murine and human neuroblastoma tumour cells suppress T cell proliferation, through increased arginase activity. Arginase II is the predominant isoform expressed and creates an arginine deplete local and systemic microenvironment. Neuroblastoma arginase activity results in inhibition of myeloid cell activation and suppression of bone marrow CD34+ progenitor proliferation. Finally we demonstrate that the arginase activity of neuroblastoma impairs NY-ESO-1 specific TCR and GD2-specific CAR engineered T cell proliferation and cytotoxicity. High arginase II expression correlates with poor survival for neuroblastoma patients. The results support the hypothesis that neuroblastoma creates an arginase-dependent immunosuppressive microenvironment in both the tumour and blood that leads to impaired immune surveillance and sub-optimal efficacy of immunotherapeutic approaches. PMID:26054597

  8. Arginase induction and activation during ischemia and reperfusion and functional consequences for the heart

    Directory of Open Access Journals (Sweden)

    Klaus-Dieter eSchlüter

    2015-03-01

    Full Text Available Induction and activation of arginase is among the fastest responses of the heart to ischemic events. Induction of arginase expression and enzyme activation under ischemic conditions shifts arginine consumption from nitric oxide formation (NO to the formation of ornithine and urea. In the heart such a switch in substrate utilisation reduces the impact of the NO/cGMP-pathway on cardiac function that requires intact electromechanical coupling but at the same time it induces ornithine-dependent pathways such as the polyamine metabolism. Both effects significantly reduce the recovery of heart function during reperfusion and thereby limits the success of reperfusion strategies. In this context, changes in arginine consumption trigger cardiac remodelling in an unfavourable way and increases the risk of arrhythmia, specifically in the initial post-ischemic period in which arginase activity is dominating. However, during the entire ischemic period arginase activation might be a meaningful adaptation that is specifically relevant for reperfusion following prolonged ischemic periods. Therefore, a precise understanding about the underlying mechanism that leads to arginase induction as well as of it’s mechanistic impact on post-ischemic hearts is required for optimizing reperfusion strategies. In this review we will summarize our current understanding of these processes and give an outlook about possible treatment options for the future.

  9. Local increase of arginase activity in lesions of patients with cutaneous leishmaniasis in Ethiopia.

    Directory of Open Access Journals (Sweden)

    Tamrat Abebe

    Full Text Available Cutaneous leishmaniasis is a vector-borne disease that is in Ethiopia mainly caused by the parasite Leishmania aethiopica. This neglected tropical disease is common in rural areas and causes serious morbidity. Persistent nonhealing cutaneous leishmaniasis has been associated with poor T cell mediated responses; however, the underlying mechanisms are not well understood.We have recently shown in an experimental model of cutaneous leishmaniasis that arginase-induced L-arginine metabolism suppresses antigen-specific T cell responses at the site of pathology, but not in the periphery. To test whether these results translate to human disease, we recruited patients presenting with localized lesions of cutaneous leishmaniasis and assessed the levels of arginase activity in cells isolated from peripheral blood and from skin biopsies. Arginase activity was similar in peripheral blood mononuclear cells (PBMCs from patients and healthy controls. In sharp contrast, arginase activity was significantly increased in lesion biopsies of patients with localized cutaneous leishmaniasis as compared with controls. Furthermore, we found that the expression levels of CD3ζ, CD4 and CD8 molecules were considerably lower at the site of pathology as compared to those observed in paired PBMCs.Our results suggest that increased arginase in lesions of patients with cutaneous leishmaniasis might play a role in the pathogenesis of the disease by impairing T cell effector functions.

  10. A Trypanosoma brucei kinesin heavy chain promotes parasite growth by triggering host arginase activity.

    Directory of Open Access Journals (Sweden)

    Géraldine De Muylder

    2013-10-01

    Full Text Available In order to promote infection, the blood-borne parasite Trypanosoma brucei releases factors that upregulate arginase expression and activity in myeloid cells.By screening a cDNA library of T. brucei with an antibody neutralizing the arginase-inducing activity of parasite released factors, we identified a Kinesin Heavy Chain isoform, termed TbKHC1, as responsible for this effect. Following interaction with mouse myeloid cells, natural or recombinant TbKHC1 triggered SIGN-R1 receptor-dependent induction of IL-10 production, resulting in arginase-1 activation concomitant with reduction of nitric oxide (NO synthase activity. This TbKHC1 activity was IL-4Rα-independent and did not mirror M2 activation of myeloid cells. As compared to wild-type T. brucei, infection by TbKHC1 KO parasites was characterized by strongly reduced parasitaemia and prolonged host survival time. By treating infected mice with ornithine or with NO synthase inhibitor, we observed that during the first wave of parasitaemia the parasite growth-promoting effect of TbKHC1-mediated arginase activation resulted more from increased polyamine production than from reduction of NO synthesis. In late stage infection, TbKHC1-mediated reduction of NO synthesis appeared to contribute to liver damage linked to shortening of host survival time.A kinesin heavy chain released by T. brucei induces IL-10 and arginase-1 through SIGN-R1 signaling in myeloid cells, which promotes early trypanosome growth and favors parasite settlement in the host. Moreover, in the late stage of infection, the inhibition of NO synthesis by TbKHC1 contributes to liver pathogenicity.

  11. Long-term dietary restriction up-regulates activity and expression of renal arginase II in aging mice.

    Science.gov (United States)

    Majaw, T; Sharma, R

    2017-06-01

    Arginase II is a mitochondrial enzyme that catalyses the hydrolysis of L-arginine into urea and ornithine. It is present in other extra-hepatic tissues that lack urea cycle. Therefore, it is plausible that arginase II has a physiological role other than urea cycle which includes polyamine, proline, glutamate synthesis and regulation of nitric oxide production. The high expression of arginase II in kidney, among extrahepatic tissues, might have an important role associated with kidney functions. The present study is aimed to determine the age-associated alteration in the activity and expression of arginase II in the kidney of mice of different ages. The effect of dietary restriction to modulate the agedependent changes of arginase II was also studied. Results showed that renal arginase II activity declines significantly with the progression of age (p less than 0.01 and p less than 0.001 in 6- and 18-month-old mice, respectively as compared to 2-month old mice) and is due to the reduction in its protein as well as the mRNA level (p less than 0.001 in both 6- and 18-month-old mice as compared to 2-month-old mice). Long-term dietary restriction for three months has significantly up-regulated arginase II activity and expression level in both 2- and 18-month-old mice (p less than 0.01 and p less than 0.001, respectively as compared to AL group). These findings clearly indicate that the reducing level of arginase II during aging might have an impact on the declining renal functions. This age-dependent down-regulation of arginase II in the kidney can be attenuated by dietary restriction which may help in the maintenance of such functions.

  12. Inhibition of herpes simplex virus multiplication by activated macrophages: a role for arginase?

    Science.gov (United States)

    Wildy, P; Gell, P G; Rhodes, J; Newton, A

    1982-07-01

    Proteose-peptone-activated mouse macrophages can prevent productive infection by herpes simplex virus in neighboring cells in vitro whether or not those cells belong to the same animal species. The effect does not require contact between the macrophages and the infected cells, may be prevented by adding extra arginine to the medium, and may be reversed when extra arginine is added 24 h after the macrophages. Arginase activity was found both intracellularly and released from the macrophages. The extracellular enzyme is quite stable; 64% activity was found after 48 h of incubation at 37 degrees C in tissue culture medium. No evidence was found that the inefficiency of virus replication in macrophages was due to self-starvation by arginase. As might be predicted macrophages can, by the same mechanism, limit productive infection by vaccinia virus.

  13. Effect of Two Ginger Varieties on Arginase Activity in Hypercholesterolemic Rats.

    Science.gov (United States)

    Akinyemi, Ayodele Jacob; Oboh, Ganiyu; Ademiluyi, Adedayo Oluwaseun; Boligon, Aline Augusti; Athayde, Margareth Linde

    2016-04-01

    Recently, ginger has been used in traditional Chinese medicine as an herbal therapy for treating several cardiovascular diseases, however, information on its mechanism of action is limited. The present study assessed the effect of two ginger varieties (Zingiber officinale and Curcuma longa) on the arginase activity, atherogenic index, levels of liver thiobarbituric acid reactive substances (TBARSs), and plasma lipids in rats fed with a high-cholesterol (2%) diet for 14 days. Following the treatment period, it was found that feeding a high-cholesterol diet to rats caused significant (p ginger and turmeric (2% and 4%) caused significant (p ginger and turmeric) inhibited arginase activity and prevented hypercholesterolemia in rats that received a high-cholesterol diet. Therefore, these activities of ginger and turmeric represent possible mechanisms underlying its use in herbal medicine to treat several cardiovascular diseases. Copyright © 2015. Published by Elsevier B.V.

  14. ASOTHEMIA EFFECT UPON THE LIVER ARGINASE ACTIVITY IN THE ACUTE KIDNEY DAMAGE

    Directory of Open Access Journals (Sweden)

    Jelena Djordjevic

    2002-07-01

    Full Text Available The acute damage of the kidney function leads to an outstanding disbalance of many homeostatic mechanisms in the organism that emerges as a consequence of the reduced glomerulic filtration and the accompanying oliguria. This conditions the emergence of asothemia, that is, the state caracterized by an increase of the level of urea, creatinine and other ureic toxins in the blood. The results of the previous exami-nations show that the acute renal insufficiency is a disturbance accompanied with ac-celerated protein catabolism. The urea is a terminal product of the protein catabolism whose synthesis is mainly taking place in the liver; that is why the research aimed at examining the liver arginase activity, terminal enzyme in the urea synthesis cycle in various experimental models of the acute renal insufficiency. The acute asothemia is experimentally caused upon the male Spraque Dawlly rats by means of two models, namely, the model of bilateral binding of the urethra (BPU and the clycerolic model. The arginase activity in the liver tissue homogenate is measured by the Porembsky and Cedra method on the basis of the liberated ornithine liberation. In the plasma of the experimental animals the level of urea and creatinine was measured for the sake of estimating the renal function. In both the models of the acute kidney damage there was a considerable increase of the urea and creatinine concentration in the plasma (p<0,001 which is followed by a significant increase of the hepatic arginase activity with respect to the control group of the animals. On the basis of the obtained results it can be conclude that asothemia in the acute renal insufficiency is followed by an in-crease in the liver arginase activity.

  15. Arginase activity in peripheral blood of patients with intestinal schistosomiasis, Wonji, Central Ethiopia.

    Science.gov (United States)

    Getaneh, A; Tamrat, A; Tadesse, K

    2015-07-01

    Morbidity and mortality caused by schistosomiasis usually results from immunopathology. But the underlying mechanisms are not yet clearly understood. Th2-type immune response is thought to be dominant during chronic schistosomiasis, and upregulation of arginase-I is one component of this milieu. A cohort study was conducted to assess arginase activity in peripheral blood of humans with intestinal schistosomiasis in Wonji-Shoa Sugar Estate, Central Ethiopia. Laboratory-confirmed 30 Schistosoma mansoni-infected patients and 18 apparently healthy controls were recruited. Faecal egg count was carried out by Kato-Katz technique. Plasma and peripheral blood mononuclear cells (PBMCs) were isolated from whole blood. Activity of arginase in plasma and PBMC lysates was measured, and results were compared with that of controls. Twenty-one of 30 patients had light infection, whereas moderate and heavy intensity infections were observed in eight and only one patient(s), respectively. A significant increase in both PBMC (patients: 59.96 + 82.99, controls: 25.44 + 24.6 mU/mg protein, P intestinal schistosomiasis. © 2015 John Wiley & Sons Ltd.

  16. Multitracer Stable Isotope Quantification of Arginase and Nitric Oxide Synthase Activity in a Mouse Model of Pseudomonas Lung Infection

    Directory of Open Access Journals (Sweden)

    Hartmut Grasemann

    2014-01-01

    Full Text Available Cystic fibrosis airways are deficient for L-arginine, a substrate for nitric oxide synthases (NOSs and arginases. The rationale for this study was to quantify NOS and arginase activity in the mouse lung. Anesthetized unventilated mice received a primed constant stable isotope intravenous infusion containing labeled L-arginine, ornithine, and citrulline. The isotopic enrichment of each of the infused isotopomers and its product amino acids were measured in plasma and organ homogenates using liquid chromatography-tandem mass spectrometry. The effect of infection was studied three days after direct tracheal instillation of Pseudomonas-coated agar beads. In the infusion model, lung infection resulted in a significant (28-fold increase in NOS activity in lung but not in trachea, kidney, liver, or plasma. Absolute rates of arginase activity in solid tissues could not be calculated in this model. In an isolated lung perfusion model used for comparison increased NOS activity in infected lungs was confirmed (28.5-fold and lung arginase activity was increased 9.7-fold. The activity of L-arginine metabolizing enzymes can be measured using stable isotope conversion in the mouse. Accumulation of L-ornithine in the whole mouse model hindered the exact quantification of arginase activity in the lung, a problem that was overcome utilizing an isolated lung perfusion model.

  17. Increase of arginase activity in old apolipoprotein-E deficient mice under Western diet associated with changes in neurovascular unit

    Directory of Open Access Journals (Sweden)

    Badaut Jérôme

    2012-06-01

    Full Text Available Abstract Aging and atherosclerosis are well-recognized risk factors for cardiac and neurovascular diseases. The Apolipoprotein E deficient (ApoE−/− mouse on a high-fat diet is a classical model of atherosclerosis, characterized by the presence of atherosclerotic plaques in extracranial vessels but not in cerebral arteries. Increase in arginase activity was shown to participate in vascular dysfunction in the peripheral arteries of atherosclerotic mice by changing the level of nitric oxide (NO. NO plays a key role in the physiological functions of the neurovascular unit (NVU. However, the regulation of arginase expression and activity in the brain was never investigated in association with changes in the NVU, ApoE deficiency and high fat diet. Fourteen-month-old ApoE−/− mice on high-fat diet exhibited deposition of lipids in the NVU, impairment of blood–brain barrier properties, astrogliosis and an increase of aquaporin 4 staining. In association with these changes, brain arginase activity was significantly increased in the old ApoE−/− mice as compared to old wild type mice, with an increase in the level of arginase type I in the blood vessels. In conclusion, aging in this classical mouse model of atherosclerosis induces an increase in the level and activity of arginase I that may impair NO synthesis and contribute to changes in the NVU leading to blood–brain barrier leakage and inflammation.

  18. Inhibitory effect of mycoplasma-released arginase. Activity in mixed-lymphocyte and tumour cell cultures

    DEFF Research Database (Denmark)

    Claesson, M H; Tscherning, T; Nissen, Mogens Holst

    1990-01-01

    inhibition can be reversed by addition of excess arginine to the culture medium. Antisera raised against non-fermenting, but not against fermenting, mycoplasma species block the inhibitory effect of MAE. SDS-PAGE separation of MAE disclosed a broad band at 60 kDa which contained arginase activity when...... assayed in MLC and cell proliferation culture. SDS-PAGE followed by western blotting and reaction with antisera raised against non-fermenting mycoplasma species demonstrated a band at 43 kDa common for these micro-organisms....

  19. Curcumin inhibits adenosine deaminase and arginase activities in cadmium-induced renal toxicity in rat kidney

    Directory of Open Access Journals (Sweden)

    Ayodele Jacob Akinyemi

    2017-04-01

    Full Text Available In this study, the effect of enzymes involved in degradation of renal adenosine and l-arginine was investigated in rats exposed to cadmium (Cd and treated with curcumin, the principal active phytochemical in turmeric rhizome. Animals were divided into six groups (n = 6: saline/vehicle, saline/curcumin 12.5 mg/kg, saline/curcumin 25 mg/kg, Cd/vehicle, Cd/curcumin 12.5 mg/kg, and Cd/curcumin 25 mg/kg. The results of this study revealed that the activities of renal adenosine deaminase and arginase were significantly increased in Cd-treated rats when compared with the control (p < 0.05. However, co-treatment with curcumin inhibits the activities of these enzymes compared with Cd-treated rats. Furthermore, Cd intoxication increased the levels of some renal biomarkers (serum urea, creatinine, and electrolytes and malondialdehyde level with a concomitant decrease in functional sulfhydryl group and nitric oxide (NO. However, co-treatment with curcumin at 12.5 mg/kg and 25 mg/kg, respectively, increases the nonenzymatic antioxidant status and NO in the kidney, with a concomitant decrease in the levels of malondialdehyde and renal biomarkers. Therefore, our results reinforce the importance of adenosine deaminase and arginase activities in Cd poisoning conditions and suggest some possible mechanisms of action by which curcumin prevent Cd-induced renal toxicity in rats.

  20. Neutrophil degranulation and immunosuppression in patients with GBM: restoration of cellular immune function by targeting arginase I.

    Science.gov (United States)

    Sippel, Trisha R; White, Jason; Nag, Kamalika; Tsvankin, Vadim; Klaassen, Marci; Kleinschmidt-DeMasters, B K; Waziri, Allen

    2011-11-15

    The source of glioblastoma (GBM)-associated immunosuppression remains multifactorial. We sought to clarify and therapeutically target myeloid cell-derived peripheral immunosuppression in patients with GBM. Direct ex vivo T-cell function, serum Arginase I (ArgI) levels, and circulating myeloid lineage populations were compared between patients with GBM and normal donors or patients with other intracranial tumors. Immunofunctional assays were conducted using bulk and sorted cell populations to explore the potential transfer of myeloid cell-mediated immunosuppression and to identify a potential mechanism for these effects. ArgI-mediated immunosuppression was therapeutically targeted in vitro through pharmacologic inhibition or arginine supplementation. We identified a significantly expanded population of circulating, degranulated neutrophils associated with elevated levels of serum ArgI and decreased T-cell CD3ζ expression within peripheral blood from patients with GBM. Sorted CD11b(+) cells from patients with GBM were found to markedly suppress normal donor T-cell function in coculture, and media harvested from mitogen-stimulated GBM peripheral blood mononuclear cell (PBMC) or GBM-associated mixed lymphoid reactions showed ArgI levels that were significantly higher than controls. Critically, T-cell suppression in both settings could be completely reversed through pharmacologic ArgI inhibition or with arginine supplementation. These data indicate that peripheral cellular immunosuppression in patients with GBM is associated with neutrophil degranulation and elevated levels of circulating ArgI, and that T-cell function can be restored in these individuals by targeting ArgI. These data identify a novel pathway of GBM-mediated suppression of cellular immunity and offer a potential therapeutic window for improving antitumor immunity in affected patients.

  1. Th2-related immune responses by the Brucella abortus cellular antigens, malate dehydrogenase, elongation factor, and arginase.

    Science.gov (United States)

    Im, Young Bin; Shim, Soojin; Park, Woo Bin; Kim, Suk; Yoo, Han Sang

    2017-09-01

    Brucellosis is an important zoonotic disease caused by Brucella species. The disease is difficult to control due to the intracellular survival of the bacterium and the lack of precise understanding of pathogenesis. Despite of continuous researches on the pathogenesis of Brucella spp. infection, there is still question on the pathogenesis, especially earlier immune response in the bacterial infection. Malate dehydrogenase (MDH), elongation factor (Tsf), and arginase (RocF), which showed serological reactivity, were purified after gene cloning, and their immune modulating activities were then analyzed in a murine model. Cytokine production profiles were investigated by stimulating RAW 264.7 cells and naïve splenocytes with the three recombinant proteins. Also, immune responses were analyzed by ELISA and an ELIspot assay after immunizing mice with the three proteins. Only TNF-α was produced in stimulated RAW 264.7 cells, whereas Th1-related cytokines, IFN-γ and IL-2, were induced in naïve splenocytes. In contrast, Th2-type immune response was more strongly induced in antigen-secreting cells in the splenocytes obtained 28 days after immunizing mice with the three proteins, as were IgM and IgG. The induction of Th2-related antibody, IgG1, was higher than the Th1-related antibody, IgG2a, in immunized mice. These results suggest that the three proteins strongly induce Th2-type immune response in vivo, even though Th1-related cytokines were produced in vitro. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Arginase activity in pathogenic and non-pathogenic species of Leishmania parasites.

    Science.gov (United States)

    Badirzadeh, Alireza; Taheri, Tahereh; Taslimi, Yasaman; Abdossamadi, Zahra; Heidari-Kharaji, Maryam; Gholami, Elham; Sedaghat, Baharehsadat; Niyyati, Maryam; Rafati, Sima

    2017-07-01

    Proliferation of Leishmania (L.) parasites depends on polyamine availability, which can be generated by the L-arginine catabolism and the enzymatic activity of arginase (ARG) of the parasites and of the mammalian hosts. In the present study, we characterized and compared the arginase (arg) genes from pathogenic L. major and L. tropica and from non-pathogenic L. tarentolae. We quantified the level of the ARG activity in promastigotes and macrophages infected with pathogenic L. major and L. tropica and non-pathogenic L. tarentolae amastigotes. The ARG's amino acid sequences of the pathogenic and non-pathogenic Leishmania demonstrated virtually 98.6% and 88% identities with the reference L. major Friedlin ARG. Higher ARG activity was observed in all pathogenic promastigotes as compared to non-pathogenic L. tarentolae. In vitro infection of human macrophage cell line (THP1) with pathogenic and non-pathogenic Leishmania spp. resulted in increased ARG activities in the infected macrophages. The ARG activities present in vivo were assessed in susceptible BALB/c and resistant C57BL/6 mice infected with L. major, L. tropica and L. tarentolae. We demonstrated that during the development of the infection, ARG is induced in both strains of mice infected with pathogenic Leishmania. However, in L. major infected BALB/c mice, the induction of ARG and parasite load increased simultaneously according to the time course of infection, whereas in C57BL/6 mice, the enzyme is upregulated solely during the period of footpad swelling. In L. tropica infected mice, the footpads' swellings were slow to develop and demonstrated minimal cutaneous pathology and ARG activity. In contrast, ARG activity was undetectable in mice inoculated with the non-pathogenic L. tarentolae. Our data suggest that infection by Leishmania parasites can increase ARG activity of the host and provides essential polyamines for parasite salvage and its replication. Moreover, the ARG of Leishmania is vital for parasite

  3. Contribution of arginase to manganese metabolism of Aspergillus niger.

    Science.gov (United States)

    Keni, Sarita; Punekar, Narayan S

    2016-02-01

    Aspects of manganese metabolism during normal and acidogenic growth of Aspergillus niger were explored. Arginase from this fungus was a Mn[II]-enzyme. The contribution of the arginase protein towards A. niger manganese metabolism was investigated using arginase knockout (D-42) and arginase over-expressing (ΔXCA-29) strains of A. niger NCIM 565. The Mn[II] contents of various mycelial fractions were found in the order: D-42 strain niger mycelia harvested from acidogenic growth media contain substantially less Mn[II] as compared to those from normal growth media. Nevertheless, acidogenic mycelia harbor considerable Mn[II] levels and a functional arginase. Altered levels of mycelial arginase protein did not significantly influence citric acid production. The relevance of arginase to cellular Mn[II] pool and homeostasis was evaluated and the results suggest that arginase regulation could occur via manganese availability.

  4. ARGINASE 2 DEFICIENCY RESULTS IN SPONTANEOUS STEATOHEPATITIS: A NOVEL LINK BETWEEN INNATE IMMUNE ACTIVATION AND HEPATIC DE NOVO LIPOGENESIS

    Science.gov (United States)

    Navarro, Laura A.; Wree, Alexander; Povero, Davide; Berk, Michael P.; Eguchi, Akiko; Ghosh, Sudakshina; Papouchado, Bettina G.; Erzurum, Serpil C.; Feldstein, Ariel E.

    2016-01-01

    BACKGROUND & AIMS Innate immune activation has been postulated as a central mechanism for disease progression from hepatic steatosis to steatohepatitis in obesity-related fatty liver disease. Arginase 2 competes with inducible nitric oxide synthase (iNOS) for its substrate and the balance between these two enzymes plays a crucial role in regulating immune responses and macrophage activation. Our aim was to test the hypothesis that arginase 2 deficiency in mice favors progression from isolated hepatic steatosis, induced by high fat feeding to steatohepatitis. METHODS Arginase 2-knockout (Arg2−/−) mice were studied for changes in liver histology and metabolic phenotype at baseline and after a short term course (7 week) feeding with a high fat (HFAT) diet. In additional experiments, Arg2−/− mice received tail vein injections of liposome-encapsulated clodronate (CLOD) over a three-week period to selectively deplete liver macrophages. RESULTS Unexpectedly, Arg2−/− mice showed profound changes in their livers at baseline characterized by significant steatosis as demonstrated with histological and biochemical analysis. These changes were independent of systemic metabolic parameters and associated with marked increase mRNA levels of genes involved in hepatic de novo lipogenesis. Liver injury and inflammation were present with elevated serum ALT, marked infiltration of F4/80 positive cells, and increased mRNA levels of inflammatory genes. HFAT feeding exacerbated these changes. Macrophage depletion after CLOD injection significantly attenuated lipid deposition and normalized lipogenic mRNA profile of livers from Arg2−/− mice. CONCLUSIONS This study identifies arginase 2 as novel link between innate immune responses, hepatic lipid deposition, and liver injury. PMID:25234945

  5. Modulation of cholinergic airway reactivity and nitric oxide production by endogenous arginase activity

    NARCIS (Netherlands)

    Meurs, Herman; Hamer, M.A M; Pethe, S; Vadon-Le Goff, S; Boucher, J.-L; Zaagsma, Hans

    1 Cholinergic airway constriction is functionally antagonized by agonist-induced constitutive nitric oxide synthase (cNOS)-derived nitric oxide (NO). Since cNOS and arginase, which hydrolyzes L-arginine to L-ornithine and urea, use L-arginine as a common substrate, competition between both enzymes

  6. Role of arginase in vessel wall remodeling

    Directory of Open Access Journals (Sweden)

    William eDurante

    2013-05-01

    Full Text Available Arginase metabolizes the semi-essential amino acid L-arginine to L-ornithine and urea. There are two distinct isoforms of arginase, arginase I and II, which are encoded by separate genes and display differences in tissue distribution, subcellular localization, and molecular regulation. Blood vessels express both arginase I and II but their distribution appears to be cell-, vessel-, and species-specific. Both isoforms of arginase are induced by numerous pathologic stimuli and contribute to vascular cell dysfunction and vessel wall remodeling in several diseases. Clinical and experimental studies have documented increases in the expression and/or activity of arginase I or II in blood vessels following arterial injury and in pulmonary and arterial hypertension, aging, and atherosclerosis. Significantly, pharmacological inhibition or genetic ablation of arginase in animals ameliorates abnormalities in vascular cells and normalizes blood vessel architecture and function in all of these pathological states. The detrimental effect of arginase in vascular remodeling is attributable to its ability to stimulate vascular smooth muscle cell and endothelial cell proliferation, and collagen deposition by promoting the synthesis of polyamines and L-proline, respectively. In addition, arginase adversely impacts arterial remodeling by directing macrophages towards an inflammatory phenotype. Moreover, the proliferative, fibrotic, and inflammatory actions of arginase in the vasculature are further amplified by its capacity to inhibit nitric oxide synthesis by competing with nitric oxide synthase for substrate, L-arginine. Pharmacologic or molecular approaches targeting specific isoforms of arginase represent a promising strategy in treating obstructive fibroproliferative vascular disease.

  7. Diclofenac inhibits tumor growth in a murine model of pancreatic cancer by modulation of VEGF levels and arginase activity.

    Directory of Open Access Journals (Sweden)

    Nina Mayorek

    Full Text Available BACKGROUND: Diclofenac is one of the oldest anti-inflammatory drugs in use. In addition to its inhibition of cyclooxygenases (COX, diclofenac potently inhibits phospholipase A(2 (PLA(2, thus yielding a broad anti-inflammatory effect. Since inflammation is an important factor in the development of pancreatic tumors we explored the potential of diclofenac to inhibit tumor growth in mice inoculated with PANCO2 cells orthotopically. METHODOLOGY/PRINCIPAL FINDINGS: We found that diclofenac treatment (30 mg/kg/bw for 11 days of mice inoculated with PANC02 cells, reduced the tumor weight by 60%, correlating with increased apoptosis of tumor cells. Since this effect was not observed in vitro on cultured PANCO2 cells, we theorized that diclofenac beneficial treatment involved other mediators present in vivo. Indeed, diclofenac drastically decreased tumor vascularization by downregulating VEGF in the tumor and in abdominal cavity fluid. Furthermore, diclofenac directly inhibited vascular sprouting ex vivo. Surprisingly, in contrast to other COX-2 inhibitors, diclofenac increased arginase activity/arginase 1 protein content in tumor stroma cells, peritoneal macrophages and white blood cells by 2.4, 4.8 and 2 fold, respectively. We propose that the subsequent arginine depletion and decrease in NO levels, both in serum and peritoneal cavity, adds to tumor growth inhibition by malnourishment and poor vasculature development. CONCLUSION/SIGNIFICANCE: In conclusion, diclofenac shows pronounced antitumoral properties in pancreatic cancer model that can contribute to further treatment development. The ability of diclofenac to induce arginase activity in tumor stroma, peritoneal macrophages and white blood cells provides a tool to study a controversial issue of pro-and antitumoral effects of arginine depletion.

  8. Diclofenac inhibits tumor growth in a murine model of pancreatic cancer by modulation of VEGF levels and arginase activity.

    Science.gov (United States)

    Mayorek, Nina; Naftali-Shani, Nili; Grunewald, Myriam

    2010-09-15

    Diclofenac is one of the oldest anti-inflammatory drugs in use. In addition to its inhibition of cyclooxygenases (COX), diclofenac potently inhibits phospholipase A(2) (PLA(2)), thus yielding a broad anti-inflammatory effect. Since inflammation is an important factor in the development of pancreatic tumors we explored the potential of diclofenac to inhibit tumor growth in mice inoculated with PANCO2 cells orthotopically. We found that diclofenac treatment (30 mg/kg/bw for 11 days) of mice inoculated with PANC02 cells, reduced the tumor weight by 60%, correlating with increased apoptosis of tumor cells. Since this effect was not observed in vitro on cultured PANCO2 cells, we theorized that diclofenac beneficial treatment involved other mediators present in vivo. Indeed, diclofenac drastically decreased tumor vascularization by downregulating VEGF in the tumor and in abdominal cavity fluid. Furthermore, diclofenac directly inhibited vascular sprouting ex vivo. Surprisingly, in contrast to other COX-2 inhibitors, diclofenac increased arginase activity/arginase 1 protein content in tumor stroma cells, peritoneal macrophages and white blood cells by 2.4, 4.8 and 2 fold, respectively. We propose that the subsequent arginine depletion and decrease in NO levels, both in serum and peritoneal cavity, adds to tumor growth inhibition by malnourishment and poor vasculature development. In conclusion, diclofenac shows pronounced antitumoral properties in pancreatic cancer model that can contribute to further treatment development. The ability of diclofenac to induce arginase activity in tumor stroma, peritoneal macrophages and white blood cells provides a tool to study a controversial issue of pro-and antitumoral effects of arginine depletion.

  9. Effects of the Hydroalcoholic Extract of Zingiber officinale on Arginase I Activity and Expression in the Retina of Streptozotocin-Induced Diabetic Rats.

    Science.gov (United States)

    Lamuchi-Deli, Nasrin; Aberomand, Mohammad; Babaahmadi-Rezaei, Hossein; Mohammadzadeh, Ghorban

    2017-04-01

    Emerging evidence suggests that an increased arginase activity is involved in vascular dysfunction in experimental animals. Zingiber officinale Roscoe, commonly known as ginger, has been widely used in the traditional medicine for treatment of diabetes. This study aimed at investigating the effects of the hydroalcoholic extract of Z. officinale on arginase I activity and expression in the retina of streptozotocin (STZ)-induced diabetic rats. In this experimental study, 16 male Wistar rats weighing 200 - 250 g were assessed. Diabetes was induced via a single intraperitoneal injection of STZ (60 mg/kg body weight). The rats were randomly allocated into four experimental groups. Untreated healthy and diabetic controls received 1.5 mL/kg distilled water. Treated diabetic rats received 200, and 400 mg/kg of the Z. officinale extract dissolved in distilled water (1.5 mL/kg). Body weight, blood glucose and insulin concentration were measured by standard methods. The arginase I activity and expression were determined by spectrophotometric and western blot analysis, respectively. Our results showed that blood glucose concentration was significantly decreased in diabetic rats treated with the extract compared to untreated diabetic controls (P officinale hydroalcoholic extract may potentially be a promising therapeutic option for treating diabetes-induced vascular disorders, possibly through reducing arginase I activity and expression in the retina.

  10. In vitro antileishmanial activity of fisetin flavonoid via inhibition of glutathione biosynthesis and arginase activity in Leishmania infantum.

    Science.gov (United States)

    Adinehbeigi, Keivan; Razi Jalali, Mohammad Hossein; Shahriari, Ali; Bahrami, Somayeh

    2017-06-01

    With the increasing emergence of drug resistant Leishmania sp. in recent years, combination therapy has been considered as a useful way to treat and control of Leishmaniasis. The present study was designed to evaluate the antileishmanial effects of the fisetin alone and combination of fisetin plus Meglumine antimoniate (Fi-MA) against Leishmania infantum. The IC50 values for fisetin were obtained 0.283 and 0.102 μM against promastigotes and amastigote forms, respectively. Meglumine antimoniate (MA, Glucantime) as control drug also revealed IC50 values of 0.247 and 0.105 μM for promastigotes and amastigotes of L. infantum, respectively. In order to determine the mode of action of fisetin and Meglumine antimoniate (MA, Glucantime), the activities of arginase (ARG), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) were measured. Moreover, intracellular glutathione (GSH) and nitric oxide (NO) levels in L. infantum-infected macrophages and L. infantum promastigotes which were treated with IC50 concentrations of fisetin, MA and Fi-MA were investigated. Our results showed that MA decreased CAT and SOD activity and increased NO levels in L. infantum-infected macrophages. In promastigotes, MA inhibited parasite SOD activity and reduced parasite NO production. The decreased levels of most of the antioxidant enzymes, accompanying by the raised level of NO in treated macrophages with MA, were observed to regain their normal profiles due to Fi-MA treatment. Furthermore, fisetin could prevent the growth of promastigotes by inhibition of ARG activity and reduction of GSH levels and NO production. In conclusion, these findings showed that fisetin improves MA side effects.

  11. p38 mitogen-activated protein kinase is involved in arginase-II-mediated eNOS-uncoupling in obesity.

    Science.gov (United States)

    Yu, Yi; Rajapakse, Angana G; Montani, Jean-Pierre; Yang, Zhihong; Ming, Xiu-Fen

    2014-07-18

    Endothelial nitric oxide synthase (eNOS)-uncoupling links obesity-associated insulin resistance and type-II diabetes to the increased incidence of cardiovascular disease. Studies have indicated that increased arginase is involved in eNOS-uncoupling through competing with the substrate L-arginine. Given that arginase-II (Arg-II) exerts some of its biological functions through crosstalk with signal transduction pathways, and that p38 mitogen-activated protein kinase (p38mapk) is involved in eNOS-uncoupling, we investigated here whether p38mapk is involved in Arg-II-mediated eNOS-uncoupling in a high fat diet (HFD)-induced obesity mouse model. Obesity was induced in wild type (WT) and Arg-II-deficient (Arg-II(-/-)) mice on C57BL/6 J background by high-fat diet (HFD, 55% fat) for 14 weeks starting from age of 7 weeks. The entire aortas were isolated and subjected to 1) immunoblotting analysis of the protein level of eNOS, Arg-II and p38mapk activation; 2) arginase activity assay; 3) endothelium-dependent and independent vasomotor responses; 4) en face staining of superoxide anion and NO production with Dihydroethidium and 4,5-Diaminofluorescein Diacetate, respectively, to assess eNOS-uncoupling. To evaluate the role of p38mapk, isolated aortas were treated with p38mapk inhibitor SB203580 (10 μmol/L, 1 h) prior to the analysis. In addition, the role of p38mapk in Arg-II-induced eNOS-uncoupling was investigated in cultured human endothelial cells overexpressing Arg-II in the absence or presence of shRNA against p38mapk. HFD enhanced Arg-II expression/activity and p38mapk activity, which was associated with eNOS-uncoupling as revealed by decreased NO and enhanced L-NAME-inhibitable superoxide in aortas of WT obese mice. In accordance, WT obese mice revealed decreased endothelium-dependent relaxations to acetylcholine despite of higher eNOS protein level, whereas Arg-II(-/-) obese mice were protected from HFD-induced eNOS-uncoupling and endothelial dysfunction, which

  12. Insulin-Like Growth Factor-I Induces Arginase Activity in Leishmania amazonensis Amastigote-Infected Macrophages through a Cytokine-Independent Mechanism

    Directory of Open Access Journals (Sweden)

    Celia Maria Vieira Vendrame

    2014-01-01

    Full Text Available Leishmania (Leishmania amazonensis exhibits peculiarities in its interactions with hosts. Because amastigotes are the primary form associated with the progression of infection, we studied the effect of insulin-like growth factor (IGF-I on interactions between L. (L. amazonensis amastigotes and macrophages. Upon stimulation of infected macrophages with IGF-I, we observed decreased nitric oxide production but increased arginase expression and activity, which lead to increased parasitism. However, stimulation of amastigote-infected macrophages with IGF-I did not result in altered cytokine levels compared to unstimulated controls. Because IGF-I is present in tissue fluids and also within macrophages, we examined the possible effect of this factor on phosphatidylserine (PS exposure on amastigotes, seen previously in tissue-derived amastigotes leading to increased parasitism. Stimulation with IGF-I induced PS exposure on amastigotes but not on promastigotes. Using a PS-liposome instead of amastigotes, we observed that the PS-liposome but not the control phosphatidylcholine-liposome led to increased arginase activity in macrophages, and this process was not blocked by anti-TGF-β antibodies. Our results suggest that in L. (L. amazonensis amastigote-infected macrophages, IGF-I induces arginase activity directly in amastigotes and in macrophages through the induction of PS exposure on amastigotes in the latter, which could lead to the alternative activation of macrophages through cytokine-independent mechanisms.

  13. Novel selective inhibitor of Leishmania (Leishmania) amazonensis arginase.

    Science.gov (United States)

    da Silva, Edson R; Boechat, Nubia; Pinheiro, Luiz C S; Bastos, Monica M; Costa, Carolina C P; Bartholomeu, Juliana C; da Costa, Talita H

    2015-11-01

    Arginase is a glycosomal enzyme in Leishmania that is involved in polyamine and trypanothione biosynthesis. The central role of arginase in Leishmania (Leishmania) amazonensis was demonstrated by the generation of two mutants: one with an arginase lacking the glycosomal addressing signal and one in which the arginase-coding gene was knocked out. Both of these mutants exhibited decreased infectivity. Thus, arginase seems to be a potential drug target for Leishmania treatment. In an attempt to search for arginase inhibitors, 29 derivatives of the [1,2,4]triazolo[1,5-a]pyrimidine system were tested against Leishmania (Leishmania) amazonensis arginase in vitro. The [1,2,4]triazolo[1,5-a]pyrimidine scaffold containing R1  = CF3 exhibited greater activity against the arginase rather than when the substituent R1  = CH3 in the 2-position. The novel compound 2-(5-methyl-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)hydrazinecarbothioamide (30) was the most potent, inhibiting arginase by a non-competitive mechanism, with the Ki and IC50 values for arginase inhibition estimated to be 17 ± 1 μm and 16.5 ± 0.5 μm, respectively. These results can guide the development of new drugs against leishmaniasis based on [1,2,4]triazolo[1,5-a]pyrimidine derivatives targeting the arginase enzyme. © 2015 John Wiley & Sons A/S.

  14. Arginase-1 deficiency.

    Science.gov (United States)

    Sin, Yuan Yan; Baron, Garrett; Schulze, Andreas; Funk, Colin D

    2015-12-01

    Arginase-1 (ARG1) deficiency is a rare autosomal recessive disorder that affects the liver-based urea cycle, leading to impaired ureagenesis. This genetic disorder is caused by 40+ mutations found fairly uniformly spread throughout the ARG1 gene, resulting in partial or complete loss of enzyme function, which catalyzes the hydrolysis of arginine to ornithine and urea. ARG1-deficient patients exhibit hyperargininemia with spastic paraparesis, progressive neurological and intellectual impairment, persistent growth retardation, and infrequent episodes of hyperammonemia, a clinical pattern that differs strikingly from other urea cycle disorders. This review briefly highlights the current understanding of the etiology and pathophysiology of ARG1 deficiency derived from clinical case reports and therapeutic strategies stretching over several decades and reports on several exciting new developments regarding the pathophysiology of the disorder using ARG1 global and inducible knockout mouse models. Gene transfer studies in these mice are revealing potential therapeutic options that can be exploited in the future. However, caution is advised in extrapolating results since the lethal disease phenotype in mice is much more severe than in humans indicating that the mouse models may not precisely recapitulate human disease etiology. Finally, some of the functions and implications of ARG1 in non-urea cycle activities are considered. Lingering questions and future areas to be addressed relating to the clinical manifestations of ARG1 deficiency in liver and brain are also presented. Hopefully, this review will spark invigorated research efforts that lead to treatments with better clinical outcomes.

  15. Arginase promotes endothelial dysfunction and hypertension in obese rats.

    Science.gov (United States)

    Johnson, Fruzsina K; Peyton, Kelly J; Liu, Xiao-Ming; Azam, Mohammed A; Shebib, Ahmad R; Johnson, Robert A; Durante, William

    2015-02-01

    This study investigated whether arginase contributes to endothelial dysfunction and hypertension in obese rats. Endothelial function and arginase expression were examined in skeletal muscle arterioles from lean and obese Zucker rats (ZRs). Arginase activity, arginine bioavailability, and blood pressure were measured in lean and obese animals. Arginase activity and expression was increased while global arginine bioavailability decreased in obese ZRs. Acetylcholine or luminal flow caused dilation of isolated skeletal muscle arterioles, but this was reduced or absent in vessels from obese ZRs. Treatment of arterioles with a nitric oxide synthase inhibitor blocked dilation in lean arterioles and eliminated differences among lean and obese vessels. In contrast, arginase inhibitors or l-arginine enhanced vasodilation in obese ZRs and abolished differences between lean and obese animals, while d-arginine had no effect. Finally, mean arterial blood pressure was significantly increased in obese ZRs. However, administration of l-arginine or arginase inhibitors lowered blood pressure in obese but not lean animals, and this was associated with an improvement in systemic arginine bioavailability. Arginase promotes endothelial dysfunction and hypertension in obesity by reducing arginine bioavailability. Therapeutic approaches targeting arginase represent a promising approach in treating obesity-related vascular disease. © 2014 The Obesity Society.

  16. Arginase inhibition prevents the development of hypertension and improves insulin resistance in obese rats.

    Science.gov (United States)

    Peyton, Kelly J; Liu, Xiao-Ming; Shebib, Ahmad R; Johnson, Fruzsina K; Johnson, Robert A; Durante, William

    2018-04-27

    This study investigated the temporal activation of arginase in obese Zucker rats (ZR) and determined if arginase inhibition prevents the development of hypertension and improves insulin resistance in these animals. Arginase activity, plasma arginine and nitric oxide (NO) concentration, blood pressure, and insulin resistance were measured in lean and obese animals. There was a chronological increase in vascular and plasma arginase activity in obese ZR beginning at 8 weeks of age. The increase in arginase activity in obese animals was associated with a decrease in insulin sensitivity and circulating levels of arginine and NO. The rise in arginase activity also preceded the increase in blood pressure in obese ZR detected at 12 weeks of age. Chronic treatment of 8-week-old obese animals with an arginase inhibitor or L-arginine for 4 weeks prevented the development of hypertension and improved plasma concentrations of arginine and NO. Arginase inhibition also improved insulin sensitivity in obese ZR while L-arginine supplementation had no effect. In conclusion, arginase inhibition prevents the development of hypertension and improves insulin sensitivity while L-arginine administration only mitigates hypertension in obese animals. Arginase represents a promising therapeutic target in ameliorating obesity-associated vascular and metabolic dysfunction.

  17. Short curcumin treatment modulates oxidative stress, arginase activity, aberrant crypt foci, and TGF-β1 and HES-1 transcripts in 1,2-dimethylhydrazine-colon carcinogenesis in mice

    International Nuclear Information System (INIS)

    Bounaama, Abdelkader; Djerdjouri, Bahia; Laroche-Clary, Audrey; Le Morvan, Valérie; Robert, Jacques

    2012-01-01

    Highlights: ► 1,2-Dimethylhydrazine (DMH) toxicity was driven by oxidative stress. ► Arginase activity correlated to aberrant crypt foci (ACF). ► Curcumin diet restored redox status and induced apoptosis of dysplastic ACF. ► Curcumin reduced arginase activity and up regulated TGF-β1 and HES-1 transcripts. -- Abstract: This study investigated the effect of short curcumin treatment, a natural antioxidant on 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci (ACF) in mice. The incidence of aberrant crypt foci (ACF) was 100%, with 54 ± 6 per colon, 10 weeks after the first DMH injection and reached 67 ± 12 per colon after 12 weeks. A high level of undifferentiated goblet cells and a weak apoptotic activity were shown in dysplastic ACF. The morphological alterations of colonic mucosa were associated to severe oxidative stress ratio with 43% increase in malondialdehyde vs. 36% decrease in GSH. DMH also increased inducible nitric synthase (iNOS) mRNA transcripts (250%), nitrites level (240%) and arginase activity (296%), leading to nitrosative stress and cell proliferation. Curcumin treatment, starting at week 10 post-DMH injection for 14 days, reduced the number of ACF (40%), iNOS expression (25%) and arginase activity (73%), and improved redox status by approximately 46%, compared to DMH-treated mice. Moreover, curcumin induced apoptosis of dysplastic ACF cells without restoring goblet cells differentiation. Interestingly, curcumin induced a parallel increase in TGF-β1 and HES-1 transcripts (42% and 26%, respectively). In conclusion, the protective effect of curcumin was driven by the reduction of arginase activity and nitrosative stress. The up regulation of TGF-β1 and HES-1 expression by curcumin suggests for the first time, a potential interplay between these signalling pathways in the chemoprotective mechanism of curcumin.

  18. Effects of Arginase Inhibition in Hypertensive Hyperthyroid Rats.

    Science.gov (United States)

    Rodríguez-Gómez, Isabel; Manuel Moreno, Juan; Jimenez, Rosario; Quesada, Andrés; Montoro-Molina, Sebastian; Vargas-Tendero, Pablo; Wangensteen, Rosemary; Vargas, Félix

    2015-12-01

    This study analyzed the effects of chronic administration of N[omega]-hydroxy-nor-l-arginine (nor-NOHA), an inhibitor of arginase, on the hemodynamic, oxidative stress, morphologic, metabolic, and renal manifestations of hyperthyroidism in rats. Four groups of male Wistar rats were used: control, nor-NOHA-treated (10 mg/kg/day), thyroxine (T4)-treated (75 μg/rat/day), and thyroxine- plus nor-NOHA-treated rats. All treatments were maintained for 4 weeks. Body weight, tail systolic blood pressure (SBP), and heart rate (HR) were recorded weekly. Finally, morphologic, metabolic, plasma, and renal variables were measured. Arginase I and II protein abundance and arginase activity were measured in aorta, heart, and kidney. The T4 group showed increased arginase I and II protein abundance, arginase activity, SBP, HR, plasma nitrates/nitrites (NOx), brainstem and urinary isoprostanes, proteinuria and cardiac and renal hypertrophy in comparison to control rats. In hyperthyroid rats, chronic nor-NOHA prevented the increase in SBP and HR and decreased proteinuria in association with an increase in plasma NOx and a decrease in brainstem and urinary isoprostanes. In normal rats, nor-NOHA treatment did not significantly change any hemodynamic, morphologic, or renal variables. Acute nor-NOHA administration did not affect renal or systemic hemodynamic variables in normal or T4-treated rats. Hyperthyroidism in rats is associated with the increased expression and activity of arginase in aorta, heart, and kidney. Chronic arginase inhibition with nor-NOHA suppresses the characteristic hemodynamic manifestations of hyperthyroidism in association with a reduced oxidative stress. These results indicate an important role for arginase pathway alterations in the cardiovascular and renal abnormalities of hyperthyroidism. © American Journal of Hypertension, Ltd 2015. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  19. Local arginase inhibition during early reperfusion mediates cardioprotection via increased nitric oxide production.

    Directory of Open Access Journals (Sweden)

    Adrian T Gonon

    Full Text Available Consumption of L-arginine contributes to reduced bioavailability of nitric oxide (NO that is critical for the development of ischemia-reperfusion injury. The aim of the study was to determine myocardial arginase expression and activity in ischemic-reperfusion myocardium and whether local inhibition of arginase within the ischemic myocardium results in increased NO production and protection against myocardial ischemia-reperfusion. Anesthetized pigs were subjected to coronary artery occlusion for 40 min followed by 4 h reperfusion. The pigs were randomized to intracoronary infusion of vehicle (n = 7, the arginase inhibitor N-hydroxy-nor-L-arginine (nor-NOHA, 2 mg/min, n = 7, the combination of nor-NOHA and the NO synthase inhibitor N(G-monomethyl-L-arginine (L-NMMA, 0.35 mg/min, n = 6 into the jeopardized myocardial area or systemic intravenous infusion of nor-NOHA (2 mg/min, n = 5 at the end of ischemia and start of reperfusion. The infarct size of the vehicle group was 80 ± 4% of the area at risk. Intracoronary nor-NOHA reduced infarct size to 46 ± 5% (P<0.01. Co-administration of L-NMMA abrogated the cardioprotective effect mediated by nor-NOHA (infarct size 72 ± 6%. Intravenous nor-NOHA did not reduce infarct size. Arginase I and II were expressed in cardiomyocytes, endothelial, smooth muscle and poylmorphonuclear cells. There was no difference in cytosolic arginase I or mitochondrial arginase II expression between ischemic-reperfused and non-ischemic myocardium. Arginase activity increased 2-fold in the ischemic-reperfused myocardium in comparison with non-ischemic myocardium. In conclusion, ischemia-reperfusion increases arginase activity without affecting cytosolic arginase I or mitochondrial arginase II expression. Local arginase inhibition during early reperfusion reduces infarct size via a mechanism that is dependent on increased bioavailability of NO.

  20. Development of novel arginase inhibitors for therapy of endothelial dysfunction

    Directory of Open Access Journals (Sweden)

    Jochen eSteppan

    2013-09-01

    Full Text Available Endothelial dysfunction and resulting vascular pathology have been identified as an early hallmark of multiple diseases, including diabetes mellitus. One of the major contributors to endothelial dysfunction is a decrease in nitric oxide (NO bioavailability, impaired NO signaling and an increase in the amount of reactive oxygen species (ROS. In the endothelium NO is produced by eNOS (endothelial nitric oxide synthase, for which L-arginine is a substrate. Arginase, an enzyme critical in the urea cycle also metabolizes L-arginine, thereby directly competing with eNOS for their common substrate and constraining its bioavailability for eNOS, thereby compromising NO production. Arginase expression and activity is upregulated in many cardiovascular diseases including ischemia reperfusion injury, hypertension, atherosclerosis, and diabetes mellitus. More importantly, since the 1990s, specific arginase inhibitors such as N-hydroxy-guanidinium or N-hydroxy-nor-L-arginine, and boronic acid derivatives, such as, 2(S-amino-6-boronohexanoic acid, and S-(2-boronoethyl-L-cysteine (BEC, that can bridge the binuclear manganese cluster of arginase have been developed. These highly potent and specific inhibitors can now be used to probe arginase function and thereby modulate the redox milieu of the cell by changing the balance between NO and ROS. Inspired by this success, drug discovery programs have recently led to the identification of α-α-disubstituted amino acid based arginase inhibitors (such as (R-2-amino-6-borono-2-(2-(piperidin-1-ylethylhexanoic acid, that are currently under early investigation as therapeutics. Finally, some investigators concentrate on identification of plant derived compounds with arginase inhibitory capability, such as piceatannol-3'-O-β-D-glucopyranoside (PG. All of these synthesized or naturally derived small molecules may represent novel therapeutics for vascular disease particularly that associated with diabetes.

  1. Arginase Inhibition Ameliorates Hepatic Metabolic Abnormalities in Obese Mice

    Science.gov (United States)

    Moon, Jiyoung; Do, Hyun Ju; Cho, Yoonsu; Shin, Min-Jeong

    2014-01-01

    Objectives We examined whether arginase inhibition influences hepatic metabolic pathways and whole body adiposity in diet-induced obesity. Methods and Results After obesity induction by a high fat diet (HFD), mice were fed either the HFD or the HFD with an arginase inhibitor, Nω-hydroxy-nor-L-arginine (nor-NOHA). Nor-NOHA significantly prevented HFD-induced increases in body, liver, and visceral fat tissue weight, and ameliorated abnormal lipid profiles. Furthermore, nor-NOHA treatment reduced lipid accumulation in oleic acid-induced hepatic steatosis in vitro. Arginase inhibition increased hepatic nitric oxide (NO) in HFD-fed mice and HepG2 cells, and reversed the elevated mRNA expression of hepatic genes in lipid metabolism. Expression of phosphorylated 5′ AMPK-activated protein kinase α was increased by arginase inhibition in the mouse livers and HepG2 cells. Conclusions Arginase inhibition ameliorated obesity-induced hepatic lipid abnormalities and whole body adiposity, possibly as a result of increased hepatic NO production and subsequent activation of metabolic pathways involved in hepatic triglyceride metabolism and mitochondrial function. PMID:25057910

  2. TNF-Mediated Restriction of Arginase 1 Expression in Myeloid Cells Triggers Type 2 NO Synthase Activity at the Site of Infection

    Directory of Open Access Journals (Sweden)

    Ulrike Schleicher

    2016-05-01

    Full Text Available Neutralization or deletion of tumor necrosis factor (TNF causes loss of control of intracellular pathogens in mice and humans, but the underlying mechanisms are incompletely understood. Here, we found that TNF antagonized alternative activation of macrophages and dendritic cells by IL-4. TNF inhibited IL-4-induced arginase 1 (Arg1 expression by decreasing histone acetylation, without affecting STAT6 phosphorylation and nuclear translocation. In Leishmania major-infected C57BL/6 wild-type mice, type 2 nitric oxide (NO synthase (NOS2 was detected in inflammatory dendritic cells or macrophages, some of which co-expressed Arg1. In TNF-deficient mice, Arg1 was hyperexpressed, causing an impaired production of NO in situ. A similar phenotype was seen in L. major-infected BALB/c mice. Arg1 deletion in hematopoietic cells protected these mice from an otherwise lethal disease, although their disease-mediating T cell response (Th2, Treg was maintained. Thus, deletion or TNF-mediated restriction of Arg1 unleashes the production of NO by NOS2, which is critical for pathogen control.

  3. Comparison of biochemical properties of liver arginase from streptozocin-induced diabetic and control mice.

    Science.gov (United States)

    Spolarics, Z; Bond, J S

    1989-11-01

    Arginase activity is elevated in livers of diabetic animals compared to controls and there is evidence that this is due in part to increased specific activity (activity/mg arginase protein). To investigate the molecular basis of this increased activity, the physicochemical and kinetic properties of hepatic arginase from diabetic and control mice were compared. Two types of arginase subunits with molecular weights of 35,000 and 38,000 were found in both the diabetic and control animals and the subunits in these animals had similar, multiple ionic forms. Kinetic parameters of purified preparations of arginase for arginine (apparent Km and Vmax values) and the thermal stability of these preparations from diabetics and controls were also similar. Furthermore, no difference was found in the distribution of arginase activity among different subcellular liver fractions. Separation of basic and acidic oligomeric forms of arginase by fast-protein liquid chromatography resulted in a slightly different distribution of activity among the forms in the normal and diabetic group. The apparent Km values for Mn2+ of the basic form of the enzyme were 25 and 33 microM for the enzyme from normal and diabetic animals, respectively; for acidic forms, for which two apparent Km values were measured, the values were 8 and 197 microM for arginase from controls and 35 and 537 microM from diabetics. These results indicate that in diabetes, while no marked changes in the physicochemical characteristics of arginase are obvious, some changes are found in the interaction of arginase with its cofactor Mn.

  4. The Involvement of Arginase and Nitric Oxide Synthase in Breast Cancer Development: Arginase and NO Synthase as Therapeutic Targets in Cancer

    Directory of Open Access Journals (Sweden)

    Nikolay Avtandilyan

    2018-01-01

    Full Text Available It is well established that, during development of malignancies, metabolic changes occur, including alterations of enzyme activities and isoenzyme expression. Arginase and nitric oxide (NO synthase (NOS are two of those enzymes considered to be involved in tumorigenesis. The goal of this article was to study the involvement of arginase and NOS in the development of different stages of breast cancer. Our results have shown that human serum arginase activity and NO (resp., and NOS activity and polyamines quantities increased in parallel with cancer stage progression and decreased after neoadjuvant chemotherapy. For breast cancer, the only isoenzyme of arginase expressed in serum before and after chemotherapy was in a cationic form. The data of Lineweaver-Burk plot with a Km value of 2 mM was calculated, which is characteristic for human liver type isoform of arginase. During electrophoresis at pH 8.9, the enzyme exhibited high electrophoretic mobility and was detected near the anode. The presented results demonstrated that arginase in human serum with breast cancer and after chemotherapy is not polymorphic. We suggest that arginase and NOS inhibition has antitumor effects on cancer development, as it can inhibit polyamines and NO levels, a precursor of cancer cell proliferation, metastasis, and tumor angiogenesis.

  5. Arginase Inhibitor in the Pharmacological Correction of Endothelial Dysfunction

    Directory of Open Access Journals (Sweden)

    Mihail V. Pokrovskiy

    2011-01-01

    Full Text Available This paper is about a way of correction of endothelial dysfunction with the inhibitor of arginase: L-norvaline. There is an imbalance between vasoconstriction and vasodilatation factors of endothelium on the basis of endothelial dysfunction. Among vasodilatation agents, nitrogen oxide plays the basic role. Amino acid L-arginine serves as a source of molecules of nitrogen oxide in an organism. Because of the high activity of arginase enzyme which catalyzes the hydrolysis of L-arginine into ornithine and urea, the bioavailability of nitrogen oxide decreases. The inhibitors of arginase suppress the activity of the given enzyme, raising and production of nitrogen oxide, preventing the development of endothelial dysfunction.

  6. Arginase expression modulates nitric oxide production in Leishmania (Leishmania) amazonensis.

    Science.gov (United States)

    Acuña, Stephanie Maia; Aoki, Juliana Ide; Laranjeira-Silva, Maria Fernanda; Zampieri, Ricardo Andrade; Fernandes, Juliane Cristina Ribeiro; Muxel, Sandra Marcia; Floeter-Winter, Lucile Maria

    2017-01-01

    Arginase is an enzyme that converts L-arginine to urea and L-ornithine, an essential substrate for the polyamine pathway supporting Leishmania (Leishmania) amazonensis replication and its survival in the mammalian host. L-arginine is also the substrate of macrophage nitric oxide synthase 2 (NOS2) to produce nitric oxide (NO) that kills the parasite. This competition can define the fate of Leishmania infection. The transcriptomic profiling identified a family of oxidoreductases in L. (L.) amazonensis wild-type (La-WT) and L. (L.) amazonensis arginase knockout (La-arg-) promastigotes and axenic amastigotes. We highlighted the identification of an oxidoreductase that could act as nitric oxide synthase-like (NOS-like), due to the following evidences: conserved domain composition, the participation of NO production during the time course of promastigotes growth and during the axenic amastigotes differentiation, regulation dependence on arginase activity, as well as reduction of NO amount through the NOS activity inhibition. NO quantification was measured by DAF-FM labeling analysis in a flow cytometry. We described an arginase-dependent NOS-like activity in L. (L.) amazonensis and its role in the parasite growth. The increased detection of NO production in the mid-stationary and late-stationary growth phases of La-WT promastigotes could suggest that this production is an important factor to metacyclogenesis triggering. On the other hand, La-arg- showed an earlier increase in NO production compared to La-WT, suggesting that NO production can be arginase-dependent. Interestingly, La-WT and La-arg- axenic amastigotes produced higher levels of NO than those observed in promastigotes. As a conclusion, our work suggested that NOS-like is expressed in Leishmania in the stationary growth phase promastigotes and amastigotes, and could be correlated to metacyclogenesis and amastigotes growth in a dependent way to the internal pool of L-arginine and arginase activity.

  7. Genetics Home Reference: arginase deficiency

    Science.gov (United States)

    ... belongs to a class of genetic diseases called urea cycle disorders. The urea cycle is a sequence of reactions ... links) Baby's First Test GeneReview: Arginase Deficiency GeneReview: Urea Cycle Disorders Overview MedlinePlus Encyclopedia: Hereditary urea cycle abnormality National ...

  8. Arginase promotes skeletal muscle arteriolar endothelial dysfunction in diabetic rats.

    Directory of Open Access Journals (Sweden)

    Fruzsina K. Johnson

    2013-05-01

    Full Text Available Endothelial dysfunction is a characteristic feature in diabetes that contributes to the development of vascular disease. Recently, arginase has been implicated in triggering endothelial dysfunction in diabetic patients and animals by competing with endothelial nitric oxide synthase for substrate L-arginine. While most studies have focused on the coronary circulation and large conduit blood vessels, the role of arginase in mediating diabetic endothelial dysfunction in other vascular beds has not been fully investigated. In the present study, we determined whether arginase contributes to endothelial dysfunction in skeletal muscle arterioles of diabetic rats. Diabetes was induced in male Sprague Dawley rats by streptozotocin injection. Four weeks after streptozotocin administration, blood glucose, glycated hemoglobin, and vascular arginase activity were significantly increased. In addition, a significant increase in arginase I and II mRNA expression was detected in gracilis muscle arterioles of diabetic rats compared to age-matched, vehicle control animals. To examine endothelial function, first-order gracilis muscle arterioles were isolated, cannulated in a pressure myograph system, exposed to graded levels of luminal flow, and internal vessel diameter measured. Increases in luminal flow (0-50µL/min caused progressive vasodilation in arterioles isolated from control, normoglycemic animals. However, flow-induced vasodilation was absent in arterioles obtained from streptozotocin-treated rats. Acute in-vitro pretreatment of blood vessels with the arginase inhibitors Nω-hydroxy-nor-L-arginine or S-(2-boronoethyl-L-cysteine restored flow-induced responses in arterioles from diabetic rats and abolished differences between diabetic and control animals. Similarly, acute in-vitro pretreatment with L-arginine returned flow-mediated vasodilation in vessels from diabetic animals to that of control rats. In contrast, D-arginine failed to restore flow

  9. Strategies to rescue the consequences of inducible arginase-1 deficiency in mice.

    Directory of Open Access Journals (Sweden)

    Laurel L Ballantyne

    Full Text Available Arginase-1 catalyzes the conversion of arginine to ornithine and urea, which is the final step of the urea cycle used to remove excess ammonia from the body. Arginase-1 deficiency leads to hyperargininemia in mice and man with severe lethal consequences in the former and progressive neurological impairment to varying degrees in the latter. In a tamoxifen-induced arginase-1 deficient mouse model, mice succumb to the enzyme deficiency within 2 weeks after inducing the knockout and retain <2 % enzyme in the liver. Standard clinical care regimens for arginase-1 deficiency (low-protein diet, the nitrogen-scavenging drug sodium phenylbutyrate, ornithine supplementation either failed to extend lifespan (ornithine or only minimally prolonged lifespan (maximum 8 days with low-protein diet and drug. A conditional, tamoxifen-inducible arginase-1 transgenic mouse strain expressing the enzyme from the Rosa26 locus modestly extended lifespan of neonatal mice, but not that of 4-week old mice, when crossed to the inducible arginase-1 knockout mouse strain. Delivery of an arginase-1/enhanced green fluorescent fusion construct by adeno-associated viral delivery (rh10 serotype with a strong cytomegalovirus-chicken β-actin hybrid promoter rescued about 30% of male mice with lifespan prolongation to at least 6 months, extensive hepatic expression and restoration of significant enzyme activity in liver. In contrast, a vector of the AAV8 serotype driven by the thyroxine-binding globulin promoter led to weaker liver expression and did not rescue arginase-1 deficient mice to any great extent. Since the induced arginase-1 deficient mouse model displays a much more severe phenotype when compared to human arginase-1 deficiency, these studies reveal that it may be feasible with gene therapy strategies to correct the various manifestations of the disorder and they provide optimism for future clinical studies.

  10. Genetic microheterogeneity and phenotypic variation of Helicobacter pylori arginase in clinical isolates

    Directory of Open Access Journals (Sweden)

    Spadafora Domenico

    2007-04-01

    Full Text Available Abstract Background Clinical isolates of the gastric pathogen Helicobacter pylori display a high level of genetic macro- and microheterogeneity, featuring a panmictic, rather than clonal structure. The ability of H. pylori to survive the stomach acid is due, in part, to the arginase-urease enzyme system. Arginase (RocF hydrolyzes L-arginine to L-ornithine and urea, and urease hydrolyzes urea to carbon dioxide and ammonium, which can neutralize acid. Results The degree of variation in arginase was explored at the DNA sequence, enzyme activity and protein expression levels. To this end, arginase activity was measured from 73 minimally-passaged clinical isolates and six laboratory-adapted strains of H. pylori. The rocF gene from 21 of the strains was cloned into genetically stable E. coli and the enzyme activities measured. Arginase activity was found to substantially vary (>100-fold in both different H. pylori strains and in the E. coli model. Western blot analysis revealed a positive correlation between activity and amount of protein expressed in most H. pylori strains. Several H. pylori strains featured altered arginase activity upon in vitro passage. Pairwise alignments of the 21 rocF genes plus strain J99 revealed extensive microheterogeneity in the promoter region and 3' end of the rocF coding region. Amino acid S232, which was I232 in the arginase-negative clinical strain A2, was critical for arginase activity. Conclusion These studies demonstrated that H. pylori arginase exhibits extensive genotypic and phenotypic variation which may be used to understand mechanisms of microheterogeneity in H. pylori.

  11. Deregulation of arginase induces bone complications in high-fat/high-sucrose diet diabetic mouse model.

    Science.gov (United States)

    Bhatta, Anil; Sangani, Rajnikumar; Kolhe, Ravindra; Toque, Haroldo A; Cain, Michael; Wong, Abby; Howie, Nicole; Shinde, Rahul; Elsalanty, Mohammed; Yao, Lin; Chutkan, Norman; Hunter, Monty; Caldwell, Ruth B; Isales, Carlos; Caldwell, R William; Fulzele, Sadanand

    2016-02-15

    A balanced diet is crucial for healthy development and prevention of musculoskeletal related diseases. Diets high in fat content are known to cause obesity, diabetes and a number of other disease states. Our group and others have previously reported that activity of the urea cycle enzyme arginase is involved in diabetes-induced dysregulation of vascular function due to decreases in nitric oxide formation. We hypothesized that diabetes may also elevate arginase activity in bone and bone marrow, which could lead to bone-related complications. To test this we determined the effects of diabetes on expression and activity of arginase, in bone and bone marrow stromal cells (BMSCs). We demonstrated that arginase 1 is abundantly present in the bone and BMSCs. We also demonstrated that arginase activity and expression in bone and bone marrow is up-regulated in models of diabetes induced by HFHS diet and streptozotocin (STZ). HFHS diet down-regulated expression of healthy bone metabolism markers (BMP2, COL-1, ALP, and RUNX2) and reduced bone mineral density, bone volume and trabecular thickness. However, treatment with an arginase inhibitor (ABH) prevented these bone-related complications of diabetes. In-vitro study of BMSCs showed that high glucose treatment increased arginase activity and decreased nitric oxide production. These effects were reversed by treatment with an arginase inhibitor (ABH). Our study provides evidence that deregulation of l-arginine metabolism plays a vital role in HFHS diet-induced diabetic complications and that these complications can be prevented by treatment with arginase inhibitors. The modulation of l-arginine metabolism in disease could offer a novel therapeutic approach for osteoporosis and other musculoskeletal related diseases. Published by Elsevier Ireland Ltd.

  12. Production of nitric oxide during graft rejection is regulated by the Th1/Th2 balance, the arginase activity, and L-arginine metabolism

    Czech Academy of Sciences Publication Activity Database

    Holáň, Vladimír; Pindjáková, Jana; Krulová, Magdalena; Neuwirth, Aleš; Frič, Jan; Zajícová, Alena

    2006-01-01

    Roč. 81, č. 12 (2006), s. 1708-1715 ISSN 0041-1337 R&D Projects: GA MZd(CZ) NR7816; GA ČR GD310/03/H147; GA MŠk 1M0506 Institutional research plan: CEZ:AV0Z50520514 Keywords : macrophages * arginase * nitric oxide Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.972, year: 2006

  13. Arginase attenuates inhibitory nonadrenergic noncholinergic nerve-induced nitric oxide generation and airway smooth muscle relaxation

    NARCIS (Netherlands)

    Maarsingh, H; Tio, MA; Zaagsma, J; Meurs, H

    2005-01-01

    Background: Recent evidence suggests that endogenous arginase activity potentiates airway responsiveness to methacholine by attenuation of agonist-induced nitric oxide (NO) production, presumably by competition with epithelial constitutive NO synthase for the common substrate, L-arginine. Using

  14. Arginase strongly impairs neuronal nitric oxide-mediated airway smooth muscle relaxation in allergic asthma

    NARCIS (Netherlands)

    Maarsingh, H; Leusink, J; Bos, I Sophie T; Zaagsma, J; Meurs, H

    2006-01-01

    Background: Using guinea pig tracheal preparations, we have recently shown that endogenous arginase activity attenuates inhibitory nonadrenergic noncholinergic (iNANC) nerve-mediated airway smooth muscle relaxation by reducing nitric oxide (NO) production - due to competition with neuronal

  15. Changes in arginase isoenzymes pattern in human hepatocellular carcinoma

    International Nuclear Information System (INIS)

    Chrzanowska, Alicja; Krawczyk, Marek; Baranczyk-Kuzma, Anna

    2008-01-01

    Hepatocellular carcinoma (HCC) is one of the most common tumors worldwide affecting preferentially patients with liver cirrhosis. The studies were performed on tissues obtained during surgery from 50 patients with HCC, 40 with liver cirrhosis and 40 control livers. It was found that arginase activity in HCC was nearly 5- and 15-fold lower than in cirrhotic and normal livers, respectively. Isoenzymes AI (so-called liver-type arginase) and AII (extrahepatic arginase) were identified by Western blotting in all studied tissues, however the amount of AI, as well as the expression of AI-mRNA were lower in HCC, in comparison with normal liver, and those of AII were significantly higher. Since HCC is arginine-dependent, and arginine is essential for cells growth, the decrease of AI may preserve this amino acid within tumor cells. Concurrently, the rise of AII can increase the level of polyamines, compounds crucial for cells proliferation. Thus, both arginase isoenzymes seem to participate in liver cancerogenesis.

  16. Inhibition of Human Arginase I by Substrate adn Product Analogues

    Energy Technology Data Exchange (ETDEWEB)

    L Di Costanzo; M Ilies; K Thorn; D Christianson

    2011-12-31

    Human arginase I is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to generate L-ornithine and urea. We demonstrate that N-hydroxy-L-arginine (NOHA) binds to this enzyme with K(d)=3.6 microM, and nor-N-hydroxy-L-arginine (nor-NOHA) binds with K(d)=517 nM (surface plasmon resonance) or K(d) approximately 50 nM (isothermal titration calorimetry). Crystals of human arginase I complexed with NOHA and nor-NOHA afford 2.04 and 1.55 A resolution structures, respectively, which are significantly improved in comparison with previously-determined structures of the corresponding complexes with rat arginase I. Higher resolution structures clarify the binding interactions of the inhibitors. Finally, the crystal structure of the complex with L-lysine (K(d)=13 microM) is reported at 1.90 A resolution. This structure confirms the importance of hydrogen bond interactions with inhibitor alpha-carboxylate and alpha-amino groups as key specificity determinants of amino acid recognition in the arginase active site.

  17. High-performance liquid chromatography method with radiochemical detection for measurement of nitric oxide synthase, arginase, and arginine decarboxylase activities

    DEFF Research Database (Denmark)

    Volke, A; Wegener, Gregers; Vasar, E

    2006-01-01

    regulate NOS activity. We aimed to develop a HPLC-based method to measure simultaneously the products of these three enzymes. Traditionally, the separation of amino acids and related compounds with HPLC has been carried out with precolumn derivatization and reverse phase chromatography. We describe here...

  18. Frequent adaptive immune responses against arginase-1

    DEFF Research Database (Denmark)

    Martinenaite, Evelina; Mortensen, Rasmus Erik Johansson; Hansen, Morten

    2018-01-01

    The enzyme arginase-1 reduces the availability of arginine to tumor-infiltrating immune cells, thus reducing T-cell functionality in the tumor milieu. Arginase-1 is expressed by some cancer cells and by immune inhibitory cells, such as myeloid-derived suppressor cells (MDSCs) and tumor-associated...

  19. ARGINASE ENZYMES IN ISOLATED AIRWAYS FROM NORMAL AND NITRIC OXIDE SYNTHASE 2-KNOCKOUT MICE EXPOSED TO OVALBUMIN

    Science.gov (United States)

    Bratt, Jennifer M.; Franzi, Lisa M.; Linderholm, Angela L.; Last, Michael S.; Kenyon, Nicholas J.; Last, Jerold A.

    2009-01-01

    Arginase has been suggested to compete with nitric oxide synthase (NOS) for their common substrate, L-arginine. To study the mechanisms underlying this interaction, we compared arginase expression in isolated airways and the consequences of inhibiting arginase activity in vivo with NO production, lung inflammation, and lung function in both C57BL/6 and NOS2 knockout mice undergoing ovalbumin-induced airway inflammation, a mouse model of asthma. Arginases I and II were measured by western blot in isolated airways from sensitized C57BL/6 mice exposed to ovalbumin aerosol. Physiological and biochemical responses---inflammation, lung compliance, airway hyperreactivity, exhaled NO concentration, arginine concentration--were compared with the responses of NOS2 knockout mice. NOS2 knockout mice had increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity. Both arginase I and arginase II were constitutively expressed in the airways of normal C57BL/6 mice. Arginase I was up-regulated approximately 8-fold in the airways of C57BL/6 mice exposed to ovalbumin. Expression of both arginase isoforms were significantly upregulated in NOS2 knockout mice exposed to ovalbumin, with about 40- and 4-fold increases in arginases I and II, respectively. Arginine concentration in isolated airways was not significantly different in any of the groups studied. Inhibition of arginase by systemic treatment of C57BL/6 mice with a competitive inhibitor, Nω-hydroxy-nor-L-arginine (nor-NOHA), significantly decreased the lung inflammatory response to ovalbumin in these animals. We conclude that NOS2 knockout mice are more sensitive to ovalbumin-induced airway inflammation and its sequelae than are C57BL/6 mice, as determined by increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity, and that these findings are strongly correlated with increased expression of both arginase isoforms in the airways of the NOS2

  20. Arginase 1 and arginase 2 variations associate with asthma, asthma severity and beta(2) agonist and steroid response

    NARCIS (Netherlands)

    Vonk, Judith M.; Postma, Dirkje S.; Maarsingh, Harm; Bruinenberg, Marcel; Koppelman, Gerard H.; Meurs, Herman

    Rationale Arginase probably plays an important role in asthma development, severity and progression. Polymorphisms in arginase 1 and arginase 2 genes have been associated with childhood asthma and FEV1 reversibility to beta(2) agonists. Objectives We investigated the association between arginase 1

  1. Obesity-induced vascular dysfunction and arterial stiffening requires endothelial cell arginase 1.

    Science.gov (United States)

    Bhatta, Anil; Yao, Lin; Xu, Zhimin; Toque, Haroldo A; Chen, Jijun; Atawia, Reem T; Fouda, Abdelrahman Y; Bagi, Zsolt; Lucas, Rudolf; Caldwell, Ruth B; Caldwell, Robert W

    2017-11-01

    Elevation of arginase activity has been linked to vascular dysfunction in diabetes and hypertension by a mechanism involving decreased nitric oxide (NO) bioavailability due to L-arginine depletion. Excessive arginase activity also can drive L-arginine metabolism towards the production of ornithine, polyamines, and proline, promoting proliferation of vascular smooth muscle cells and collagen formation, leading to perivascular fibrosis. We hypothesized that there is a specific involvement of arginase 1 expression within the vascular endothelial cells in this pathology. To test this proposition, we used models of type 2 diabetes and metabolic syndrome. Studies were performed using wild type (WT), endothelial-specific arginase 1 knockout (EC-A1-/-) and littermate controls(A1con) mice fed high fat-high sucrose (HFHS) or normal diet (ND) for 6 months and isolated vessels exposed to palmitate-high glucose (PA/HG) media. Some WT mice or isolated vessels were treated with an arginase inhibitor, ABH [2-(S)-amino-6-boronohexanoic acid. In WT mice, the HFHS diet promoted increases in body weight, fasting blood glucose, and post-prandial insulin levels along with arterial stiffening and fibrosis, elevated blood pressure, decreased plasma levels of L-arginine, and elevated L-ornithine. The HFHS diet or PA/HG treatment also induced increases in vascular arginase activity along with oxidative stress, reduced vascular NO levels, and impaired endothelial-dependent vasorelaxation. All of these effects except obesity and hypercholesterolemia were prevented or significantly reduced by endothelial-specific deletion of arginase 1 or ABH treatment. Vascular dysfunctions in diet-induced obesity are prevented by deletion of arginase 1 in vascular endothelial cells or arginase inhibition. These findings indicate that upregulation of arginase 1 expression/activity in vascular endothelial cells has an integral role in diet-induced cardiovascular dysfunction and metabolic syndrome. Published

  2. Augmentation of arginase 1 expression by exposure to air pollution exacerbates the airways hyperresponsiveness in murine models of asthma

    Directory of Open Access Journals (Sweden)

    Amatullah Hajera

    2011-02-01

    Full Text Available Abstract Background Arginase overexpression contributes to airways hyperresponsiveness (AHR in asthma. Arginase expression is further augmented in cigarette smoking asthmatics, suggesting that it may be upregulated by environmental pollution. Thus, we hypothesize that arginase contributes to the exacerbation of respiratory symptoms following exposure to air pollution, and that pharmacologic inhibition of arginase would abrogate the pollution-induced AHR. Methods To investigate the role of arginase in the air pollution-induced exacerbation of airways responsiveness, we employed two murine models of allergic airways inflammation. Mice were sensitized to ovalbumin (OVA and challenged with nebulized PBS (OVA/PBS or OVA (OVA/OVA for three consecutive days (sub-acute model or 12 weeks (chronic model, which exhibit inflammatory cell influx and remodeling/AHR, respectively. Twenty-four hours after the final challenge, mice were exposed to concentrated ambient fine particles plus ozone (CAP+O3, or HEPA-filtered air (FA, for 4 hours. After the CAP+O3 exposures, mice underwent tracheal cannulation and were treated with an aerosolized arginase inhibitor (S-boronoethyl-L-cysteine; BEC or vehicle, immediately before determination of respiratory function and methacholine-responsiveness using the flexiVent®. Lungs were then collected for comparison of arginase activity, protein expression, and immunohistochemical localization. Results Compared to FA, arginase activity was significantly augmented in the lungs of CAP+O3-exposed OVA/OVA mice in both the sub-acute and chronic models. Western blotting and immunohistochemical staining revealed that the increased activity was due to arginase 1 expression in the area surrounding the airways in both models. Arginase inhibition significantly reduced the CAP+O3-induced increase in AHR in both models. Conclusions This study demonstrates that arginase is upregulated following environmental exposures in murine models of

  3. Increased levels of circulating arginase I in overweight compared to normal weight adolescents.

    Science.gov (United States)

    Jung, Christian; Figulla, Hans R; Lichtenauer, Michael; Franz, Marcus; Pernow, John

    2014-02-01

    Overweight and the metabolic syndrome have become major problems, especially in children and adolescents. Obesity at a young age increases the risk for cardiovascular diseases and diabetes mellitus later in life. An early event in the development of cardiovascular disease is endothelial dysfunction which is found in obese young individuals. Increased activity of the enzyme arginase has been described as a central mechanism for endothelial dysfunction, especially in patients with diabetes mellitus. The aim of the study was to determine plasma levels of arginase in overweight adolescents. Sixty-six male German adolescents (age: 15.2 ± 1.1 years old) were included. Thirty-one of them were overweight (>90th age-specific weight percentile). Plasma arginase I and tumor necrosis factor alpha (TNFα) were determined. In addition, clinical data were recorded and anthropometrical measurements of obesity were performed. Overweight adolescents had a higher systolic blood pressure, lower high-density lipoprotein and increased levels of high-sensitive C-reactive protein (CRP). Circulating arginase I was elevated in overweight adolescents (95.8 ± 68.2 ng/ml) compared to normal weight adolescents (39.3 ± 26.9 ng/ml, p obesity. There was no difference between the two groups regarding TNFα. We demonstrate that arginase I levels are increased in obese adolescents. Knowing the important role for arginase in endothelial dysfunction, elevated levels of arginase I may represent a link between obesity, endothelial dysfunction and related comorbidities. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. Restoring Ureagenesis in Hepatocytes by CRISPR/Cas9-mediated Genomic Addition to Arginase-deficient Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Lee, Patrick C; Truong, Brian; Vega-Crespo, Agustin; Gilmore, W Blake; Hermann, Kip; Angarita, Stephanie Ak; Tang, Jonathan K; Chang, Katherine M; Wininger, Austin E; Lam, Alex K; Schoenberg, Benjamen E; Cederbaum, Stephen D; Pyle, April D; Byrne, James A; Lipshutz, Gerald S

    2016-11-29

    Urea cycle disorders are incurable enzymopathies that affect nitrogen metabolism and typically lead to hyperammonemia. Arginase deficiency results from a mutation in Arg1, the enzyme regulating the final step of ureagenesis and typically results in developmental disabilities, seizures, spastic diplegia, and sometimes death. Current medical treatments for urea cycle disorders are only marginally effective, and for proximal disorders, liver transplantation is effective but limited by graft availability. Advances in human induced pluripotent stem cell research has allowed for the genetic modification of stem cells for potential cellular replacement therapies. In this study, we demonstrate a universally-applicable CRISPR/Cas9-based strategy utilizing exon 1 of the hypoxanthine-guanine phosphoribosyltransferase locus to genetically modify and restore arginase activity, and thus ureagenesis, in genetically distinct patient-specific human induced pluripotent stem cells and hepatocyte-like derivatives. Successful strategies restoring gene function in patient-specific human induced pluripotent stem cells may advance applications of genetically modified cell therapy to treat urea cycle and other inborn errors of metabolism.

  5. Molecular cloning and nucleotide sequence of cDNA for human liver arginase

    International Nuclear Information System (INIS)

    Haraguchi, Y.; Takiguchi, M.; Amaya, Y.; Kawamoto, S.; Matsuda, I.; Mori, M.

    1987-01-01

    Arginase (EC3.5.3.1) catalyzes the last step of the urea cycle in the liver of ureotelic animals. Inherited deficiency of the enzyme results in argininemia, an autosomal recessive disorder characterized by hyperammonemia. To facilitate investigation of the enzyme and gene structures and to elucidate the nature of the mutation in argininemia, the authors isolated cDNA clones for human liver arginase. Oligo(dT)-primed and random primer human liver cDNA libraries in λ gt11 were screened using isolated rat arginase cDNA as a probe. Two of the positive clones, designated λ hARG6 and λ hARG109, contained an overlapping cDNA sequence with an open reading frame encoding a polypeptide of 322 amino acid residues (predicted M/sub r/, 34,732), a 5'-untranslated sequence of 56 base pairs, a 3'-untranslated sequence of 423 base pairs, and a poly(A) segment. Arginase activity was detected in Escherichia coli cells transformed with the plasmid carrying λ hARG6 cDNA insert. RNA gel blot analysis of human liver RNA showed a single mRNA of 1.6 kilobases. The predicted amino acid sequence of human liver arginase is 87% and 41% identical with those of the rat liver and yeast enzymes, respectively. There are several highly conserved segments among the human, rat, and yeast enzymes

  6. A protective effect of the laminated layer on Echinococcus granulosus survival dependent on upregulation of host arginase.

    Science.gov (United States)

    Amri, Manel; Touil-Boukoffa, Chafia

    2015-09-01

    The role of nitric oxide (NO) in host defense against Echinococcus granulosus larvae was previously reported. However, NO production by NOS2 (inducible NO synthase) is counteracted by the expression of Arginase. In the present study, our aim is to evaluate the involvement of the laminated layer (external layer of parasitic cyst) in Arginase induction and the protoscoleces (living and infective part of the cyst) survival. Our in vitro results indicate that this cystic compound increases the Arginase activity in macrophages. Moreover, C-type lectin receptors (CLRs) with specificity for mannan and the TGF-β are implicated in this effect as shown after adding Mannan and Anti-TGFβ. Interestingly, the laminated layer increases protoscoleces survival in macrophages-parasite co-cultures. Our results indicate that the laminated layer protects E. granulosus against the NOS2 protective response through Arginase pathway, a hallmark of M2 macrophages. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Arginase 1: an unexpected mediator of pulmonary capillary barrier dysfunction in models of acute lung injury

    Directory of Open Access Journals (Sweden)

    Rudolf eLucas

    2013-08-01

    Full Text Available The integrity of epithelial and endothelial barriers in the lower airspaces of the lungs has to be tightly regulated, in order to prevent leakage and to assure efficient gas exchange between the alveoli and capillaries. Both G- and G+ bacterial toxins, such as LPS and pneumolysin, respectively, can be released in high concentrations within the pulmonary compartments upon antibiotic treatment of patients suffering from acute respiratory distress syndrome (ARDS or severe pneumonia. These toxins are able to impair endothelial barrier function, either directly, or indirectly, by induction of pro-inflammatory mediators and neutrophil sequestration. Toxin-induced endothelial hyperpermeability can involve myosin light chain phosphorylation and/or microtubule rearrangement. Endothelial nitric oxide synthase (eNOS was proposed to be a guardian of basal barrier function, since eNOS knock-out mice display an impaired expression of inter-endothelial junction proteins and as such an increased vascular permeability, as compared to wild type mice. The enzyme arginase, the activity of which can be regulated by the redox status of the cell, exists in two isoforms - arginase 1 (cytosolic and arginase 2 (mitochondrial - both of which can be expressed in lung microvascular endothelial cells. Upon activation, arginase competes with eNOS for the substrate L-arginine, as such impairing eNOS-dependent NO generation and promoting ROS generation by the enzyme. This mini-review will discuss recent findings regarding the interaction between bacterial toxins and arginase during acute lung injury and will as such address the role of arginase in bacterial toxin-induced pulmonary endothelial barrier dysfunction.

  8. Highly selective apo-arginase based method for sensitive enzymatic assay of manganese (II) and cobalt (II) ions

    Science.gov (United States)

    Stasyuk, Nataliya; Gayda, Galina; Zakalskiy, Andriy; Zakalska, Oksana; Errachid, Abdelhamid; Gonchar, Mykhailo

    2018-03-01

    A novel enzymatic method of manganese (II) and cobalt (II) ions assay, based on using apo-enzyme of Mn2 +-dependent recombinant arginase I (arginase) and 2,3-butanedione monoxime (DMO) as a chemical reagent is proposed. The principle of the method is the evaluation of the activity of L-arginine-hydrolyzing of arginase holoenzyme after the specific binding of Mn2 + or Co2 + with apo-arginase. Urea, which is the product of enzymatic hydrolysis of L-arginine (Arg), reacts with DMO and the resulted compound is detected by both fluorometry and visual spectrophotometry. Thus, the content of metal ions in the tested samples can be determined by measuring the level of urea generated after enzymatic hydrolysis of Arg by reconstructed arginase holoenzyme in the presence of tested metal ions. The linearity range of the fluorometric apo-arginase-DMO method in the case of Mn2 + assay is from 4 pM to 1.10 nM with a limit of detection of 1 pM Mn2 +, whereas the linearity range of the present method in the case of Co2 + assay is from 8 pM to 45 nM with a limit of detection of 2.5 pM Co2 +. The proposed method being highly sensitive, selective, valid and low-cost, may be useful to monitor Mn2 + and Co2 + content in clinical laboratories, food industry and environmental control service.

  9. Arginase and Arginine Decarboxylase - Where Do the Putative Gate Keepers of Polyamine Synthesis Reside in Rat Brain?

    Directory of Open Access Journals (Sweden)

    Daniela Peters

    Full Text Available Polyamines are important regulators of basal cellular functions but also subserve highly specific tasks in the mammalian brain. With this respect, polyamines and the synthesizing and degrading enzymes are clearly differentially distributed in neurons versus glial cells and also in different brain areas. The synthesis of the diamine putrescine may be driven via two different pathways. In the "classical" pathway urea and carbon dioxide are removed from arginine by arginase and ornithine decarboxylase. The alternative pathway, first removing carbon dioxide by arginine decarboxlyase and then urea by agmatinase, may serve the same purpose. Furthermore, the intermediate product of the alternative pathway, agmatine, is an endogenous ligand for imidazoline receptors and may serve as a neurotransmitter. In order to evaluate and compare the expression patterns of the two gate keeper enzymes arginase and arginine decarboxylase, we generated polyclonal, monospecific antibodies against arginase-1 and arginine decarboxylase. Using these tools, we immunocytochemically screened the rat brain and compared the expression patterns of both enzymes in several brain areas on the regional, cellular and subcellular level. In contrast to other enzymes of the polyamine pathway, arginine decarboxylase and arginase are both constitutively and widely expressed in rat brain neurons. In cerebral cortex and hippocampus, principal neurons and putative interneurons were clearly labeled for both enzymes. Labeling, however, was strikingly different in these neurons with respect to the subcellular localization of the enzymes. While with antibodies against arginine decarboxylase the immunosignal was distributed throughout the cytoplasm, arginase-like immunoreactivity was preferentially localized to Golgi stacks. Given the apparent congruence of arginase and arginine decarboxylase distribution with respect to certain cell populations, it seems likely that the synthesis of agmatine

  10. Helminth-induced arginase-1 exacerbates lung inflammation and disease severity in tuberculosis

    Science.gov (United States)

    Monin, Leticia; Griffiths, Kristin L.; Lam, Wing Y.; Gopal, Radha; Kang, Dongwan D.; Ahmed, Mushtaq; Rajamanickam, Anuradha; Cruz-Lagunas, Alfredo; Zúñiga, Joaquín; Babu, Subash; Kolls, Jay K.; Mitreva, Makedonka; Rosa, Bruce A.; Ramos-Payan, Rosalio; Morrison, Thomas E.; Murray, Peter J.; Rangel-Moreno, Javier; Pearce, Edward J.; Khader, Shabaana A.

    2015-01-01

    Parasitic helminth worms, such as Schistosoma mansoni, are endemic in regions with a high prevalence of tuberculosis (TB) among the population. Human studies suggest that helminth coinfections contribute to increased TB susceptibility and increased rates of TB reactivation. Prevailing models suggest that T helper type 2 (Th2) responses induced by helminth infection impair Th1 immune responses and thereby limit Mycobacterium tuberculosis (Mtb) control. Using a pulmonary mouse model of Mtb infection, we demonstrated that S. mansoni coinfection or immunization with S. mansoni egg antigens can reversibly impair Mtb-specific T cell responses without affecting macrophage-mediated Mtb control. Instead, S. mansoni infection resulted in accumulation of high arginase-1–expressing macrophages in the lung, which formed type 2 granulomas and exacerbated inflammation in Mtb-infected mice. Treatment of coinfected animals with an antihelminthic improved Mtb-specific Th1 responses and reduced disease severity. In a genetically diverse mouse population infected with Mtb, enhanced arginase-1 activity was associated with increased lung inflammation. Moreover, in patients with pulmonary TB, lung damage correlated with increased serum activity of arginase-1, which was elevated in TB patients coinfected with helminths. Together, our data indicate that helminth coinfection induces arginase-1–expressing type 2 granulomas, thereby increasing inflammation and TB disease severity. These results also provide insight into the mechanisms by which helminth coinfections drive increased susceptibility, disease progression, and severity in TB. PMID:26571397

  11. Arginase attenuates inhibitory nonadrenergic noncholinergic nerve-induced nitric oxide generation and airway smooth muscle relaxation

    Directory of Open Access Journals (Sweden)

    Meurs Herman

    2005-03-01

    Full Text Available Abstract Background Recent evidence suggests that endogenous arginase activity potentiates airway responsiveness to methacholine by attenuation of agonist-induced nitric oxide (NO production, presumably by competition with epithelial constitutive NO synthase for the common substrate, L-arginine. Using guinea pig tracheal open-ring preparations, we now investigated the involvement of arginase in the modulation of neuronal nitric oxide synthase (nNOS-mediated relaxation induced by inhibitory nonadrenergic noncholinergic (iNANC nerve stimulation. Methods Electrical field stimulation (EFS; 150 mA, 4 ms, 4 s, 0.5 – 16 Hz-induced relaxation was measured in tracheal preparations precontracted to 30% with histamine, in the presence of 1 μM atropine and 3 μM indomethacin. The contribution of NO to the EFS-induced relaxation was assessed by the nonselective NOS inhibitor L-NNA (0.1 mM, while the involvement of arginase activity in the regulation of EFS-induced NO production and relaxation was investigated by the effect of the specific arginase inhibitor nor-NOHA (10 μM. Furthermore, the role of substrate availability to nNOS in EFS-induced relaxation was measured in the presence of various concentrations of exogenous L-arginine. Results EFS induced a frequency-dependent relaxation, ranging from 6.6 ± 0.8% at 0.5 Hz to 74.6 ± 1.2% at 16 Hz, which was inhibited with the NOS inhibitor L-NNA by 78.0 ± 10.5% at 0.5 Hz to 26.7 ± 7.7% at 8 Hz (P Conclusion The results indicate that endogenous arginase activity attenuates iNANC nerve-mediated airway relaxation by inhibition of NO generation, presumably by limiting L-arginine availability to nNOS.

  12. Over-expression of zmarg encoding an arginase improves grain production in maize

    International Nuclear Information System (INIS)

    Hong, D.; Tian, Y.; Meng, X.; Zhang, P.

    2016-01-01

    Arginase, as one of the three key enzymes in nitrogen catabolism, the physiological role of Arg catabolism in cereal crops has not been fully clarified. Studies have shown that arginase-encoding genes play a key role in providing nitrogen to developing seedlings in many plant species.Yield is a primary trait in many crop breeding programs, which can be increased by modification of genes related to photosynthesis, nitrogen assimilation, carbon distribution, plant architecture, and transcriptional networks controlling plant development. In the present study, a maize arginase gene ZmARG was cloned and introduced into maize inbred lines by Agrobacterium tumefaciens- mediated transformation. Putative transgenic plants were confirmed by PCR, Southern blotting RT-PCR analysis. The expression of the ZmARG gene increased arginase activity in several tissues in transgenic lines. Transgenic maize plants had significantly higher ear weight and 100-seed weight as compared with wild-type control. Our results suggested that ZmARG was a potential target gene for crop yield improvement. (author)

  13. Inducible arginase 1 deficiency in mice leads to hyperargininemia and altered amino acid metabolism.

    Directory of Open Access Journals (Sweden)

    Yuan Yan Sin

    Full Text Available Arginase deficiency is a rare autosomal recessive disorder resulting from a loss of the liver arginase isoform, arginase 1 (ARG1, which is the final step in the urea cycle for detoxifying ammonia. ARG1 deficiency leads to hyperargininemia, characterized by progressive neurological impairment, persistent growth retardation and infrequent episodes of hyperammonemia. Using the Cre/loxP-directed conditional gene knockout system, we generated an inducible Arg1-deficient mouse model by crossing "floxed" Arg1 mice with CreER(T2 mice. The resulting mice (Arg-Cre die about two weeks after tamoxifen administration regardless of the starting age of inducing the knockout. These treated mice were nearly devoid of Arg1 mRNA, protein and liver arginase activity, and exhibited symptoms of hyperammonemia. Plasma amino acid analysis revealed pronounced hyperargininemia and significant alterations in amino acid and guanidino compound metabolism, including increased citrulline and guanidinoacetic acid. Despite no alteration in ornithine levels, concentrations of other amino acids such as proline and the branched-chain amino acids were reduced. In summary, we have generated and characterized an inducible Arg1-deficient mouse model exhibiting several pathologic manifestations of hyperargininemia. This model should prove useful for exploring potential treatment options of ARG1 deficiency.

  14. Unique hepatic cytosolic arginase evolved independently in ureogenic freshwater air-breathing teleost, Heteropneustes fossilis.

    Directory of Open Access Journals (Sweden)

    Shilpee Srivastava

    Full Text Available Hepatic cytosolic arginase (ARG I, an enzyme of the urea cycle operating in the liver of ureotelic animals, is reported to be present in an ammoniotelic freshwater air-breathing teleost, Heteropneustes fossilis which has ureogenic potential. Antibodies available against mammalian ARG I showed no cross reactivity with the H. fossilis ARG I. We purified unique ARG I from H. fossilis liver. Purified ARG I is a homotrimer with molecular mass 75 kDa and subunit molecular mass of 24 kDa. The pI value of the enzyme was 8.5. It showed maximum activity at pH 10.5 and 55°C. The Km of purified enzyme for L-arginine was 2.65±0.39 mM. L-ornithine and N(ω-hydroxy-L-arginine showed inhibition of the ARG I activity, with Ki values 0.52±0.02mM and 0.08±0.006mM, respectively. Antibody raised against the purified fish liver ARG I showed exclusive specificity, and has no cross reactivity against fish liver ARG II and mammalian liver ARG I and ARG II. We found another isoform of arginase bound to the outer membrane of the mitochondria which was released by 150-200 mM KCl in the extraction medium. This isoform was immunologically different from the soluble cytosolic and mitochondrial arginase. The results of present study support that hepatic cytosolic arginase evolved in this ureogenic freshwater teleost, H. fossilis. Phylogenetic analysis confirms an independent evolution event that occurred much after the evolution of the cytosolic arginase of ureotelic vertebrates.

  15. Mathematical analysis of complex cellular activity

    CERN Document Server

    Bertram, Richard; Teka, Wondimu; Vo, Theodore; Wechselberger, Martin; Kirk, Vivien; Sneyd, James

    2015-01-01

    This book contains two review articles on mathematical physiology that deal with closely related topics but were written and can be read independently. The first article reviews the basic theory of calcium oscillations (common to almost all cell types), including spatio-temporal behaviors such as waves. The second article uses, and expands on, much of this basic theory to show how the interaction of cytosolic calcium oscillators with membrane ion channels can result in highly complex patterns of electrical spiking. Through these examples one can see clearly how multiple oscillatory processes interact within a cell, and how mathematical methods can be used to understand such interactions better. The two reviews provide excellent examples of how mathematics and physiology can learn from each other, and work jointly towards a better understanding of complex cellular processes. Review 1: Richard Bertram, Joel Tabak, Wondimu Teka, Theodore Vo, Martin Wechselberger: Geometric Singular Perturbation Analysis of Burst...

  16. Arginase induction by sodium phenylbutyrate in mouse tissues and human cell lines.

    Science.gov (United States)

    Kern, R M; Yang, Z; Kim, P S; Grody, W W; Iyer, R K; Cederbaum, S D

    2007-01-01

    Hyperargininemia is a urea cycle disorder caused by mutations in the gene for arginase I (AI) resulting in elevated blood arginine and ammonia levels. Sodium phenylacetate and a precursor, sodium phenylbutyrate (NaPB) have been used to lower ammonia, conjugating glutamine to produce phenylacetylglutamine which is excreted in urine. The elevated arginine levels induce the second arginase (AII) in patient kidney and kidney tissue culture. It has been shown that NaPB increases expression of some target genes and we tested its effect on arginase induction. Eight 9-week old male mice fed on chow containing 7.5 g NaPB/kg rodent chow and drank water with 10 g NaPB/L, and four control mice had a normal diet. After one week all mice were sacrificed. The arginase specific activities for control and NaPB mice, respectively, were 38.2 and 59.4 U/mg in liver, 0.33 and 0.42 U/mg in kidney, and 0.29 and 1.19 U/mg in brain. Immunoprecipitation of arginase in each tissue with AI and AII antibodies showed the activity induced by NaPB is mostly AI. AII may also be induced in kidney. AI accounts for the fourfold increased activity in brain. In some cell lines, NaPB increased arginase activity up to fivefold depending on dose (1-5 mM) and exposure time (2-5 days); control and NaPB activities, respectively, are: erythroleukemia, HEL, 0.06 and 0.31 U/mg, and K562, 0.46 and 1.74 U/mg; embryonic kidney, HEK293, 1.98 and 3.58 U/mg; breast adenocarcinoma, MDA-MB-468, 1.11 and 4.06 U/mg; and prostate adenocarcinoma, PC-3, 0.55 and 3.20 U/mg. In MDA-MB-468 and HEK most, but not all, of the induced activity is AI. These studies suggest that NaPB may induce AI when used to treat urea cycle disorders. It is relatively less useful in AI deficiency, although it could have some effect in those patients with missense mutations.

  17. Arginase strongly impairs neuronal nitric oxide-mediated airway smooth muscle relaxation in allergic asthma

    Directory of Open Access Journals (Sweden)

    Zaagsma Johan

    2006-01-01

    Full Text Available Abstract Background Using guinea pig tracheal preparations, we have recently shown that endogenous arginase activity attenuates inhibitory nonadrenergic noncholinergic (iNANC nerve-mediated airway smooth muscle relaxation by reducing nitric oxide (NO production – due to competition with neuronal NO-synthase (nNOS for the common substrate, L-arginine. Furthermore, in a guinea pig model of allergic asthma, airway arginase activity is markedly increased after the early asthmatic reaction (EAR, leading to deficiency of agonist-induced, epithelium-derived NO and subsequent airway hyperreactivity. In this study, we investigated whether increased arginase activity after the EAR affects iNANC nerve-derived NO production and airway smooth muscle relaxation. Methods Electrical field stimulation (EFS; 150 mA, 4 ms, 4 s, 0.5 – 16 Hz-induced relaxation was measured in tracheal open-ring preparations precontracted to 30% with histamine in the presence of 1 μM atropine and 3 μM indomethacin. The contribution of NO to EFS-induced relaxation was assessed by the nonselective NOS inhibitor Nω-nitro-L-arginine (L-NNA, 100 μM, while the involvement of arginase activity in the regulation of EFS-induced NO production and relaxation was investigated by the effect of the specific arginase inhibitor Nω-hydroxy-nor-L-arginine (nor-NOHA, 10 μM. Furthermore, the role of substrate availability to nNOS was measured in the presence of exogenous L-arginine (5.0 mM. Results At 6 h after ovalbumin-challenge (after the EAR, EFS-induced relaxation (ranging from 3.2 ± 1.1% at 0.5 Hz to 58.5 ± 2.2% at 16 Hz was significantly decreased compared to unchallenged controls (7.1 ± 0.8% to 75.8 ± 0.7%; P P P Conclusion The results clearly demonstrate that increased arginase activity after the allergen-induced EAR contributes to a deficiency of iNANC nerve-derived NO and decreased airway smooth muscle relaxation, presumably via increased substrate competition with nNOS.

  18. Cerebral changes occurring in arginase and dimethylarginine dimethylaminohydrolase (DDAH in a rat model of sleeping sickness.

    Directory of Open Access Journals (Sweden)

    Donia Amrouni

    2011-03-01

    Full Text Available Involvement of nitric oxide (NO in the pathophysiology of human African trypanosomiasis (HAT was analyzed in a HAT animal model (rat infected with Trypanosoma brucei brucei. With this model, it was previously reported that trypanosomes were capable of limiting trypanocidal properties carried by NO by decreasing its blood concentration. It was also observed that brain NO concentration, contrary to blood, increases throughout the infection process. The present approach analyses the brain impairments occurring in the regulations exerted by arginase and N(G, N(G-dimethylarginine dimethylaminohydrolase (DDAH on NO Synthases (NOS. In this respect: (i cerebral enzymatic activities, mRNA and protein expression of arginase and DDAH were determined; (ii immunohistochemical distribution and morphometric parameters of cells expressing DDAH-1 and DDAH-2 isoforms were examined within the diencephalon; (iii amino acid profiles relating to NOS/arginase/DDAH pathways were established.Arginase and DDAH activities together with mRNA (RT-PCR and protein (western-blot expressions were determined in diencephalic brain structures of healthy or infected rats at various days post-infection (D5, D10, D16, D22. While arginase activity remained constant, that of DDAH increased at D10 (+65% and D16 (+51% in agreement with western-blot and amino acids data (liquid chromatography tandem-mass spectrometry. Only DDAH-2 isoform appeared to be up-regulated at the transcriptional level throughout the infection process. Immunohistochemical staining further revealed that DDAH-1 and DDAH-2 are contained within interneurons and neurons, respectively.In the brain of infected animals, the lack of change observed in arginase activity indicates that polyamine production is not enhanced. Increases in DDAH-2 isoform may contribute to the overproduction of NO. These changes are at variance with those reported in the periphery. As a whole, the above processes may ensure additive protection

  19. Reporters to monitor cellular MMP12 activity

    Science.gov (United States)

    Cobos-Correa, Amanda; Mall, Marcus A.; Schultz, Carsten

    2010-02-01

    Macrophage elastase, also called MMP12, belongs to a family of proteolytic enzymes whose best known physiological function is the remodeling of the extracellular matrix. Under certain pathological conditions, including inflammation, chronic overexpression of MMP12 has been observed and its elevated proteolytic activity has been suggested to be the cause of pulmonary emphysema. However, it was until recently impossible to monitor the activity of MMP12 under disease conditions, mainly due to a lack of detection methods. Recent development of new reporters for monitoring MMP12 activity in living cells, such as LaRee1, provided novel insights into the pathobiology of MMP12 in pulmonary inflammation.1 In the future, these reporters might contribute to improved diagnosis and in finding better treatments for chronic inflammatory lung diseases and emphysema. Our approach for visualizing MMP12 activity is based on peptidic, membrane-targeted FRET (Foerster Resonance Energy Transfer) reporters. Here we describe a set of new reporters containing different fluorophore pairs as well as modifications in the membrane-targeting lipid moiety. We studied the influence of these modifications on reporter performance and the reporter mobility on live cell membranes by FRAP (fluorescence recovery after photobleaching). Finally, we generated several new fluorescently labeled MMP inhibitors based on the peptidic reporter structures as prototypes for future tools to inhibit and monitor MMP activity at the same time.

  20. Mechanisms of Diabetes-Induced Endothelial Cell Senescence: Role of Arginase 1

    Directory of Open Access Journals (Sweden)

    Esraa Shosha

    2018-04-01

    Full Text Available We have recently found that diabetes-induced premature senescence of retinal endothelial cells is accompanied by NOX2-NADPH oxidase-induced increases in the ureohydrolase enzyme arginase 1 (A1. Here, we used genetic strategies to determine the specific involvement of A1 in diabetes-induced endothelial cell senescence. We used A1 knockout mice and wild type mice that were rendered diabetic with streptozotocin and retinal endothelial cells (ECs exposed to high glucose or transduced with adenovirus to overexpress A1 for these experiments. ABH [2(S-Amino-6-boronohexanoic acid] was used to inhibit arginase activity. We used Western blotting, immunolabeling, quantitative PCR, and senescence associated β-galactosidase (SA β-Gal activity to evaluate senescence. Analyses of retinal tissue extracts from diabetic mice showed significant increases in mRNA expression of the senescence-related proteins p16INK4a, p21, and p53 when compared with non-diabetic mice. SA β-Gal activity and p16INK4a immunoreactivity were also increased in retinal vessels from diabetic mice. A1 gene deletion or pharmacological inhibition protected against the induction of premature senescence. A1 overexpression or high glucose treatment increased SA β-Gal activity in cultured ECs. These results demonstrate that A1 is critically involved in diabetes-induced senescence of retinal ECs. Inhibition of arginase activity may therefore be an effective therapeutic strategy to alleviate diabetic retinopathy by preventing premature senescence.

  1. Angiotensin II-induced arterial thickening, fibrosis and stiffening involves elevated arginase function.

    Directory of Open Access Journals (Sweden)

    Anil Bhatta

    Full Text Available Arterial stiffness (AS is an independent risk factor for cardiovascular morbidity/mortality. Smooth muscle cell (SMC proliferation and increased collagen synthesis are key features in development of AS. Arginase (ARG, an enzyme implicated in many cardiovascular diseases, can compete with nitric oxide (NO synthase for their common substrate, L-arginine. Increased arginase can also provide ornithine for synthesis of polyamines via ornithine decarboxylase (ODC and proline/collagen via ornithine aminotransferase (OAT, leading to vascular cell proliferation and collagen formation, respectively. We hypothesized that elevated arginase activity is involved in Ang II-induced arterial thickening, fibrosis, and stiffness and that limiting its activity can prevent these changes.We tested this by studies in mice lacking one copy of the ARG1 gene that were treated with angiotensin II (Ang II, 4 weeks. Studies were also performed in rat aortic Ang II-treated SMC. In WT mice treated with Ang II, we observed aortic stiffening (pulse wave velocity and aortic and coronary fibrosis and thickening that were associated with increases in ARG1 and ODC expression/activity, proliferating cell nuclear antigen, hydroxyproline levels, and collagen 1 protein expression. ARG1 deletion prevented each of these alterations. Furthermore, exposure of SMC to Ang II (1 μM, 48 hrs increased ARG1 expression, ARG activity, ODC mRNA and activity, cell proliferation, collagen 1 protein expression and hydroxyproline content. Treatment with ABH prevented these changes.Arginase 1 is crucially involved in Ang II-induced SMC proliferation and arterial fibrosis and stiffness and represents a promising therapeutic target.

  2. L-arginine and Arginase Products Potentiate Dexmedetomidine-induced Contractions in the Rat Aorta.

    Science.gov (United States)

    Wong, Emily S W; Man, Ricky Y K; Ng, Kwok F J; Leung, Susan W S; Vanhoutte, Paul M

    2018-03-01

    dexmedetomidine by L-arginine depends on arginase activity and the production of urea and ornithine.

  3. Vasomotor Regulation of Coronary Microcirculation by Oxidative Stress: Role of Arginase

    Directory of Open Access Journals (Sweden)

    Lih eKuo

    2013-08-01

    Full Text Available Overproduction of reactive oxygen species, i.e., oxidative stress, is associated with the activation of redox signaling pathways linking to inflammatory insults and cardiovascular diseases by impairing endothelial function and consequently blood flow dysregulation due to microvascular dysfunction. This review focuses on the regulation of vasomotor function in the coronary microcirculation by endothelial nitric oxide (NO during oxidative stress and inflammation related to the activation of L-arginine consuming enzyme arginase. Superoxide produced in the vascular wall compromises vasomotor function by not only scavenging endothelium-derived NO but also inhibiting prostacyclin synthesis due to formation of peroxynitrite. The upregulation of arginase contributes to the deficiency of endothelial NO and microvascular dysfunction in various vascular diseases by initiating or following oxidative stress and inflammation. Hydrogen peroxide, a diffusible and stable oxidizing agent, exerts vasodilator function and plays important roles in the physiological regulation of coronary blood flow. In occlusive coronary ischemia, the release of hydrogen peroxide from the microvasculature helps to restore vasomotor function of coronary collateral microvessels with exercise training. However, excessive production and prolonged exposure of microvessels to hydrogen peroxide impairs NO-mediated endothelial function by reducing L-arginine availability through hydroxyl radical-dependent upregulation of arginase. The redox signaling can be a double-edged sword in the microcirculation, which helps tissue survival in one way by improving vasomotor regulation and elicits oxidative stress and tissue injury in the other way by causing vascular dysfunction. The impact of vascular arginase on the development of vasomotor dysfunction associated with angiotensin II receptor activation, hypertension, ischemia-reperfusion, hypercholesterolemia and inflammatory insults is discussed.

  4. DNA damage and decrease of cellular oxidase activity in piglet ...

    African Journals Online (AJOL)

    DNA damage and decrease of cellular oxidase activity in piglet sertoli cells exposed to gossypol. Ming Zhang, Hui Yuan, Zuping He, Liyun Yuan, Jine Yi, Sijun Deng, Li Zhu, Chengzhi Guo, Yin Lu, Jing Wu, Lixin Wen, Qiang Wei, Liqun Xue ...

  5. Transplantation of Gene-Edited Hepatocyte-like Cells Modestly Improves Survival of Arginase-1-Deficient Mice

    Directory of Open Access Journals (Sweden)

    Yuan Yan Sin

    2018-03-01

    Full Text Available Progress in gene editing research has been accelerated by utilizing engineered nucleases in combination with induced pluripotent stem cell (iPSC technology. Here, we report transcription activator-like effector nuclease (TALEN-mediated reincorporation of Arg1 exons 7 and 8 in iPSCs derived from arginase-1-deficient mice possessing Arg1Δ alleles lacking these terminal exons. The edited cells could be induced to differentiate into hepatocyte-like cells (iHLCs in vitro and were subsequently used for transplantation into our previously described (Sin et al., PLoS ONE 2013 tamoxifen-inducible Arg1-Cre arginase-1-deficient mouse model. While successful gene-targeted repair was achieved in iPSCs containing Arg1Δ alleles, only minimal restoration of urea cycle function could be observed in the iHLC-transplanted mice compared to control mice, and survival in this lethal model was extended by up to a week in some mice. The partially rescued phenotype may be due to inadequate regenerative capacity of arginase-1-expressing cells in the correct metabolic zones. Technical hurdles exist and will need to be overcome for gene-edited iPSC to iHLC rescue of arginase-1 deficiency, a rare urea cycle disorder.

  6. T cell-macrophage interaction in arginase-mediated resistance to herpes simplex virus.

    Science.gov (United States)

    Bonina, L; Nash, A A; Arena, A; Leung, K N; Wildy, P

    1984-09-01

    Peritoneal macrophages activated by-products derived from a herpes simplex virus-specific helper T cell clone were used to investigate intrinsic and extrinsic resistance mechanisms to herpes simplex virus type 1 infection in vitro. T cell-activated macrophages produced fewer infective centres, indicating enhanced intrinsic resistance, and markedly reduced the growth of virus in a permissive cell line. The reduction in virus growth correlated with the depletion of arginine in the support medium, presumably resulting from increased arginase production by activated macrophages. The significance of these findings for antiviral immunity in vivo is discussed.

  7. Cellular reprogramming through mitogen-activated protein kinases

    Directory of Open Access Journals (Sweden)

    Justin eLee

    2015-10-01

    Full Text Available Mitogen-activated protein kinase (MAPK cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554 in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression – including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding and degradation steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

  8. Ureogenesis in Aantarctic birds-Blood levels of nitrogen compounds and liver and kidney arginase in penguins

    Directory of Open Access Journals (Sweden)

    Edson Rodrigues

    1998-03-01

    Full Text Available A study was carried out on the levels and the kinetics of liver and kidney arginase from Pygoscelis penguins, the gentoo Pygoscelis papua, the chinstrap, Pygoscelis antarctica, and the Adelie, Pygoscelis adeliae. Higher values of blood urea were found in the gentoo penguins in the native state when compared with specimens maintained in the fasting state for 24 hours. In the chinstrap penguin Pygoscelis antarctica the average value for blood urea was 1.5 times higher in the native state than in the fasting condition. In the native gentoo penguin P. papua the relative increase in the blood urea concentration is as high as 3.5 times in regard to the levels found in the fasting state. In regard to the blood levels of uric acid, the difference between the native state and the fasting state is 2.0 times for P. antarctica and 4.8 times for P. papua. Specific activities of arginase assayed in penguin liver were 561 mU/mg protein and 208mU/mg protein for adult P. antarctica and P. papua respectively. Kinetic studies with arginase from penguin liver homogenates showed Km values for L-arginine of 16.0±2.0mM at pH9.5. Arginase from birds possesses in general high Km values (between 100-200mM. It seems then that the high protein diet and the high levels of blood urea of penguins are a consequence of the levels of hepatic arginase and the high affinity of this enzyme toward its substrate.

  9. Crystal structures of Leishmania mexicana arginase complexed with α,α-disubstituted boronic amino-acid inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Hai, Yang; Christianson, David W.

    2016-03-16

    Leishmaniaarginase is a potential drug target for the treatment of leishmaniasis because this binuclear manganese metalloenzyme initiatesde novopolyamine biosynthesis by catalyzing the hydrolysis of L-arginine to generate L-ornithine and urea. The product L-ornithine subsequently undergoes decarboxylation to yield putrescine, which in turn is utilized for spermidine biosynthesis. Polyamines such as spermidine are essential for the growth and survival of the parasite, so inhibition of enzymes in the polyamine-biosynthetic pathway comprises an effective strategy for treating parasitic infections. To this end, two X-ray crystal structures ofL. mexicanaarginase complexed with α,α-disubstituted boronic amino-acid inhibitors based on the molecular scaffold of 2-(S)-amino-6-boronohexanoic acid are now reported. Structural comparisons with human and parasitic arginase complexes reveal interesting differences in the binding modes of the additional α-substituents,i.e.the D side chains, of these inhibitors. Subtle differences in the three-dimensional contours of the outer active-site rims among arginases from different species lead to different conformations of the D side chains and thus different inhibitor-affinity trends. The structures suggest that it is possible to maintain affinity while fine-tuning intermolecular interactions of the D side chain of α,α-disubstituted boronic amino-acid inhibitors in the search for isozyme-specific and species-specific arginase inhibitors.

  10. Study of Stevia rebaudiana Bertoni antioxidant activities and cellular properties.

    Science.gov (United States)

    Bender, Cecilia; Graziano, Sara; Zimmermann, Benno F

    2015-01-01

    The aim of our study was to determine the antioxidant activities, cytotoxicity and proliferative properties in Stevia rebaudiana leaves and stems. Leaves extracts exhibited a higher antioxidant activity than stems extract, through oxygen radical absorbance capacity (ORAC) and cellular antioxidant activity (CAA) assays. Stevioside and rebaudioside A, the main sweetening metabolites in stevia leaves, exhibited a low ORAC value in comparison with plant extracts, while did not elicit any CAA. Stevia rebaudiana did not exhibit toxicity against HepG2 (hepatocellular carcinoma) human cells. No proliferative nor catalase modulations were observed in cells treated with such extracts. Our findings support the promising role of stevia that, apart from its sweetness, can act as a source of antioxidants, even at the intracellular level. This activity makes S. rebaudiana crude extract an interesting resource of natural sweetness with antioxidant properties which may find numerous applications in foods and nutritional supplements industries.

  11. Cell activation and cellular-cellular interactions during hemodialysis: effect of dialyzer membrane.

    Science.gov (United States)

    Sirolli, V; Ballone, E; Di Stante, S; Amoroso, L; Bonomini, M

    2002-06-01

    During hemodialysis (HD), circulating blood cells can be activated and also engage in dynamic interplay. These phenomena may be important factors behind dialysis membrane bio(in)compatibility. In the present prospective cross-over study, we have used flow cytometry to evaluate the influence of different dialysis membranes on the activation of circulating blood cells (leukocytes, platelets) and their dynamic interactions (formation of circulating platelet-leukocyte and platelet-erythrocyte aggregates) during in vivo HD. Each patient (n = 10) was treated with dialyzers containing membranes of cellulose diacetate, polysulfone and ethylenevinylalcohol (EVAL) in a randomized order. Upregulation of adhesion receptor expression (CD15s, CD11b/CD18) occurred mainly with the cellulosic membrane, though an increase in CD11b/CD18 circulating on neutrophils was also found with both synthetic membranes. Circulating activated platelets (P-selectin/CD63-positive platelets) increased during HD sessions with cellulose diacetate and polysulfone. An increased formation of platelet-neutrophil aggregates was found at 15 and 30 min during dialysis with cellulose diacetate and polysulfone but not with EVAL. Platelet-erythrocyte aggregates also increased with cellulose diacetate and at 15 min with polysulfone as well. Generally in concomitance with the increase in platelet-neutrophil coaggregates, there was an increased hydrogen peroxide production by neutrophils. The results of this study indicate that cellular mechanisms can be activated during HD largely depending on the membrane material, EVAL causing less reactivity than the other two membranes. It appears that each dialysis membrane has multiple and different characteristics that may contribute to interactions with blood components. Our results also indicate that derivatizing cellulose (cellulose diacetate) may be a useful way to improve the biocompatibility of the cellulose polymer and that there may be great variability in the

  12. Inhibition and Kinetic Studies of Tortoise (Kinixys erosa) Liver arginase

    African Journals Online (AJOL)

    The effect of amino acid on tortoise liver arginase showed that L-lysine, L-valine, L-serine, L-aspartic acid and L aspartic acid had significant inhibitory effect on the enzyme but proline and glutamic acid showed slight inhibition. Ethylenediaminetetraacetic acid (EDTA), citrate, ascorbic acid, boric acid and sodium borate ...

  13. Modulation of hyaluronan synthase activity in cellular membrane fractions.

    Science.gov (United States)

    Vigetti, Davide; Genasetti, Anna; Karousou, Evgenia; Viola, Manuela; Clerici, Moira; Bartolini, Barbara; Moretto, Paola; De Luca, Giancarlo; Hascall, Vincent C; Passi, Alberto

    2009-10-30

    Hyaluronan (HA), the only non-sulfated glycosaminoglycan, is involved in morphogenesis, wound healing, inflammation, angiogenesis, and cancer. In mammals, HA is synthesized by three homologous HA synthases, HAS1, HAS2, and HAS3, that polymerize the HA chain using UDP-glucuronic acid and UDP-N-acetylglucosamine as precursors. Since the amount of HA is critical in several pathophysiological conditions, we developed a non-radioactive assay for measuring the activity of HA synthases (HASs) in eukaryotic cells and addressed the question of HAS activity during intracellular protein trafficking. We prepared three cellular fractions: plasma membrane, cytosol (containing membrane proteins mainly from the endoplasmic reticulum and Golgi), and nuclei. After incubation with UDP-sugar precursors, newly synthesized HA was quantified by polyacrylamide gel electrophoresis of fluorophore-labeled saccharides and high performance liquid chromatography. This new method measured HAS activity not only in the plasma membrane fraction but also in the cytosolic membranes. This new technique was used to evaluate the effects of 4-methylumbeliferone, phorbol 12-myristate 13-acetate, interleukin 1beta, platelet-derived growth factor BB, and tunicamycin on HAS activities. We found that HAS activity can be modulated by post-translational modification, such as phosphorylation and N-glycosylation. Interestingly, we detected a significant increase in HAS activity in the cytosolic membrane fraction after tunicamycin treatment. Since this compound is known to induce HA cable structures, this result links HAS activity alteration with the capability of the cell to promote HA cable formation.

  14. Exploring cellular memory molecules marking competent and active transcriptions

    Directory of Open Access Journals (Sweden)

    Liu De-Pei

    2007-05-01

    Full Text Available Abstract Background Development in higher eukaryotes involves programmed gene expression. Cell type-specific gene expression is established during this process and is inherited in succeeding cell cycles. Higher eukaryotes have evolved elegant mechanisms by which committed gene-expression states are transmitted through numerous cell divisions. Previous studies have shown that both DNase I-sensitive sites and the basal transcription factor TFIID remain on silenced mitotic chromosomes, suggesting that certain trans-factors might act as bookmarks, maintaining the information and transmitting it to the next generation. Results We used the mouse globin gene clusters as a model system to examine the retention of active information on M-phase chromosomes and its contribution to the persistence of transcriptional competence of these gene clusters in murine erythroleukemia cells. In cells arrested in mitosis, the erythroid-specific activator NF-E2p45 remained associated with its binding sites on the globin gene loci, while the other major erythroid factor, GATA-1, was removed from chromosome. Moreover, despite mitotic chromatin condensation, the distant regulatory regions and promoters of transcriptionally competent globin gene loci are marked by a preserved histone code consisting in active histone modifications such as H3 acetylation, H3-K4 dimethylation and K79 dimethylation. Further analysis showed that other active genes are also locally marked by the preserved active histone code throughout mitotic inactivation of transcription. Conclusion Our results imply that certain kinds of specific protein factors and active histone modifications function as cellular memory markers for both competent and active genes during mitosis, and serve as a reactivated core for the resumption of transcription when the cells exit mitosis.

  15. Active Cellular Mechanics and its Consequences for Animal Development

    Science.gov (United States)

    Noll, Nicholas B.

    A central goal of developmental biology is to understand how an organism shapes itself, a process referred to as morphogenesis. While the molecular components critical to determining the initial body plan have been well characterized, the control of the subsequent dynamics of cellular rearrangements which ultimately shape the organism are far less understood. A major roadblock to a more complete picture of morphogenesis is the inability to measure tissue-scale mechanics throughout development and thus answer fundamental questions: How is the mechanical state of the cell regulated by local protein expression and global pattering? In what way does stress feedback onto the larger developmental program? In this dissertation, we begin to approach these questions through the introduction and analysis of a multi-scale model of epithelial mechanics which explicitly connects cytoskeletal protein activity to tissue-level stress. In Chapter 2, we introduce the discrete Active Tension Network (ATN) model of cellular mechanics. ATNs are tissues that satisfy two primary assumptions: that the mechanical balance of cells is dominated by cortical tension and that myosin actively remodels the actin cytoskeleton in a stress-dependent manner. Remarkably, the interplay of these features allows for angle-preserving, i.e. 'isogonal', dilations or contractions of local cell geometry that do not generate stress. Asymptotically this model is stabilized provided there is mechanical feedback on expression of myosin within the cell; we take this to be a strong prediction to be tested. The ATN model exposes a fundamental connection between equilibrium cell geometry and its underlying force network. In Chapter 3, we relax the tension-net approximation and demonstrate that at equilibrium, epithelial tissues with non-uniform pressure have non-trivial geometric constraints that imply the network is described by a weighted `dual' triangulation. We show that the dual triangulation encodes all

  16. Late onset arginase deficiency presenting with encephalopathy and midbrain hyperintensity

    Directory of Open Access Journals (Sweden)

    Boby Varkey Maramattom

    2016-01-01

    Full Text Available Urea cycle disorders (UCD are very rare metabolic disorders that present with encephalopathy and hyperammonemia. Of the UCDs, Arginase deficiency (ARD is the rarest and presents in childhood with a progressive spastic diplegia or seizures. Acute presentation in adulthood is extremely unusual. [1] We present the first case of adult onset ARD presenting with encephalopathy and diffusion weighted MRI findings that resembled a moustache in the midbrain.

  17. Anesthetic Management of a Pediatric Patient with Arginase Deficiency

    Directory of Open Access Journals (Sweden)

    Abdulkadir Atım

    2011-09-01

    Full Text Available Arginase deficiency is an autosomal recessive disorder of the urea cycle in which a defect in conversion of arginine to urea and ornithine leads to hyperammonemia. Patients with urea cycle disorders may show increased protein catabolism due to inadequate intake of energy, protein and essential amino acids; infections, fever and surgery. A 12-year-old girl with arginase deficiency, ASA II who weighed 40 kg was scheduled for bilateral adductor, quadriceps and gastrocnemius tenotomies. She had mental retardation, spasticity and flexion posture of thelower limbs. Metabolic homeostasis was restored with appropriate diet. Successful anesthetic management allowed the patient to be discharged 48 hours after surgery. Increased levels of arginine and ammonia during or after surgery may lead to serious complications such as hypotension, cerebral edema, convulsions, hypothermia and spasticity. Thus special attention must be given to metabolic homeostasis and nutrition of the patients with arginase deficiency in the perioperative period. Primary goals should be to minimize stress levels by effective anxiolysis, provide an adequate amount of protein-free energy with proper fluid management and to obtain an effective preemptive and postoperative analgesia. In addition to a high level of knowledge, successful anesthesia requires professional communication among nursing staff, dietitians, pediatric metabolism specialist, surgeon and anesthesiologist.

  18. l-Arginine Uptake by Cationic Amino Acid Transporter Promotes Intra-Macrophage Survival of Leishmania donovani by Enhancing Arginase-Mediated Polyamine Synthesis

    Directory of Open Access Journals (Sweden)

    Abhishek Mandal

    2017-07-01

    Full Text Available The survival of intracellular protozoan parasite, Leishmania donovani, the causative agent of Indian visceral leishmaniasis (VL, depends on the activation status of macrophages. l-Arginine, a semi-essential amino acid plays a crucial regulatory role for activation of macrophages. However, the role of l-arginine transport in VL still remains elusive. In this study, we demonstrated that intra-macrophage survival of L. donovani depends on the availability of extracellular l-arginine. Infection of THP-1-derived macrophage/human monocyte-derived macrophage (hMDM with Leishmania, resulted in upregulation of l-arginine transport. While investigating the involvement of the transporters, we observed that Leishmania survival was greatly impaired when the transporters were blocked either using inhibitor or siRNA-mediated downregulation. CAT-2 was found to be the main isoform associated with l-arginine transport in L. donovani-infected macrophages. l-arginine availability and its transport regulated the host arginase in Leishmania infection. Arginase and inducible nitric oxide synthase (iNOS expression were reciprocally regulated when assayed using specific inhibitors and siRNA-mediated downregulation. Interestingly, induction of iNOS expression and nitric oxide production were observed in case of inhibition of arginase in infected macrophages. Furthermore, inhibition of l-arginine transport as well as arginase resulted in decreased polyamine production, limiting parasite survival inside macrophages. l-arginine availability and transport regulated Th1/Th2 cytokine levels in case of Leishmania infection. Upregulation of l-arginine transport, induction of host arginase, and enhanced polyamine production were correlated with increased level of IL-10 and decreased level of IL-12 and TNF-α in L. donovani-infected macrophages. Our findings provide clear evidence for targeting the metabolism of l-arginine and l-arginine-metabolizing enzymes as an important

  19. Competitive metabolism of L-arginine: arginase as a therapeutic target in asthma☆

    Science.gov (United States)

    Bratt, Jennifer M.; Zeki, Amir A.; Last, Jerold A.; Kenyon, Nicholas J.

    2011-01-01

    Exhaled breath nitric oxide (NO) is an accepted asthma biomarker. Lung concentrations of NO and its amino acid precursor, L-arginine, are regulated by the relative expressions of the NO synthase (NOS) and arginase isoforms. Increased expression of arginase I and NOS2 occurs in murine models of allergic asthma and in biopsies of asthmatic airways. Although clinical trials involving the inhibition of NO-producing enzymes have shown mixed results, small molecule arginase inhibitors have shown potential as a therapeutic intervention in animal and cell culture models. Their transition to clinical trials is hampered by concerns regarding their safety and potential toxicity. In this review, we discuss the paradigm of arginase and NOS competition for their substrate L-arginine in the asthmatic airway. We address the functional role of L-arginine in inflammation and the potential role of arginase inhibitors as therapeutics. PMID:23554705

  20. Unusual hepatic mitochondrial arginase in an Indian air-breathing teleost, Heteropneustes fossilis: purification and characterization.

    Science.gov (United States)

    Srivastava, Shilpee; Ratha, B K

    2013-02-01

    A functional urea cycle with both cytosolic (ARG I) and mitochondrial (ARG II) arginase activity is present in the liver of an ureogenic air-breathing teleost, Heteropneustes fossilis. Antibodies against mammalian ARG II showed no cross-reactivity with the H. fossilis ARG II. ARG II was purified to homogeneity from H. fossilis liver. Purified ARG II showed a native molecular mass of 96 kDa. SDS-PAGE showed a major band at 48 kDa. The native enzyme, therefore, appears to be a homodimer. The pI value of the enzyme was 7.5. The purified enzyme showed maximum activity at pH 10.5 and 55 °C. The K(m) of purified ARG II for l-arginine was 5.25±1.12 mM. L-Ornithine and N(ω)-hydroxy-L-arginine showed mixed inhibition with K(i) values 2.16±0.08 and 0.02±0.004 mM respectively. Mn(+2) and Co(+2) were effective activators of arginase activity. Antibody raised against purified H. fossilis ARG II did not cross-react with fish ARG I, and mammalian ARG I and ARG II. Western blot with the antibodies against purified H. fossilis hepatic ARG II showed cross reactivity with a 96 kDa band on native PAGE and a 48 kDa band on SDS-PAGE. The molecular, immunological and kinetic properties suggest uniqueness of the hepatic mitochondrial ARG II in H. fossilis. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Genetic Algorithm Calibration of Probabilistic Cellular Automata for Modeling Mining Permit Activity

    Science.gov (United States)

    Louis, S.J.; Raines, G.L.

    2003-01-01

    We use a genetic algorithm to calibrate a spatially and temporally resolved cellular automata to model mining activity on public land in Idaho and western Montana. The genetic algorithm searches through a space of transition rule parameters of a two dimensional cellular automata model to find rule parameters that fit observed mining activity data. Previous work by one of the authors in calibrating the cellular automaton took weeks - the genetic algorithm takes a day and produces rules leading to about the same (or better) fit to observed data. These preliminary results indicate that genetic algorithms are a viable tool in calibrating cellular automata for this application. Experience gained during the calibration of this cellular automata suggests that mineral resource information is a critical factor in the quality of the results. With automated calibration, further refinements of how the mineral-resource information is provided to the cellular automaton will probably improve our model.

  2. Antifungal activity of redox-active benzaldehydes that target cellular antioxidation

    Directory of Open Access Journals (Sweden)

    Mahoney Noreen

    2011-05-01

    Full Text Available Abstract Background Disruption of cellular antioxidation systems should be an effective method for control of fungal pathogens. Such disruption can be achieved with redox-active compounds. Natural phenolic compounds can serve as potent redox cyclers that inhibit microbial growth through destabilization of cellular redox homeostasis and/or antioxidation systems. The aim of this study was to identify benzaldehydes that disrupt the fungal antioxidation system. These compounds could then function as chemosensitizing agents in concert with conventional drugs or fungicides to improve antifungal efficacy. Methods Benzaldehydes were tested as natural antifungal agents against strains of Aspergillus fumigatus, A. flavus, A. terreus and Penicillium expansum, fungi that are causative agents of human invasive aspergillosis and/or are mycotoxigenic. The yeast Saccharomyces cerevisiae was also used as a model system for identifying gene targets of benzaldehydes. The efficacy of screened compounds as effective chemosensitizers or as antifungal agents in formulations was tested with methods outlined by the Clinical Laboratory Standards Institute (CLSI. Results Several benzaldehydes are identified having potent antifungal activity. Structure-activity analysis reveals that antifungal activity increases by the presence of an ortho-hydroxyl group in the aromatic ring. Use of deletion mutants in the oxidative stress-response pathway of S. cerevisiae (sod1Δ, sod2Δ, glr1Δ and two mitogen-activated protein kinase (MAPK mutants of A. fumigatus (sakAΔ, mpkCΔ, indicates antifungal activity of the benzaldehydes is through disruption of cellular antioxidation. Certain benzaldehydes, in combination with phenylpyrroles, overcome tolerance of A. fumigatus MAPK mutants to this agent and/or increase sensitivity of fungal pathogens to mitochondrial respiration inhibitory agents. Synergistic chemosensitization greatly lowers minimum inhibitory (MIC or fungicidal (MFC

  3. Asymmetric dimethylarginine (ADMA) elevation and arginase up-regulation contribute to endothelial dysfunction related to insulin resistance in rats and morbidly obese humans.

    Science.gov (United States)

    El Assar, Mariam; Angulo, Javier; Santos-Ruiz, Marta; Ruiz de Adana, Juan Carlos; Pindado, María Luz; Sánchez-Ferrer, Alberto; Hernández, Alberto; Rodríguez-Mañas, Leocadio

    2016-06-01

    expression determination by RT-PCR revealed not only the decreased expression of ADMA degrading enzyme dimethylarginine dimethylaminohydrolase (DDAH)1/2 in IR-MO microarteries, but also increased expression of arginase-2. Arginase inhibition improved endothelial vasodilatation in IR-MO. Analysis of endothelial vasodilatation in a non-obese IR model (fructose-fed rat) confirmed an elevation of circulating and aortic ADMA concentrations, as well as reduced DDAH aortic content and increased aortic arginase activity in IR. Improvement of endothelial vasodilatation in IR rats by l-arginine supplementation and arginase inhibition provided functional corroboration. These results demonstrate that increased ADMA and up-regulated arginase contribute to endothelial dysfunction as determined by the presence of IR in human obesity, most probably by compromising arginine availability. The results provide novel insights regarding the mechanisms of endothelial dysfunction related to obesity and IR and establish potential therapeutic targets for intervention. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

  4. Pomegranate Extracts and Cancer Prevention: Molecular and Cellular Activities

    Science.gov (United States)

    Syed, Deeba N.; Chamcheu, Jean-Christopher; Adhami, Vaqar M.; Mukhtar, Hasan

    2014-01-01

    There is increased appreciation by the scientific community that dietary phytochemicals can be potential weapons in the fight against cancer. Emerging data has provided new insights into the molecular and cellular framework needed to establish novel mechanism-based strategies for cancer prevention by selective bioactive food components. The unique chemical composition of the pomegranate fruit, rich in antioxidant tannins and flavonoids has drawn the attention of many investigators. Polyphenol rich fractions derived from the pomegranate fruit have been studied for their potential chemopreventive and/or cancer therapeutic effects in several animal models. Although data from in vitro and in vivo studies look convincing, well designed clinical trials in humans are needed to ascertain whether pomegranate can become part of our armamentarium against cancer. This review summarizes the available literature on the effects of pomegranate against various cancers. PMID:23094914

  5. Targeting arginase-II protects mice from high-fat-diet-induced hepatic steatosis through suppression of macrophage inflammation.

    Science.gov (United States)

    Liu, Chang; Rajapakse, Angana G; Riedo, Erwin; Fellay, Benoit; Bernhard, Marie-Claire; Montani, Jean-Pierre; Yang, Zhihong; Ming, Xiu-Fen

    2016-02-05

    Nonalcoholic fatty liver disease (NAFLD) associates with obesity and type 2 diabetes. Hypoactive AMP-activated protein kinase (AMPK), hyperactive mammalian target of rapamycin (mTOR) signaling, and macrophage-mediated inflammation are mechanistically linked to NAFLD. Studies investigating roles of arginase particularly the extrahepatic isoform arginase-II (Arg-II) in obesity-associated NAFLD showed contradictory results. Here we demonstrate that Arg-II(-/-) mice reveal decreased hepatic steatosis, macrophage infiltration, TNF-α and IL-6 as compared to the wild type (WT) littermates fed high fat diet (HFD). A higher AMPK activation (no difference in mTOR signaling), lower levels of lipogenic transcription factor SREBP-1c and activity/expression of lipogenic enzymes were observed in the Arg-II(-/-) mice liver. Moreover, release of TNF-α and IL-6 from bone marrow-derived macrophages (BMM) of Arg-II(-/-) mice is decreased as compared to WT-BMM. Conditioned medium from Arg-II(-/-)-BMM exhibits weaker activity to facilitate triglyceride synthesis paralleled with lower expression of SREBP-1c and SCD-1 and higher AMPK activation in hepatocytes as compared to that from WT-BMM. These effects of BMM conditioned medium can be neutralized by neutralizing antibodies against TNF-α and IL-6. Thus, Arg-II-expressing macrophages facilitate diet-induced NAFLD through TNF-α and IL-6 in obesity.

  6. The AP-1 Transcription Factor c-Jun Promotes Arthritis by Regulating Cyclooxygenase-2 and Arginase-1 Expression in Macrophages.

    Science.gov (United States)

    Hannemann, Nicole; Jordan, Jutta; Paul, Sushmita; Reid, Stephen; Baenkler, Hanns-Wolf; Sonnewald, Sophia; Bäuerle, Tobias; Vera, Julio; Schett, Georg; Bozec, Aline

    2017-05-01

    Activation of proinflammatory macrophages is associated with the inflammatory state of rheumatoid arthritis. Their polarization and activation are controlled by transcription factors such as NF-κB and the AP-1 transcription factor member c-Fos. Surprisingly, little is known about the role of the AP-1 transcription factor c-Jun in macrophage activation. In this study, we show that mRNA and protein levels of c-Jun are increased in macrophages following pro- or anti-inflammatory stimulations. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment cluster analyses of microarray data using wild-type and c-Jun-deleted macrophages highlight the central function of c-Jun in macrophages, in particular for immune responses, IL production, and hypoxia pathways. Mice deficient for c-Jun in macrophages show an amelioration of inflammation and bone destruction in the serum-induced arthritis model. In vivo and in vitro gene profiling, together with chromatin immunoprecipitation analysis of macrophages, revealed direct activation of the proinflammatory factor cyclooxygenase-2 and indirect inhibition of the anti-inflammatory factor arginase-1 by c-Jun. Thus, c-Jun regulates the activation state of macrophages and promotes arthritis via differentially regulating cyclooxygenase-2 and arginase-1 levels. Copyright © 2017 by The American Association of Immunologists, Inc.

  7. Cellular Links between Neuronal Activity and Energy Homeostasis

    OpenAIRE

    Shetty, Pavan K.; Galeffi, Francesca; Turner, Dennis A.

    2012-01-01

    Neuronal activity, astrocytic responses to this activity, and energy homeostasis are linked together during baseline, conscious conditions, and short-term rapid activation (as occurs with sensory or motor function). Nervous system energy homeostasis also varies during long-term physiological conditions (i.e., development and aging) and with adaptation to pathological conditions, such as ischemia or low glucose. Neuronal activation requires increased metabolism (i.e., ATP generation) which lea...

  8. Cellular Cholesterol Directly Activates Smoothened in Hedgehog Signaling

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Pengxiang; Nedelcu, Daniel; Watanabe, Miyako; Jao, Cindy; Kim, Youngchang; Liu, Jing; Salic, Adrian

    2016-08-01

    In vertebrates, sterols are necessary for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Sterols activate the membrane protein Smoothened by binding its extracellular, cysteine-rich domain (CRD). Major unanswered questions concern the nature of the endogenous, activating sterol and the mechanism by which it regulates Smoothened. We report crystal structures of CRD complexed with sterols and alone, revealing that sterols induce a dramatic conformational change of the binding site, which is sufficient for Smoothened activation and is unique among CRD-containing receptors. We demonstrate that Hedgehog signaling requires sterol binding to Smoothened and define key residues for sterol recognition and activity. We also show that cholesterol itself binds and activates Smoothened. Furthermore, the effect of oxysterols is abolished in Smoothened mutants that retain activation by cholesterol and Hedgehog. We propose that the endogenous Smoothened activator is cholesterol, not oxysterols, and that vertebrate Hedgehog signaling controls Smoothened by regulating its access to cholesterol.

  9. The Influence of nonuniform activity distribution on cellular dosimetry

    International Nuclear Information System (INIS)

    Naling, Song; Yuan, Tian; Liangan, Zhang; Guangfu, Dai

    2008-01-01

    S value is an important parameter in determination of absorbed dose in nuclear medicine and radiobiology. The distribution of radioactivity shows significant influence on the S value especially in microdosimetry. In present work, a semi Monte Carlo Model is developed to calculate the microdosimetric cellular S value for different micro-distributions of radioactivity, i.e. uniform, linear increase, linear decrease, exponential increase, exponential decrease and centroid distribution. Emission of alpha particles is simulated by Monte Carlo model and the energy imparted to the target volume is calculated by the analytical Continuous Slowing Down Approximation (CSDA) model and the spline interpolation of range-energy relationship. We calculate tables of S values for 213 Po and 210 Po with various dimensions and most important with various possible micro-distributions of radioactivity, such as linear increase, linear decrease, exponential increase and exponential decrease. Then we compare the S values from cell to cell of uniform distribution with the Hamacher's results to test the feasibility of our model. S values of some nonuniform micro-distributions are compared to the corresponding data of the uniform distribution. The possible sources of these differences are theoretical analyzed. (author)

  10. Neural, Cellular and Molecular Mechanisms of Active Forgetting

    Science.gov (United States)

    Medina, Jorge H.

    2018-01-01

    The neurobiology of memory formation attracts much attention in the last five decades. Conversely, the rules that govern and the mechanisms underlying forgetting are less understood. In addition to retroactive interference, retrieval-induced forgetting and passive decay of time, it has been recently demonstrated that the nervous system has a diversity of active and inherent processes involved in forgetting. In Drosophila, some operate mainly at an early stage of memory formation and involves dopamine (DA) neurons, specific postsynaptic DA receptor subtypes, Rac1 activation and induces rapid active forgetting. In mammals, others regulate forgetting and persistence of seemingly consolidated memories and implicate the activity of DA receptor subtypes and AMPA receptors in the hippocampus (HP) and related structures to activate parallel signaling pathways controlling active time-dependent forgetting. Most of them may involve plastic changes in synaptic and extrasynaptic receptors including specific removal of GluA2 AMPA receptors. Forgetting at longer timescales might also include changes in adult neurogenesis in the dentate gyrus (DG) of the HP. Therefore, based on relevance or value considerations neuronal circuits may regulate in a time-dependent manner what is formed, stored, and maintained and what is forgotten. PMID:29467630

  11. Cellular Links between Neuronal Activity and Energy Homeostasis.

    Science.gov (United States)

    Shetty, Pavan K; Galeffi, Francesca; Turner, Dennis A

    2012-01-01

    Neuronal activity, astrocytic responses to this activity, and energy homeostasis are linked together during baseline, conscious conditions, and short-term rapid activation (as occurs with sensory or motor function). Nervous system energy homeostasis also varies during long-term physiological conditions (i.e., development and aging) and with adaptation to pathological conditions, such as ischemia or low glucose. Neuronal activation requires increased metabolism (i.e., ATP generation) which leads initially to substrate depletion, induction of a variety of signals for enhanced astrocytic function, and increased local blood flow and substrate delivery. Energy generation (particularly in mitochondria) and use during ATP hydrolysis also lead to considerable heat generation. The local increases in blood flow noted following neuronal activation can both enhance local substrate delivery but also provides a heat sink to help cool the brain and removal of waste by-products. In this review we highlight the interactions between short-term neuronal activity and energy metabolism with an emphasis on signals and factors regulating astrocyte function and substrate supply.

  12. Cellular Links Between Neuronal Activity and Energy Homeostasis

    Directory of Open Access Journals (Sweden)

    Pavan K Shetty

    2012-03-01

    Full Text Available Neuronal activity, astrocytic responses to this activity, and energy homeostasis are linked together during baseline, conscious conditions, and short-term rapid activation (as occurs with sensory or motor function. Nervous system energy homeostasis also varies during long-term physiological conditions (ie, development and aging and with adaptation to pathological conditions, such as ischemia or low glucose. Neuronal activation requires increased metabolism (i.e., ATP generation which leads initially to substrate depletion, induction of a variety of signals for enhanced astrocytic function, and increased local blood flow and substrate delivery. Energy generation (particularly in mitochondria and use during ATP hydrolysis also lead to considerable heat generation. The local increases in blood flow noted following neuronal activation can both enhance local substrate delivery but also provides a heat sink to help cool the brain and removal of waste byproducts. In this review we highlight the interactions between short-term neuronal activity and energy metabolism with an emphasis on signals and factors regulating astrocyte function and substrate supply.

  13. Arginase up-regulation and eNOS uncoupling contribute to impaired endothelium-dependent vasodilation in a rat model of intrauterine growth restriction.

    Science.gov (United States)

    Grandvuillemin, Isabelle; Buffat, Christophe; Boubred, Farid; Lamy, Edouard; Fromonot, Julien; Charpiot, Philippe; Simoncini, Stephanie; Sabatier, Florence; Dignat-George, Françoise; Peyter, Anne-Christine; Simeoni, Umberto; Yzydorczyk, Catherine

    2018-05-09

    Individuals born after intrauterine growth restriction (IUGR) are at increased risk of developing cardiovascular diseases in adulthood, notably hypertension (HTN). Alterations in the vascular system, particularly impaired endothelium-dependent vasodilation, may play an important role in long-term effects of IUGR. Whether such vascular dysfunction precedes HTN has not been fully established in individuals born after IUGR. Moreover, the intimate mechanisms of altered endothelium-dependent vasodilation remain incompletely elucidated. We therefore investigated, using a rat model of IUGR, whether impaired endothelium-dependent relaxation precedes the development of HTN and whether key components of the L-Arginine-nitric oxide (NO) pathway are involved in its pathogenesis. Pregnant rats were fed with a control (CTRL, 23% casein) or low-protein diet (LP, 9% casein) to induce IUGR. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography in 5- and 8-week-old male offspring. Aortic rings were isolated to investigate relaxation to acetylcholine, NO production, eNOS protein content, arginase activity, and superoxide anion production. SBP was not different at 5 weeks, but significantly increased in 8-week-old LP vs. CRTL offspring. In 5-week-old LP vs. CRTL males, endothelium-dependent vasorelaxation was significantly impaired, but restored by pre-incubation with L-Arginine or the arginase inhibitor BEC; NO production was significantly reduced, but restored by L-Arginine pretreatment; total eNOS protein, dimer/monomer ratio, and arginase activity were significantly increased; superoxide anion production was significantly enhanced, but normalized by pretreatment with the NOS inhibitor L-NNA. In this model, IUGR leads to early-impaired endothelium-dependent vasorelaxation, resulting from arginase up-regulation and eNOS uncoupling, which precedes the development of HTN.

  14. Mitochondrial Control and Guidance of Cellular Activities of T Cells

    Directory of Open Access Journals (Sweden)

    Ping-Chih Ho

    2017-04-01

    Full Text Available Immune cells protect us against infection and cancer cells, as well as functioning during healing processes to support tissue repairing and regeneration. These behaviors require that upon stimulation from immune activation the appropriate subsets of immune cells are generated. In addition to activation-induced signaling cascades, metabolic reprogramming (profound changes in metabolic pathways also provides a novel form of regulation to control the formation of desirable immune responses. Immune cells encounter various nutrient compositions by circulating in bloodstream and infiltrating into peripheral tissues; therefore, proper engagement of metabolic pathways is critical to fulfill the metabolic demands of immune cells. Metabolic pathways are tightly regulated mainly via mitochondrial dynamics and the activities of the tricarboxylic acid cycle and the electron transport chain. In this review, we will discuss how metabolic reprogramming influences activation, effector functions, and lineage polarization in T cells, with a particular focus on mitochondria-regulated metabolic checkpoints. Additionally, we will further explore how in various diseases deregulation and manipulation of mitochondrial regulation can occur and be exploited. Furthermore, we will discuss how this knowledge can facilitate the design of immunotherapies.

  15. DNA supercoiling: changes during cellular differentiation and activation of chromatin transcription

    International Nuclear Information System (INIS)

    Luchnik, A.N.; Bakayev, V.V.; Glaser, V.M.; Moscow State Univ., USSR)

    1983-01-01

    In this paper it is reported that elastic DNA torsional tension has been observed in a fraction of isolated SV40 minichromosomes, which are shown to be transcriptionally active, and that the number of DNA topological (titratable superhelical) turns in closed superhelical loops of nuclear DNA decreases during cellular differentiation, which, we propose, may be responsible for the coordinate switch in transcription of genes controlling cellular proliferation. 37 references, 6 figures, 2 tables

  16. Modulation of hyaluronan synthase activity in cellular membrane fractions

    OpenAIRE

    Vigetti, Davide; Genasetti, A; Karousou, Evgenia; Viola, Manuela; Clerici, M; Bartolini, B; Moretto, Paola; DE LUCA, Giancarlo; Hascall, Vc; Passi, Alberto

    2009-01-01

    Hyaluronan (HA), the only non-sulfated glycosaminoglycan, is involved in morphogenesis, wound healing, inflammation, angiogenesis, and cancer. In mammals, HA is synthesized by three homologous HA synthases, HAS1, HAS2, and HAS3, that polymerize the HA chain using UDP-glucuronic acid and UDP-N-acetylglucosamine as precursors. Since the amount of HA is critical in several pathophysiological conditions, we developed a non-radioactive assay for measuring the activity of HA synthases (HASs) in euk...

  17. Antioxidant activity of puha (Sonchus oleraceus L.) as assessed by the cellular antioxidant activity (CAA) assay.

    Science.gov (United States)

    McDowell, Arlene; Thompson, Scott; Stark, Mirjam; Ou, Zong-Quan; Gould, Kevin S

    2011-12-01

    There is considerable interest in antioxidant dietary components that can be protective against degenerative diseases in humans. Puha (Sonchus oleraceus L.) is a rich source of polyphenols, and exhibits strong antioxidant activity as measured by the 2,2-diphenylpicrylhydrazyl (DPPH) assay. However, the potential of puha to protect against degenerative diseases requires that low molecular weight antioxidants (LMWA) are absorbed by, and active in, human cells. The cellular antioxidant activity (CAA) assay was used to investigate the antioxidant activity of puha leaf extracts. Preparation methods of freezing and freeze-drying reduced the total polyphenolic content compared with fresh puha, but did not affect the LMWA potential as determined by the DPPH assay. The IC(50) values were 0.012 ± 0.003 mg/mL and 0.010 ± 0.005 mg/mL for freeze-dried and fresh puha leaves, respectively. Using the CAA assay, it was shown that LMWAs from foliar extracts of puha were effectively absorbed into HepG2 cells, and exerted antioxidant activity at levels comparable to those of extracts from blueberry fruits, the much-touted antioxidant superfood. Methylene blue staining of HepG2 cells indicated that puha extracts were not cytotoxic at concentrations below 100 mg DW/mL. The data indicate the potential of puha as a nutraceutical supplement for human health. Copyright © 2011 John Wiley & Sons, Ltd.

  18. DMPD: Regulation of nitric oxide synthesis and apoptosis by arginase and argininerecycling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17513437 Regulation of nitric oxide synthesis and apoptosis by arginase and arginin...tion of nitric oxide synthesis and apoptosis by arginase and argininerecycling. A...erecycling. Mori M. J Nutr. 2007 Jun;137(6 Suppl 2):1616S-1620S. (.png) (.svg) (.html) (.csml) Show Regulation of nitric oxide synthe...sis and apoptosis by arginase and argininerecycling. PubmedID 17513437 Title Regula

  19. Asymmetric dimethylarginine (ADMA) elevation and arginase up‐regulation contribute to endothelial dysfunction related to insulin resistance in rats and morbidly obese humans

    Science.gov (United States)

    El Assar, Mariam; Angulo, Javier; Santos‐Ruiz, Marta; Ruiz de Adana, Juan Carlos; Pindado, María Luz; Sánchez‐Ferrer, Alberto; Hernández, Alberto

    2016-01-01

    IR score and negatively with pD2 for bradykinin. Gene expression determination by RT‐PCR revealed not only the decreased expression of ADMA degrading enzyme dimethylarginine dimethylaminohydrolase (DDAH)1/2 in IR‐MO microarteries, but also increased expression of arginase‐2. Arginase inhibition improved endothelial vasodilatation in IR‐MO. Analysis of endothelial vasodilatation in a non‐obese IR model (fructose‐fed rat) confirmed an elevation of circulating and aortic ADMA concentrations, as well as reduced DDAH aortic content and increased aortic arginase activity in IR. Improvement of endothelial vasodilatation in IR rats by l‐arginine supplementation and arginase inhibition provided functional corroboration. These results demonstrate that increased ADMA and up‐regulated arginase contribute to endothelial dysfunction as determined by the presence of IR in human obesity, most probably by compromising arginine availability. The results provide novel insights regarding the mechanisms of endothelial dysfunction related to obesity and IR and establish potential therapeutic targets for intervention. PMID:26840628

  20. Detection of silent cells, synchronization and modulatory activity in developing cellular networks.

    NARCIS (Netherlands)

    Hjorth, J.J.J.; Dawitz, J.; Kroon, T.; da Silva Dias Pires, J.H.; Dassen, V.J.; Berkhout, J.A.; Emperador Melero, J.; Nadadhur, A.G.; Alevra, M.; Toonen, R.F.G.; Heine, V.M.; Mansvelder, H.D.; Meredith, R.M.

    2016-01-01

    Developing networks in the immature nervous system and in cellular cultures are characterized by waves of synchronous activity in restricted clusters of cells. Synchronized activity in immature networks is proposed to regulate many different developmental processes, from neuron growth and cell

  1. Cellular Mechanisms of Calcium-Mediated Triggered Activity

    Science.gov (United States)

    Song, Zhen

    Life-threatening cardiac arrhythmias continue to pose a major health problem. Ventricular fibrillation, which is a complex form of electrical wave turbulence in the lower chambers of the heart, stops the heart from pumping and is the largest cause of natural death in the United States. Atrial fibrillation, a related form of wave turbulence in the upper heart chambers, is in turn the most common arrhythmia diagnosed in clinical practice. Despite extensive research to date, mechanisms of cardiac arrhythmias remain poorly understood. It is well established that both spatial disorder of the refractory period of heart cells and triggered activity (TA) jointly contribute to the initiation and maintenance of arrhythmias. TA broadly refers to the abnormal generation of a single or a sequence of abnormal excitation waves from a small submillimeter region of the heart in the interval of time between two normal waves generated by the heart's natural pacemaker (the sinoatrial node). TA has been widely investigated experimentally and occurs in several pathological conditions where the intracellular concentration of free Ca2+ ions in heart cells becomes elevated. Under such conditions, Ca2+ can be spontaneously released from intracellular stores, thereby driving an electrogenic current that exchanges 3Na+ ions for one Ca2+ ion across the cell membrane. This current in turn depolarizes the membrane of heart cells after a normal excitation. If this calcium-mediated "delayed after depolarization'' (DAD) is sufficiently large, it can generate an action potential. While the arrhythmogenic importance of spontaneous Ca2+ release and DADs is well appreciated, the conditions under which they occur in heart pathologies remain poorly understood. Calcium overload is only one factor among several other factors that can promote DADs, including sympathetic nerve stimulation, different expression levels of membrane ion channels and calcium handling proteins, and different mutations of those

  2. Activation of the hypothalamic-pituitary-adrenal stress axis induces cellular oxidative stress

    Directory of Open Access Journals (Sweden)

    Jereme G. Spiers

    2015-01-01

    Full Text Available Glucocorticoids released from the adrenal gland in response to stress-induced activation of the hypothalamic-pituitary-adrenal (HPA axis induce activity in the cellular reduction-oxidation (redox system. The redox system is a ubiquitous chemical mechanism allowing the transfer of electrons between donor/acceptors and target molecules during oxidative phosphorylation while simultaneously maintaining the overall cellular environment in a reduced state. The objective of this review is to present an overview of the current literature discussing the link between HPA axis-derived glucocorticoids and increased oxidative stress, particularly focussing on the redox changes observed in the hippocampus following glucocorticoid exposure.

  3. Detection of silent cells, synchronization and modulatory activity in developing cellular networks.

    Science.gov (United States)

    Hjorth, Johannes J J; Dawitz, Julia; Kroon, Tim; Pires, Johny; Dassen, Valerie J; Berkhout, Janna A; Emperador Melero, Javier; Nadadhur, Aish G; Alevra, Mihai; Toonen, Ruud F; Heine, Vivi M; Mansvelder, Huibert D; Meredith, Rhiannon M

    2016-04-01

    Developing networks in the immature nervous system and in cellular cultures are characterized by waves of synchronous activity in restricted clusters of cells. Synchronized activity in immature networks is proposed to regulate many different developmental processes, from neuron growth and cell migration, to the refinement of synapses, topographic maps, and the mature composition of ion channels. These emergent activity patterns are not present in all cells simultaneously within the network and more immature "silent" cells, potentially correlated with the presence of silent synapses, are prominent in different networks during early developmental periods. Many current network analyses for detection of synchronous cellular activity utilize activity-based pixel correlations to identify cellular-based regions of interest (ROIs) and coincident cell activity. However, using activity-based correlations, these methods first underestimate or ignore the inactive silent cells within the developing network and second, are difficult to apply within cell-dense regions commonly found in developing brain networks. In addition, previous methods may ignore ROIs within a network that shows transient activity patterns comprising both inactive and active periods. We developed analysis software to semi-automatically detect cells within developing neuronal networks that were imaged using calcium-sensitive reporter dyes. Using an iterative threshold, modulation of activity was tracked within individual cells across the network. The distribution pattern of both inactive and active, including synchronous cells, could be determined based on distance measures to neighboring cells and according to different anatomical layers. © 2015 Wiley Periodicals, Inc.

  4. Cellular degradation activity is maintained during aging in long-living queen bees.

    Science.gov (United States)

    Hsu, Chin-Yuan; Qiu, Jiantai Timothy; Chan, Yu-Pei

    2016-11-01

    Queen honeybees (Apis mellifera) have a much longer lifespan than worker bees. Whether cellular degradation activity is involved in the longevity of queen bees is unknown. In the present study, cellular degradation activity was evaluated in the trophocytes and oenocytes of young and old queen bees. The results indicated that (i) 20S proteasome activity and the size of autophagic vacuoles decreased with aging, and (ii) there were no significant differences between young and old queen bees with regard to 20S proteasome expression or efficiency, polyubiquitin aggregate expression, microtubule-associated protein 1 light chain 3-II (LC3-II) expression, 70 kDa heat shock cognate protein (Hsc70) expression, the density of autophagic vacuoles, p62/SQSTM1 expression, the activity or density of lysosomes, or molecular target of rapamycin expression. These results indicate that cellular degradation activity maintains a youthful status in the trophocytes and oenocytes of queen bees during aging and that cellular degradation activity is involved in maintaining the longevity of queen bees.

  5. Cellular telephones measure activity and lifespace in community-dwelling adults: proof of principle.

    Science.gov (United States)

    Schenk, Ana Katrin; Witbrodt, Bradley C; Hoarty, Carrie A; Carlson, Richard H; Goulding, Evan H; Potter, Jane F; Bonasera, Stephen J

    2011-02-01

    To describe a system that uses off-the-shelf sensor and telecommunication technologies to continuously measure individual lifespace and activity levels in a novel way. Proof of concept involving three field trials of 30, 30, and 21 days. Omaha, Nebraska, metropolitan and surrounding rural region. Three participants (48-year-old man, 33-year-old woman, and 27-year-old male), none with any functional limitations. Cellular telephones were used to detect in-home position and in-community location and to measure physical activity. Within the home, cellular telephones and Bluetooth transmitters (beacons) were used to locate participants at room-level resolution. Outside the home, the same cellular telephones and global positioning system (GPS) technology were used to locate participants at a community-level resolution. Physical activity was simultaneously measured using the cellular telephone accelerometer. This approach had face validity to measure activity and lifespace. More importantly, this system could measure the spatial and temporal organization of these metrics. For example, an individual's lifespace was automatically calculated across multiple time intervals. Behavioral time budgets showing how people allocate time to specific regions within the home were also automatically generated. Mobile monitoring shows much promise as an easily deployed system to quantify activity and lifespace, important indicators of function, in community-dwelling adults. © 2011, Copyright the Authors. Journal compilation © 2011, The American Geriatrics Society.

  6. Cellular and Molecular Pathways Leading to External Root Resorption

    Science.gov (United States)

    Iglesias-Linares, A.; Hartsfield, J.K.

    2016-01-01

    External apical root resorption during orthodontic treatment implicates specific molecular pathways that orchestrate nonphysiologic cellular activation. To date, a substantial number of in vitro and in vivo molecular, genomic, and proteomic studies have supplied data that provide new insights into root resorption. Recent mechanisms and developments reviewed here include the role of the cellular component—specifically, the balance of CD68+, iNOS+ M1- and CD68+, CD163+ M2-like macrophages associated with root resorption and root surface repair processes linked to the expression of the M1-associated proinflammatory cytokine tumor necrosis factor, inducible nitric oxide synthase, the M1 activator interferon γ, the M2 activator interleukin 4, and M2-associated anti-inflammatory interleukin 10 and arginase I. Insights into the role of mesenchymal dental pulp cells in attenuating dentin resorption in homeostasis are also reviewed. Data on recently deciphered molecular pathways are reviewed at the level of (1) clastic cell adhesion in the external apical root resorption process and the specific role of α/β integrins, osteopontin, and related extracellular matrix proteins; (2) clastic cell fusion and activation by the RANKL/RANK/OPG and ATP-P2RX7-IL1 pathways; and (3) regulatory mechanisms of root resorption repair by cementum at the proteomic and transcriptomic levels. PMID:27811065

  7. Selective transcription and cellular proliferation induced by PDGF require histone deacetylase activity

    International Nuclear Information System (INIS)

    Catania, Annunziata; Iavarone, Carlo; Carlomagno, Stella M.; Chiariello, Mario

    2006-01-01

    Histone deacetylases (HDACs) are key regulatory enzymes involved in the control of gene expression and their inhibition by specific drugs has been widely correlated to cell cycle arrest, terminal differentiation, and apoptosis. Here, we investigated whether HDAC activity was required for PDGF-dependent signal transduction and cellular proliferation. Exposure of PDGF-stimulated NIH3T3 fibroblasts to the HDAC inhibitor trichostatin A (TSA) potently repressed the expression of a group of genes correlated to PDGF-dependent cellular growth and pro-survival activity. Moreover, we show that TSA interfered with STAT3-dependent transcriptional activity induced by PDGF. Still, neither phosphorylation nor nuclear translocation and DNA-binding in vitro and in vivo of STAT3 were affected by using TSA to interfere with PDGF stimulation. Finally, TSA treatment resulted in the suppression of PDGF-dependent cellular proliferation without affecting cellular survival of NIH3T3 cells. Our data indicate that inhibition of HDAC activity antagonizes the mitogenic effect of PDGF, suggesting that these drugs may specifically act on the expression of STAT-dependent, PDGF-responsive genes

  8. Monocyte Activation in Immunopathology: Cellular Test for Development of Diagnostics and Therapy

    Directory of Open Access Journals (Sweden)

    Ekaterina A. Ivanova

    2016-01-01

    Full Text Available Several highly prevalent human diseases are associated with immunopathology. Alterations in the immune system are found in such life-threatening disorders as cancer and atherosclerosis. Monocyte activation followed by macrophage polarization is an important step in normal immune response to pathogens and other relevant stimuli. Depending on the nature of the activation signal, macrophages can acquire pro- or anti-inflammatory phenotypes that are characterized by the expression of distinct patterns of secreted cytokines and surface antigens. This process is disturbed in immunopathologies resulting in abnormal monocyte activation and/or bias of macrophage polarization towards one or the other phenotype. Such alterations could be used as important diagnostic markers and also as possible targets for the development of immunomodulating therapy. Recently developed cellular tests are designed to analyze the phenotype and activity of living cells circulating in patient’s bloodstream. Monocyte/macrophage activation test is a successful example of cellular test relevant for atherosclerosis and oncopathology. This test demonstrated changes in macrophage activation in subclinical atherosclerosis and breast cancer and could also be used for screening a panel of natural agents with immunomodulatory activity. Further development of cellular tests will allow broadening the scope of their clinical implication. Such tests may become useful tools for drug research and therapy optimization.

  9. Correlation between proliferative activity and cellular thickness of human mesenchymal stem cells

    International Nuclear Information System (INIS)

    Katsube, Yoshihiro; Hirose, Motohiro; Nakamura, Chikashi; Ohgushi, Hajime

    2008-01-01

    A cell's shape is known to be related to its proliferative activity. In particular, large and flat mammalian adult stem cells seem to show slow proliferation, however using quantitative analysis to prove the phenomenon is difficult. We measured the proliferation and cellular thickness of human mesenchymal stem cells (MSCs) by atomic force microscopy and found that MSCs with high proliferative activity were thick while those with low proliferative activity were thin, even though these MSCs were early passage cells. Further, low proliferative MSCs contained many senescence-associated β-galactosidase positive cells together with high senescence-associated gene expression. These findings suggest that the measurement of cellular thickness is useful for estimating the proliferative activity of human MSCs and is expected to be a practical tool for MSC applications in regenerative medicine

  10. Hepatitis C virus NS2 protein activates cellular cyclic AMP-dependent pathways

    International Nuclear Information System (INIS)

    Kim, Kyoung Mi; Kwon, Shi-Nae; Kang, Ju-Il; Lee, Song Hee; Jang, Sung Key; Ahn, Byung-Yoon; Kim, Yoon Ki

    2007-01-01

    Chronic infection of the hepatitis C virus (HCV) leads to liver cirrhosis and cancer. The mechanism leading to viral persistence and hepatocellular carcinoma, however, has not been fully understood. In this study, we show that the HCV infection activates cellular cAMP-dependent pathways. Expression of a luciferase reporter gene controlled by a basic promoter with the cAMP response element (CRE) was significantly elevated in human hepatoma Huh-7 cells infected with the HCV JFH1. Analysis with viral subgenomic replicons indicated that the HCV NS2 protein is responsible for the effect. Furthermore, the level of cellular transcripts whose stability is known to be regulated by cAMP was specifically reduced in cells harboring NS2-expressing replicons. These results allude to the HCV NS2 protein having a novel function of regulating cellular gene expression and proliferation through the cAMP-dependent pathway

  11. Arginase II expressed in cancer-associated fibroblasts indicates tissue hypoxia and predicts poor outcome in patients with pancreatic cancer.

    Directory of Open Access Journals (Sweden)

    Yoshinori Ino

    Full Text Available An adequate level of arginine in the tissue microenvironment is essential for T cell activity and survival. Arginine levels are regulated by the arginine-catabolizing enzyme, arginase (ARG. It has been reported that arginase II (ARG2, one of two ARGs, is aberrantly expressed in prostate cancer cells, which convert arginine into ornithine, resulting in a lack of arginine that weakens tumor-infiltrating lymphocytes and renders them dysfunctional. However, immune suppression mediated by ARG2-expressing cancer cells in lung cancer has not been observed. Here we studied the expression of ARG2 in pancreatic ductal carcinoma (PDC tissue clinicopathologically by examining over 200 cases of PDC. In contrast to prostate cancer, ARG2 expression was rarely demonstrated in PDC cells by immunohistochemistry, and instead ARG2 was characteristically expressed in α-smooth muscle actin-positive cancer-associated fibroblasts (CAFs, especially those located within and around necrotic areas in PDC. The presence of ARG2-expressing CAFs was closely correlated with shorter overall survival (OS; P  = 0.003 and disease-free survival (DFS; P  = 0.0006. Multivariate Cox regression analysis showed that the presence of ARG2-expressing CAFs in PDC tissue was an independent predictor of poorer OS (hazard ratio [HR]  = 1.582, P  = 0.007 and DFS (HR  = 1.715, P  = 0.001 in PDC patients. In addition to the characteristic distribution of ARG2-expressing CAFs, such CAFs co-expressed carbonic anhydrase IX, SLC2A1, or HIF-1α, markers of hypoxia, in PDC tissue. Furthermore, in vitro experiments revealed that cultured fibroblasts extracted from PDC tissue expressed the ARG2 transcript after exposure to hypoxia, which had arginase activity. These results indicate that cancer cell-mediated immune suppression through ARG2 expression is not a general event and that the presence of ARG2-expressing CAFs is an indicator of poor prognosis, as well as hypoxia, in PDC

  12. Autophagy Facilitates IFN-γ-induced Jak2-STAT1 Activation and Cellular Inflammation*

    Science.gov (United States)

    Chang, Yu-Ping; Tsai, Cheng-Chieh; Huang, Wei-Ching; Wang, Chi-Yun; Chen, Chia-Ling; Lin, Yee-Shin; Kai, Jui-In; Hsieh, Chia-Yuan; Cheng, Yi-Lin; Choi, Pui-Ching; Chen, Shun-Hua; Chang, Shih-Ping; Liu, Hsiao-Sheng; Lin, Chiou-Feng

    2010-01-01

    Autophagy is regulated for IFN-γ-mediated antimicrobial efficacy; however, its molecular effects for IFN-γ signaling are largely unknown. Here, we show that autophagy facilitates IFN-γ-activated Jak2-STAT1. IFN-γ induces autophagy in wild-type but not in autophagy protein 5 (Atg5−/−)-deficient mouse embryonic fibroblasts (MEFs), and, autophagy-dependently, IFN-γ induces IFN regulatory factor 1 and cellular inflammatory responses. Pharmacologically inhibiting autophagy using 3-methyladenine, a known inhibitor of class III phosphatidylinositol 3-kinase, confirms these effects. Either Atg5−/− or Atg7−/− MEFs are, independent of changes in IFN-γ receptor expression, resistant to IFN-γ-activated Jak2-STAT1, which suggests that autophagy is important for IFN-γ signal transduction. Lentivirus-based short hairpin RNA for Atg5 knockdown confirmed the importance of autophagy for IFN-γ-activated STAT1. Without autophagy, reactive oxygen species increase and cause SHP2 (Src homology-2 domain-containing phosphatase 2)-regulated STAT1 inactivation. Inhibiting SHP2 reversed both cellular inflammation and the IFN-γ-induced activation of STAT1 in Atg5−/− MEFs. Our study provides evidence that there is a link between autophagy and both IFN-γ signaling and cellular inflammation and that autophagy, because it inhibits the expression of reactive oxygen species and SHP2, is pivotal for Jak2-STAT1 activation. PMID:20592027

  13. Antiproliferative Activity and Cellular Uptake of Evodiamine and Rutaecarpine Based on 3D Tumor Models

    Directory of Open Access Journals (Sweden)

    Hui Guo

    2016-07-01

    Full Text Available Evodiamine (EVO and rutaecarpine (RUT are promising anti-tumor drug candidates. The evaluation of the anti-proliferative activity and cellular uptake of EVO and RUT in 3D multicellular spheroids of cancer cells would better recapitulate the native situation and thus better reflect an in vivo response to the treatment. Herein, we employed the 3D culture of MCF-7 and SMMC-7721 cells based on hanging drop method and evaluated the anti-proliferative activity and cellular uptake of EVO and RUT in 3D multicellular spheroids, and compared the results with those obtained from 2D monolayers. The drugs’ IC50 values were significantly increased from the range of 6.4–44.1 μM in 2D monolayers to 21.8–138.0 μM in 3D multicellular spheroids, which may be due to enhanced mass barrier and reduced drug penetration in 3D models. The fluorescence of EVO and RUT was measured via fluorescence spectroscopy and the cellular uptake of both drugs was characterized in 2D tumor models. The results showed that the cellular uptake concentrations of RUT increased with increasing drug concentrations. However, the EVO concentrations uptaken by the cells showed only a small change with increasing drug concentrations, which may be due to the different solubility of EVO and Rut in solvents. Overall, this study provided a new vision of the anti-tumor activity of EVO and RUT via 3D multicellular spheroids and cellular uptake through the fluorescence of compounds.

  14. Dynamic Simulation of 1D Cellular Automata in the Active aTAM.

    Science.gov (United States)

    Jonoska, Nataša; Karpenko, Daria; Seki, Shinnosuke

    2015-07-01

    The Active aTAM is a tile based model for self-assembly where tiles are able to transfer signals and change identities according to the signals received. We extend Active aTAM to include deactivation signals and thereby allow detachment of tiles. We show that the model allows a dynamic simulation of cellular automata with assemblies that do not record the entire computational history but only the current updates of the states, and thus provide a way for (a) algorithmic dynamical structural changes in the assembly and (b) reusable space in self-assembly. The simulation is such that at a given location the sequence of tiles that attach and detach corresponds precisely to the sequence of states the synchronous cellular automaton generates at that location.

  15. Multiple-channel detection of cellular activities by ion-sensitive transistors

    Science.gov (United States)

    Machida, Satoru; Shimada, Hideto; Motoyama, Yumi

    2018-04-01

    An ion-sensitive field-effect transistor to record cellular activities was demonstrated. This field-effect transistor (bio transistor) includes cultured cells on the gate insulator instead of gate electrode. The bio transistor converts a change in potential underneath the cells into variation of the drain current when ion channels open. The bio transistor has high detection sensitivity to even minute variations in potential utilizing a subthreshold swing region. To open ion channels, a reagent solution (acetylcholine) was added to a human-originating cell cultured on the bio transistor. The drain current was successfully decreased with the addition of acetylcholine. Moreover, we attempted to detect the opening of ion channels using a multiple-channel measurement circuit containing several bio transistors. As a consequence, the drain current distinctly decreased only after the addition of acetylcholine. We confirmed that this measurement system including bio transistors enables to observation of cellular activities sensitively and simultaneously.

  16. AMP-activated protein kinase reduces inflammatory responses and cellular senescence in pulmonary emphysema.

    Science.gov (United States)

    Cheng, Xiao-Yu; Li, Yang-Yang; Huang, Cheng; Li, Jun; Yao, Hong-Wei

    2017-04-04

    Current drug therapy fails to reduce lung destruction of chronic obstructive pulmonary disease (COPD). AMP-activated protein kinase (AMPK) has emerged as an important integrator of signals that control energy balance and lipid metabolism. However, there are no studies regarding the role of AMPK in reducing inflammatory responses and cellular senescence during the development of emphysema. Therefore, we hypothesize that AMPK reduces inflammatroy responses, senescence, and lung injury. To test this hypothesis, human bronchial epithelial cells (BEAS-2B) and small airway epithelial cells (SAECs) were treated with cigarette smoke extract (CSE) in the presence of a specific AMPK activator (AICAR, 1 mM) and inhibitor (Compound C, 5 μM). Elastase injection was performed to induce mouse emphysema, and these mice were treated with a specific AMPK activator metformin as well as Compound C. AICAR reduced, whereas Compound C increased CSE-induced increase in IL-8 and IL-6 release and expression of genes involved in cellular senescence. Knockdown of AMPKα1/α2 increased expression of pro-senescent genes (e.g., p16, p21, and p66shc) in BEAS-2B cells. Prophylactic administration of an AMPK activator metformin (50 and 250 mg/kg) reduced while Compound C (4 and 20 mg/kg) aggravated elastase-induced airspace enlargement, inflammatory responses and cellular senescence in mice. This is in agreement with therapeutic effect of metformin (50 mg/kg) on airspace enlargement. Furthermore, metformin prophylactically protected against but Compound C further reduced mitochondrial proteins SOD2 and SIRT3 in emphysematous lungs. In conclusion, AMPK reduces abnormal inflammatory responses and cellular senescence, which implicates as a potential therapeutic target for COPD/emphysema.

  17. Free-Radical-Scavenging, Antityrosinase, and Cellular Melanogenesis Inhibitory Activities of Synthetic Isoflavones.

    Science.gov (United States)

    Lu, Tzy-Ming; Ko, Horng-Huey; Ng, Lean-Teik; Hsieh, Yen-Pin

    2015-06-01

    In this study, we examined the potential of synthetic isoflavones for application in cosmeceuticals. Twenty-five isoflavones were synthesized and their capacities of free-radical-scavenging and mushroom tyrosinase inhibition, as well as their impact on cell viability of B16F10 murine melanoma cells and HaCaT human keratinocytes were evaluated. Isoflavones that showed significant mushroom tyrosinase inhibitory activities were further studied on reduction of cellular melanin formation and antityrosinase activities in B16F10 melanocytes in vitro. Among the isoflavones tested, 6-hydroxydaidzein (2) was the strongest scavenger of both ABTS(.+) and DPPH(.) radicals with SC50 values of 11.3 ± 0.3 and 9.4 ± 0.1 μM, respectively. Texasin (20) exhibited the most potent inhibition of mushroom tyrosinase (IC50 14.9 ± 4.5 μM), whereas retusin (17) showed the most efficient inhibition both of cellular melanin formation and antityrosinase activity in B16F10 melanocytes, respectively. In summary, both retusin (17) and texasin (20) exhibited potent free-radical-scavenging capacities as well as efficient inhibition of cellular melanogenesis, suggesting that they are valuable hit compounds with potential for advanced cosmeceutical development. Copyright © 2015 Verlag Helvetica Chimica Acta AG, Zürich.

  18. Influence of βS-Globin Haplotypes and Hydroxyurea on Arginase I Levels in Sickle Cell Disease

    Directory of Open Access Journals (Sweden)

    J. A. Moreira

    2016-01-01

    Full Text Available Introduction. Sickle cell disease (SCD is characterized by hemoglobin S homozygosity, leading to hemolysis and vasoocclusion. The hemolysis releases arginase I, an enzyme that decreases the bioavailability of nitric oxide, worsening the symptoms. The different SCD haplotypes are related to clinical symptoms and varied hemoglobin F (HbF concentration. The aim of this study was to evaluate the impact of the βS gene haplotypes and HbF concentration on arginase I levels in SCD patients. Methods. Fifty SCD adult patients were enrolled in the study and 20 blood donors composed the control group. Arginase I was measured by ELISA. The βS haplotypes were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP. Statistical analyses were performed with GraphPad Prism program and the significance level was p<0.05. Results. Significant increase was observed in the arginase I levels in SCD patients compared to the control group (p<0.0001. The comparison between the levels of arginase I in three haplotypes groups showed a difference between the Bantu/Bantu × Bantu/Benin groups; Bantu/Bantu × Benin/Benin, independent of HU dosage. An inverse correlation with the arginase I levels and HbF concentration was observed. Conclusion. The results support the hypothesis that arginase I is associated with HbF concentration, also measured indirectly by the association with haplotypes.

  19. Novel cellular bouton structure activated by ATP in the vascular wall of porcine retinal arterioles.

    Science.gov (United States)

    Misfeldt, Mikkel Wölck; Aalkjaer, Christian; Simonsen, Ulf; Bek, Toke

    2010-12-01

    The retinal blood flow is regulated by the tone of resistance arterioles, which is influenced by purinergic compounds such as adenosine and adenosine 5'-triphosphate (ATP) released from the retinal tissue. However, it is unknown what cellular elements in the perivascular retina are responsible for the effect of purines on the tone of retinal arterioles. Porcine retinal arterioles were loaded with the calcium-sensitive fluorophore Oregon green. The vessels were mounted in a confocal myograph for simultaneous recordings of tone and calcium activity in cells of the vascular wall during stimulation with ATP and adenosine, with and without modifiers of these compounds. Additionally, immunohistochemistry was used to localize elements with calcium activity in the vascular wall. Hyperfluorescence indicating calcium activity was recorded in a population of abundant round boutons interspersed in a network of vimentin-positive processes located immediately external to the smooth muscle cell layer but internal to the perivascular glial cells. These structures showed calcium activity when the vessel was relaxed with ATP but not when it was relaxed with adenosine. Ryanodine reduced calcium activity in the boutons, whereas the ATP antagonist adenosine-5'-O-(α, β- methylene diphosphate) reduced calcium activity in both the boutons and vascular tone. The vasodilating effect of purines in porcine retinal tissue involves ATP-dependent calcium activity in a layer of cellular boutons located external to the vascular smooth muscle cells and internal to the perivascular glial cells.

  20. Active cellular sensing with quantum dots: Transitioning from research tool to reality; a review

    Energy Technology Data Exchange (ETDEWEB)

    Delehanty, James B., E-mail: james.delehanty@nrl.navy.mil [Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, DC 20375 (United States); Susumu, Kimihiro, E-mail: susumu@ccs.nrl.navy.mil [Optical Sciences Division, Code 5611, U.S. Naval Research Laboratory, Washington, DC 20375 (United States); Manthe, Rachel L., E-mail: rmanthe@umd.edu [Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, DC 20375 (United States); Fischell Department of Bioengineering, School of Engineering, University of Maryland College Park, College Park, MD 20742 (United States); Algar, W. Russ, E-mail: russ.algar.ctr.ca@nrl.navy.mil [Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, DC 20375 (United States); College of Science, George Mason University, Fairfax, VA 22030 (United States); Medintz, Igor L., E-mail: igor.medintz@nrl.navy.mil [Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, DC 20375 (United States)

    2012-10-31

    Highlights: Black-Right-Pointing-Pointer Quantum dots (QDs) have evolved beyond mere cellular labeling reagents. Black-Right-Pointing-Pointer Significant advances have been made in QD materials, surface coatings and bioconjugation. Black-Right-Pointing-Pointer Cellular targeting/delivery has been achieved using polymers, peptides, proteins. Black-Right-Pointing-Pointer Numerous QD-based sensing applications: extracellular, membrane, intracellular. - Abstract: The application of luminescent semiconductor quantum dots (QDs) within a wide range of biological imaging and sensing formats is now approaching its 15th year. The unique photophysical properties of these nanomaterials have long been envisioned as having the potential to revolutionize biosensing within cellular studies that rely on fluorescence. However, it is only now that these materials are making the transition towards accomplishing this goal. With the idea of understanding how to actively incorporate QDs into different types of cellular biosensing, we review the progress in many of the areas relevant to achieving this goal. This includes the synthesis of the QDs themselves, with an emphasis on minimizing potential toxicity, along with the general methods for making these nanocrystalline structures stable in aqueous media. We next survey some methods for conjugating QDs to biomolecules to allow them to participate in active biosensing. Lastly, we extensively review many of the applications where QDs have been demonstrated in an active role in cellular biosensing. These formats cover a wide range of possibilities including where the QDs have contributed to: monitoring the cell's interaction with its extracellular environment; elucidating the complex molecular interplay that characterizes the plasma membrane; understanding how cells continuously endocytose and exocytose materials across the cellular membrane; visualizing organelle trafficking; and, perhaps most importantly, monitoring the intracellular

  1. Active cellular sensing with quantum dots: Transitioning from research tool to reality; a review

    International Nuclear Information System (INIS)

    Delehanty, James B.; Susumu, Kimihiro; Manthe, Rachel L.; Algar, W. Russ; Medintz, Igor L.

    2012-01-01

    Highlights: ► Quantum dots (QDs) have evolved beyond mere cellular labeling reagents. ► Significant advances have been made in QD materials, surface coatings and bioconjugation. ► Cellular targeting/delivery has been achieved using polymers, peptides, proteins. ► Numerous QD-based sensing applications: extracellular, membrane, intracellular. - Abstract: The application of luminescent semiconductor quantum dots (QDs) within a wide range of biological imaging and sensing formats is now approaching its 15th year. The unique photophysical properties of these nanomaterials have long been envisioned as having the potential to revolutionize biosensing within cellular studies that rely on fluorescence. However, it is only now that these materials are making the transition towards accomplishing this goal. With the idea of understanding how to actively incorporate QDs into different types of cellular biosensing, we review the progress in many of the areas relevant to achieving this goal. This includes the synthesis of the QDs themselves, with an emphasis on minimizing potential toxicity, along with the general methods for making these nanocrystalline structures stable in aqueous media. We next survey some methods for conjugating QDs to biomolecules to allow them to participate in active biosensing. Lastly, we extensively review many of the applications where QDs have been demonstrated in an active role in cellular biosensing. These formats cover a wide range of possibilities including where the QDs have contributed to: monitoring the cell's interaction with its extracellular environment; elucidating the complex molecular interplay that characterizes the plasma membrane; understanding how cells continuously endocytose and exocytose materials across the cellular membrane; visualizing organelle trafficking; and, perhaps most importantly, monitoring the intracellular presence of target molecules such as nucleic acids, nutrients, cofactors, and ions or, alternatively

  2. Arginase Inhibition Reverses Monocrotaline-Induced Pulmonary Hypertension

    Directory of Open Access Journals (Sweden)

    Christian Jung

    2017-07-01

    Full Text Available Pulmonary hypertension (PH is a heterogeneous disorder associated with a poor prognosis. Thus, the development of novel treatment strategies is of great interest. The enzyme arginase (Arg is emerging as important player in PH development. The aim of the current study was to determine the expression of ArgI and ArgII as well as the effects of Arg inhibition in a rat model of PH. PH was induced in 35 Sprague–Dawley rats by monocrotaline (MCT, 60 mg/kg as single-dose. There were three experimental groups: sham-treated controls (control group, n = 11, MCT-induced PH (MCT group, n = 11 and MCT-induced PH treated with the Arg inhibitor Nω-hydroxy-nor-l-arginine (nor-NOHA; MCT/NorNoha group, n = 13. ArgI and ArgII expression was determined by immunohistochemistry and Western blot. Right ventricular systolic pressure (RVPsys was measured and lung tissue remodeling was determined. Induction of PH resulted in an increase in RVPsys (81 ± 16 mmHg compared to the control group (41 ± 15 mmHg, p = 0.002 accompanied by a significant elevation of histological sum-score (8.2 ± 2.4 in the MCT compared to 1.6 ± 1.6 in the control group, p < 0.001. Both, ArgI and ArgII were relevantly expressed in lung tissue and there was a significant increase in the MCT compared to the control group (p < 0.01. Arg inhibition resulted in a significant reduction of RVPsys to 52 ± 19 mmHg (p = 0.006 and histological sum-score to 5.8 ± 1.4 compared to the MCT group (p = 0.022. PH leads to increased expression of Arg. Arg inhibition leads to reduction of RVPsys and diminished lung tissue remodeling and therefore represents a potential treatment strategy in PH.

  3. Surface Immobilization of Human Arginase-1 with an Engineered Ice Nucleation Protein Display System in E. coli.

    Directory of Open Access Journals (Sweden)

    Zhen Zhang

    Full Text Available Ice nucleation protein (INP is frequently used as a surface anchor for protein display in gram-negative bacteria. Here, MalE and TorA signal peptides, and three charged polypeptides, 6×Lys, 6×Glu and 6×Asp, were anchored to the N-terminus of truncated INP (InaK-N to improve its surface display efficiency for human Arginase1 (ARG1. Our results indicated that the TorA signal peptide increased the surface translocation of non-protein fused InaK-N and human ARG1 fused InaK-N (InaK-N/ARG1 by 80.7% and 122.4%, respectively. Comparably, the MalE signal peptide decreased the display efficiencies of both the non-protein fused InaK-N and InaK-N/ARG1. Our results also suggested that the 6×Lys polypeptide significantly increased the surface display efficiency of K6-InaK-N/ARG1 by almost 2-fold, while also practically abolishing the surface translocation of non-protein fused InaK-N, indicating the interesting roles of charged polypeptides in bacteria surface display systems. Cell surface-immobilized K6-InaK-N/ARG1 presented an arginase activity of 10.7 U/OD600 under the optimized conditions of 40°C, pH 10.0 and 1 mM Mn2+, which could convert more than 95% of L-Arginine (L-Arg to L-Ornithine (L-Orn in 16 hours. The engineered InaK-Ns expanded the INP surface display system, which aided in the surface immobilization of human ARG1 in E. coli cells.

  4. Active cell-matrix coupling regulates cellular force landscapes of cohesive epithelial monolayers

    Science.gov (United States)

    Zhao, Tiankai; Zhang, Yao; Wei, Qiong; Shi, Xuechen; Zhao, Peng; Chen, Long-Qing; Zhang, Sulin

    2018-03-01

    Epithelial cells can assemble into cohesive monolayers with rich morphologies on substrates due to competition between elastic, edge, and interfacial effects. Here we present a molecularly based thermodynamic model, integrating monolayer and substrate elasticity, and force-mediated focal adhesion formation, to elucidate the active biochemical regulation over the cellular force landscapes in cohesive epithelial monolayers, corroborated by microscopy and immunofluorescence studies. The predicted extracellular traction and intercellular tension are both monolayer size and substrate stiffness dependent, suggestive of cross-talks between intercellular and extracellular activities. Our model sets a firm ground toward a versatile computational framework to uncover the molecular origins of morphogenesis and disease in multicellular epithelia.

  5. The Stability, Sustained Release and Cellular Antioxidant Activity of Curcumin Nanoliposomes

    Directory of Open Access Journals (Sweden)

    Xing Chen

    2015-08-01

    Full Text Available Curcumin is a multifunctional and natural agent considered to be pharmacologically safe. However, its application in the food and medical industry is greatly limited by its poor water solubility, physicochemical instability and inadequate bioavailability. Nanoliposome encapsulation could significantly enhance the solubility and stability of curcumin. Curcumin nanoliposomes exhibited good physicochemical properties (entrapment efficiency = 57.1, particle size = 68.1 nm, polydispersity index = 0.246, and zeta potential = −3.16 mV. Compared with free curcumin, curcumin nanoliposomes exhibited good stability against alkaline pH and metal ions as well as good storage stability at 4 °C. Curcumin nanoliposomes also showed good sustained release properties. Compared with free curcumin, curcumin nanoliposomes presented an equal cellular antioxidant activity, which is mainly attributed to its lower cellular uptake as detected by fluorescence microscopy and flow cytometry. This study provide theoretical and practical guides for the further application of curcumin nanoliposomes.

  6. Opportunistic activation of TRP receptors by endogenous lipids: exploiting lipidomics to understand TRP receptor cellular communication.

    Science.gov (United States)

    Bradshaw, Heather B; Raboune, Siham; Hollis, Jennifer L

    2013-03-19

    Transient receptor potential channels (TRPs) form a large family of ubiquitous non-selective cation channels that function as cellular sensors and in many cases regulate intracellular calcium. Identification of the endogenous ligands that activate these TRP receptors is still under intense investigation with the majority of these channels still remaining "orphans." That these channels respond to a variety of external stimuli (e.g. plant-derived lipids, changes in temperature, and changes in pH) provides a framework for their abilities as cellular sensors, however, the mechanism of direct activation is still under much debate and research. In the cases where endogenous ligands (predominately lipids) have shown direct activation of a channel, multiple ligands have been shown to activate the same channel suggesting that these receptors are "promiscuous" in nature. Lipidomics of a growing class of endogenous lipids, N-acyl amides, the most famous of which is N-arachidonoyl ethanolamine (the endogenous cannabinoid, Anandamide) is providing a novel set of ligands that have been shown to activate some members of the TRP family and have the potential to deorphanize many more. Here it is argued that activation of TRPV receptors, a subset of the larger family of TRPs, by multiple endogenous lipids that are structurally analogous is a model system to drive our understanding that many TRP receptors are not promiscuous, but are more characteristically "opportunistic" in nature; exploiting the structural similarity and biosynthesis of a narrow range of analogous endogenous lipids. In addition, this manuscript will compare the activation properties of TRPC5 to the activity profile of an "orphan" lipid, N-palmitoyl glycine; further demonstrating that lipidomics aimed at expanding our knowledge of the family of N-acyl amides has the potential to provide novel avenues of research for TRP receptors. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Ion channel signaling influences cellular proliferation and phagocyte activity during axolotl tail regeneration.

    Science.gov (United States)

    Franklin, Brandon M; Voss, S Randal; Osborn, Jeffrey L

    2017-08-01

    Little is known about the potential for ion channels to regulate cellular behaviors during tissue regeneration. Here, we utilized an amphibian tail regeneration assay coupled with a chemical genetic screen to identify ion channel antagonists that altered critical cellular processes during regeneration. Inhibition of multiple ion channels either partially (anoctamin1/Tmem16a, anoctamin2/Tmem16b, K V 2.1, K V 2.2, L-type Ca V channels and H/K ATPases) or completely (GlyR, GABA A R, K V 1.5 and SERCA pumps) inhibited tail regeneration. Partial inhibition of tail regeneration by blocking the calcium activated chloride channels, anoctamin1&2, was associated with a reduction of cellular proliferation in tail muscle and mesenchymal regions. Inhibition of anoctamin 1/2 also altered the post-amputation transcriptional response of p44/42 MAPK signaling pathway genes, including decreased expression of erk1/erk2. We also found that complete inhibition via voltage gated K + channel blockade was associated with diminished phagocyte recruitment to the amputation site. The identification of H + pumps as required for axolotl tail regeneration supports findings in Xenopus and Planaria models, and more generally, the conservation of ion channels as regulators of tissue regeneration. This study provides a preliminary framework for an in-depth investigation of the mechanistic role of ion channels and their potential involvement in regulating cellular proliferation and other processes essential to wound healing, appendage regeneration, and tissue repair. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Telomerase activity and cellular aging might be positively modified by a yoga-based lifestyle intervention.

    Science.gov (United States)

    Kumar, Shiv Basant; Yadav, Rashmi; Yadav, Raj Kumar; Tolahunase, Madhuri; Dada, Rima

    2015-06-01

    Recent studies showed that a brief yoga-based lifestyle intervention was efficacious in reducing levels of oxidative stress and cellular aging in obese men. The objective of this case report was to assess the efficacy of this intervention in reducing the levels of biochemical markers of cellular ageing, oxidative stress, and inflammation at baseline (day 0), at the end of active intervention (day 10), and follow-up at day 90. Single case report from a prospective ongoing study with pre-post design assessing the level of various markers of cellular aging. Integral Health Clinic, an outpatient facility conducting meditation and yoga-based lifestyle intervention programs for management of chronic diseases. A 31-year-old man with class I obesity (body-mass index, 29.5 kg/m(2)) who presented to the medicine outpatient department at All India Institute of Medical Sciences, New Delhi, India, with a history of fatigue, difficulty losing weight, and lack of motivation. He noted a marked decrease in his energy level, particularly in the afternoon. A pretested intervention program included asanas (postures), pranayama (breathing exercises), stress management, group discussions, lectures, and individualized advice. From baseline (day 0) to day 90, the activity of telomerase and levels of β-endorphins, plasma cortisol, and interleukin-6 increased, and a sustained reduction in oxidative stress markers, such as reactive oxygen species and 8-hydroxy-2-deoxy-guanosine levels. Adopting yoga/meditation-based lifestyle modification causes reversal of markers of aging, mainly oxidative stress, telomerase activity, and oxidative DNA damage. This may not only delay aging and prolong a youthful healthy life but also delay or prevent onset of several lifestyle-related diseases, of which oxidative stress and inflammation are the chief cause. This report suggests this simple lifestyle intervention may be therapeutic for oxidative DNA damage and oxidative stress.

  9. Concurrent measurement of cellular turbidity and hemoglobin to evaluate the antioxidant activity of plants.

    Science.gov (United States)

    Bellik, Yuva; Iguer-Ouada, Mokrane

    2016-01-01

    In past decades, a multitude of analytical methods for measuring antioxidant activity of plant extracts has been developed. However, when using methods to determine hemoglobin released from human erythrocytes treated with ginger extracts, we found hemoglobin concentrations were significantly higher than in untreated control samples. This suggests in the presence of antioxidants that measuring hemoglobin alone is not sufficient to determine hemolysis. We show concurrent measurement of erythrocyte concentration and hemoglobin is essential in such assays, and describe a new protocol based on simultaneous measurement of cellular turbidity and hemoglobin. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Role of excision repair in postradiation recovery of biological activity of cellular DNA Bacillus subtilis

    International Nuclear Information System (INIS)

    Filippov, V.D.

    1976-01-01

    DNA extracted from UV-irradiated prototroph cells of Bacillus subtilis uvr + (45 sec. of UV light, 20% survivals) has a lowered transforming activity (TA) of markers purB and metB, and a lowered ratio TA pur/TA met. During the subsequent incubation of uvr + cells in glucose-salt medium free of nitrogen sources the TA of markers and the ratio between them increase. No increase is observed during the postradiation incubation under the same conditions or in a nutrition medium of uvr cells, deficient in escision of pyrimidine dimers. The increment of DNA begins approsimately in 30 min. after the beginning of incubation of irradiated uvr cells in nutrition medium. On the basis of these facts it is concluded that neither the replication of damaged DNA nor the postreplication repair, but only excision repair, can provide the recovery of biological (transforming) activity of cellular DNA in Bac. subtilis. The system given might be a suitable model for testing compounds which affect the activity of this process. The well-known inhibitors of dark repair, caffeine, proflavine to inhibit reversibly the initial steps of the process/ and especially acriflavine, delay the recovery of markers of cellular DNA in irradiated uvr + cells. Caffeine is proved to inhibit reversibly the initial steps of the process

  11. Effect of Cellular Location of Human Carboxylesterase 2 on CPT-11 Hydrolysis and Anticancer Activity.

    Directory of Open Access Journals (Sweden)

    Yuan-Ting Hsieh

    Full Text Available CPT-11 is an anticancer prodrug that is clinically used for the treatment of metastatic colorectal cancer. Hydrolysis of CPT-11 by human carboxylesterase 2 (CE2 generates SN-38, a topoisomerase I inhibitor that is the active anti-tumor agent. Expression of CE2 in cancer cells is under investigation for the tumor-localized activation of CPT-11. CE2 is normally expressed in the endoplasmic reticulum of cells but can be engineered to direct expression of active enzyme on the plasma membrane or as a secreted form. Although previous studies have investigated different locations of CE2 expression in cancer cells, it remains unclear if CE2 cellular location affects CPT-11 anticancer activity. In the present study, we directly compared the influence of CE2 cellular location on substrate hydrolysis and CPT-11 cytotoxicity. We linked expression of CE2 and enhanced green fluorescence protein (eGFP via a foot-and-mouth disease virus 2A (F2A peptide to facilitate fluorescence-activated cell sorting to achieve similar expression levels of ER-located, secreted or membrane-anchored CE2. Soluble CE2 was detected in the medium of cells that expressed secreted and membrane-anchored CE2, but not in cells that expressed ER-retained CE2. Cancer cells that expressed all three forms of CE2 were more sensitive to CPT-11 as compared to unmodified cancer cells, but the membrane-anchored and ER-retained forms of CE2 were consistently more effective than secreted CE2. We conclude that expression of CE2 in the ER or on the membrane of cancer cells is suitable for enhancing CPT-11 anticancer activity.

  12. Different cellular effects of four anti-inflammatory eye drops on human corneal epithelial cells: independent in active components

    OpenAIRE

    Qu, Mingli; Wang, Yao; Yang, Lingling; Zhou, Qingjun

    2011-01-01

    Purpose To evaluate and compare the cellular effects of four commercially available anti-inflammatory eye drops and their active components on human corneal epithelial cells (HCECs) in vitro. Methods The cellular effects of four eye drops (Bromfenac Sodium Hydrate Eye Drops, Pranoprofen Eye Drops, Diclofenac Sodium Eye Drops, and Tobramycin & Dex Eye Drops) and their corresponding active components were evaluated in an HCEC line with five in vitro assays. Cell proliferation and migration were...

  13. Chronic innate immune activation of TBK1 suppresses mTORC1 activity and dysregulates cellular metabolism.

    Science.gov (United States)

    Hasan, Maroof; Gonugunta, Vijay K; Dobbs, Nicole; Ali, Aktar; Palchik, Guillermo; Calvaruso, Maria A; DeBerardinis, Ralph J; Yan, Nan

    2017-01-24

    Three-prime repair exonuclease 1 knockout (Trex1 -/- ) mice suffer from systemic inflammation caused largely by chronic activation of the cyclic GMP-AMP synthase-stimulator of interferon genes-TANK-binding kinase-interferon regulatory factor 3 (cGAS-STING-TBK1-IRF3) signaling pathway. We showed previously that Trex1-deficient cells have reduced mammalian target of rapamycin complex 1 (mTORC1) activity, although the underlying mechanism is unclear. Here, we performed detailed metabolic analysis in Trex1 -/- mice and cells that revealed both cellular and systemic metabolic defects, including reduced mitochondrial respiration and increased glycolysis, energy expenditure, and fat metabolism. We also genetically separated the inflammatory and metabolic phenotypes by showing that Sting deficiency rescued both inflammatory and metabolic phenotypes, whereas Irf3 deficiency only rescued inflammation on the Trex1 -/- background, and many metabolic defects persist in Trex1 -/- Irf3 -/- cells and mice. We also showed that Leptin deficiency (ob/ob) increased lipogenesis and prolonged survival of Trex1 -/- mice without dampening inflammation. Mechanistically, we identified TBK1 as a key regulator of mTORC1 activity in Trex1 -/- cells. Together, our data demonstrate that chronic innate immune activation of TBK1 suppresses mTORC1 activity, leading to dysregulated cellular metabolism.

  14. Human immunodeficiency virus-induced pathology favored by cellular transmission and activation

    International Nuclear Information System (INIS)

    Lewis, D.E.; Yoffe, B.; Bosworth, C.G.; Hollinger, F.B.; Rich, R.R.

    1988-01-01

    Epidemiological data suggest that transmission of human immunodeficiency virus (HIV) occurs primarily by transference of virally infected cells. However, the efficiency of lytic productive infection induced by HIV after transmission of cell-associated virus vs. free virus is difficult to assess. The present studies compare the extent of depletion of CD4+ (helper/inducer) T cells after mixing uninfected cells with either free HIV or irradiated HIV-infected allogeneic or autologous cells in vitro. Rapid CD4+ cellular depletion occurred only in cultures containing allogeneic infected cells or after addition of a nonspecific T cell activation signal to cultures with autologous infected cells. These in vitro observations strongly support the epidemiological implication that interactions between infected and uninfected cells are the most efficient means of transmission and HIV-induced cytopathology in vivo. They also provide direct support for the concept that immunological stimulation by foreign cells infected with HIV dramatically increases the likelihood of transmission. These in vitro observations suggest a model for the acquisition of HIV in vivo and the role of cellular activation in dissemination of the virus to uninfected cells in an infected individual

  15. Sensitivity-Enhanced Wearable Active Voiceprint Sensor Based on Cellular Polypropylene Piezoelectret.

    Science.gov (United States)

    Li, Wenbo; Zhao, Sheng; Wu, Nan; Zhong, Junwen; Wang, Bo; Lin, Shizhe; Chen, Shuwen; Yuan, Fang; Jiang, Hulin; Xiao, Yongjun; Hu, Bin; Zhou, Jun

    2017-07-19

    Wearable active sensors have extensive applications in mobile biosensing and human-machine interaction but require good flexibility, high sensitivity, excellent stability, and self-powered feature. In this work, cellular polypropylene (PP) piezoelectret was chosen as the core material of a sensitivity-enhanced wearable active voiceprint sensor (SWAVS) to realize voiceprint recognition. By virtue of the dipole orientation control method, the air layers in the piezoelectret were efficiently utilized, and the current sensitivity was enhanced (from 1.98 pA/Hz to 5.81 pA/Hz at 115 dB). The SWAVS exhibited the superiorities of high sensitivity, accurate frequency response, and excellent stability. The voiceprint recognition system could make correct reactions to human voices by judging both the password and speaker. This study presented a voiceprint sensor with potential applications in noncontact biometric recognition and safety guarantee systems, promoting the progress of wearable sensor networks.

  16. Carbon nanotube-based sensing devices for human Arginase-1 detection

    Directory of Open Access Journals (Sweden)

    S. Baldo

    2016-03-01

    Full Text Available A new carbon nanotube-based device for detection of Arginase 1 (ARG-1 was produced. Multi-walled carbon nanotubes (MWCNTs were deposited between electrodes by dielectrophoresis (DEP in an accurate and reproducible way. This deposition method has the advantages of low cost and room temperature conditions and therefore, can be used on different kinds of substrates (silicon, glass, plastics allowing for large scale production of chemical or biological sensors. Scanning electrical microscope (SEM and electrical characterization have been performed on the biosensors before and after protein exposure. The devices were tested in the present work for the detection of ARG-1. They show high sensitivity and reproducibility, and can be easily and suitably modified to detect other proteins. Keywords: Carbon nanotube, Biosensor, Arginase, Dielectrophoresis, Biomarker, Protein

  17. Cellular Activation of the Self-Quenched Fluorescent Reporter Probe in Tumor Microenvironment

    Directory of Open Access Journals (Sweden)

    Alexei A. Bogdanov, Jr.

    2002-01-01

    Full Text Available The effect of intralysosomal proteolysis of near-infrared fluorescent (NIRF self-quenched macromolecular probe (PGC-Cy5.5 has been previously reported and used for tumor imaging. Here we demonstrate that proteolysis can be detected noninvasively in vivo at the cellular level. A codetection of GFP fluorescence (using two-photon excitation and NIRF was performed in tumor-bearing animals injected with PGC-Cy5.5. In vivo microscopy of tumor cells in subdermal tissue layers (up to 160 μm showed a strong Cy5.5 dequenching effect in GFP-negative cells. This observation was corroborated by flow cytometry, sorting, and reverse transcription polymerase chain reaction analysis of tumor-isolated cells. Both GFP-positive (81% total and GFP-negative (19% total populations contained Cy5.5-positive cells. The GFP-negative cells were confirmed to be host mouse cells by the absence of rat cathepsin mRNA signal. The subfraction of GFPnegative cells (2.5-3.0% had seven times higher NIRF intensity than the majority of GFP-positive or GFPnegative cells (372 and 55 AU, respectively. Highly NIRF-positive, FP-negative cells were CD45-and MAC3-positive. Our results indicate that: 1 intracellular proteolysis can be imaged in vivo at the cellular level using cathepsin-sensitive probes; 2 tumor-recruited cells of hematopoetic origin participate most actively in uptake and degradation of long-circulating macromolecular probes.

  18. Characteristics of Middle School Students Learning Actions in Outdoor Mathematical Activities with the Cellular Phone

    Science.gov (United States)

    Daher, Wajeeh; Baya'a, Nimer

    2012-01-01

    Learning in the cellular phone environment enables utilizing the multiple functions of the cellular phone, such as mobility, availability, interactivity, verbal and voice communication, taking pictures or recording audio and video, measuring time and transferring information. These functions together with mathematics-designated cellular phone…

  19. Endothelin-1 activation of ETB receptors leads to a reduced cellular proliferative rate and an increased cellular footprint

    International Nuclear Information System (INIS)

    Wilson, Jamie L.; Taylor, Linda; Polgar, Peter

    2012-01-01

    Endothelin-1 (ET-1) is a vasoactive peptide which signals through two G-protein coupled receptors, endothelin receptor A (ETA) and B (ETB). We determined that ET-1 activation of its ETB receptor in stably cDNA transfected CHO cells leads to a 55% reduction in cell number by end-point cell counting and a 35% decrease in cell growth by a real-time cell-substrate impedance-based assay after 24 h of cell growth. When CHO ETB cells were synchronized in the late G1 cell cycle phase, ET-1 delayed their S phase progression compared to control by 30% as determined by [ 3 H]-thymidine incorporation. On the other hand, no such delay was observed during late G2/M to G1 transit when cells were treated with ET-1 after release from mitotic arrest. Using the cell-substrate impedance-based assay, we observed that ET-1 induces opposing morphological changes in CHO ETA and CHO ETB cells with ETB causing an increase in the cell footprint and ETA a decrease. Likewise, in pulmonary artery smooth muscle cells, which express both ETA and ETB receptors, ET-1 induces an ETA-dependent contraction and an ETB dependent dilation. These results are shedding light on a possible beneficial role for ETB in diseases involving ET-1 dysfunction such as pulmonary hypertension. -- Highlights: ► ET- hinders cell proliferation in CHO cells transfected with ETB. ► ET-1 also decreases the rate of DNA synthesis in CHO ETB cells. ► JNK and PI3K appear to be involved in this reduction of DNA synthesis. ► ETB activation in CHO ETB cells and hSMCs leads to dilatory morphological changes. ► In CHO ETA and hSMCs, ETA activation leads to constrictive morphological changes.

  20. Endothelin-1 activation of ETB receptors leads to a reduced cellular proliferative rate and an increased cellular footprint

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, Jamie L.; Taylor, Linda; Polgar, Peter, E-mail: peterp@bu.edu

    2012-06-10

    Endothelin-1 (ET-1) is a vasoactive peptide which signals through two G-protein coupled receptors, endothelin receptor A (ETA) and B (ETB). We determined that ET-1 activation of its ETB receptor in stably cDNA transfected CHO cells leads to a 55% reduction in cell number by end-point cell counting and a 35% decrease in cell growth by a real-time cell-substrate impedance-based assay after 24 h of cell growth. When CHO ETB cells were synchronized in the late G1 cell cycle phase, ET-1 delayed their S phase progression compared to control by 30% as determined by [{sup 3}H]-thymidine incorporation. On the other hand, no such delay was observed during late G2/M to G1 transit when cells were treated with ET-1 after release from mitotic arrest. Using the cell-substrate impedance-based assay, we observed that ET-1 induces opposing morphological changes in CHO ETA and CHO ETB cells with ETB causing an increase in the cell footprint and ETA a decrease. Likewise, in pulmonary artery smooth muscle cells, which express both ETA and ETB receptors, ET-1 induces an ETA-dependent contraction and an ETB dependent dilation. These results are shedding light on a possible beneficial role for ETB in diseases involving ET-1 dysfunction such as pulmonary hypertension. -- Highlights: Black-Right-Pointing-Pointer ET- hinders cell proliferation in CHO cells transfected with ETB. Black-Right-Pointing-Pointer ET-1 also decreases the rate of DNA synthesis in CHO ETB cells. Black-Right-Pointing-Pointer JNK and PI3K appear to be involved in this reduction of DNA synthesis. Black-Right-Pointing-Pointer ETB activation in CHO ETB cells and hSMCs leads to dilatory morphological changes. Black-Right-Pointing-Pointer In CHO ETA and hSMCs, ETA activation leads to constrictive morphological changes.

  1. Cucurbitacin IIb exhibits anti-inflammatory activity through modulating multiple cellular behaviors of mouse lymphocytes.

    Directory of Open Access Journals (Sweden)

    Yao Wang

    Full Text Available Cucurbitacin IIb (CuIIb is one of the major active compounds in Hemsleyadine tablets which have been used for clinical treatment of bacillary dysentery, enteritis and acute tonsilitis. However, its action mechanism has not been completely understood. This study aimed to explore the anti-inflammatory activity of CuIIb and its underlying mechanism in mitogen-activated lymphocytes isolated from mouse mesenteric lymph nodes. The results showed that CuIIb inhibited the proliferation of concanavalin A (Con A-activated lymphocytes in a time- and dose-dependent manner. CuIIb treatment arrested their cell cycle in S and G2/M phases probably due to the disruption of the actin cytoskeleton and the modulation of p27(Kip1 and cyclin levels. Moreover, the surface expression of activation markers CD69 and CD25 on Con A-activated CD3(+ T lymphocytes was suppressed by CuIIb treatment. Both Con A- and phorbol ester plus ionomycin-induced expression of TNF-α, IFN-γ and IL-6 proteins was attenuated upon exposure to CuIIb. Mechanistically, CuIIb treatment suppressed the phosphorylation of JNK and Erk1/2 but not p38 in Con A-activated lymphocytes. Although CuIIb unexpectedly enhanced the phosphorylation of IκB and NF-κB (p65, it blocked the nuclear translocation of NF-κB (p65. In support of this, CuIIb significantly decreased the mRNA levels of IκBα and TNF-α, two target genes of NF-κB, in Con A-activated lymphocytes. In addition, CuIIb downregulated Con A-induced STAT3 phosphorylation and increased cell apoptosis. Collectively, these results suggest that CuIIb exhibits its anti-inflammatory activity through modulating multiple cellular behaviors and signaling pathways, leading to the suppression of the adaptive immune response.

  2. Dual fluorescent molecular substrates selectively report the activation, sustainability and reversibility of cellular PKB/Akt activity.

    Science.gov (United States)

    Shen, Duanwen; Bai, Mingfeng; Tang, Rui; Xu, Baogang; Ju, Xiaoming; Pestell, Richard G; Achilefu, Samuel

    2013-01-01

    Using a newly developed near-infrared (NIR) dye that fluoresces at two different wavelengths (dichromic fluorescence, DCF), we discovered a new fluorescent substrate for Akt, also known as protein kinase B, and a method to quantitatively report this enzyme's activity in real time. Upon insulin activation of cellular Akt, the enzyme multi-phosphorylated a single serine residue of a diserine DCF substrate in a time-dependent manner, culminating in monophospho- to triphospho-serine products. The NIR DCF probe was highly selective for the Akt1 isoform, which was demonstrated using Akt1 knockout cells derived from MMTV-ErbB2 transgenic mice. The DCF mechanism provides unparalleled potential to assess the stimulation, sustainability, and reversibility of Akt activation longitudinally. Importantly, NIR fluorescence provides a pathway to translate findings from cells to living organisms, a condition that could eventually facilitate the use of these probes in humans.

  3. The cellular distribution of extracellular superoxide dismutase in macrophages is altered by cellular activation but unaffected by the natural occurring R213G substitution

    DEFF Research Database (Denmark)

    Gottfredsen, Randi Heidemann; Goldstrohm, David; Hartney, John

    2014-01-01

    and associated with the cell surface via the extracellular matrix (ECM)-binding region. Upon cellular activation induced by lipopolysaccharide, EC-SOD is relocated and detected both in the cell culture medium and in lipid raft structures. Although the secreted material presented a significantly reduced ligand......-binding capacity, this could not be correlated to proteolytic removal of the ECM-binding region, because the integrity of the material recovered from the medium was comparable to that of the cell surface-associated protein. The naturally occurring R213G amino acid substitution located in the ECM-binding region...

  4. Bacterial RNA induces myocyte cellular dysfunction through the activation of PKR

    Science.gov (United States)

    Bleiblo, Farag; Michael, Paul; Brabant, Danielle; Ramana, Chilakamarti V.; Tai, TC; Saleh, Mazen; Parrillo, Joseph E.; Kumar, Anand

    2012-01-01

    Severe sepsis and the ensuing septic shock are serious life threatening conditions. These diseases are triggered by the host's over exuberant systemic response to the infecting pathogen. Several surveillance mechanisms have evolved to discriminate self from foreign RNA and accordingly trigger effective cellular responses to target the pathogenic threats. The RNA-dependent protein kinase (PKR) is a key component of the cytoplasmic RNA sensors involved in the recognition of viral double-stranded RNA (dsRNA). Here, we identify bacterial RNA as a distinct pathogenic pattern recognized by PKR. Our results indicate that natural RNA derived from bacteria directly binds to and activates PKR. We further show that bacterial RNA induces human cardiac myocyte apoptosis and identify the requirement for PKR in mediating this response. In addition to bacterial immunity, the results presented here may also have implications in cardiac pathophysiology. PMID:22833816

  5. Arginase treatment prevents the recovery of canine lymphoma and osteosarcoma cells resistant to the toxic effects of prolonged arginine deprivation.

    Science.gov (United States)

    Wells, James W; Evans, Christopher H; Scott, Milcah C; Rütgen, Barbara C; O'Brien, Timothy D; Modiano, Jaime F; Cvetkovic, Goran; Tepic, Slobodan

    2013-01-01

    Rapidly growing tumor cells require a nutrient-rich environment in order to thrive, therefore, restricting access to certain key amino acids, such as arginine, often results in the death of malignant cells, which frequently display defective cell cycle check-point control. Healthy cells, by contrast, become quiescent and remain viable under arginine restriction, displaying full recovery upon return to arginine-rich conditions. The use of arginase therapy to restrict available arginine for selectively targeting malignant cells is currently under investigation in human clinical trials. However, the suitability of this approach for veterinary uses is unexplored. As a prelude to in vivo studies in canine malignancies, we examined the in vitro effects of arginine-deprivation on canine lymphoid and osteosarcoma cell lines. Two lymphoid and 2 osteosarcoma cell lines were unable to recover following 6 days of arginine deprivation, but all remaining cell lines displayed full recovery upon return to arginine-rich culture conditions. These remaining cell lines all proved susceptible to cell death following the addition of arginase to the cultures. The lymphoid lines were particularly sensitive to arginase, becoming unrecoverable after just 3 days of treatment. Two of the osteosarcoma lines were also susceptible over this time-frame; however the other 3 lines required 6-8 days of arginase treatment to prevent recovery. In contrast, adult progenitor cells from the bone marrow of a healthy dog were able to recover fully following 9 days of culture in arginase. Over 3 days in culture, arginase was more effective than asparaginase in inducing the death of lymphoid lines. These results strongly suggest that short-term arginase treatment warrants further investigation as a therapy for lymphoid malignancies and osteosarcomas in dogs.

  6. Arginase treatment prevents the recovery of canine lymphoma and osteosarcoma cells resistant to the toxic effects of prolonged arginine deprivation.

    Directory of Open Access Journals (Sweden)

    James W Wells

    Full Text Available Rapidly growing tumor cells require a nutrient-rich environment in order to thrive, therefore, restricting access to certain key amino acids, such as arginine, often results in the death of malignant cells, which frequently display defective cell cycle check-point control. Healthy cells, by contrast, become quiescent and remain viable under arginine restriction, displaying full recovery upon return to arginine-rich conditions. The use of arginase therapy to restrict available arginine for selectively targeting malignant cells is currently under investigation in human clinical trials. However, the suitability of this approach for veterinary uses is unexplored. As a prelude to in vivo studies in canine malignancies, we examined the in vitro effects of arginine-deprivation on canine lymphoid and osteosarcoma cell lines. Two lymphoid and 2 osteosarcoma cell lines were unable to recover following 6 days of arginine deprivation, but all remaining cell lines displayed full recovery upon return to arginine-rich culture conditions. These remaining cell lines all proved susceptible to cell death following the addition of arginase to the cultures. The lymphoid lines were particularly sensitive to arginase, becoming unrecoverable after just 3 days of treatment. Two of the osteosarcoma lines were also susceptible over this time-frame; however the other 3 lines required 6-8 days of arginase treatment to prevent recovery. In contrast, adult progenitor cells from the bone marrow of a healthy dog were able to recover fully following 9 days of culture in arginase. Over 3 days in culture, arginase was more effective than asparaginase in inducing the death of lymphoid lines. These results strongly suggest that short-term arginase treatment warrants further investigation as a therapy for lymphoid malignancies and osteosarcomas in dogs.

  7. Effect of arginase inhibition on ischemia-reperfusion injury in patients with coronary artery disease with and without diabetes mellitus.

    Directory of Open Access Journals (Sweden)

    Oskar Kövamees

    Full Text Available Arginase competes with nitric oxide synthase for their common substrate L-arginine. Up-regulation of arginase in coronary artery disease (CAD and diabetes mellitus may reduce nitric oxide bioavailability contributing to endothelial dysfunction and ischemia-reperfusion injury. Arginase inhibition reduces infarct size in animal models. Therefore the aim of the current study was to investigate if arginase inhibition protects from endothelial dysfunction induced by ischemia-reperfusion in patients with CAD with or without type 2 diabetes (NCT02009527.Male patients with CAD (n = 12 or CAD + type 2 diabetes (n = 12, were included in this cross-over study with blinded evaluation. Endothelium-dependent vasodilatation was assessed by flow-mediated dilatation (FMD of the radial artery before and after 20 min ischemia-reperfusion during intra-arterial infusion of the arginase inhibitor (Nω-hydroxy-nor-L-arginine, 0.1 mg/min or saline.The forearm ischemia-reperfusion was well tolerated. Endothelium-independent vasodilatation was assessed by sublingual nitroglycerin. Ischemia-reperfusion decreased FMD in patients with CAD from 12.7±5.2% to 7.9±4.0% during saline administration (P<0.05. Nω-hydroxy-nor-L-arginine administration prevented the decrease in FMD in the CAD group (10.3±4.3% at baseline vs. 11.5±3.6% at reperfusion. Ischemia-reperfusion did not significantly reduce FMD in patients with CAD + type 2 diabetes. However, FMD at reperfusion was higher following nor-NOHA than following saline administration in both groups (P<0.01. Endothelium-independent vasodilatation did not differ between the occasions.Inhibition of arginase protects against endothelial dysfunction caused by ischemia-reperfusion in patients with CAD. Arginase inhibition may thereby be a promising therapeutic strategy in the treatment of ischemia-reperfusion injury.

  8. Tetraspanin CD9 modulates human lymphoma cellular proliferation via histone deacetylase activity

    Energy Technology Data Exchange (ETDEWEB)

    Herr, Michael J. [Vascular Biology Center of Excellence, The University of Tennessee Health Science Center, Memphis, TN 38163 (United States); Department of Medicine, The University of Tennessee Health Science Center, Memphis, TN 38163 (United States); Department of Molecular Sciences, The University of Tennessee Health Science Center, Memphis, TN 38163 (United States); Department of Surgery, The University of Tennessee Health Science Center, Memphis, TN 38163 (United States); Longhurst, Celia M.; Baker, Benjamin [Vascular Biology Center of Excellence, The University of Tennessee Health Science Center, Memphis, TN 38163 (United States); Homayouni, Ramin [Department of Biology, Bioinformatics Program, University of Memphis, Memphis, TN 38152 (United States); Speich, Henry E.; Kotha, Jayaprakash [Vascular Biology Center of Excellence, The University of Tennessee Health Science Center, Memphis, TN 38163 (United States); Jennings, Lisa K., E-mail: ljennings@uthsc.edu [Vascular Biology Center of Excellence, The University of Tennessee Health Science Center, Memphis, TN 38163 (United States); Department of Medicine, The University of Tennessee Health Science Center, Memphis, TN 38163 (United States); Department of Molecular Sciences, The University of Tennessee Health Science Center, Memphis, TN 38163 (United States); Department of Surgery, The University of Tennessee Health Science Center, Memphis, TN 38163 (United States); Department of Biology, Bioinformatics Program, University of Memphis, Memphis, TN 38152 (United States)

    2014-05-16

    Highlights: • CD9 is differentially expressed in human Burkitt’s lymphoma cells. • We found that CD9 expression promotes these cells proliferation. • CD9 expression also increases HDAC activity. • HDAC inhibition decreased both cell proliferation and importantly CD9 expression. • CD9 may dictate HDAC efficacy and play a role in HDAC regulation. - Abstract: Non-Hodgkin Lymphoma (NHL) is a type of hematological malignancy that affects two percent of the overall population in the United States. Tetraspanin CD9 is a cell surface protein that has been thoroughly demonstrated to be a molecular facilitator of cellular phenotype. CD9 expression varies in two human lymphoma cell lines, Raji and BJAB. In this report, we investigated the functional relationship between CD9 and cell proliferation regulated by histone deacetylase (HDAC) activity in these two cell lines. Introduction of CD9 expression in Raji cells resulted in significantly increased cell proliferation and HDAC activity compared to Mock transfected Raji cells. The increase in CD9–Raji cell proliferation was significantly inhibited by HDAC inhibitor (HDACi) treatment. Pretreatment of BJAB cells with HDAC inhibitors resulted in a significant decrease in endogenous CD9 mRNA and cell surface expression. BJAB cells also displayed decreased cell proliferation after HDACi treatment. These results suggest a significant relationship between CD9 expression and cell proliferation in human lymphoma cells that may be modulated by HDAC activity.

  9. Dermal quercetin smartCrystals®: Formulation development, antioxidant activity and cellular safety.

    Science.gov (United States)

    Hatahet, T; Morille, M; Hommoss, A; Dorandeu, C; Müller, R H; Bégu, S

    2016-05-01

    Flavonoids are natural plant pigments, which possess high antioxidative and antiradical activities. However, their poor water solubility led to a limited bioavailability. To overcome this major hurdle, quercetin nanocrystals were produced implementing smartCrystals® technology. This process combines bead milling and subsequent high-pressure homogenization at relatively low pressure (300bar). To test the possibility to develop a dermal formulation from quercetin smartCrystals®, quercetin nanosuspensions were admixed to Lutrol® F127 and hydroxythylcellulose nonionic gels. The physicochemical properties (morphology, size and charge), saturation solubility, dissolution velocity and the antioxidant properties (DPPH assay) as well as the cellular interaction of the produced quercetin smartCrystals® were studied and compared to crude quercetin powder. Quercetin smartCrystals® showed a strong increase in the saturation solubility and the dissolution velocity (7.6 fold). SmartCrystals® loaded or not into gels proved to be physically stable over a period of three months at 25°C. Interestingly, in vitro DPPH assay confirmed the preservation of quercetin antioxidative properties after nanonization. In parallel, the nanocrystalline form did not display cellular toxicity, even at high concentration (50μg/ml), as assayed on an epithelial cell line (VERO cells). In addition, the nanocrystalline form confirmed a protective activity for VERO cells against hydrogen peroxide induced toxicity in vitro. This new formulation presents a promising approach to deliver quercetin efficiently to skin in well-tolerated formulations. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Insulin-like growth factor-I, physical activity, and control of cellular anabolism.

    Science.gov (United States)

    Nindl, Bradley C

    2010-01-01

    The underlying mechanisms responsible for mediating the beneficial outcomes of exercise undoubtedly are many, but the insulin-like growth factor-I (IGF-I) system is emerging as an important and central hormonal axis that plays a significant role concerning cellular anabolism. This introductory article summarizes the intent and the content for papers presented as part of a 2008 American College of Sports Medicine national symposium entitled "Insulin-like Growth Factor-I, Physical Activity, and Control of Cellular Anabolism." The individual authors and their papers are as follows: Jan Frystyk authoring "The relationship between exercise and the growth hormone/insulin-like growth factor-I axis," Greg Adams authoring "IGF-I signaling in skeletal muscle and the potential for cytokine interactions," and Brad Nindl authoring "Insulin-like growth factor-I as a biomarker of health, fitness, and training status." These papers focus on 1) different assay methodologies for IGF-I within the paradigm of exercise studies, 2) research demonstrating that intracellular signaling components associated with several proinflammatory cytokines have the potential to interact with anabolic signaling processes in skeletal muscle, and 3) an overview of IGF-I as a biomarker related to exercise training, muscle and bone remodeling, body composition, cognition, and cancer. When summed in total, the contribution that these papers will make will undoubtedly involve bringing attention to the vast regulatory complexity of the IGF-I system and will hopefully convince the reader that the IGF-I system warrants further detailed scientific inquiry to resolve many unanswered questions and paradoxical experimental findings. The IGF-I system remains one of the most intriguing and captivating marvels of human physiology that seems central in mediating numerous adaptations from physical activity.

  11. Probiotic Properties and Cellular Antioxidant Activity of Lactobacillus plantarum MA2 Isolated from Tibetan Kefir Grains.

    Science.gov (United States)

    Tang, Wei; Li, Chao; He, Zengguo; Pan, Fen; Pan, Shuo; Wang, Yanping

    2017-11-20

    Lactobacillus plantarum MA2 was isolated from traditional Chinese Tibetan kefir grains. Its antioxidant properties had been demonstrated in vitro and in vivo previously. In the present study, the probiotic characteristics of this strain were further evaluated by investigating its acid and bile salt tolerances, cell surface hydrophobicity, and autoaggregation, respectively. In addition, the cellular antioxidant activity (CAA) assay was applied to test the antioxidant capacity of the isolate in different growth phases. Same method was also used to evaluate the antioxidant capacity of its fermentation supernatant, cell-free extract, and intact cell quantitatively. The results of probiotic characteristic tests showed that MA2 could survive at pH 2.5 and 0.3% bile salt. Meanwhile, the measurements of cell surface hydrophobicity and autoaggregation were 45.29 ± 2.15 and 6.30 ± 0.34%, respectively. The results of cellular antioxidant activity tests indicated that MA2 had high antioxidant potential. The CAA value of logarithmic phase cell-free extract of MA2 (39,450.00 ± 424.05 μmol quercetin equivalents/100 g sample) was significantly higher than that in stationary phase cell-free extract (3395.98 ± 126.06 μmol quercetin equivalents/100 g sample) and that of fermentation supernatant in logarithmic phase (2174.41 ± 224.47 μmol quercetin equivalents/100 g sample) (p < 0.05). The CAA method was successively applied to evaluate the antioxidant capacity of MA2 in this study, which suggests that it could be used as a useful method for lactic acid bacteria antioxidant potential evaluation.

  12. Sleep deprivation and activation of morning levels of cellular and genomic markers of inflammation.

    Science.gov (United States)

    Irwin, Michael R; Wang, Minge; Campomayor, Capella O; Collado-Hidalgo, Alicia; Cole, Steve

    2006-09-18

    Inflammation is associated with increased risk of cardiovascular disorders, arthritis, diabetes mellitus, and mortality. The effects of sleep loss on the cellular and genomic mechanisms that contribute to inflammatory cytokine activity are not known. In 30 healthy adults, monocyte intracellular proinflammatory cytokine production was repeatedly assessed during the day across 3 baseline periods and after partial sleep deprivation (awake from 11 pm to 3 am). We analyzed the impact of sleep loss on transcription of proinflammatory cytokine genes and used DNA microarray analyses to characterize candidate transcription-control pathways that might mediate the effects of sleep loss on leukocyte gene expression. In the morning after a night of sleep loss, monocyte production of interleukin 6 and tumor necrosis factor alpha was significantly greater compared with morning levels following uninterrupted sleep. In addition, sleep loss induced a more than 3-fold increase in transcription of interleukin 6 messenger RNA and a 2-fold increase in tumor necrosis factor alpha messenger RNA. Bioinformatics analyses suggested that the inflammatory response was mediated by the nuclear factor kappaB inflammatory signaling system as well as through classic hormone and growth factor response pathways. Sleep loss induces a functional alteration of the monocyte proinflammatory cytokine response. A modest amount of sleep loss also alters molecular processes that drive cellular immune activation and induce inflammatory cytokines; mapping the dynamics of sleep loss on molecular signaling pathways has implications for understanding the role of sleep in altering immune cell physiologic characteristics. Interventions that target sleep might constitute new strategies to constrain inflammation with effects on inflammatory disease risk.

  13. Genetic Targeting of Arginase-II in Mouse Prevents Renal Oxidative Stress and Inflammation in Diet-Induced Obesity.

    Science.gov (United States)

    Huang, Ji; Rajapakse, Angana; Xiong, Yuyan; Montani, Jean-Pierre; Verrey, François; Ming, Xiu-Fen; Yang, Zhihong

    2016-01-01

    Obesity is associated with development and progression of chronic kidney disease (CKD). Recent evidence demonstrates that enhanced levels of the L-arginine:ureahydrolase, including the two isoenzymes arginase-I (Arg-I) and arginase-II (Arg-II) in vascular endothelial cells promote uncoupling of endothelial nitric oxide synthase (eNOS), leading to increased superoxide radical anion and decreased NO production thereby endothelial dysfunction. Arg-II but not Arg-I is abundantly expressed in kidney and the role of Arg-II in CKD is uncertain and controversial. We aimed to investigate the role of Arg-II in renal damage associated with diet-induced obesity mouse model. Wild type (WT) C57BL/6 mice and mice deficient in Arg-II gene (Arg-II -/- ) were fed with either a normal chow (NC) or a high-fat-diet (HFD) for 14 weeks (starting at the age of 7 weeks) to induce obesity. In WT mice, HFD feeding caused frequent renal lipid accumulation, enhancement of renal reactive oxygen species (ROS) levels which could be attenuated by a NOS inhibitor, suggesting uncoupling of NOS in kidney. HFD feeding also significantly augmented renal Arg-II expression and activity. All the alterations in the kidney under HFD feeding were reduced in Arg-II -/- mice. Moreover, mesangial expansion as analyzed by Periodic Acid Schiff (PAS) staining and renal expression of vascular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in HFD-fed WT mouse assessed by immunoblotting were reduced in the HFD-fed Arg-II -/- mice, although there was no significant difference in body weight and renal weight/body weight ratio between the WT and Arg-II -/- mice. Thus, Arg-II expression/activity is enhanced in kidney of diet-induced obesity mice. Genetic targeting of Arg-II prevents renal damage associated with obesity, suggesting an important role of Arg-II in obesity-associated renal disease development.

  14. Genetic Targeting of Arginase-II in Mouse Prevents Renal Oxidative Stress and Inflammation in Diet-Induced Obesity

    Directory of Open Access Journals (Sweden)

    Ji Huang

    2016-11-01

    Full Text Available Obesity is associated with development and progression of chronic kidney disease (CKD. Recent evidence demonstrates that enhanced levels of the L-arginine:ureahydrolase, including the two isoenzymes arginase-I (Arg-I and arginase-II (Arg-II in vascular endothelial cells promote uncoupling of endothelial nitric oxide synthase (eNOS, leading to increased superoxide radical anion and decreased NO production thereby endothelial dysfunction. Arg-II but not Arg-I is abundantly expressed in kidney and the role of Arg-II in CKD is uncertain and controversial. We aimed to investigate the role of Arg-II in renal damage associated with diet-induced obesity mouse model. Wild type (WT C57BL/6 mice and mice deficient in Arg-II gene (Arg-II-/- were fed with either a normal chow (NC or a high-fat-diet (HFD for 14 weeks (starting at the age of 7 weeks to induce obesity. In WT mice, HFD feeding caused frequent renal lipid accumulation, enhancement of renal ROS levels which could be attenuated by a NOS inhibitor, suggesting uncoupling of NOS in kidney. HFD feeding also significantly augmented renal Arg-II expression and activity. All the alterations in the kidney under HFD feeding were reduced in Arg-II-/- mice. Moreover, mesangial expansion as analysed by Periodic Acid Schiff (PAS staining and renal expression of vascular adhesion molecule-1 (VCAM-1 and intercellular adhesion molecule-1 (ICAM-1 in HFD-fed WT mouse assessed by immunoblotting were reduced in the HFD-fed Arg-II-/- mice, although there was no significant difference in body weight and renal weight/body weight ratio between the WT and Arg-II-/- mice. Thus, Arg-II expression/activity is enhanced in kidney of diet-induced obesity mice. Genetic targeting of Arg-II prevents renal damage associated with obesity, suggesting an important role of Arg-II in obesity-associated renal disease development.

  15. Metavanadate causes cellular accumulation of copper and decreased lysyl oxidase activity

    International Nuclear Information System (INIS)

    Cui, Changtai T.; Uriu-Adams, Janet Y.; Tchaparian, Eskouhie H.; Keen, Carl L.; Rucker, Robert B.

    2004-01-01

    Selected indices of copper metabolism in weanling rats and fibroblast cultures were progressively altered in response to increased levels of sodium metavanadate. In diets, vanadium was added in amounts ranging from 0 to 80 μg V/g of diet, that is, 0-1.6 μmol V/g of diet. In fibroblast cultures, vanadium ranged from 0 to 400 nmol V/ml. The inhibition of P-ATPase-7A activity by metavanadate, important to copper egress from cells, was a primary focus. In skin, and tendon, the copper concentration was increased in response to increased dietary levels of metavanadate, whereas lysyl oxidase activity, a secreted cuproprotein, was reduced. The reduction in lysyl oxidase activity was also accompanied by reduced redox cycling potential of isolated fractions of lysyl oxidase, presumably due to reduced lysyltyrosyl quinone (LTQ) formation at the active site of lysyl oxidase. In contrast, liver copper concentrations and plasma ceruloplasmin activity were not affected by metavanadate exposure. However, semicarbazide-sensitive benzylamine oxidase (SCBO) activity, which was taken as an indirect measure of vascular adhesive protein-1 (VAP-1), was increased. In cultured fibroblasts, cellular copper was also increased and lysyl oxidase decreased in response to metavanadate. Moreover, the steady-state levels of atp7a and lysyl oxidase mRNAs were not affected by addition of metavanadate to culture medium up to 200 nmol/ml. Taken together, these data suggest that pathways involving copper egress and lysyl oxidase activation are particularly sensitive to metavanadate exposure through processes that are predominately posttranslational

  16. Relationship between peroxisome proliferator-activated receptor alpha activity and cellular concentration of 14 perfluoroalkyl substances in HepG2 cells.

    Science.gov (United States)

    Rosenmai, Anna Kjerstine; Ahrens, Lutz; le Godec, Théo; Lundqvist, Johan; Oskarsson, Agneta

    2018-02-01

    Peroxisome proliferator-activated receptor alpha (PPARα) is a molecular target for perfluoroalkyl substances (PFASs). Little is known about the cellular uptake of PFASs and how it affects the PPARα activity. We investigated the relationship between PPARα activity and cellular concentration in HepG2 cells of 14 PFASs, including perfluoroalkyl carboxylates (PFCAs), perfluoroalkyl sulfonates and perfluorooctane sulfonamide (FOSA). Cellular concentrations were determined by high-performance liquid chromatography-tandem mass spectrometry and PPARα activity was determined in transiently transfected cells by reporter gene assay. Cellular uptake of the PFASs was low (0.04-4.1%) with absolute cellular concentrations in the range 4-2500 ng mg -1 protein. Cellular concentration of PFCAs increased with perfluorocarbon chain length up to perfluorododecanoate. PPARα activity of PFCAs increased with chain length up to perfluorooctanoate. The maximum induction of PPARα activity was similar for short-chain (perfluorobutanoate and perfluoropentanoate) and long-chain PFCAs (perfluorododecanoate and perfluorotetradecanoate) (approximately twofold). However, PPARα activities were induced at lower cellular concentrations for the short-chain homologs compared to the long-chain homologs. Perfluorohexanoate, perfluoroheptanoate, perfluorooctanoate, perfluorononanoate (PFNA) and perfluorodecanoate induced PPARα activities >2.5-fold compared to controls. The concentration-response relationships were positive for all the tested compounds, except perfluorooctane sulfonate PFOS and FOSA, and were compound-specific, as demonstrated by differences in the estimated slopes. The relationships were steeper for PFCAs with chain lengths up to and including PFNA than for the other studied PFASs. To our knowledge, this is the first report establishing relationships between PPARα activity and cellular concentration of a broad range of PFASs. Copyright © 2017 John Wiley & Sons, Ltd.

  17. Activation of the cellular unfolded protein response by recombinant adeno-associated virus vectors.

    Directory of Open Access Journals (Sweden)

    Balaji Balakrishnan

    Full Text Available The unfolded protein response (UPR is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER. In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α, activating transcription factor 6 (ATF6 and PKR-like ER kinase (PERK in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold and PERK (up to 8 fold genes 12-48 hours after infection with self-complementary (scAAV2 but less prominent with single-stranded (ssAAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold while AAV6 vectors induced a significant increase on all the three major UPR pathways [6-16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5-2 fold in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively. However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer.

  18. A case study of technology-enhanced active learning in introductory cellular biology

    Science.gov (United States)

    Chacon Diaz, Lucia Bernardette

    Science teaching and learning in higher education has been evolving over the years to encourage student retention in STEM fields and reduce student attrition. As novel pedagogical practices emerge in the college science classroom, research on the effectiveness of such approaches must be undertaken. The following research applied a case study research design in order to evaluate the experiences of college students in a TEAL classroom. This case study was conducted during the 2017 Summer Cellular and Organismal Biology course at a four-year Hispanic Serving Institution located in the Southwest region of the United States. The main components evaluated were students' exam performance, self-efficacy beliefs, and behaviors and interactions in the Technology-Enhanced Active Learning (TEAL) classroom. The findings suggest that students enrolled in a TEAL classroom are equally capable of answering high and low order thinking questions. Additionally, students are equally confident in answering high and low order thinking items related to cellular biology. In the TEAL classroom, student-student interactions are encouraged and collaborative behaviors are exhibited. Gender and ethnicity do not influence self-efficacy beliefs in students in the TEAL room, and the overall class average of self-efficacy beliefs tended to be higher compared to exam performance. Based on the findings of this case study, TEAL classrooms are greatly encouraged in science higher education in order to facilitate learning and class engagement for all students. Providing students with the opportunity to expand their academic talents in the science classroom accomplishes a crucial goal in STEM higher education.

  19. Special Issue: Redox Active Natural Products and Their Interaction with Cellular Signalling Pathways

    Directory of Open Access Journals (Sweden)

    Claus Jacob

    2014-11-01

    Full Text Available During the last decade, research into natural products has experienced a certain renaissance. The urgent need for more and more effective antibiotics in medicine, the demand for ecologically friendly plant protectants in agriculture, “natural” cosmetics and the issue of a sustainable and healthy nutrition in an ageing society have fuelled research into Nature’s treasure chest of “green gold”. Here, redox active secondary metabolites from plants, fungi, bacteria and other (micro-organisms often have been at the forefront of the most interesting developments. These agents provide powerful means to interfere with many, probably most cellular signaling pathways in humans, animals and lower organisms, and therefore can be used to protect, i.e., in form of antioxidants, and to frighten off or even kill, i.e., in form of repellants, antibiotics, fungicides and selective, often catalytic “sensor/effector” anticancer agents. Interestingly, whilst natural product research dates back many decades, in some cases even centuries, and compounds such as allicin and various flavonoids have been investigated thoroughly in the past, it has only recently become possible to investigate their precise interactions and mode(s of action inside living cells. Here, fluorescent staining and labelling on the one side, and appropriate detection, either qualitatively under the microscope or quantitatively in flow cytometers and plate readers, on the other, enable researchers to obtain the various pieces of information necessary to construct a fairly complete puzzle of how such compounds act and interact in living cells. Complemented by the more traditional activity assays and Western Blots, and increasingly joined by techniques such as proteomics, chemogenetic screening and mRNA profiling, these cell based bioanalytical techniques form a powerful platform for “intracellular diagnostics”. In the case of redox active compounds, especially of Reactive Sulfur

  20. Controlling cellular P-TEFb activity by the HIV-1 transcriptional transactivator Tat.

    Directory of Open Access Journals (Sweden)

    Lisa Muniz

    Full Text Available The human immunodeficiency virus 1 (HIV-1 transcriptional transactivator (Tat is essential for synthesis of full-length transcripts from the integrated viral genome by RNA polymerase II (Pol II. Tat recruits the host positive transcription elongation factor b (P-TEFb to the HIV-1 promoter through binding to the transactivator RNA (TAR at the 5'-end of the nascent HIV transcript. P-TEFb is a general Pol II transcription factor; its cellular activity is controlled by the 7SK small nuclear RNA (snRNA and the HEXIM1 protein, which sequester P-TEFb into transcriptionally inactive 7SK/HEXIM/P-TEFb snRNP. Besides targeting P-TEFb to HIV transcription, Tat also increases the nuclear level of active P-TEFb through promoting its dissociation from the 7SK/HEXIM/P-TEFb RNP by an unclear mechanism. In this study, by using in vitro and in vivo RNA-protein binding assays, we demonstrate that HIV-1 Tat binds with high specificity and efficiency to an evolutionarily highly conserved stem-bulge-stem motif of the 5'-hairpin of human 7SK snRNA. The newly discovered Tat-binding motif of 7SK is structurally and functionally indistinguishable from the extensively characterized Tat-binding site of HIV TAR and importantly, it is imbedded in the HEXIM-binding elements of 7SK snRNA. We show that Tat efficiently replaces HEXIM1 on the 7SK snRNA in vivo and therefore, it promotes the disassembly of the 7SK/HEXIM/P-TEFb negative transcriptional regulatory snRNP to augment the nuclear level of active P-TEFb. This is the first demonstration that HIV-1 specifically targets an important cellular regulatory RNA, most probably to promote viral transcription and replication. Demonstration that the human 7SK snRNA carries a TAR RNA-like Tat-binding element that is essential for the normal transcriptional regulatory function of 7SK questions the viability of HIV therapeutic approaches based on small drugs blocking the Tat-binding site of HIV TAR.

  1. Integrin Beta 3 Regulates Cellular Senescence by Activating the TGF-β Pathway

    Directory of Open Access Journals (Sweden)

    Valentina Rapisarda

    2017-03-01

    Full Text Available Cellular senescence is an important in vivo mechanism that prevents the propagation of damaged cells. However, the precise mechanisms regulating senescence are not well characterized. Here, we find that ITGB3 (integrin beta 3 or β3 is regulated by the Polycomb protein CBX7. β3 expression accelerates the onset of senescence in human primary fibroblasts by activating the transforming growth factor β (TGF-β pathway in a cell-autonomous and non-cell-autonomous manner. β3 levels are dynamically increased during oncogene-induced senescence (OIS through CBX7 Polycomb regulation, and downregulation of β3 levels overrides OIS and therapy-induced senescence (TIS, independently of its ligand-binding activity. Moreover, cilengitide, an αvβ3 antagonist, has the ability to block the senescence-associated secretory phenotype (SASP without affecting proliferation. Finally, we show an increase in β3 levels in a subset of tissues during aging. Altogether, our data show that integrin β3 subunit is a marker and regulator of senescence.

  2. Cellular and antitumor activity of a new diethylene glycol benzoporphyrin derivative (lemuteporfin).

    Science.gov (United States)

    Boch, Ron; Canaan, Alice J; Cho, Angela; Dolphin, David D; Hong, Lina; Jain, Ashok K; North, John R; Richter, Anna M; Smits, Claire; Sternberg, Ethan D

    2006-01-01

    A newly synthesized diethylene glycol functionalized chlorin-type photosensitizer, lemuteporfin, was characterized for use in photodynamic therapy (PDT) in a panel of in vitro and in vivo test systems. The photosensitizer was highly potent, killing cells at low nanomolar concentrations upon exposure to activating light. The cellular uptake of lemuteporfin was rapid, with maximum levels reached within 20 min. Mitogen-activated lymphoid cells accumulated more of the lemuteporfin than their quiescent equivalents, supporting selectivity. Photosensitizer fluorescence in the skin increased rapidly within the first few minutes following intravenous administration to mice, then decreased over the next 24 h. Skin photosensitivity reactions indicated rapid clearance of the photosensitizer. Intravenous doses as low as 1.4 micromol/kg combined with exposure to 50 J/cm2 red light suppressed tumor growth in a mouse model. In conclusion, this new benzoporphyrin was found to be an effective photosensitizer, showing rapid uptake and clearance both in vitro and in vivo. This rapid photosensitization of tumors could be useful in therapies requiring a potent, rapidly accumulating photosensitizer, while minimizing the potential for skin photosensitivity reactions to sunlight following treatment.

  3. Arylamine N-acetyltransferase activity in bronchial epithelial cells and its inhibition by cellular oxidants

    International Nuclear Information System (INIS)

    Dairou, Julien; Petit, Emile; Ragunathan, Nilusha; Baeza-Squiban, Armelle; Marano, Francelyne; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2009-01-01

    Bronchial epithelial cells express xenobiotic-metabolizing enzymes (XMEs) that are involved in the biotransformation of inhaled toxic compounds. The activities of these XMEs in the lung may modulate respiratory toxicity and have been linked to several diseases of the airways. Arylamine N-acetyltransferases (NAT) are conjugating XMEs that play a key role in the biotransformation of aromatic amine pollutants such as the tobacco-smoke carcinogens 4-aminobiphenyl (4-ABP) and β-naphthylamine (β-NA). We show here that functional human NAT1 or its murine counterpart Nat2 are present in different lung epithelial cells i.e. Clara cells, type II alveolar cells and bronchial epithelial cells, thus indicating that inhaled aromatic amines may undergo NAT-dependent biotransformation in lung epithelium. Exposure of these cells to pathophysiologically relevant amounts of oxidants known to contribute to lung dysfunction, such as H 2 O 2 or peroxynitrite, was found to impair the NAT1/Nat2-dependent cellular biotransformation of aromatic amines. Genetic and non genetic impairment of intracellular NAT enzyme activities has been suggested to compromise the important detoxification pathway of aromatic amine N-acetylation and subsequently to contribute to an exacerbation of untoward effects of these pollutants on health. Our study suggests that oxidative/nitroxidative stress in lung epithelial cells, due to air pollution and/or inflammation, could contribute to local and/or systemic dysfunctions through the alteration of the functions of pulmonary NAT enzymes.

  4. Scavenger receptor B1 facilitates macrophage uptake of silver nanoparticles and cellular activation

    Energy Technology Data Exchange (ETDEWEB)

    Aldossari, Abdullah A.; Shannahan, Jonathan H. [The University of Colorado Anschutz Medical Campus, Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences (United States); Podila, Ramakrishna [Clemson University, Department of Physics and Astronomy (United States); Brown, Jared M., E-mail: jared.brown@ucdenver.edu [The University of Colorado Anschutz Medical Campus, Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences (United States)

    2015-07-15

    Due to increased use of silver nanoparticles (AgNPs) for their antimicrobial activity, concerns have risen regarding potential adverse human health effects. Scavenger receptor B1 (SR-B1), a major receptor for high-density lipoprotein (HDL), is expressed by macrophages and has also been reported to play a role in recognition of negatively charged particles. We, therefore, hypothesized that SR-B1 mediates macrophage uptake of AgNPs and inflammatory activation. To test this hypothesis, we exposed a mouse macrophage cell line RAW264.7 (RAW) and bone marrow-derived macrophages (BMDM) to 20 nm citrate-suspended AgNPs. To verify the role of the SR-B1 receptor, we utilized a SR-B1 inhibitor (Blt2). In vitro studies demonstrated uptake of AgNPs and HDL-coated AgNPs by macrophages which were significantly reduced following pretreatment with Blt2. Inflammatory cytokine arrays revealed that macrophages exposed to AgNPs up-regulated expression of Tnf-α, Oncostatin m (OSM), Ccl4, Il17f, Ccl7, and Ccl2, whereas Il16 was found to be down-regulated. Macrophage activation was observed following AgNP and HDL-coated AgNP exposure as measured by OSM protein production and increased surface expression of CD86. These markers of activation were reduced with Blt2 pretreatment. The in vitro findings were confirmed in vivo through pulmonary instillation of AgNPs in mice. Pulmonary instillation of AgNPs resulted in a recruitment of inflammatory cells that were reduced in SR-B1-deficient mice or following Blt2 pretreatment. This study suggests that SR-B1 plays a major role in cellular recognition of AgNPs and the induction of cell responses that could contribute to inflammation caused by AgNP exposure.

  5. Scavenger receptor B1 facilitates macrophage uptake of silver nanoparticles and cellular activation

    Science.gov (United States)

    Aldossari, Abdullah A.; Shannahan, Jonathan H.; Podila, Ramakrishna; Brown, Jared M.

    2015-07-01

    Due to increased use of silver nanoparticles (AgNPs) for their antimicrobial activity, concerns have risen regarding potential adverse human health effects. Scavenger receptor B1 (SR-B1), a major receptor for high-density lipoprotein (HDL), is expressed by macrophages and has also been reported to play a role in recognition of negatively charged particles. We, therefore, hypothesized that SR-B1 mediates macrophage uptake of AgNPs and inflammatory activation. To test this hypothesis, we exposed a mouse macrophage cell line RAW264.7 (RAW) and bone marrow-derived macrophages (BMDM) to 20 nm citrate-suspended AgNPs. To verify the role of the SR-B1 receptor, we utilized a SR-B1 inhibitor (Blt2). In vitro studies demonstrated uptake of AgNPs and HDL-coated AgNPs by macrophages which were significantly reduced following pretreatment with Blt2. Inflammatory cytokine arrays revealed that macrophages exposed to AgNPs up-regulated expression of Tnf- α, Oncostatin m (OSM), Ccl4, Il17f, Ccl7, and Ccl2, whereas Il16 was found to be down-regulated. Macrophage activation was observed following AgNP and HDL-coated AgNP exposure as measured by OSM protein production and increased surface expression of CD86. These markers of activation were reduced with Blt2 pretreatment. The in vitro findings were confirmed in vivo through pulmonary instillation of AgNPs in mice. Pulmonary instillation of AgNPs resulted in a recruitment of inflammatory cells that were reduced in SR-B1-deficient mice or following Blt2 pretreatment. This study suggests that SR-B1 plays a major role in cellular recognition of AgNPs and the induction of cell responses that could contribute to inflammation caused by AgNP exposure.

  6. Different cellular effects of four anti-inflammatory eye drops on human corneal epithelial cells: independent in active components.

    Science.gov (United States)

    Qu, Mingli; Wang, Yao; Yang, Lingling; Zhou, Qingjun

    2011-01-01

    To evaluate and compare the cellular effects of four commercially available anti-inflammatory eye drops and their active components on human corneal epithelial cells (HCECs) in vitro. The cellular effects of four eye drops (Bromfenac Sodium Hydrate Eye Drops, Pranoprofen Eye Drops, Diclofenac Sodium Eye Drops, and Tobramycin & Dex Eye Drops) and their corresponding active components were evaluated in an HCEC line with five in vitro assays. Cell proliferation and migration were measured using 3-(4,5)-dimethylthiahiazo (-z-y1)-3 5-di-phenytetrazoliumromide (MTT) assay and transwell migration assay. Cell damage was determined with the lactate dehydrogenase (LDH) assay. Cell viability and median lethal time (LT₅₀) were measured by 7-amino-actinomycin D (7-AAD) staining and flow cytometry analysis. Cellular effects after exposure of HCECs to the four anti-inflammatory eye drops were concentration dependent. The differences of cellular toxicity on cell proliferation became significant at lower concentrations (Eye Drops showed significant increasing effects on cell damage and viability when compared with the other three solutions. Tobramycin & Dex Eye Drops inhibited the migration of HCECs significantly. Tobramycin & Dex Eye Drops showed the quickest effect on cell viability: the LT₅₀ was 3.28, 9.23, 10.38, and 23.80 min for Tobramycin & Dex Eye Drops, Diclofenac Sodium Eye Drops, Pranoprofen Eye Drops, and Bromfenac Sodium Hydrate Eye Drops, respectively. However, the comparisons of cellular toxicity revealed significant differences between the eye drops and their active components under the same concentration. The corneal epithelial toxicity differences among the active components of the four eye drops became significant as higher concentration (>0.020%). The four anti-inflammatory eye drops showed different cellular effects on HCECs, and the toxicity was not related with their active components, which provides new reference for the clinical application and drug

  7. Effect of inhomogeneous activity distributions and airway geometry on cellular doses in radon lung dosimetry

    International Nuclear Information System (INIS)

    Szoke, Istvan; Balashazy, Imre; Farkas, Arpad; Hofmann, Werner

    2007-01-01

    The human tracheobronchial system has a very complex structure including cylindrical airway ducts connected by airway bifurcation units. The deposition of the inhaled aerosols within the airways exhibits a very inhomogeneous pattern. The formation of deposition hot spots near the carinal ridge has been confirmed by experimental and computational fluid and particle dynamics (CFPD) methods. In spite of these observations, current radon lung dosimetry models apply infinitely long cylinders as models of the airway system and assume uniform deposition of the inhaled radon progenies along the airway walls. The aim of this study is to investigate the effect of airway geometry and non-uniform activity distributions within bronchial bifurcations on cellular dose distributions. In order to answer these questions, the nuclear doses of the bronchial epithelium were calculated in three different irradiation situations. (1) First, CFPD methods were applied to calculate the distribution of the deposited alpha-emitting nuclides in a numerically constructed idealized airway bifurcation. (2) Second, the deposited radionuclides were randomly distributed along the surface of the above-mentioned geometry. (3) Finally, calculations were made in cylindrical geometries corresponding to the parent and daughter branches of the bifurcation geometry assuming random nuclide activity distribution. In all three models, the same 218 Po and 214 Po surface activities per tissue volumes were assumed. Two conclusions can be drawn from this analysis: (i) average nuclear doses are very similar in all three cases (minor differences can be attributed to differences in the linear energy transfer (LET) spectra) and (ii) dose distributions are significantly different in all three cases, with the highest doses at the carinal ridge in case 3. (authors)

  8. Sleep deprivation and divergent toll-like receptor-4 activation of cellular inflammation in aging.

    Science.gov (United States)

    Carroll, Judith E; Carrillo, Carmen; Olmstead, Richard; Witarama, Tuff; Breen, Elizabeth C; Yokomizo, Megumi; Seeman, Teresa; Irwin, Michael R

    2015-02-01

    Sleep disturbance and aging are associated with increases in inflammation, as well as increased risk of infectious disease. However, there is limited understanding of the role of sleep loss on age-related differences in immune responses. This study examines the effects of sleep deprivation on toll-like receptor activation of monocytic inflammation in younger compared to older adults. Community-dwelling adults (n = 70) who were categorized as younger (25-39 y old, n = 21) and older (60-84 y old, n = 49) participants, underwent a sleep laboratory-based experimental partial sleep deprivation (PSD) protocol including adaptation, an uninterrupted night of sleep, sleep deprivation (sleep restricted to 03:00-07:00), and recovery. Blood samples were obtained each morning to measure toll-like receptor-4 activation of monocyte intracellular production of the inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Partial sleep deprivation induced a significant increase in the production of IL-6 and/or TNF-α that persisted after a night of recovery sleep (F(2,121.2) = 3.8, P sleep loss, such that younger adults had an increase in inflammatory cytokine production that was not present in older adults (F(2,121.2) = 4.0, P sleep loss. Whereas sleep loss increases cellular inflammation in younger adults and may contribute to inflammatory disorders, blunted toll-like receptor activation in older adults may increase the risk of infectious disease seen with aging. © 2015 Associated Professional Sleep Societies, LLC.

  9. Akita spontaneously type 1 diabetic mice exhibit elevated vascular arginase and impaired vascular endothelial and nitrergic function.

    Science.gov (United States)

    Toque, Haroldo A; Nunes, Kenia P; Yao, Lin; Xu, Zhimin; Kondrikov, Dmitry; Su, Yunchao; Webb, R Clinton; Caldwell, Ruth B; Caldwell, R William

    2013-01-01

    Elevated arginase (Arg) activity is reported to be involved in diabetes-induced vascular endothelial dysfunction. It can reduce L-arginine availability to nitric oxide (NO) synthase (NOS) and NO production. Akita mice, a genetic non-obese type 1 diabetes model, recapitulate human diabetes. We determined the role of Arg in a time-course of diabetes-associated endothelial dysfunction in aorta and corpora cavernosa (CC) from Akita mice. Endothelium-dependent relaxation, Arg and NOS activity, and protein expression levels of Arg and constitutive NOS were assessed in aortas and CC from Akita and non-diabetic wild type (WT) mice at 4, 12 and 24 wks of age. Systolic blood pressure (SBP) was assessed by tail cuff. In aorta and CC, Akita mice exhibited a progressive impairment of vascular endothelial and nitrergic function increased Arg activity and expression (Arg1 in aorta and both Arg1 and Arg2 in CC) compared with that of age-matched WT mice. Treatment of aorta and CC from Akita mice with an Arg inhibitor (BEC or ABH) reduced diabetes-induced elevation of Arg activity and restored endothelial and nitrergic function. Reduced levels of phospho-eNOS at Ser(1177) (in aorta and CC) and nNOS expression (in CC) were observed in Akita mice at 12 and 24 wks. Akita mice also had decreased NOS activity in aorta and CC at 12 and 24 wks that was restored by BEC treatment. Further, Akita mice exhibited moderately increased SBP at 24 wks and increased sensitivity to PE-induced contractions in aorta and sympathetic nerve stimulation in CC at 12 and 24 wks. Over 24 wks of diabetes in Akita mice, both aortic and cavernosal tissues exhibited increased Arg activity/expression, contributing to impaired endothelial and nitrergic function and reduced NO production. Our findings demonstrate involvement of Arg activity in diabetes-induced impairment of vascular function in Akita mouse.

  10. Akita spontaneously type 1 diabetic mice exhibit elevated vascular arginase and impaired vascular endothelial and nitrergic function.

    Directory of Open Access Journals (Sweden)

    Haroldo A Toque

    Full Text Available Elevated arginase (Arg activity is reported to be involved in diabetes-induced vascular endothelial dysfunction. It can reduce L-arginine availability to nitric oxide (NO synthase (NOS and NO production. Akita mice, a genetic non-obese type 1 diabetes model, recapitulate human diabetes. We determined the role of Arg in a time-course of diabetes-associated endothelial dysfunction in aorta and corpora cavernosa (CC from Akita mice.Endothelium-dependent relaxation, Arg and NOS activity, and protein expression levels of Arg and constitutive NOS were assessed in aortas and CC from Akita and non-diabetic wild type (WT mice at 4, 12 and 24 wks of age. Systolic blood pressure (SBP was assessed by tail cuff. In aorta and CC, Akita mice exhibited a progressive impairment of vascular endothelial and nitrergic function increased Arg activity and expression (Arg1 in aorta and both Arg1 and Arg2 in CC compared with that of age-matched WT mice. Treatment of aorta and CC from Akita mice with an Arg inhibitor (BEC or ABH reduced diabetes-induced elevation of Arg activity and restored endothelial and nitrergic function. Reduced levels of phospho-eNOS at Ser(1177 (in aorta and CC and nNOS expression (in CC were observed in Akita mice at 12 and 24 wks. Akita mice also had decreased NOS activity in aorta and CC at 12 and 24 wks that was restored by BEC treatment. Further, Akita mice exhibited moderately increased SBP at 24 wks and increased sensitivity to PE-induced contractions in aorta and sympathetic nerve stimulation in CC at 12 and 24 wks.Over 24 wks of diabetes in Akita mice, both aortic and cavernosal tissues exhibited increased Arg activity/expression, contributing to impaired endothelial and nitrergic function and reduced NO production. Our findings demonstrate involvement of Arg activity in diabetes-induced impairment of vascular function in Akita mouse.

  11. Abnormalities in the cellular phase of blood fibrinolytic activity in systemic lupus erythematosus and in venous thromboembolism

    International Nuclear Information System (INIS)

    Moroz, L.A.; MacLean, L.D.; Langleben, D.

    1986-01-01

    Fibrinolytic activities of whole blood and plasma were determined by 125 I-fibrin radiometric assay in 16 normal subjects, and in 11 patients with systemic lupus erythematosus (SLE), 14 with progressive systemic sclerosis (PSS), 23 with venous thromboembolic disease, and 20 patients awaiting elective surgery. Mean whole blood and plasma activities for patients with PSS, and for those awaiting elective surgery, were similar to normal values, as was the mean plasma activity in patients with SLE. However, mean whole blood activity in SLE was significantly decreased compared with normals (p less than 0.05), with mean plasma activity accounting for 44% of mean whole blood activity (compared with 17% in normal subjects), representing a 67% decrease in mean calculated cellular phase activity in SLE, when compared with normals. Since the numbers of cells (neutrophils, monocytes) possibly involved in cellular activity were not decreased, the findings suggest a functional defect in fibrinolytic activity of one or more blood cell types in SLE. An additional finding was the participation of the cellular phase as well as the well-known plasma phase of blood in the fibrinolytic response to thromboembolism

  12. Intestinal Permeability and Cellular Antioxidant Activity of Phenolic Compounds from Mango (Mangifera indica cv. Ataulfo Peels

    Directory of Open Access Journals (Sweden)

    Ramón Pacheco-Ordaz

    2018-02-01

    Full Text Available Mango (Mangifera indica cv. Ataulfo peel contains bound phenolics that may be released by alkaline or acid hydrolysis and may be converted into less complex molecules. Free phenolics from mango cv. Ataulfo peel were obtained using a methanolic extraction, and their cellular antioxidant activity (CAA and permeability were compared to those obtained for bound phenolics released by alkaline or acid hydrolysis. Gallic acid was found as a simple phenolic acid after alkaline hydrolysis along with mangiferin isomers and quercetin as aglycone and glycosides. Only gallic acid, ethyl gallate, mangiferin, and quercetin were identified in the acid fraction. The acid and alkaline fractions showed the highest CAA (60.5% and 51.5% when tested at 125 µg/mL. The value of the apparent permeability coefficient (Papp across the Caco-2/HT-29 monolayer of gallic acid from the alkaline fraction was higher (2.61 × 10−6 cm/s than in the other fractions and similar to that obtained when tested pure (2.48 × 10−6 cm/s. In conclusion, mango peels contain bound phenolic compounds that, after their release, have permeability similar to pure compounds and exert an important CAA. This finding can be applied in the development of nutraceuticals using this important by-product from the mango processing industry.

  13. Continuous cellularization of calcium phosphate hybrid scaffolds induced by plasma polymer activation

    International Nuclear Information System (INIS)

    Bergemann, Claudia; Cornelsen, Matthias; Quade, Antje; Laube, Thorsten; Schnabelrauch, Matthias; Rebl, Henrike; Weißmann, Volker; Seitz, Hermann; Nebe, Barbara

    2016-01-01

    The generation of hybrid materials based on β-tricalcium phosphate (TCP) and various biodegradable polymers like poly(L-lactide-co-D,L-lactide) (PLA) represents a common approach to overcoming the disadvantages of pure TCP devices. These disadvantages lie in TCP's mechanical properties, such as brittleness. The positive characteristic of PLA — improvement of compressive strength of calcium phosphate scaffolds – is diametrically opposed to its cell attractiveness. Therefore, the objective of this work was to optimize osteoblast migration and cellularization inside a three-dimensionally (3D) printed, PLA polymer stabilized TCP hybrid scaffold by a plasma polymer process depositing amino groups via allylamine. MG-63 osteoblastic cells inside the 10 mm hybrid scaffold were dynamically cultivated for 14 days in a 3D model system integrated in a perfusion reactor. The whole TCP/PLA hybrid scaffold was continuously colonized due to plasma polymerized allylamine activation inducing the migration potential of osteoblasts. - Highlights: • Mechanical stabilization of β-tricalcium phosphate scaffolds by PLA infiltration • Hybrid scaffolds with higher cell attraction due to plasma polymerized allylamine • 3D perfusion in vitro model for observation of cell migration inside scaffolds • Enhanced cell migration within plasma polymer coated TCP hybrid scaffolds

  14. Ceruloplasmin Oxidation, a Feature of Parkinson's Disease CSF, Inhibits Ferroxidase Activity and Promotes Cellular Iron Retention

    KAUST Repository

    Olivieri, S.

    2011-12-14

    Parkinson\\'s disease is a neurodegenerative disorder characterized by oxidative stress and CNS iron deposition. Ceruloplasmin is an extracellular ferroxidase that regulates cellular iron loading and export, and hence protects tissues from oxidative damage. Using two-dimensional electrophoresis, we investigated ceruloplasmin patterns in the CSF of human Parkinson\\'s disease patients. Parkinson\\'s disease ceruloplasmin profiles proved more acidic than those found in healthy controls and in other human neurological diseases (peripheral neuropathies, amyotrophic lateral sclerosis, and Alzheimer\\'s disease); degrees of acidity correlated with patients\\' pathological grading. Applying an unsupervised pattern recognition procedure to the two-dimensional electrophoresis images, we identified representative pathological clusters. In vitro oxidation of CSF in two-dimensional electrophoresis generated a ceruloplasmin shift resembling that observed in Parkinson\\'s disease and co-occurred with an increase in protein carbonylation. Likewise, increased protein carbonylation was observed in Parkinson\\'s disease CSF, and the same modification was directly identified in these samples on ceruloplasmin. These results indicate that ceruloplasmin oxidation contributes to pattern modification in Parkinson\\'s disease. From the functional point of view, ceruloplasmin oxidation caused a decrease in ferroxidase activity, which in turn promotes intracellular iron retention in neuronal cell lines as well as in primary neurons, which are more sensitive to iron accumulation. Accordingly, the presence of oxidized ceruloplasmin in Parkinson\\'s disease CSF might be used as a marker for oxidative damage and might provide new insights into the underlying pathological mechanisms.

  15. TWO-DIMENSIONAL CELLULAR AUTOMATON MODEL FOR THE EVOLUTION OF ACTIVE REGION CORONAL PLASMAS

    Energy Technology Data Exchange (ETDEWEB)

    López Fuentes, Marcelo [Instituto de Astronomía y Física del Espacio, CONICET-UBA, CC. 67, Suc. 28, 1428 Buenos Aires (Argentina); Klimchuk, James A., E-mail: lopezf@iafe.uba.ar [NASA Goddard Space Flight Center, Code 671, Greenbelt, MD 20771 (United States)

    2015-02-01

    We study a two-dimensional cellular automaton (CA) model for the evolution of coronal loop plasmas. The model is based on the idea that coronal loops are made of elementary magnetic strands that are tangled and stressed by the displacement of their footpoints by photospheric motions. The magnetic stress accumulated between neighbor strands is released in sudden reconnection events or nanoflares that heat the plasma. We combine the CA model with the Enthalpy Based Thermal Evolution of Loops model to compute the response of the plasma to the heating events. Using the known response of the X-Ray Telescope on board Hinode, we also obtain synthetic data. The model obeys easy-to-understand scaling laws relating the output (nanoflare energy, temperature, density, intensity) to the input parameters (field strength, strand length, critical misalignment angle). The nanoflares have a power-law distribution with a universal slope of –2.5, independent of the input parameters. The repetition frequency of nanoflares, expressed in terms of the plasma cooling time, increases with strand length. We discuss the implications of our results for the problem of heating and evolution of active region coronal plasmas.

  16. Continuous cellularization of calcium phosphate hybrid scaffolds induced by plasma polymer activation

    Energy Technology Data Exchange (ETDEWEB)

    Bergemann, Claudia [University Medical Center Rostock, Cell Biology, Schillingallee 69, D-18057 Rostock (Germany); Cornelsen, Matthias [University of Rostock, Fluid Technology and Microfluidics, Justus-von-Liebig Weg 6, D-18059 Rostock (Germany); Quade, Antje [Leibniz-Institute for Plasma Science and Technology (INP), Felix-Hausdorff-Str. 2, D-17489 Greifswald (Germany); Laube, Thorsten; Schnabelrauch, Matthias [INNOVENT e.V., Biomaterials Department, Pruessingstrasse 27B, D-07745 Jena (Germany); Rebl, Henrike [University Medical Center Rostock, Cell Biology, Schillingallee 69, D-18057 Rostock (Germany); Weißmann, Volker [Institute for Polymer Technologies (IPT) e.V., Alter Holzhafen 19, D-23966 Wismar (Germany); Seitz, Hermann [University of Rostock, Fluid Technology and Microfluidics, Justus-von-Liebig Weg 6, D-18059 Rostock (Germany); Nebe, Barbara, E-mail: barbara.nebe@med.uni-rostock.de [University Medical Center Rostock, Cell Biology, Schillingallee 69, D-18057 Rostock (Germany)

    2016-02-01

    The generation of hybrid materials based on β-tricalcium phosphate (TCP) and various biodegradable polymers like poly(L-lactide-co-D,L-lactide) (PLA) represents a common approach to overcoming the disadvantages of pure TCP devices. These disadvantages lie in TCP's mechanical properties, such as brittleness. The positive characteristic of PLA — improvement of compressive strength of calcium phosphate scaffolds – is diametrically opposed to its cell attractiveness. Therefore, the objective of this work was to optimize osteoblast migration and cellularization inside a three-dimensionally (3D) printed, PLA polymer stabilized TCP hybrid scaffold by a plasma polymer process depositing amino groups via allylamine. MG-63 osteoblastic cells inside the 10 mm hybrid scaffold were dynamically cultivated for 14 days in a 3D model system integrated in a perfusion reactor. The whole TCP/PLA hybrid scaffold was continuously colonized due to plasma polymerized allylamine activation inducing the migration potential of osteoblasts. - Highlights: • Mechanical stabilization of β-tricalcium phosphate scaffolds by PLA infiltration • Hybrid scaffolds with higher cell attraction due to plasma polymerized allylamine • 3D perfusion in vitro model for observation of cell migration inside scaffolds • Enhanced cell migration within plasma polymer coated TCP hybrid scaffolds.

  17. Intestinal Permeability and Cellular Antioxidant Activity of Phenolic Compounds from Mango (Mangifera indica cv. Ataulfo) Peels

    Science.gov (United States)

    Pacheco-Ordaz, Ramón; González-Aguilar, Gustavo A.

    2018-01-01

    Mango (Mangifera indica cv. Ataulfo) peel contains bound phenolics that may be released by alkaline or acid hydrolysis and may be converted into less complex molecules. Free phenolics from mango cv. Ataulfo peel were obtained using a methanolic extraction, and their cellular antioxidant activity (CAA) and permeability were compared to those obtained for bound phenolics released by alkaline or acid hydrolysis. Gallic acid was found as a simple phenolic acid after alkaline hydrolysis along with mangiferin isomers and quercetin as aglycone and glycosides. Only gallic acid, ethyl gallate, mangiferin, and quercetin were identified in the acid fraction. The acid and alkaline fractions showed the highest CAA (60.5% and 51.5%) when tested at 125 µg/mL. The value of the apparent permeability coefficient (Papp) across the Caco-2/HT-29 monolayer of gallic acid from the alkaline fraction was higher (2.61 × 10−6 cm/s) than in the other fractions and similar to that obtained when tested pure (2.48 × 10−6 cm/s). In conclusion, mango peels contain bound phenolic compounds that, after their release, have permeability similar to pure compounds and exert an important CAA. This finding can be applied in the development of nutraceuticals using this important by-product from the mango processing industry. PMID:29419800

  18. Intestinal Permeability and Cellular Antioxidant Activity of Phenolic Compounds from Mango (Mangifera indica cv. Ataulfo) Peels.

    Science.gov (United States)

    Pacheco-Ordaz, Ramón; Antunes-Ricardo, Marilena; Gutiérrez-Uribe, Janet A; González-Aguilar, Gustavo A

    2018-02-08

    Mango ( Mangifera indica cv. Ataulfo) peel contains bound phenolics that may be released by alkaline or acid hydrolysis and may be converted into less complex molecules. Free phenolics from mango cv. Ataulfo peel were obtained using a methanolic extraction, and their cellular antioxidant activity (CAA) and permeability were compared to those obtained for bound phenolics released by alkaline or acid hydrolysis. Gallic acid was found as a simple phenolic acid after alkaline hydrolysis along with mangiferin isomers and quercetin as aglycone and glycosides. Only gallic acid, ethyl gallate, mangiferin, and quercetin were identified in the acid fraction. The acid and alkaline fractions showed the highest CAA (60.5% and 51.5%) when tested at 125 µg/mL. The value of the apparent permeability coefficient (Papp) across the Caco-2/HT-29 monolayer of gallic acid from the alkaline fraction was higher (2.61 × 10 -6 cm/s) than in the other fractions and similar to that obtained when tested pure (2.48 × 10 -6 cm/s). In conclusion, mango peels contain bound phenolic compounds that, after their release, have permeability similar to pure compounds and exert an important CAA. This finding can be applied in the development of nutraceuticals using this important by-product from the mango processing industry.

  19. Evidence of parasexual activity in "asexual amoebae" Cochliopodium spp. (Amoebozoa): extensive cellular and nuclear fusion.

    Science.gov (United States)

    Tekle, Yonas I; Anderson, O Roger; Lecky, Ariel F

    2014-09-01

    The majority of microbial eukaryotes have long been considered asexual, though new evidence indicates sex, or sexual-like (parasexual) behaviors that deviate from the usual union of two gametes, among other variant aspects. Over a dozen amoebozoans are implicated to have sexual stages. However, the exact mechanism by which sex occurs in these lineages remains elusive. This is mainly due to the diverse quality and cryptic nature of their life cycle. In this study we present evidence of some previously unreported aspects of the life cycle of an amoeba, Cochliopodium, that undergoes unusual intraspecific interactions using light microscopy and immunocytochemistry. Similar to other amoebozoans, Cochliopodium, is considered asexual with no published reports of sex or parasexuality. We also investigated environmental conditions that govern the observed intraspecific interactions. Both light microscopic and immunocytochemistry evidence demonstrates Cochliopodium undergoes cellular fusion (plasmogamy) and nuclear fusion (karyogamy). Large plasmodia eventually undergo karyogamy and contain large fused, polyploid, nuclei. These are observed to fragment, subsequently, by karyotomy (nuclear fission) and cytoplasmic fission to yield uninucleated amoebae. This process could lead to a non-meiotic, parasexual exchange of chromosomes in Cochliopodium. These findings strongly suggest that Cochliopodium is involved in parasexual activity and should no longer be considered strictly asexual. Copyright © 2014 Elsevier GmbH. All rights reserved.

  20. Cellular variability of RpoS expression underlies subpopulation activation of an integrative and conjugative element.

    Directory of Open Access Journals (Sweden)

    Ryo Miyazaki

    Full Text Available Conjugative transfer of the integrative and conjugative element ICEclc in the bacterium Pseudomonas knackmussii is the consequence of a bistable decision taken in some 3% of cells in a population during stationary phase. Here we study the possible control exerted by the stationary phase sigma factor RpoS on the bistability decision. The gene for RpoS in P. knackmussii B13 was characterized, and a loss-of-function mutant was produced and complemented. We found that, in absence of RpoS, ICEclc transfer rates and activation of two key ICEclc promoters (P(int and P(inR decrease significantly in cells during stationary phase. Microarray and gene reporter analysis indicated that the most direct effect of RpoS is on P(inR, whereas one of the gene products from the P(inR-controlled operon (InrR transmits activation to P(int and other ICEclc core genes. Addition of a second rpoS copy under control of its native promoter resulted in an increase of the proportion of cells expressing the P(int and P(inR promoters to 18%. Strains in which rpoS was replaced by an rpoS-mcherry fusion showed high mCherry fluorescence of individual cells that had activated P(int and P(inR, whereas a double-copy rpoS-mcherry-containing strain displayed twice as much mCherry fluorescence. This suggested that high RpoS levels are a prerequisite for an individual cell to activate P(inR and thus ICEclc transfer. Double promoter-reporter fusions confirmed that expression of P(inR is dominated by extrinsic noise, such as being the result of cellular variability in RpoS. In contrast, expression from P(int is dominated by intrinsic noise, indicating it is specific to the ICEclc transmission cascade. Our results demonstrate how stochastic noise levels of global transcription factors can be transduced to a precise signaling cascade in a subpopulation of cells leading to ICE activation.

  1. Activation of human natural killer cells by the soluble form of cellular prion protein

    Energy Technology Data Exchange (ETDEWEB)

    Seong, Yeon-Jae [Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of); Hafis Clinic, Seoul (Korea, Republic of); Sung, Pil Soo; Jang, Young-Soon; Choi, Young Joon [Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of); Park, Bum-Chan [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Park, Su-Hyung [Laboratory of Translational Immunology and Vaccinology, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of); Park, Young Woo [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Shin, Eui-Cheol, E-mail: ecshin@kaist.ac.kr [Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of)

    2015-08-21

    Cellular prion protein (PrP{sup C}) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP{sup C} in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP{sup C} protein on human natural killer (NK) cells. Recombinant soluble PrP{sup C} protein was generated by fusion of human PrP{sup C} with the Fc portion of human IgG{sub 1} (PrP{sup C}-Fc). PrP{sup C}-Fc binds to the surface of human NK cells, particularly to CD56{sup dim} NK cells. PrP{sup C}-Fc induced the production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP{sup C}-Fc facilitated the IL-15-induced proliferation of NK cells. PrP{sup C}-Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP{sup C}-Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways. - Highlights: • Recombinant soluble PrP{sup C} (PrP{sup C}-Fc) was generated by fusion of human PrP{sup C} with IgG1 Fc portion. • PrP{sup C}-Fc protein induces the production of cytokines and degranulation from human NK cells. • PrP{sup C}-Fc protein enhances the IL-15-induced proliferation of human NK cells. • PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways.

  2. Activation of human natural killer cells by the soluble form of cellular prion protein

    International Nuclear Information System (INIS)

    Seong, Yeon-Jae; Sung, Pil Soo; Jang, Young-Soon; Choi, Young Joon; Park, Bum-Chan; Park, Su-Hyung; Park, Young Woo; Shin, Eui-Cheol

    2015-01-01

    Cellular prion protein (PrP C ) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP C in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP C protein on human natural killer (NK) cells. Recombinant soluble PrP C protein was generated by fusion of human PrP C with the Fc portion of human IgG 1 (PrP C -Fc). PrP C -Fc binds to the surface of human NK cells, particularly to CD56 dim NK cells. PrP C -Fc induced the production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP C -Fc facilitated the IL-15-induced proliferation of NK cells. PrP C -Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP C -Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP C -Fc protein activates human NK cells via the ERK and JNK signaling pathways. - Highlights: • Recombinant soluble PrP C (PrP C -Fc) was generated by fusion of human PrP C with IgG1 Fc portion. • PrP C -Fc protein induces the production of cytokines and degranulation from human NK cells. • PrP C -Fc protein enhances the IL-15-induced proliferation of human NK cells. • PrP C -Fc protein activates human NK cells via the ERK and JNK signaling pathways

  3. Ceruloplasmin Oxidation, a Feature of Parkinson's Disease CSF, Inhibits Ferroxidase Activity and Promotes Cellular Iron Retention

    KAUST Repository

    Olivieri, S.; Conti, A.; Iannaccone, S.; Cannistraci, C. V.; Campanella, A.; Barbariga, M.; Codazzi, F.; Pelizzoni, I.; Magnani, G.; Pesca, M.; Franciotta, D.; Cappa, S. F.; Alessio, M.

    2011-01-01

    Parkinson's disease is a neurodegenerative disorder characterized by oxidative stress and CNS iron deposition. Ceruloplasmin is an extracellular ferroxidase that regulates cellular iron loading and export, and hence protects tissues from oxidative

  4. Spatio-temporal analysis of brain electrical activity in epilepsy based on cellular nonlinear networks

    Science.gov (United States)

    Gollas, Frank; Tetzlaff, Ronald

    2009-05-01

    Epilepsy is the most common chronic disorder of the nervous system. Generally, epileptic seizures appear without foregoing sign or warning. The problem of detecting a possible pre-seizure state in epilepsy from EEG signals has been addressed by many authors over the past decades. Different approaches of time series analysis of brain electrical activity already are providing valuable insights into the underlying complex dynamics. But the main goal the identification of an impending epileptic seizure with a sufficient specificity and reliability, has not been achieved up to now. An algorithm for a reliable, automated prediction of epileptic seizures would enable the realization of implantable seizure warning devices, which could provide valuable information to the patient and time/event specific drug delivery or possibly a direct electrical nerve stimulation. Cellular Nonlinear Networks (CNN) are promising candidates for future seizure warning devices. CNN are characterized by local couplings of comparatively simple dynamical systems. With this property these networks are well suited to be realized as highly parallel, analog computer chips. Today available CNN hardware realizations exhibit a processing speed in the range of TeraOps combined with low power consumption. In this contribution new algorithms based on the spatio-temporal dynamics of CNN are considered in order to analyze intracranial EEG signals and thus taking into account mutual dependencies between neighboring regions of the brain. In an identification procedure Reaction-Diffusion CNN (RD-CNN) are determined for short segments of brain electrical activity, by means of a supervised parameter optimization. RD-CNN are deduced from Reaction-Diffusion Systems, which usually are applied to investigate complex phenomena like nonlinear wave propagation or pattern formation. The Local Activity Theory provides a necessary condition for emergent behavior in RD-CNN. In comparison linear spatio

  5. Comparison of home and away-from-home physical activity using accelerometers and cellular network-based tracking devices.

    Science.gov (United States)

    Ramulu, Pradeep Y; Chan, Emilie S; Loyd, Tara L; Ferrucci, Luigi; Friedman, David S

    2012-08-01

    Measuring physical at home and away from home is essential for assessing health and well-being, and could help design interventions to increase physical activity. Here, we describe how physical activity at home and away from home can be quantified by combining information from cellular network-based tracking devices and accelerometers. Thirty-five working adults wore a cellular network-based tracking device and an accelerometer for 6 consecutive days and logged their travel away from home. Performance of the tracking device was determined using the travel log for reference. Tracking device and accelerometer data were merged to compare physical activity at home and away from home. The tracking device detected 98.6% of all away-from-home excursions, accurately measured time away from home and demonstrated few prolonged signal drop-out periods. Most physical activity took place away from home on weekdays, but not on weekends. Subjects were more physically active per unit of time while away from home, particularly on weekends. Cellular network-based tracking devices represent an alternative to global positioning systems for tracking location, and provide information easily integrated with accelerometers to determine where physical activity takes place. Promoting greater time spent away from home may increase physical activity.

  6. Hyaluronan Hybrid Cooperative Complexes as a Novel Frontier for Cellular Bioprocesses Re-Activation.

    Directory of Open Access Journals (Sweden)

    Antonietta Stellavato

    Full Text Available Hyaluronic Acid (HA-based dermal formulations have rapidly gained a large consensus in aesthetic medicine and dermatology. HA, highly expressed in the Extracellular Matrix (ECM, acts as an activator of biological cascades, stimulating cell migration and proliferation, and operating as a regulator of the skin immune surveillance, through specific interactions with its receptors. HA may be used in topical formulations, as dermal inducer, for wound healing. Moreover, intradermal HA formulations (injectable HA provide an attractive tool to counteract skin aging (e.g., facial wrinkles, dryness, and loss of elasticity and restore normal dermal functions, through simple and minimally invasive procedures. Biological activity of a commercially available hyaluronic acid, Profhilo®, based on NAHYCO™ technology, was compared to H-HA or L-HA alone. The formation of hybrid cooperative complexes was confirmed by the sudden drop in η0 values in the rheological measurements. Besides, hybrid cooperative complexes proved stable to hyaluronidase (BTH digestion. Using in vitro assays, based on keratinocytes, fibroblasts cells and on the Phenion® Full Thickness Skin Model 3D, hybrid cooperative complexes were compared to H-HA, widely used in biorevitalization procedures, and to L-HA, recently proposed as the most active fraction modulating the inflammatory response. Quantitative real-time PCR analyses were accomplished for the transcript quantification of collagens and elastin. Finally immunofluorescence staining permitted to evaluate the complete biosynthesis of all the molecules investigated. An increase in the expression levels of type I and type III collagen in fibroblasts and type IV and VII collagen in keratinocytes were found with the hybrid cooperative complexes, compared to untreated cells (CTR and to the H-HA and L-HA treatments. The increase in elastin expression found in both cellular model and in the Phenion® Full Thickness Skin Model 3D also at

  7. Resistance to degradation and cellular distribution are important features for the antitumor activity of gomesin.

    Directory of Open Access Journals (Sweden)

    Marcus V Buri

    Full Text Available Many reports have shown that antimicrobial peptides exhibit anticancer abilities. Gomesin (Gm exhibits potent cytotoxic activity against cancer cells by a membrane pore formation induced after well-orchestrated intracellular mechanisms. In this report, the replacements of the Cys by Ser or Thr, and the use D-amino acids in the Gm structure were done to investigate the importance of the resistance to degradation of the molecule with its cytotoxicity. [Thr(2,6,11,15]-Gm, and [Ser(2,6,11,15]-Gm exhibits low cytotoxicity, and low resistance to degradation, and after 24 h are present in localized area near to the membrane. Conversely, the use of D-amino acids in the analogue [D-Thr(2,6,11,15]-D-Gm confers resistance to degradation, increases its potency, and maintained this peptide spread in the cytosol similarly to what happens with Gm. Replacements of Cys by Thr and Gln by L- or D-Pro ([D-Thr(2,6,11,15, Pro(9]-D-Gm, and [Thr(2,6,11,15, D-Pro(9]-Gm, which induced a similar β-hairpin conformation, also increase their resistance to degradation, and cytotoxicity, but after 24 h they are not present spread in the cytosol, exhibiting lower cytotoxicity in comparison to Gm. Additionally, chloroquine, a lysosomal enzyme inhibitor potentiated the effect of the peptides. Furthermore, the binding and internalization of peptides was determined, but a direct correlation among these factors was not observed. However, cholesterol ablation, which increase fluidity of cellular membrane, also increase cytotoxicity and internalization of peptides. β-hairpin spatial conformation, and intracellular localization/target, and the capability of entry are important properties of gomesin cytotoxicity.

  8. Resistance to degradation and cellular distribution are important features for the antitumor activity of gomesin.

    Science.gov (United States)

    Buri, Marcus V; Domingues, Tatiana M; Paredes-Gamero, Edgar J; Casaes-Rodrigues, Rafael L; Rodrigues, Elaine Guadelupe; Miranda, Antonio

    2013-01-01

    Many reports have shown that antimicrobial peptides exhibit anticancer abilities. Gomesin (Gm) exhibits potent cytotoxic activity against cancer cells by a membrane pore formation induced after well-orchestrated intracellular mechanisms. In this report, the replacements of the Cys by Ser or Thr, and the use D-amino acids in the Gm structure were done to investigate the importance of the resistance to degradation of the molecule with its cytotoxicity. [Thr(2,6,11,15)]-Gm, and [Ser(2,6,11,15)]-Gm exhibits low cytotoxicity, and low resistance to degradation, and after 24 h are present in localized area near to the membrane. Conversely, the use of D-amino acids in the analogue [D-Thr(2,6,11,15)]-D-Gm confers resistance to degradation, increases its potency, and maintained this peptide spread in the cytosol similarly to what happens with Gm. Replacements of Cys by Thr and Gln by L- or D-Pro ([D-Thr(2,6,11,15), Pro(9)]-D-Gm, and [Thr(2,6,11,15), D-Pro(9)]-Gm), which induced a similar β-hairpin conformation, also increase their resistance to degradation, and cytotoxicity, but after 24 h they are not present spread in the cytosol, exhibiting lower cytotoxicity in comparison to Gm. Additionally, chloroquine, a lysosomal enzyme inhibitor potentiated the effect of the peptides. Furthermore, the binding and internalization of peptides was determined, but a direct correlation among these factors was not observed. However, cholesterol ablation, which increase fluidity of cellular membrane, also increase cytotoxicity and internalization of peptides. β-hairpin spatial conformation, and intracellular localization/target, and the capability of entry are important properties of gomesin cytotoxicity.

  9. Resistance to Degradation and Cellular Distribution are Important Features for the Antitumor Activity of Gomesin

    Science.gov (United States)

    Buri, Marcus V.; Domingues, Tatiana M.; Paredes-Gamero, Edgar J.; Casaes-Rodrigues, Rafael L.; Rodrigues, Elaine Guadelupe; Miranda, Antonio

    2013-01-01

    Many reports have shown that antimicrobial peptides exhibit anticancer abilities. Gomesin (Gm) exhibits potent cytotoxic activity against cancer cells by a membrane pore formation induced after well-orchestrated intracellular mechanisms. In this report, the replacements of the Cys by Ser or Thr, and the use D-amino acids in the Gm structure were done to investigate the importance of the resistance to degradation of the molecule with its cytotoxicity. [Thr2,6,11,15]-Gm, and [Ser2,6,11,15]-Gm exhibits low cytotoxicity, and low resistance to degradation, and after 24 h are present in localized area near to the membrane. Conversely, the use of D-amino acids in the analogue [D-Thr2,6,11,15]-D-Gm confers resistance to degradation, increases its potency, and maintained this peptide spread in the cytosol similarly to what happens with Gm. Replacements of Cys by Thr and Gln by L- or D-Pro ([D-Thr2,6,11,15, Pro9]-D-Gm, and [Thr2,6,11,15, D-Pro9]-Gm), which induced a similar β-hairpin conformation, also increase their resistance to degradation, and cytotoxicity, but after 24 h they are not present spread in the cytosol, exhibiting lower cytotoxicity in comparison to Gm. Additionally, chloroquine, a lysosomal enzyme inhibitor potentiated the effect of the peptides. Furthermore, the binding and internalization of peptides was determined, but a direct correlation among these factors was not observed. However, cholesterol ablation, which increase fluidity of cellular membrane, also increase cytotoxicity and internalization of peptides. β-hairpin spatial conformation, and intracellular localization/target, and the capability of entry are important properties of gomesin cytotoxicity. PMID:24312251

  10. Lunar Dust and Lunar Simulant Activation, Monitoring, Solution and Cellular Toxicity Properties

    Science.gov (United States)

    Wallace, William; Jeevarajan, A. S.

    2009-01-01

    During the Apollo missions, many undesirable situations were encountered that must be mitigated prior to returning humans to the moon. Lunar dust (that part of the lunar regolith less than 20 microns in diameter) was found to produce several problems with mechanical equipment and could have conceivably produced harmful physiological effects for the astronauts. For instance, the abrasive nature of the dust was found to cause malfunctions of various joints and seals of the spacecraft and suits. Additionally, though efforts were made to exclude lunar dust from the cabin of the lunar module, a significant amount of material nonetheless found its way inside. With the loss of gravity correlated with ascent from the lunar surface, much of the finer fraction of this dust began to float and was inhaled by the astronauts. The short visits tothe Moon during Apollo lessened exposure to the dust, but the plan for future lunar stays of up to six months demands that methods be developed to minimize the risk of dust inhalation. The guidelines for what constitutes "safe" exposure will guide the development of engineering controls aimed at preventing the presence of dust in the lunar habitat. This work has shown the effects of grinding on the activation level of lunar dust, the changes in dissolution properties of lunar simulant, and the production of cytokines by cellular systems. Grinding of lunar dust leads to the production of radicals in solution and increased dissolution of lunar simulant in buffers of different pH. Additionally, ground lunar simulant has been shown to promote the production of IL-6 and IL-8, pro-inflammatory cytokines, by alveolar epithelial cells. These results provide evidence of the need for further studies on these materials prior to returning to the lunar surface.

  11. Nicotinamide phosphoribosyltransferase delays cellular senescence by upregulating SIRT1 activity and antioxidant gene expression in mouse cells.

    Science.gov (United States)

    Khaidizar, Fiqri D; Nakahata, Yasukazu; Kume, Akira; Sumizawa, Kyosuke; Kohno, Kenji; Matsui, Takaaki; Bessho, Yasumasa

    2017-12-01

    Senescent cells accumulate in tissues of aged animals and deteriorate tissue functions. The elimination of senescent cells from aged mice not only attenuates progression of already established age-related disorders, but also extends median lifespan. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in mammalian NAD + salvage pathway, has shown a protective effect on cellular senescence of human primary cells. However, it still remains unclear how NAMPT has a protective impact on aging in vitro and in vivo. In this study, we found that primary mouse embryonic fibroblast (MEF) cells undergo progressive decline of NAMPT and NAD + contents during serial passaging before becoming senescent. Furthermore, we showed that constitutive Nampt over-expression increases cellular NAD + content and delays cellular senescence of MEF cells in vitro. We further found that constitutive Nampt over-expression increases SIRT1 activity, increases the expression of antioxidant genes, superoxide dismutase 2 and catalase and promotes resistance against oxidative stress. These findings suggest that Nampt over-expression in MEF cells delays cellular senescence by the mitigation of oxidative stress via the upregulation of superoxide dismutase 2 and catalase gene expressions by SIRT1 activation. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  12. Research of the method of pseudo-random number generation based on asynchronous cellular automata with several active cells

    Directory of Open Access Journals (Sweden)

    Bilan Stepan

    2017-01-01

    Full Text Available To date, there are many tasks that are aimed at studying the dynamic changes in physical processes. These tasks do not give advance known result. The solution of such problems is based on the construction of a dynamic model of the object. Successful structural and functional implementation of the object model can give a positive result in time. This approach uses the task of constructing artificial biological objects. To solve such problems, pseudo-random number generators are used, which also find wide application for information protection tasks. Such generators should have good statistical properties and give a long repetition period of the generated pseudo-random bit sequence. This work is aimed at improving these characteristics. The paper considers the method of forming pseudo-random sequences of numbers on the basis of aperiodic cellular automata with two active cells. A pseudo-random number generator is proposed that generates three bit sequences. The first two bit sequences are formed by the corresponding two active cells in the cellular automaton. The third bit sequence is the result of executing the XOR function over the bits of the first two sequences and it has better characteristics compared to them. The use of cellular automata with two active cells allowed to improve the statistical properties of the formed bit sequence, as well as its repetition period. This is proved by using graphical tests for generators built based on cellular automata using the neighborhoods of von Neumann and Moore. The tests showed high efficiency of the generator based on an asynchronous cellular automaton with the neighborhood of Moore. The proposed pseudo-random number generators have good statistical properties, which makes it possible to use them in information security systems, as well as for simulation tasks of various dynamic processes.

  13. Overexpression of (His)6-tagged human arginase I in Saccharomyces cerevisiae and enzyme purification using metal affinity chromatography

    Czech Academy of Sciences Publication Activity Database

    Zakalskiy, A. E.; Zakalska, O. M.; Rzhepetskyy, Y. A.; Potocka, N.; Stasyk, O. V.; Horák, Daniel; Gonchar, M. V.

    2012-01-01

    Roč. 81, č. 1 (2012), s. 63-68 ISSN 1046-5928 R&D Projects: GA ČR GA203/09/1242 Institutional research plan: CEZ:AV0Z40500505 Keywords : human arginase I * (His)6-tag * Saccharomyces cerevisiae Subject RIV: CD - Macromolecular Chemistry Impact factor: 1.429, year: 2012

  14. Early obesity leads to increases in hepatic arginase I and related systemic changes in nitric oxide and L-arginine metabolism in mice.

    Science.gov (United States)

    Ito, Tatsuo; Kubo, Masayuki; Nagaoka, Kenjiro; Funakubo, Narumi; Setiawan, Heri; Takemoto, Kei; Eguchi, Eri; Fujikura, Yoshihisa; Ogino, Keiki

    2018-02-01

    Obesity is a risk factor for vascular endothelial cell dysfunction characterized by low-grade, chronic inflammation. Increased levels of arginase I and concomitant decreases in L-arginine bioavailability are known to play a role in the pathogenesis of vascular endothelial cell dysfunction. In the present study, we focused on changes in the systemic expression of arginase I as well as L-arginine metabolism in the pre-disease state of early obesity prior to the onset of atherosclerosis. C57BL/6 mice were fed a control diet (CD; 10% fat) or high-fat diet (HFD; 60% fat) for 8 weeks. The mRNA expression of arginase I in the liver, adipose tissue, aorta, and muscle; protein expression of arginase I in the liver and plasma; and systemic levels of L-arginine bioavailability and NO 2 - were assessed. HFD-fed mice showed early obesity without severe disease symptoms. Arginase I mRNA and protein expression levels in the liver were significantly higher in HFD-fed obese mice than in CD-fed mice. Arginase I levels were slightly increased, whereas L-arginine levels were significantly reduced, and these changes were followed by reductions in NO 2 - levels. Furthermore, hepatic arginase I levels positively correlated with plasma arginase I levels and negatively correlated with L-arginine bioavailability in plasma. These results suggested that increases in the expression of hepatic arginase I and reductions in plasma L-arginine and NO 2 - levels might lead to vascular endothelial dysfunction in the pre-disease state of early obesity.

  15. Simvastatin Inhibits Goblet Cell Hyperplasia and Lung Arginase in a Mouse Model of Allergic Asthma: A Novel Treatment for Airway Remodeling?

    Science.gov (United States)

    Zeki, Amir A.; Bratt, Jennifer M.; Rabowsky, Michelle; Last, Jerold A.; Kenyon, Nicholas J.

    2010-01-01

    Airway remodeling in asthma contributes to airway hyperreactivity, loss of lung function, and persistent symptoms. Current therapies do not adequately treat the structural airway changes associated with asthma. The statins are cholesterol-lowering drugs that inhibit the enzyme 3-hydroxy-3-methyl-glutaryl-CoA reductase, the rate-limiting step of cholesterol biosynthesis in the mevalonate pathway. These drugs have been associated with improved respiratory health and ongoing clinical trials are testing their therapeutic potential in asthma. We hypothesized that simvastatin treatment of ovalbumin-exposed mice would attenuate early features of airway remodeling, by a mevalonate-dependent mechanism. BALB/c mice were initially sensitized to ovalbumin, and then exposed to 1% ovalbumin aerosol for 2 weeks after sensitization for a total of six exposures. Simvastatin (40 mg/kg) or simvastatin plus mevalonate (20 mg/kg) were injected intraperitoneally before each ovalbumin exposure. Treatment with simvastatin attenuated goblet cell hyperplasia, arginase-1 protein expression, and total arginase enzyme activity, but did not alter airway hydroxyproline content or transforming growth factor-β1. Inhibition of goblet cell hyperplasia by simvastatin was mevalonate-dependent. No appreciable changes to airway smooth muscle cells were observed in any of the control or treatment groups. In conclusion, in an acute mouse model of allergic asthma, simvastatin inhibited early hallmarks of airway remodeling, indicators that can lead to airway thickening and fibrosis. Statins are potentially novel treatments for airway remodeling in asthma. Further studies utilizing sub-chronic or chronic allergen exposure models are needed to extend these initial findings. PMID:21078495

  16. Proteomic Identification of Oxidized Proteins in Entamoeba histolytica by Resin-Assisted Capture: Insights into the Role of Arginase in Resistance to Oxidative Stress.

    Directory of Open Access Journals (Sweden)

    Preeti Shahi

    2016-01-01

    Full Text Available Entamoeba histolytica is an obligate protozoan parasite of humans, and amebiasis, an infectious disease which targets the intestine and/or liver, is the second most common cause of human death due to a protozoan after malaria. Although amebiasis is usually asymptomatic, E. histolytica has potent pathogenic potential. During host infection, the parasite is exposed to reactive oxygen species that are produced and released by cells of the innate immune system at the site of infection. The ability of the parasite to survive oxidative stress (OS is essential for a successful invasion of the host. Although the effects of OS on the regulation of gene expression in E. histolytica and the characterization of some proteins whose function in the parasite's defense against OS have been previously studied, our knowledge of oxidized proteins in E. histolytica is lacking. In order to fill this knowledge gap, we performed a large-scale identification and quantification of the oxidized proteins in oxidatively stressed E. histolytica trophozoites using resin-assisted capture coupled to mass spectrometry. We detected 154 oxidized proteins (OXs and the functions of some of these proteins were associated with antioxidant activity, maintaining the parasite's cytoskeleton, translation, catalysis, and transport. We also found that oxidation of the Gal/GalNAc impairs its function and contributes to the inhibition of E. histolytica adherence to host cells. We also provide evidence that arginase, an enzyme which converts L-arginine into L-ornithine and urea, is involved in the protection of the parasite against OS. Collectively, these results emphasize the importance of OS as a critical regulator of E. histolytica's functions and indicate a new role for arginase in E. histolytica's resistance to OS.

  17. Activation Mechanism of LRRK2 and Its Cellular Functions in Parkinson's Disease

    NARCIS (Netherlands)

    Rosenbusch, Katharina E.; Kortholt, Arjan

    2016-01-01

    Human LRRK2 (Leucine-Rich Repeat Kinase 2) has been associated with both familial and idiopathic Parkinson's disease (PD). Although several LRRK2 mediated pathways and interaction partners have been identified, the cellular functions of LRRK2 and LRRK2 mediated progression of PD are still only

  18. Profiling cellular bioenergetics, glutathione levels, and caspase activities in stomach biopsies of patients with upper gastrointestinal symptoms

    Science.gov (United States)

    Alfazari, Ali S; Al-Dabbagh, Bayan; Al-Dhaheri, Wafa; Taha, Mazen S; Chebli, Ahmad A; Fontagnier, Eva M; Koutoubi, Zaher; Kochiyi, Jose; Karam, Sherif M; Souid, Abdul-Kader

    2015-01-01

    AIM: To measure biochemical parameters in stomach biopsies and test their suitability as diagnostic biomarkers for gastritis and precancerous lesions. METHODS: Biopsies were obtained from the stomachs of two groups of patients (n = 40) undergoing fiber-optic endoscopy due to upper gastrointestinal symptoms. In the first group (n = 17), only the corpus region was examined. Biopsies were processed for microscopic examination and measurement of mitochondrial O2 consumption (cellular respiration), cellular adenosine triphosphate (ATP), glutathione (GSH), and caspase activity. In the second group of patients (n = 23), both corpus and antral regions were studied. Some biopsies were processed for microscopic examination, while the others were used for measurements of cellular respiration and GSH level. RESULTS: Microscopic examinations of gastric corpus biopsies from 17 patients revealed normal mucosae in 8 patients, superficial gastritis in 7 patients, and chronic atrophic gastritis in 1 patient. In patients with normal histology, the rate (mean ± SD) of cellular respiration was 0.17 ± 0.02 μmol/L O2 min-1 mg-1, ATP content was 487 ± 493 pmol/mg, and GSH was 469 ± 98 pmol/mg. Caspase activity was detected in 3 out of 8 specimens. The values of ATP and caspase activity were highly variable. The presence of superficial gastritis had insignificant effects on the measured biomarkers. In the patient with atrophic gastritis, cellular respiration was high and ATP was relatively low, suggesting uncoupling oxidative phosphorylation. In the second cohort of patients, the examined biopsies showed either normal or superficial gastritis. The rate of cellular respiration (O2. μmol/L min-1 mg-1) was slightly higher in the corpus than the antrum (0.18 ± 0.05 vs 0.15 ± 0.04, P = 0.019). The value of GSH was about the same in both tissues (310 ± 135 vs 322 ± 155, P = 0.692). CONCLUSION: The corpus mucosa was metabolically more active than the antrum tissue. The data in this

  19. LRRK2 Kinase Activity and Biology are Not Uniformly Predicted by its Autophosphorylation and Cellular Phosphorylation Site Status

    Directory of Open Access Journals (Sweden)

    April eReynolds

    2014-06-01

    Full Text Available Missense mutations in the Leucine Rich Repeat protein Kinase 2 (LRRK2 gene are the most common genetic predisposition to develop Parkinson’s disease (PD LRRK2 is a large multi-domain phosphoprotein with a GTPase domain and a serine/threonine protein kinase domain whose activity is implicated in neuronal toxicity; however the precise mechanism is unknown. LRRK2 autophosphorylates on several serine/threonine residues across the enzyme and is found constitutively phosphorylated on Ser910, Ser935, Ser955 and Ser973, which are proposed to be regulated by upstream kinases. Here we investigate the phosphoregulation at these sites by analyzing the effects of disease-associated mutations Arg1441Cys, Arg1441Gly, Ala1442Pro, Tyr1699Cys, Ile2012Thr, Gly2019Ser, and Ile2020Thr. We also studied alanine substitutions of phosphosite serines 910, 935, 955 and 973 and specific LRRK2 inhibition on autophosphorylation of LRRK2 Ser1292, Thr1491, Thr2483 and phosphorylation at the cellular sites. We found that mutants in the Roc-COR domains, including Arg1441Cys, Arg1441His, Ala1442Pro and Tyr1699Cys, can positively enhance LRRK2 kinase activity while concomitantly inducing the dephosphorylation of the cellular sites. Mutation of the cellular sites individually did not affect LRRK2 intrinsic kinase activity; however, Ser910/935/955/973Ala mutations trended toward increased kinase activity of LRRK2. Increased cAMP levels did not lead to increased LRRK2 cellular site phosphorylation, 14-3-3 binding or kinase activity. In cells, inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser1292 by Calyculin A and okadaic acid sensitive phosphatases, while the cellular sites are dephosphorylated by Calyculin A sensitive phosphatases. These findings indicate that comparative analysis of both Ser1292 and Ser910/935/955/973 phosphorylation sites will provide important and distinct measures of LRRK2 kinase and biological activity in vitro and in vivo.

  20. Progress of research on activation function of NK cell exposed to low dose radiation in adoptive cellular immunotherapy

    International Nuclear Information System (INIS)

    Pan Xiaosong; Shi Yujia; Yao Yimin; Xu Hong; Liu Fenju

    2009-01-01

    Natural killer cells is an important immunological factor in killing malignant cells. Low dose radiation can enhance proliferation and biological activity of NK cell. The involvement of P38MAPK signal pathway and endogenous glutathione induced by LDR may be the probable mechanism. Natural killer cell, especially adherent natural killer cell, is the preferential choice for adoptive cellular immunotherapy, which has a remarkable foreground in malignancy therapy.(authors)

  1. Adsorption of cellular peptides of Microcystis aeruginosa and two herbicides onto activated carbon. Effect of surface charge and interactions

    Czech Academy of Sciences Publication Activity Database

    Hnaťuková, Petra; Kopecká, Ivana; Pivokonský, Martin

    2011-01-01

    Roč. 45, č. 11 (2011), s. 3359-3368 ISSN 0043-1354 R&D Projects: GA AV ČR IAA200600902; GA ČR GPP105/10/P515 Institutional research plan: CEZ:AV0Z20600510 Keywords : cellular organic matter * granular activated carbon * molecular weight distribution * surface charge * cyanobacterial peptides Subject RIV: BK - Fluid Dynamics Impact factor: 4.865, year: 2011

  2. A cardiac electrical activity model based on a cellular automata system in comparison with neural network model.

    Science.gov (United States)

    Khan, Muhammad Sadiq Ali; Yousuf, Sidrah

    2016-03-01

    Cardiac Electrical Activity is commonly distributed into three dimensions of Cardiac Tissue (Myocardium) and evolves with duration of time. The indicator of heart diseases can occur randomly at any time of a day. Heart rate, conduction and each electrical activity during cardiac cycle should be monitor non-invasively for the assessment of "Action Potential" (regular) and "Arrhythmia" (irregular) rhythms. Many heart diseases can easily be examined through Automata model like Cellular Automata concepts. This paper deals with the different states of cardiac rhythms using cellular automata with the comparison of neural network also provides fast and highly effective stimulation for the contraction of cardiac muscles on the Atria in the result of genesis of electrical spark or wave. The specific formulated model named as "States of automaton Proposed Model for CEA (Cardiac Electrical Activity)" by using Cellular Automata Methodology is commonly shows the three states of cardiac tissues conduction phenomena (i) Resting (Relax and Excitable state), (ii) ARP (Excited but Absolutely refractory Phase i.e. Excited but not able to excite neighboring cells) (iii) RRP (Excited but Relatively Refractory Phase i.e. Excited and able to excite neighboring cells). The result indicates most efficient modeling with few burden of computation and it is Action Potential during the pumping of blood in cardiac cycle.

  3. Changes in the cellular energy state affect the activity of the bacterial phosphotransferase system

    DEFF Research Database (Denmark)

    Rohwer, J.M.; Jensen, Peter Ruhdal; Shinohara, Y.

    1996-01-01

    The effect of different cellular free-energy states on the uptake of methyl alfa-D-glucopyranoside, an analoque of glucose, by Escherichia coli phosphoenolpyruvate:carbohydrate phosphotransferase system was investigated. The intracellular ATP/ADP ratio was varied by changing the expression...... of the atp operon, which codes for the H+-ATPase, or by adding an uncoupler of oxidative phosphorylation or an inhibitor of respiration. Corresponding initial phosphotransferase uptake rates were determined using an improved uptake assay that works with growing cells in steady state. The results show...... that the initial uptake rate was decreased under conditions of lowered intracellular ATP/ADP ratios, irrespective of which method was used to change the cellular energy state.. When either the expression of the atp operon was changed or 2,4-dinitrophenol was added to wild-type cells, the relationship between...

  4. Bacterial RNA induces myocyte cellular dysfunction through the activation of PKR

    OpenAIRE

    Bleiblo, Farag; Michael, Paul; Brabant, Danielle; Ramana, Chilakamarti V.; Tai, TC; Saleh, Mazen; Parrillo, Joseph E.; Kumar, Anand; Kumar, Aseem

    2012-01-01

    Severe sepsis and the ensuing septic shock are serious life threatening conditions. These diseases are triggered by the host's over exuberant systemic response to the infecting pathogen. Several surveillance mechanisms have evolved to discriminate self from foreign RNA and accordingly trigger effective cellular responses to target the pathogenic threats. The RNA-dependent protein kinase (PKR) is a key component of the cytoplasmic RNA sensors involved in the recognition of viral double-strande...

  5. Adjuvant activity of peanut, cottonseed and rice oils on cellular and humoral response

    Directory of Open Access Journals (Sweden)

    Erika Freitas

    2013-04-01

    Full Text Available The potentiality of the usage of vegetable oils such as soybean, corn, olive, sesame, murici seed, rapeseed, linseed, rice and cashew nuts as adjuvant of the humoral and cellular immune response has been recently shown. In the present work, besides of evaluating the adjuvant action of peanut, cottonseed and rice oils on humoral and cellular immune responses against ovalbumin (OVA we also evaluated the protective immune response induced by Leishmania antigens. The peanut oil significantly increased the synthesis of anti-ovalbumin antibodies in the primary response, but it did not favor cellular response. Concerning mice immunized with L. amazonensis antigens emulsified with peanut oil exacerbated skin lesions and lymph node parasite load what suggests stimulation of the Th2 immune response and down regulation of Th1 response. The cottonseed oil was shown to have adjuvant effect to the humoral response, stimulating a secondary response and also favored the delayed-type hypersensitivity (DTH response to OVA. The rice oil stimulated a strong DTH reaction to OVA and enhanced the synthesis of antibodies after the third dose. Mice immunized with L. amazonensis antigens emulsified with rice oil or cotton seed oil were protected from developing skin lesions and lymph node parasite load. These results emphasize the interest and importance of the vegetable oils as tools in different procedures of immunization and their differential role in relation to the other adjuvant under usage.

  6. High-Concentrate Diet-Induced Change of Cellular Metabolism Leads to Decreases of Immunity and Imbalance of Cellular Activities in Rumen Epithelium

    Directory of Open Access Journals (Sweden)

    Zhongyan Lu

    2018-03-01

    Full Text Available Background/Aims: In animals, the immune and cellular processes of tissue largely depend on the status of local metabolism. However, in the rumen epithelium, how the cellular metabolism affects epithelial immunity, and cellular processes, when the diet is switched from energy-rich to energy-excess status, with regard to animal production and health, have not as yet been reported. Methods: RNA-seq was applied to compare the biological processes altered by an increase of dietary concentration from 10% to 35% with those altered by an increase of dietary concentration from 35% to 65% (dietary concentrate: the non-grass component in diet, including corn, soya bean meal and additive. High concentrate diet composed of 35% grass, 55% corn, 8% soya bean meal and 2% additive. In addition to the functional analysis of enriched genes in terms of metabolism, the immune system, and cellular process, the highly correlated genes to the enriched metabolism genes were identified, and the function and signaling pathways related to the differentially expressed neighbors were compared among the groups. Results: The variation trends of molar proportions of ruminal SCFAs and those of enriched pathways belonging to metabolism, immune system, and cellular process were altered with the change of diets. With regard to metabolism, lipid metabolism and amino acid metabolism were most affected. According to the correlation analysis, both innate and adaptive immune responses were promoted by the metabolism genes enriched under the 65% concentrate diet. However, the majority of immune responses were suppressed under the 35% concentrate diet. Moreover, the exclusive upregulation of cell growth and dysfunction of cellular transport and catabolism were induced by the metabolism genes enriched under the 65% concentrate diet. On the contrary, a balanced regulation of cellular processes was detected under the 35% concentrate diet. Conclusions: These results indicated that the

  7. High-Concentrate Diet-Induced Change of Cellular Metabolism Leads to Decreases of Immunity and Imbalance of Cellular Activities in Rumen Epithelium.

    Science.gov (United States)

    Lu, Zhongyan; Shen, Hong; Shen, Zanming

    2018-01-01

    In animals, the immune and cellular processes of tissue largely depend on the status of local metabolism. However, in the rumen epithelium, how the cellular metabolism affects epithelial immunity, and cellular processes, when the diet is switched from energy-rich to energy-excess status, with regard to animal production and health, have not as yet been reported. RNA-seq was applied to compare the biological processes altered by an increase of dietary concentration from 10% to 35% with those altered by an increase of dietary concentration from 35% to 65% (dietary concentrate: the non-grass component in diet, including corn, soya bean meal and additive. High concentrate diet composed of 35% grass, 55% corn, 8% soya bean meal and 2% additive). In addition to the functional analysis of enriched genes in terms of metabolism, the immune system, and cellular process, the highly correlated genes to the enriched metabolism genes were identified, and the function and signaling pathways related to the differentially expressed neighbors were compared among the groups. The variation trends of molar proportions of ruminal SCFAs and those of enriched pathways belonging to metabolism, immune system, and cellular process were altered with the change of diets. With regard to metabolism, lipid metabolism and amino acid metabolism were most affected. According to the correlation analysis, both innate and adaptive immune responses were promoted by the metabolism genes enriched under the 65% concentrate diet. However, the majority of immune responses were suppressed under the 35% concentrate diet. Moreover, the exclusive upregulation of cell growth and dysfunction of cellular transport and catabolism were induced by the metabolism genes enriched under the 65% concentrate diet. On the contrary, a balanced regulation of cellular processes was detected under the 35% concentrate diet. These results indicated that the alterations of cellular metabolism promote the alterations in cellular

  8. Multi-cellular natural killer (NK) cell clusters enhance NK cell activation through localizing IL-2 within the cluster

    Science.gov (United States)

    Kim, Miju; Kim, Tae-Jin; Kim, Hye Mi; Doh, Junsang; Lee, Kyung-Mi

    2017-01-01

    Multi-cellular cluster formation of natural killer (NK) cells occurs during in vivo priming and potentiates their activation to IL-2. However, the precise mechanism underlying this synergy within NK cell clusters remains unclear. We employed lymphocyte-laden microwell technologies to modulate contact-mediated multi-cellular interactions among activating NK cells and to quantitatively assess the molecular events occurring in multi-cellular clusters of NK cells. NK cells in social microwells, which allow cell-to-cell contact, exhibited significantly higher levels of IL-2 receptor (IL-2R) signaling compared with those in lonesome microwells, which prevent intercellular contact. Further, CD25, an IL-2R α chain, and lytic granules of NK cells in social microwells were polarized toward MTOC. Live cell imaging of lytic granules revealed their dynamic and prolonged polarization toward neighboring NK cells without degranulation. These results suggest that IL-2 bound on CD25 of one NK cells triggered IL-2 signaling of neighboring NK cells. These results were further corroborated by findings that CD25-KO NK cells exhibited lower proliferation than WT NK cells, and when mixed with WT NK cells, underwent significantly higher level of proliferation. These data highlights the existence of IL-2 trans-presentation between NK cells in the local microenvironment where the availability of IL-2 is limited.

  9. Telmisartan enhances mitochondrial activity and alters cellular functions in human coronary artery endothelial cells via AMP-activated protein kinase pathway.

    Science.gov (United States)

    Kurokawa, Hirofumi; Sugiyama, Seigo; Nozaki, Toshimitsu; Sugamura, Koichi; Toyama, Kensuke; Matsubara, Junichi; Fujisue, Koichiro; Ohba, Keisuke; Maeda, Hirofumi; Konishi, Masaaki; Akiyama, Eiichi; Sumida, Hitoshi; Izumiya, Yasuhiro; Yasuda, Osamu; Kim-Mitsuyama, Shokei; Ogawa, Hisao

    2015-04-01

    Mitochondrial dysfunction plays an important role in cellular senescence and impaired function of vascular endothelium, resulted in cardiovascular diseases. Telmisartan is a unique angiotensin II type I receptor blocker that has been shown to prevent cardiovascular events in high risk patients. AMP-activated protein kinase (AMPK) plays a critical role in mitochondrial biogenesis and endothelial function. This study assessed whether telmisartan enhances mitochondrial function and alters cellular functions via AMPK in human coronary artery endothelial cells (HCAECs). In cultured HCAECs, telmisartan significantly enhanced mitochondrial activity assessed by mitochondrial reductase activity and intracellular ATP production and increased the expression of mitochondria related genes. Telmisartan prevented cellular senescence and exhibited the anti-apoptotic and pro-angiogenic properties. The expression of genes related anti-oxidant and pro-angiogenic properties were increased by telmisartan. Telmisartan increased endothelial NO synthase and AMPK phosphorylation. Peroxisome proliferator-activated receptor gamma signaling was not involved in telmisartan-induced improvement of mitochondrial function. All of these effects were abolished by inhibition of AMPK. Telmisartan enhanced mitochondrial activity and exhibited anti-senescence effects and improving endothelial function through AMPK in HCAECs. Telmisartan could provide beneficial effects on vascular diseases via enhancement of mitochondrial activity and modulating endothelial function through AMPK activation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  10. Cellular Imaging at 1.5 T: Detecting Cells in Neuroinflammation using Active Labeling with Superparamagnetic Iron Oxide

    Directory of Open Access Journals (Sweden)

    Ayman J. Oweida

    2004-04-01

    Full Text Available The ability to visualize cell infiltration in experimental autoimmune encephalomyelitis (EAE, a well-known animal model for multiple sclerosis in humans, was investigated using a clinical 1.5-T magnetic resonance imaging (MRI scanner, a custom-built, high-strength gradient coil insert, a 3-D fast imaging employing steady-state acquisition (FIESTA imaging sequence and a superparamagnetic iron oxide (SPIO contrast agent. An “active labeling” approach was used with SPIO administered intravenously during inflammation in EAE. Our results show that small, discrete regions of signal void corresponding to iron accumulation in EAE brain can be detected using FIESTA at 1.5 T. This work provides early evidence that cellular abnormalities that are the basis of diseases can be probed using cellular MRI and supports our earlier work which indicates that tracking of iron-labeled cells will be possible using clinical MR scanners.

  11. Community structure, cellular rRNA content, and activity of sulfate-reducing bacteria in marine Arctic sediments

    DEFF Research Database (Denmark)

    Ravenschlag, K.; Sahm, K.; Knoblauch, C.

    2000-01-01

    The community structure of sulfate-reducing bacteria (SRB) of a marine Arctic sediment (Smeerenburg-fjorden, Svalbard) a-as characterized by both fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization by using group- and genus-specific 16S rRNA-targeted oligonucleotide probes...... that FISH and rRNA slot blot hybridization gave comparable results. Furthermore, a combination of the two methods allowed us to calculate specific cellular rRNA contents with respect to localization in the sediment profile. The rRNA contents of Desulfosarcina-Desulfococcus cells were highest in the first 5...... mm of the sediment (0.9 and 1.4 fg, respectively) and decreased steeply with depth, indicating that maximal metabolic activity occurred close to the surface, Based on SRB cell numbers, cellular sulfate reduction rates were calculated. The rates were highest in the surface layer (0.14 fmol cell(-1...

  12. Comparative effect of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction on antioxidant enzymes activity in cellular ageing of human diploid fibroblasts.

    Science.gov (United States)

    Makpol, Suzana; Yeoh, Thong Wei; Ruslam, Farah Adilah Che; Arifin, Khaizurin Tajul; Yusof, Yasmin Anum Mohd

    2013-08-16

    Human diploid fibroblasts (HDFs) undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular ageing. Even though beneficial effects of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction (TRF) have been reported, ongoing studies in relation to ageing is of interest to determine possible protective effects that may reverse the effect of ageing. The aim of this study was to evaluate the effect of P. betle, C. vulgaris and TRF in preventing cellular ageing of HDFs by determining the activity of antioxidant enzymes viz.; catalase, superoxide dismutase (SOD) and glutathione peroxidase. Different passages of HDFs were treated with P. betle, C. vulgaris and TRF for 24 h prior to enzymes activity determination. Senescence-associated beta-galactosidase (SA β-gal) expression was assayed to validate cellular ageing. In cellular ageing of HDFs, catalase and glutathione peroxidase activities were reduced, but SOD activity was heightened during pre-senescence. P. betle exhibited the strongest antioxidant activity by reducing SA β-gal expression, catalase activities in all age groups, and SOD activity. TRF exhibited a strong antioxidant activity by reducing SA β-gal expression, and SOD activity in senescent HDFs. C. vulgaris extract managed to reduce SOD activity in senescent HDFs. P. betle, C. vulgaris, and TRF have the potential as anti-ageing entities which compensated the role of antioxidant enzymes in cellular ageing of HDFs.

  13. Comparative effect of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction on antioxidant enzymes activity in cellular ageing of human diploid fibroblasts

    Science.gov (United States)

    2013-01-01

    Background Human diploid fibroblasts (HDFs) undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular ageing. Even though beneficial effects of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction (TRF) have been reported, ongoing studies in relation to ageing is of interest to determine possible protective effects that may reverse the effect of ageing. The aim of this study was to evaluate the effect of P. betle, C. vulgaris and TRF in preventing cellular ageing of HDFs by determining the activity of antioxidant enzymes viz.; catalase, superoxide dismutase (SOD) and glutathione peroxidase. Methods Different passages of HDFs were treated with P. betle, C. vulgaris and TRF for 24 h prior to enzymes activity determination. Senescence-associated beta-galactosidase (SA β-gal) expression was assayed to validate cellular ageing. Results In cellular ageing of HDFs, catalase and glutathione peroxidase activities were reduced, but SOD activity was heightened during pre-senescence. P. betle exhibited the strongest antioxidant activity by reducing SA β-gal expression, catalase activities in all age groups, and SOD activity. TRF exhibited a strong antioxidant activity by reducing SA β-gal expression, and SOD activity in senescent HDFs. C. vulgaris extract managed to reduce SOD activity in senescent HDFs. Conclusion P. betle, C. vulgaris, and TRF have the potential as anti-ageing entities which compensated the role of antioxidant enzymes in cellular ageing of HDFs. PMID:23948056

  14. Activation of Actuating Hydrogels with WS2 Nanosheets for Biomimetic Cellular Structures and Steerable Prompt Deformation.

    Science.gov (United States)

    Zong, Lu; Li, Xiankai; Han, Xiangsheng; Lv, Lili; Li, Mingjie; You, Jun; Wu, Xiaochen; Li, Chaoxu

    2017-09-20

    Macroscopic soft actuation is intrinsic to living organisms in nature, including slow deformation (e.g., contraction, bending, twisting, and curling) of plants motivated by microscopic swelling and shrinking of cells, and rapid motion of animals (e.g., deformation of jellyfish) motivated by cooperative nanoscale movement of motor proteins. These actuation behaviors, with an exceptional combination of tunable speed and programmable deformation direction, inspire us to design artificial soft actuators for broad applications in artificial muscles, nanofabrication, chemical valves, microlenses, soft robotics, etc. However, so far artificial soft actuators have been typically produced on the basis of poly(N-isopropylacrylamide) (PNiPAM), whose deformation is motived by volumetric shrinkage and swelling in analogue to plant cells, and exhibits sluggish actuation kinetics. In this study, alginate-exfoliated WS 2 nanosheets were incorporated into ice-template-polymerized PNiPAM hydrogels with the cellular microstructures which mimic plant cells, yet the prompt steerable actuation of animals. Because of the nanosheet-reinforced pore walls formed in situ in freezing polymerization and reasonable hierarchical water channels, this cellular hybrid hydrogel achieves super deformation speed (on the order of magnitude of 10° s), controllable deformation direction, and high near-infrared light responsiveness, offering an unprecedented platform of artificial muscles for various soft robotics and devices (e.g., rotator, microvalve, aquatic swimmer, and water-lifting filter).

  15. Antioxidant Activity of Lawsonia inermis Extracts Inhibits Chromium(VI-Induced Cellular and DNA Toxicity

    Directory of Open Access Journals (Sweden)

    Gunjan Guha

    2011-01-01

    Full Text Available Hexavalent chromium Cr(VI is a very strong oxidant which consequently causes high cytotoxicity through oxidative stress. Prevention of Cr(VI-induced cellular damage has been sought in this study in aqueous and methanolic extracts of Lawsonia inermis Linn. (Lythraceae, commonly known as Henna. The extracts showed significant (P < .05 potential in scavenging free radicals (DPPH• and ABTS•+ and Fe3+, and in inhibiting lipid peroxidation. DNA damage caused by exposure of pBR322 to Cr(VI-UV is markedly inhibited by both extracts in varying degrees. A distinct decline in Cr(VI-induced cytotoxicity was noticed in MDA-MB-435S (human breast carcinoma cells with an increase in dosage of both extracts individually. Furthermore, both extracts proved to contain a high content of phenolic compounds which were found to have a strong and significant (P < .05 positive correlation to the radical scavenging potential, lipid peroxidation inhibition capacity and cyto-protective efficiency against Cr(VI-induced oxidative cellular damage. HPLC analysis identified some of the major phenolic compounds in both extracts, which might be responsible for the antioxidant potential and the properties of DNA and cyto-protection. This study contributes to the search for natural resources that might yield potent therapeutic drugs against Cr(VI-induced oxidative cell damage.

  16. Membrane plasmalogen composition and cellular cholesterol regulation: a structure activity study

    Directory of Open Access Journals (Sweden)

    Su-Myat Khine K

    2010-06-01

    Full Text Available Abstract Background Disrupted cholesterol regulation leading to increased circulating and membrane cholesterol levels is implicated in many age-related chronic diseases such as cardiovascular disease (CVD, Alzheimer's disease (AD, and cancer. In vitro and ex vivo cellular plasmalogen deficiency models have been shown to exhibit impaired intra- and extra-cellular processing of cholesterol. Furthermore, depleted brain plasmalogens have been implicated in AD and serum plasmalogen deficiencies have been linked to AD, CVD, and cancer. Results Using plasmalogen deficient (NRel-4 and plasmalogen sufficient (HEK293 cells we investigated the effect of species-dependent plasmalogen restoration/augmentation on membrane cholesterol processing. The results of these studies indicate that the esterification of cholesterol is dependent upon the amount of polyunsaturated fatty acid (PUFA-containing ethanolamine plasmalogen (PlsEtn present in the membrane. We further elucidate that the concentration-dependent increase in esterified cholesterol observed with PUFA-PlsEtn was due to a concentration-dependent increase in sterol-O-acyltransferase-1 (SOAT1 levels, an observation not reproduced by 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA reductase inhibition. Conclusion The present study describes a novel mechanism of cholesterol regulation that is consistent with clinical and epidemiological studies of cholesterol, aging and disease. Specifically, the present study describes how selective membrane PUFA-PlsEtn enhancement can be achieved using 1-alkyl-2-PUFA glycerols and through this action reduce levels of total and free cholesterol in cells.

  17. Human papillomavirus 16E6 and NFX1-123 potentiate notch signaling and differentiation without activating cellular arrest

    Energy Technology Data Exchange (ETDEWEB)

    Vliet-Gregg, Portia A.; Hamilton, Jennifer R. [Center for Global Infectious Disease Research, Seattle Children' s Research Institute, 1900 Ninth Ave., Seattle, WA 98101 (United States); Katzenellenbogen, Rachel A., E-mail: rkatzen@uw.edu [Center for Global Infectious Disease Research, Seattle Children' s Research Institute, 1900 Ninth Ave., Seattle, WA 98101 (United States); Department of Pediatrics, Division of Adolescent Medicine, University of Washington, Seattle WA (United States)

    2015-04-15

    High-risk human papillomavirus (HR HPV) oncoproteins bind host cell proteins to dysregulate and uncouple apoptosis, senescence, differentiation, and growth. These pathways are important for both the viral life cycle and cancer development. HR HPV16 E6 (16E6) interacts with the cellular protein NFX1-123, and they collaboratively increase the growth and differentiation master regulator, Notch1. In 16E6 expressing keratinocytes (16E6 HFKs), the Notch canonical pathway genes Hes1 and Hes5 were increased with overexpression of NFX1-123, and their expression was directly linked to the activation or blockade of the Notch1 receptor. Keratinocyte differentiation genes Keratin 1 and Keratin 10 were also increased, but in contrast their upregulation was only indirectly associated with Notch1 receptor stimulation and was fully unlinked to growth arrest, increased p21{sup Waf1/CIP1}, or decreased proliferative factor Ki67. This leads to a model of 16E6, NFX1-123, and Notch1 differently regulating canonical and differentiation pathways and entirely uncoupling cellular arrest from increased differentiation. - Highlights: • 16E6 and NFX1-123 increased the Notch canonical pathway through Notch1. • 16E6 and NFX1-123 increased the differentiation pathway indirectly through Notch1. • 16E6 and NFX1-123 increased differentiation gene expression without growth arrest. • Increased NFX1-123 with 16E6 may create an ideal cellular phenotype for HPV.

  18. Measurement of whole body cellular and collagen nitrogen, potassium, and other elements by neutron activation and whole body counting

    International Nuclear Information System (INIS)

    James, H.M.; Fabricius, P.J.; Dykes, P.W.

    1987-01-01

    Whole body nitrogen can be measured by neutron activation analysis with an acceptable radiation dose; it is an index of body protein which, in normal subjects, is 65% cellular protein and 35% extracellular connective collagen. Whole body potassium can be measured by whole body counting without irradiating the subject; it is an index of body cell mass. We measured whole body nitrogen, potassium, extracellular water, intracellular water, and fat-folds. The differences between 37 malnourished patients and five normal subjects suggested that the patients had 9 kg less cell mass than normal, but no difference in extracellular mass. Measurements were made on eight patients before and after 14 days of total parenteral nutrition; balance of nitrogen intake and excretion also was measured. The changes were consistent with mean increases of 3 kg of cellular mass and 3 kg of fat with no change of extracellular mass. The accuracy and sensitivity of the whole body measurements need further confirmation for use in patients with changing body composition. Where tissue wasting is largely from the cellular compartment, potassium could be a more sensitive index of wasting than nitrogen. Multielement analysis of nitrogen, potassium, chlorine, and carbon will probably be valuable in elucidating body composition in malnutrition

  19. Reconstitution of the cellular response to DNA damage in vitro using damage-activated extracts from mammalian cells

    International Nuclear Information System (INIS)

    Roper, Katherine; Coverley, Dawn

    2012-01-01

    In proliferating mammalian cells, DNA damage is detected by sensors that elicit a cellular response which arrests the cell cycle and repairs the damage. As part of the DNA damage response, DNA replication is inhibited and, within seconds, histone H2AX is phosphorylated. Here we describe a cell-free system that reconstitutes the cellular response to DNA double strand breaks using damage-activated cell extracts and naïve nuclei. Using this system the effect of damage signalling on nuclei that do not contain DNA lesions can be studied, thereby uncoupling signalling and repair. Soluble extracts from G1/S phase cells that were treated with etoposide before isolation, or pre-incubated with nuclei from etoposide-treated cells during an in vitro activation reaction, restrain both initiation and elongation of DNA replication in naïve nuclei. At the same time, H2AX is phosphorylated in naïve nuclei in a manner that is dependent upon the phosphatidylinositol 3-kinase-like protein kinases. Notably, phosphorylated H2AX is not focal in naïve nuclei, but is evident throughout the nucleus suggesting that in the absence of DNA lesions the signal is not amplified such that discrete foci can be detected. This system offers a novel screening approach for inhibitors of DNA damage response kinases, which we demonstrate using the inhibitors wortmannin and LY294002. -- Highlights: ► A cell free system that reconstitutes the response to DNA damage in the absence of DNA lesions. ► Damage-activated extracts impose the cellular response to DNA damage on naïve nuclei. ► PIKK-dependent response impacts positively and negatively on two separate fluorescent outputs. ► Can be used to screen for inhibitors that impact on the response to damage but not on DNA repair. ► LY294002 and wortmannin demonstrate the system's potential as a pathway focused screening approach.

  20. Coordination of early cellular reactions during activation of bone resorption in the rat mandible periosteum: An immunohistochemical study

    Directory of Open Access Journals (Sweden)

    Bassam Hassan

    2017-10-01

    Full Text Available The activation step of bone remodeling remains poorly characterized. Activation comprises determination of the site to be remodeled, osteoclast precursor recruitment, their migration to the site of remodeling, and differentiation. These actions involve different compartments and cell types. The aim of this study was to investigate events and cell types involved during activation. We used a bone remodeling model in rats where extractions of the upper jaw molars initiate remodeling of the antagonist lower jaw (mandible cortex along the periosteum. In this model osteoclastic resorption peaks 4 days after extractions. We previously reported that mast cell activation in the periosteum fibrous compartment is an early event of activation, associated with recruitment of circulating monocyte osteoclast precursors. By using immunohistochemistry, we observed 9 hours after induction a spatially oriented expression of InterCellular Adhesion Molecule-1 in the vessels that was inhibited by antagonists of histamine receptors 1 and 2. It was followed at 12 hours by the recruitment of ED1+ monocytes. In parallel, at 9 hours, Vascular Cellular Adhesion Molecule-1+ fibroblast-like cells scattered in the fibrous compartment of the periosteum between the vessels and the osteogenic compartment increased; these cells may be implicated in osteoclast precursor migration. Receptor Activator of NF KappaB Ligand+ cells increased at 12 hours in the osteogenic compartment and reached a peak at 18 hours. At 24 hours the numbers of osteogenic cells and subjacent osteocytes expressing semaphorin 3a, a repulsive for osteoclast precursors, decreased before returning to baseline at 48 hours. These data show that during activation the two periosteum compartments and several cell types are coordinated to recruit and guide osteoclast precursors towards the bone surface. Keywords: Biological sciences, Cell biology, Physiology, Dentistry

  1. The yeast mitogen-activated protein kinase Slt2 is involved in the cellular response to genotoxic stress

    Directory of Open Access Journals (Sweden)

    Soriano-Carot María

    2012-02-01

    Full Text Available Abstract Background The maintenance of genomic integrity is essential for cell viability. Complex signalling pathways (DNA integrity checkpoints mediate the response to genotoxic stresses. Identifying new functions involved in the cellular response to DNA-damage is crucial. The Saccharomyces cerevisiae SLT2 gene encodes a member of the mitogen-activated protein kinase (MAPK cascade whose main function is the maintenance of the cell wall integrity. However, different observations suggest that SLT2 may also have a role related to DNA metabolism. Results This work consisted in a comprehensive study to connect the Slt2 protein to genome integrity maintenance in response to genotoxic stresses. The slt2 mutant strain was hypersensitive to a variety of genotoxic treatments, including incubation with hydroxyurea (HU, methylmetanosulfonate (MMS, phleomycin or UV irradiation. Furthermore, Slt2 was activated by all these treatments, which suggests that Slt2 plays a central role in the cellular response to genotoxic stresses. Activation of Slt2 was not dependent on the DNA integrity checkpoint. For MMS and UV, Slt2 activation required progression through the cell cycle. In contrast, HU also activated Slt2 in nocodazol-arrested cells, which suggests that Slt2 may respond to dNTP pools alterations. However, neither the protein level of the distinct ribonucleotide reductase subunits nor the dNTP pools were affected in a slt2 mutant strain. An analysis of the checkpoint function revealed that Slt2 was not required for either cell cycle arrest or the activation of the Rad53 checkpoint kinase in response to DNA damage. However, slt2 mutant cells showed an elongated bud and partially impaired Swe1 degradation after replicative stress, indicating that Slt2 could contribute, in parallel with Rad53, to bud morphogenesis control after genotoxic stresses. Conclusions Slt2 is activated by several genotoxic treatments and is required to properly cope with DNA damage. Slt

  2. Impaired CK1 delta activity attenuates SV40-induced cellular transformation in vitro and mouse mammary carcinogenesis in vivo.

    Directory of Open Access Journals (Sweden)

    Heidrun Hirner

    Full Text Available Simian virus 40 (SV40 is a powerful tool to study cellular transformation in vitro, as well as tumor development and progression in vivo. Various cellular kinases, among them members of the CK1 family, play an important role in modulating the transforming activity of SV40, including the transforming activity of T-Ag, the major transforming protein of SV40, itself. Here we characterized the effects of mutant CK1δ variants with impaired kinase activity on SV40-induced cell transformation in vitro, and on SV40-induced mammary carcinogenesis in vivo in a transgenic/bi-transgenic mouse model. CK1δ mutants exhibited a reduced kinase activity compared to wtCK1δ in in vitro kinase assays. Molecular modeling studies suggested that mutation N172D, located within the substrate binding region, is mainly responsible for impaired mutCK1δ activity. When stably over-expressed in maximal transformed SV-52 cells, CK1δ mutants induced reversion to a minimal transformed phenotype by dominant-negative interference with endogenous wtCK1δ. To characterize the effects of CK1δ on SV40-induced mammary carcinogenesis, we generated transgenic mice expressing mutant CK1δ under the control of the whey acidic protein (WAP gene promoter, and crossed them with SV40 transgenic WAP-T-antigen (WAP-T mice. Both WAP-T mice as well as WAP-mutCK1δ/WAP-T bi-transgenic mice developed breast cancer. However, tumor incidence was lower and life span was significantly longer in WAP-mutCK1δ/WAP-T bi-transgenic animals. The reduced CK1δ activity did not affect early lesion formation during tumorigenesis, suggesting that impaired CK1δ activity reduces the probability for outgrowth of in situ carcinomas to invasive carcinomas. The different tumorigenic potential of SV40 in WAP-T and WAP-mutCK1δ/WAP-T tumors was also reflected by a significantly different expression of various genes known to be involved in tumor progression, specifically of those involved in wnt-signaling and DNA

  3. Cascading reaction of arginase and urease on a graphene-based FET for ultrasensitive, real-time detection of arginine.

    Science.gov (United States)

    Berninger, Teresa; Bliem, Christina; Piccinini, Esteban; Azzaroni, Omar; Knoll, Wolfgang

    2018-09-15

    Herein, a biosensor based on a reduced graphene oxide field effect transistor (rGO-FET) functionalized with the cascading enzymes arginase and urease was developed for the detection of L-arginine. Arginase and urease were immobilized on the rGO-FET sensing surface via electrostatic layer-by-layer assembly using polyethylenimine (PEI) as cationic building block. The signal transduction mechanism is based on the ability of the cascading enzymes to selectively perform chemical transformations and prompt local pH changes, that are sensitively detected by the rGO-FET. In the presence of L-arginine, the transistors modified with (PEI/urease(arginase)) multilayers showed a shift in the Dirac point due to the change in the local pH close to the graphene surface, produced by the catalyzed urea hydrolysis. The transistors were able to monitor L-arginine in the 10-1000 μM linear range with a LOD of 10 μM, displaying a fast response and a good long-term stability. The sensor showed stereospecificity and high selectivity in the presence of non-target amino acids. Taking into account the label-free, real-time measurement capabilities and the easily quantifiable, electronic output signal, this biosensor offers advantages over state-of-the-art L-arginine detection methods. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  4. Sirtuin 7 promotes cellular survival following genomic stress by attenuation of DNA damage, SAPK activation and p53 response

    Energy Technology Data Exchange (ETDEWEB)

    Kiran, Shashi; Oddi, Vineesha [Laboratory of Cancer Biology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, Telangana, 500001 (India); Ramakrishna, Gayatri, E-mail: gayatrirama1@gmail.com [Laboratory of Cancer Biology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, Telangana, 500001 (India); Laboratory of Cancer Cell Biology, Department of Research, Institute of Liver and Biliary Sciences, Delhi 110070 (India)

    2015-02-01

    Maintaining the genomic integrity is a constant challenge in proliferating cells. Amongst various proteins involved in this process, Sirtuins play a key role in DNA damage repair mechanisms in yeast as well as mammals. In the present work we report the role of one of the least explored Sirtuin viz., SIRT7, under conditions of genomic stress when treated with doxorubicin. Knockdown of SIRT7 sensitized osteosarcoma (U2OS) cells to DNA damage induced cell death by doxorubicin. SIRT7 overexpression in NIH3T3 delayed cell cycle progression by causing delay in G1 to S transition. SIRT7 overexpressing cells when treated with low dose of doxorubicin (0.25 µM) showed delayed onset of senescence, lesser accumulation of DNA damage marker γH2AX and lowered levels of growth arrest markers viz., p53 and p21 when compared to doxorubicin treated control GFP expressing cells. Resistance to DNA damage following SIRT7 overexpression was also evident by EdU incorporation studies where cellular growth arrest was significantly delayed. When treated with higher dose of doxorubicin (>1 µM), SIRT7 conferred resistance to apoptosis by attenuating stress activated kinases (SAPK viz., p38 and JNK) and p53 response thereby shifting the cellular fate towards senescence. Interestingly, relocalization of SIRT7 from nucleolus to nucleoplasm together with its co-localization with SAPK was an important feature associated with DNA damage. SIRT7 mediated resistance to doxorubicin induced apoptosis and senescence was lost when p53 level was restored by nutlin treatment. Overall, we propose SIRT7 attenuates DNA damage, SAPK activation and p53 response thereby promoting cellular survival under conditions of genomic stress. - Highlights: • Knockdown of SIRT7 sensitized cells to DNA damage induced apoptosis. • SIRT7 delayed onset of premature senescence by attenuating DNA damage response. • Overexpression of SIRT7 delayed cell cycle progression by delaying G1/S transition. • Upon DNA damage SIRT

  5. Determining antioxidant activities of lactobacilli cell-free supernatants by cellular antioxidant assay: a comparison with traditional methods.

    Directory of Open Access Journals (Sweden)

    Jiali Xing

    Full Text Available Antioxidant activity of lactic acid bacteria is associated with multiple health-protective effects. Traditional indexes of chemical antioxidant activities poorly reflect the antioxidant effects of these bacteria in vivo. Cellular antioxidant activity (CAA assay was used in this study to determine the antioxidant activity of cell-free supernatants (CFSs of 10 Lactobacillus strains. The performance of the CAA assay was compared with that of four chemical antioxidant activity assays, namely, DPPH radical scavenging, hydroxyl radical scavenging (HRS, reducing power (RP, and inhibition of linoleic acid peroxidation (ILAP. Results of the CAA assay were associated with those of DPPH and ILAP assays, but not with those of RP and HRS assays. The inter- and intra-specific antioxidant activities of CFS were characterized by chemical and CAA assays. L. rhamnosus CCFM 1107 displayed a high antioxidative effect similar to positive control L. rhamnosus GG ATCC 53103 in all of the assays. The CAA assay is a potential method for the detection of antioxidant activities of lactobacilli CFSs.

  6. Cellular activation of hypothalamic hypocretin/orexin neurons facilitates short-term spatial memory in mice.

    Science.gov (United States)

    Aitta-Aho, Teemu; Pappa, Elpiniki; Burdakov, Denis; Apergis-Schoute, John

    2016-12-01

    The hypothalamic hypocretin/orexin (HO) system holds a central role in the regulation of several physiological functions critical for food-seeking behavior including mnemonic processes for effective foraging behavior. It is unclear however whether physiological increases in HO neuronal activity can support such processes. Using a designer rM3Ds receptor activation approach increasing HO neuronal activity resulted in improved short-term memory for novel locations. When tested on a non-spatial novelty object recognition task no significant difference was detected between groups indicating that hypothalamic HO neuronal activation can selectively facilitate short-term spatial memory for potentially supporting memory for locations during active exploration. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. State-dependent cellular activity patterns of the cat paraventricular hypothalamus measured by reflectance imaging

    DEFF Research Database (Denmark)

    Kristensen, Morten Pilgaard; Rector, D M; Poe, G R

    1996-01-01

    Activity within the cat paraventricular hypothalamus (PVH) during sleep and waking states was measured by quantifying intrinsic tissue reflectivity. A fiber optic probe consisting of a 1.0 mm coherent image conduit, surrounded by plastic fibers which conducted 660 nm source light, was attached...... to a charge-coupled device camera, and positioned over the PVH in five cats. Electrodes for assessing state variables, including electroencephalographic activity, eye movement, and somatic muscle tone were also placed. After surgical recovery, reflected light intensity was measured continuously at 2.5 Hz...... changes with behavioral state in a regionally specific manner, and that overall activity increases during quiet sleep, and is even more enhanced in active sleep. PVH activation could be expected to stimulate pituitary release of adrenocorticotropic hormone (ACTH) and affect input to autonomic regulatory...

  8. Discovery of novel, high potent, ABC type PTP1B inhibitors with TCPTP selectivity and cellular activity.

    Science.gov (United States)

    Liu, Peihong; Du, Yongli; Song, Lianhua; Shen, Jingkang; Li, Qunyi

    2016-08-08

    Protein tyrosine phosphatase 1B (PTP1B) as a key negative regulator of both insulin and leptin receptor pathways has been an attractive therapeutic target for the treatment of type 2 diabetes mellitus (T2DM) and obesity. With the goal of enhancing potency and selectivity of the PTP1B inhibitors, a series of methyl salicylate derivatives as ABC type PTP1B inhibitors (P1-P7) were discovered. More importantly, compound P6 exhibited high potent inhibitory activity (IC50 = 50 nM) for PTP1B with 15-fold selectivity over T-cell PTPase (TCPTP). Further studies on cellular activities revealed that compound P6 could enhance insulin-mediated insulin receptor β (IRβ) phosphorylation and insulin-stimulated glucose uptake. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  9. Hyperglycemia- and hyperinsulinemia-induced insulin resistance causes alterations in cellular bioenergetics and activation of inflammatory signaling in lymphatic muscle.

    Science.gov (United States)

    Lee, Yang; Fluckey, James D; Chakraborty, Sanjukta; Muthuchamy, Mariappan

    2017-07-01

    Insulin resistance is a well-known risk factor for obesity, metabolic syndrome (MetSyn) and associated cardiovascular diseases, but its mechanisms are undefined in the lymphatics. Mesenteric lymphatic vessels from MetSyn or LPS-injected rats exhibited impaired intrinsic contractile activity and associated inflammatory changes. Hence, we hypothesized that insulin resistance in lymphatic muscle cells (LMCs) affects cell bioenergetics and signaling pathways that consequently alter contractility. LMCs were treated with different concentrations of insulin or glucose or both at various time points to determine insulin resistance. Onset of insulin resistance significantly impaired glucose uptake, mitochondrial function, oxygen consumption rates, glycolysis, lactic acid, and ATP production in LMCs. Hyperglycemia and hyperinsulinemia also impaired the PI3K/Akt while enhancing the ERK/p38MAPK/JNK pathways in LMCs. Increased NF-κB nuclear translocation and macrophage chemoattractant protein-1 and VCAM-1 levels in insulin-resistant LMCs indicated activation of inflammatory mechanisms. In addition, increased phosphorylation of myosin light chain-20, a key regulator of lymphatic muscle contraction, was observed in insulin-resistant LMCs. Therefore, our data elucidate the mechanisms of insulin resistance in LMCs and provide the first evidence that hyperglycemia and hyperinsulinemia promote insulin resistance and impair lymphatic contractile status by reducing glucose uptake, altering cellular metabolic pathways, and activating inflammatory signaling cascades.-Lee, Y., Fluckey, J. D., Chakraborty, S., Muthuchamy, M. Hyperglycemia- and hyperinsulinemia-induced insulin resistance causes alterations in cellular bioenergetics and activation of inflammatory signaling in lymphatic muscle. © FASEB.

  10. Defining the action spectrum of potential PGC-1α activators on a mitochondrial and cellular level in vivo.

    Science.gov (United States)

    Hofer, Annette; Noe, Natalie; Tischner, Christin; Kladt, Nikolay; Lellek, Veronika; Schauß, Astrid; Wenz, Tina

    2014-05-01

    Previous studies have demonstrated a therapeutic benefit of pharmaceutical PGC-1α activation in cellular and murine model of disorders linked to mitochondrial dysfunction. While in some cases, this effect seems to be clearly associated with boosting of mitochondrial function, additional alterations as well as tissue- and cell-type-specific effects might play an important role. We initiated a comprehensive analysis of the effects of potential PGC-1α-activating drugs and pharmaceutically targeted the PPAR (bezafibrate, rosiglitazone), AMPK (AICAR, metformin) and Sirt1 (resveratrol) pathways in HeLa cells, neuronal cells and PGC-1α-deficient MEFs to get insight into cell type specificity and PGC-1α dependence of their working action. We used bezafibrate as a model drug to assess the effect on a tissue-specific level in a murine model. Not all analyzed drugs activate the PGC pathway or alter mitochondrial protein levels. However, they all affect supramolecular assembly of OXPHOS complexes and OXPHOS protein stability. In addition, a clear drug- and cell-type-specific influence on several cellular stress pathways as well as on post-translational modifications could be demonstrated, which might be relevant to fully understand the action of the analyzed drugs in the disease state. Importantly, the effect on the activation of mitochondrial biogenesis and stress response program upon drug treatment is PGC-1α dependent in MEFs demonstrating not only the pleiotropic effects of this molecule but points also to the working mechanism of the analyzed drugs. The definition of the action spectrum of the different drugs forms the basis for a defect-specific compensation strategy and a future personalized therapeutic approach.

  11. Integrin specificity and enhanced cellular activities associated with surfaces presenting a recombinant fibronectin fragment compared to RGD supports.

    Science.gov (United States)

    Petrie, Timothy A; Capadona, Jeffrey R; Reyes, Catherine D; García, Andrés J

    2006-11-01

    Biomimetic strategies focusing on presenting short bioadhesive oligopeptides, including the arginine-glycine-aspartic acid (RGD) motif present in numerous adhesive proteins, on a non-fouling support have emerged as promising approaches to improve cellular activities and healing responses. Nevertheless, these bio-inspired strategies are limited by low activity of the oligopeptides compared to the native ligand due to the absence of complementary or modulatory domains. In the present analysis, we generated well-defined biointerfaces presenting RGD-based ligands of increasing complexity to directly compare their biological activities in terms of cell adhesion strength, integrin binding and signaling. Mixed self-assembled monolayers of alkanethiols on gold were optimized to engineer robust supports that present anchoring groups for ligand tethering within a non-fouling, protein adsorption-resistant background. Controlled bioadhesive interfaces were generated by tethering adhesive ligands via standard peptide chemistry. On a molar basis, biointerfaces functionalized with the FNIII7-10 recombinant fragment presenting the RGD and PHSRN adhesive motifs in the correct structural context exhibited significantly higher adhesion strength, FAK activation, and cell proliferation rate than supports presenting RGD ligand or RGD-PHSRN, an oligopeptide presenting these two sites separated by a polyglycine linker. Moreover, FNIII7-10-functionalized surfaces displayed specificity for alpha5beta1 integrin, while cell adhesion to supports presenting RGD or RGD-PHSRN was primarily mediated by alphavbeta3 integrin. These results are significant to the rational engineering of bioactive materials that convey integrin binding specificity for directed cellular and tissue responses in biomedical and biotechnological applications.

  12. Mapping social behavior-induced brain activation at cellular resolution in the mouse

    Science.gov (United States)

    Kim, Yongsoo; Venkataraju, Kannan Umadevi; Pradhan, Kith; Mende, Carolin; Taranda, Julian; Turaga, Srinivas C.; Arganda-Carreras, Ignacio; Ng, Lydia; Hawrylycz, Michael J.; Rockland, Kathleen; Seung, H. Sebastian; Osten, Pavel

    2014-01-01

    Understanding how brain activation mediates behaviors is a central goal of systems neuroscience. Here we apply an automated method for mapping brain activation in the mouse in order to probe how sex-specific social behaviors are represented in the male brain. Our method uses the immediate early gene c-fos, a marker of neuronal activation, visualized by serial two-photon tomography: the c-fos-GFP-positive neurons are computationally detected, their distribution is registered to a reference brain and a brain atlas, and their numbers are analyzed by statistical tests. Our results reveal distinct and shared female and male interaction-evoked patterns of male brain activation representing sex discrimination and social recognition. We also identify brain regions whose degree of activity correlates to specific features of social behaviors and estimate the total numbers and the densities of activated neurons per brain areas. Our study opens the door to automated screening of behavior-evoked brain activation in the mouse. PMID:25558063

  13. Ex vivo activity quantification in micrometastases at the cellular scale using the α-camera technique

    DEFF Research Database (Denmark)

    Chouin, Nicolas; Lindegren, Sture; Frost, Sofia H L

    2013-01-01

    Targeted α-therapy (TAT) appears to be an ideal therapeutic technique for eliminating malignant circulating, minimal residual, or micrometastatic cells. These types of malignancies are typically infraclinical, complicating the evaluation of potential treatments. This study presents a method of ex...... vivo activity quantification with an α-camera device, allowing measurement of the activity taken up by tumor cells in biologic structures a few tens of microns....

  14. Domain-Specific Activation of Death-Associated Intracellular Signalling Cascades by the Cellular Prion Protein in Neuroblastoma Cells.

    Science.gov (United States)

    Vilches, Silvia; Vergara, Cristina; Nicolás, Oriol; Mata, Ágata; Del Río, José A; Gavín, Rosalina

    2016-09-01

    The biological functions of the cellular prion protein remain poorly understood. In fact, numerous studies have aimed to determine specific functions for the different protein domains. Studies of cellular prion protein (PrP(C)) domains through in vivo expression of molecules carrying internal deletions in a mouse Prnp null background have provided helpful data on the implication of the protein in signalling cascades in affected neurons. Nevertheless, understanding of the mechanisms underlying the neurotoxicity induced by these PrP(C) deleted forms is far from complete. To better define the neurotoxic or neuroprotective potential of PrP(C) N-terminal domains, and to overcome the heterogeneity of results due to the lack of a standardized model, we used neuroblastoma cells to analyse the effects of overexpressing PrP(C) deleted forms. Results indicate that PrP(C) N-terminal deleted forms were properly processed through the secretory pathway. However, PrPΔF35 and PrPΔCD mutants led to death by different mechanisms sharing loss of alpha-cleavage and activation of caspase-3. Our data suggest that both gain-of-function and loss-of-function pathogenic mechanisms may be associated with N-terminal domains and may therefore contribute to neurotoxicity in prion disease. Dissecting the molecular response induced by PrPΔF35 may be the key to unravelling the physiological and pathological functions of the prion protein.

  15. UPF1 silenced cellular model systems for screening of read-through agents active on β039 thalassemia point mutation.

    Science.gov (United States)

    Salvatori, Francesca; Pappadà, Mariangela; Breveglieri, Giulia; D'Aversa, Elisabetta; Finotti, Alessia; Lampronti, Ilaria; Gambari, Roberto; Borgatti, Monica

    2018-05-15

    Nonsense mutations promote premature translational termination, introducing stop codons within the coding region of mRNAs and causing inherited diseases, including thalassemia. For instance, in β 0 39 thalassemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, ribosomal read-through molecules, such as aminoglycoside antibiotics, have been tested on mRNAs carrying premature stop codons. These findings have introduced new hopes for the development of a pharmacological approach to the β 0 39 thalassemia therapy. While several strategies, designed to enhance translational read-through, have been reported to inhibit NMD efficiency concomitantly, experimental tools for systematic analysis of mammalian NMD inhibition by translational read-through are lacking. We developed a human cellular model of the β 0 39 thalassemia mutation with UPF-1 suppressed and showing a partial NMD suppression. This novel cellular model could be used for the screening of molecules exhibiting preferential read-through activity allowing a great rescue of the mutated transcripts.

  16. Activity against Mycobacterium tuberculosis with concomitant induction of cellular immune responses by a tetraaza-macrocycle with acetate pendant arms.

    Science.gov (United States)

    David, S; Ordway, D; Arroz, M J; Costa, J; Delgado, R

    2001-01-01

    The novel tetraaza-macrocyclic compound 3,7,11-tris(carboxymethyl)-3,7,11,17-tetraaza-bicyclo[11.3.1]heptadeca-1(17),13,15-triene, abbreviated as ac3py14, was investigated for its activity against Mycobacterium tuberculosis and for induction of protective cellular immune responses. Perspective results show that ac3py14 and its Fe3+ 1:1 complex, [Fe(ac3py14)], inhibited radiometric growth of several strains of M. tuberculosis. Inhibition with 25 microg/mL varied from 99% for H37Rv to 80% and above for multiple drug-resistant clinical isolates. The capacity of ac3py14 to elicit a beneficial immune response without cellular apoptosis was assessed and compared to the effects of virulent M. tuberculosis. The present study produces evidence that after stimulation with ac3py14 there was significant production of interferon gamma (IFN-gamma), whereas the production of interleukin-5 (IL-5) remained low, and there was development of a memory population (CD45RO). The level of binding of Annexin V, a marker of apoptosis, was not sufficient to result in toxic effects toward alphabeta and gammadelta T cells and CD14+ macrophages. This preliminary study is the first report of a compound that simultaneously exerts an inhibitory effect against M. tuberculosis and induces factors associated with protective immune responses.

  17. Abalone Protein Hydrolysates: Preparation, Angiotensin I Converting Enzyme Inhibition and Cellular Antioxidant Activity.

    Science.gov (United States)

    Park, Soo Yeon; Je, Jae-Young; Hwang, Joung-Youl; Ahn, Chang-Bum

    2015-09-01

    Abalone protein was hydrolyzed by enzymatic hydrolysis and the optimal enzyme/substrate (E/S) ratios were determined. Abalone protein hydrolysates (APH) produced by Protamex at E/S ratio of 1:100 showed angiotensin I converting enzyme inhibitory activity with IC50 of 0.46 mg/mL, and APH obtained by Flavourzyme at E/S ratio of 1:100 possessed the oxygen radical absorbance capacity value of 457.6 μM trolox equivalent/mg sample. Flavourzyme abalone protein hydrolysates (FAPH) also exhibited H2O2 scavenging activity with IC50 of 0.48 mg/mL and Fe(2+) chelating activity with IC50 of 2.26 mg/mL as well as high reducing power. FAPH significantly (P<0.05) protected H2O2-induced hepatic cell damage in cultured hepatocytes, and the cell viability was restored to 90.27% in the presence of FAPH. FAPH exhibited 46.20% intracellular ROS scavenging activity and 57.89% lipid peroxidation inhibition activity in cultured hepatocytes. Overall, APH may be useful as an ingredient for functional foods.

  18. Interplay between cellular activity and three-dimensional scaffold-cell constructs with different foam structure processed by electron beam melting.

    Science.gov (United States)

    Nune, Krishna C; Misra, R Devesh K; Gaytan, Sara M; Murr, Lawrence E

    2015-05-01

    The cellular activity, biological response, and consequent integration of scaffold-cell construct in the physiological system are governed by the ability of cells to adhere, proliferate, and biomineralize. In this regard, we combine cellular biology and materials science and engineering to fundamentally elucidate the interplay between cellular activity and interconnected three-dimensional foamed architecture obtained by a novel process of electron beam melting and computational tools. Furthermore, the organization of key proteins, notably, actin, vinclulin, and fibronectin, involved in cellular activity and biological functions and relationship with the structure was explored. The interconnected foamed structure with ligaments was favorable to cellular activity that includes cell attachment, proliferation, and differentiation. The primary rationale for favorable modulation of cellular functions is that the foamed structure provided a channel for migration and communication between cells leading to highly mineralized extracellular matrix (ECM) by the differentiating osteoblasts. The filopodial interaction amongst cells on the ligaments was a governing factor in the secretion of ECM, with consequent influence on maturation and mineralization. © 2014 Wiley Periodicals, Inc.

  19. Regulation of Ras exchange factors and cellular localization of Ras activation by lipid messengers in T cells

    Directory of Open Access Journals (Sweden)

    Jesse E. Jun

    2013-09-01

    Full Text Available The Ras-MAPK signaling pathway is highly conserved throughout evolution and is activated downstream of a wide range of receptor stimuli. Ras guanine nucleotide exchange factors (RasGEFs catalyze GTP loading of Ras and play a pivotal role in regulating receptor-ligand induced Ras activity. In T cells, three families of functionally important RasGEFs are expressed: RasGRF, RasGRP, and SOS-family GEFs.Early on it was recognized that Ras activation is critical for T cell development and that the RasGEFs play an important role herein. More recent work has revealed that nuances in Ras activation appear to significantly impact T cell development and selection. These nuances include distinct biochemical patterns of analog versus digital Ras activation, differences in cellular localization of Ras activation, and intricate interplays between the RasGEFs during distinct T cell developmental stages as revealed by various new mouse models. In many instances, the exact nature of these nuances in Ras activation or how these may result from fine-tuning of the RasGEFs is not understood.One large group of biomolecules critically involved in the control of Ras-GEFs´functions are lipid second messengers. Multiple, yet distinct lipid products are generated following T cell receptor (TCR stimulation and bind to different domains in the RasGRP and SOS RasGEFs to facilitate the activation of the membrane-anchored Ras GTPases. In this review we highlight how different lipid-based elements are generated by various enzymes downstream of the TCR and other receptors and how these dynamic and interrelated lipid products may fine-tune Ras activation by RasGEFs in developing T cells.

  20. Preterm labor--modeling the uterine electrical activity from cellular level to surface recording.

    Science.gov (United States)

    Rihana, S; Marque, C

    2008-01-01

    Uterine electrical activity is correlated to the appearance of uterine contractions. forceful contractions appear at the end of term. Therefore, understanding the genesis and the propagation of uterine electrical activity may provide an efficient tool to diagnose preterm labor. Moreover, the control of uterine excitability seems to have important consequences in the control of preterm labor. Modeling the electrical activity in uterine tissue is thus an important step in understanding physiological uterine contractile mechanisms and to permit uterine EMG simulation. Our model presented in this paper, incorporates ion channel models at the cell level, the reaction diffusion equations at the tissue level and the spatiotemporal integration at the uterine EMG reconstructed level. This model validates some key physiological observation hypotheses concerning uterine excitability and propagation.

  1. Vaginal Fornix Discharge Cellularity and Its Leukocyte Esterase Activity for Diagnosis of Endometritis in Dairy Cows

    Directory of Open Access Journals (Sweden)

    Abolfazl HAJIBEMANI

    2016-01-01

    Full Text Available The objective of the present study was to evaluate the application of some strip test markers (i.e., leukocyte esterase (LE activity, protein, nitrate and pH for diagnosis of endometritis in dairy cows using vaginal fornix discharge. Also, the total white blood cell count (t-WBC/l of this secretion and degenerative changes of neutrophils in cervical cytology were used as alternative methods to predict progression of the endometritis severity. Holstein cows (n=215 between 30-40 days in milk (DIM were included and examined. Giemsa-stained smear was prepared from cervical mucus. Cervical cytology test was considered as reference screening method for the detection of subclinical endometritis. The LE activity and t-WBC in the vaginal fornix discharge of subclinical endometritis cows were significantly higher than those from healthy cows. Sensitivity and specificity were 78% and 73% for LE10 activity (10 minutes after contacting with discharges and 60% and 69% for t-WBC (cut off point=210 cells/l for diagnosis of subclinical endometritis, respectively. There was a good agreement between LE10 activity, t-WBC and cervical cytology test with a Kappa coefficient of 0.4 and 0.42, respectively (P<0.0001. Total WBC count in discharge and degenerative neutrophils (DN percentages increase simultaneously with the degree and severity of endometritis. There was a highly significant (P<0.01 correlation between t-WBC and some reagent strip test markers (LE activity, protein and nitrate in clear discharge of studied cows. In conclusion, the present results suggest the LE activity and t-WBC in vaginal fornix discharge could be used as non-invasive reliable and valid methods for screening of subclinical endometritis in postpartum dairy herds.

  2. State-dependent cellular activity patterns of the cat paraventricular hypothalamus measured by reflectance imaging

    DEFF Research Database (Denmark)

    Kristensen, Morten Pilgaard; Rector, D M; Poe, G R

    1996-01-01

    Activity within the cat paraventricular hypothalamus (PVH) during sleep and waking states was measured by quantifying intrinsic tissue reflectivity. A fiber optic probe consisting of a 1.0 mm coherent image conduit, surrounded by plastic fibers which conducted 660 nm source light, was attached...... to a charge-coupled device camera, and positioned over the PVH in five cats. Electrodes for assessing state variables, including electroencephalographic activity, eye movement, and somatic muscle tone were also placed. After surgical recovery, reflected light intensity was measured continuously at 2.5 Hz...

  3. Arginase-II Promotes Tumor Necrosis Factor-α Release From Pancreatic Acinar Cells Causing β-Cell Apoptosis in Aging.

    Science.gov (United States)

    Xiong, Yuyan; Yepuri, Gautham; Necetin, Sevil; Montani, Jean-Pierre; Ming, Xiu-Fen; Yang, Zhihong

    2017-06-01

    Aging is associated with glucose intolerance. Arginase-II (Arg-II), the type-II L -arginine-ureahydrolase, is highly expressed in pancreas. However, its role in regulation of pancreatic β-cell function is not known. Here we show that female (not male) mice deficient in Arg-II (Arg-II -/- ) are protected from age-associated glucose intolerance and reveal greater glucose induced-insulin release, larger islet size and β-cell mass, and more proliferative and less apoptotic β-cells compared with the age-matched wild-type (WT) controls. Moreover, Arg-II is mainly expressed in acinar cells and is upregulated with aging, which enhances p38 mitogen-activated protein kinase (p38 MAPK) activation and release of tumor necrosis factor-α (TNF-α). Accordingly, conditioned medium of isolated acinar cells from old WT (not Arg-II -/- ) mice contains higher TNF-α levels than the young mice and stimulates β-cell apoptosis and dysfunction, which are prevented by a neutralizing anti-TNF-α antibody. In acinar cells, our study demonstrates an age-associated Arg-II upregulation, which promotes TNF-α release through p38 MAPK leading to β-cell apoptosis, insufficient insulin secretion, and glucose intolerance in female rather than male mice. © 2017 by the American Diabetes Association.

  4. Comparison of the free and bound phenolic profiles and cellular antioxidant activities of litchi pulp extracts from different solvents

    Science.gov (United States)

    2014-01-01

    Background The phenolic contents and antioxidant activities of fruits could be underestimated if the bound phenolic compounds are not considered. In the present study, the extraction efficiencies of various solvents were investigated in terms of the total content of the free and bound phenolic compounds, as well as the phenolic profiles and antioxidant activities of the extracts. Methods Five different solvent mixtures were used to extract the free phenolic compounds from litchi pulp. Alkaline and acidic hydrolysis methods were compared for the hydrolysis of bound phenolic compounds from litchi pulp residue. The phenolic compositions of the free and bound fractions from the litchi pulp were identified using HPLC-DAD. The antioxidant activities of the litchi pulp extracts were determined by oxygen radical absorbance capacity (ORAC) and cellular antioxidant activity (CAA) assays. Results Of the solvents tested, aqueous acetone extracted the largest amount of total free phenolic compounds (210.7 mg GAE/100 g FW) from litchi pulp, followed sequentially by aqueous mixtures of methanol, ethanol and ethyl acetate, and water itself. The acid hydrolysis method released twice as many bound phenolic compounds as the alkaline hydrolysis method. Nine phenolic compounds were detected in the aqueous acetone extract. In contrast, not all of these compounds were found in the other four extracts. The classification and content of the bound phenolic compounds released by the acid hydrolysis method were higher than those achieved by the alkaline hydrolysis. The aqueous acetone extract showing the highest ORAC value (3406.9 μmol TE/100 g FW) for the free phenolic extracts. For the CAA method, however, the aqueous acetone and methanol extracts (56.7 and 55.1 μmol QE/100 g FW) showed the highest levels of activity of the five extracts tested. The ORAC and CAA values of the bound phenolic compounds obtained by acid hydrolysis were 2.6- and 1.9-fold higher than those obtained using the

  5. Control of ADAM17 activity by regulation of its cellular localisation

    Science.gov (United States)

    Lorenzen, Inken; Lokau, Juliane; Korpys, Yvonne; Oldefest, Mirja; Flynn, Charlotte M.; Künzel, Ulrike; Garbers, Christoph; Freeman, Matthew; Grötzinger, Joachim; Düsterhöft, Stefan

    2016-01-01

    An important, irreversible step in many signalling pathways is the shedding of membrane-anchored proteins. A Disintegrin And Metalloproteinase (ADAM) 17 is one of the major sheddases involved in a variety of physiological and pathophysiological processes including regeneration, differentiation, and cancer progression. This central role in signalling implies that ADAM17 activity has to be tightly regulated, including at the level of localisation. Most mature ADAM17 is localised intracellularly, with only a small amount at the cell surface. We found that ADAM17 is constitutively internalised by clathrin-coated pits and that physiological stimulators such as GPCR ligands induce ADAM17-mediated shedding, but do not alter the cell-surface abundance of the protease. In contrast, the PKC-activating phorbol ester PMA, often used as a strong inducer of ADAM17, causes not only proteolysis by ADAM17 but also a rapid increase of the mature protease at the cell surface. This is followed by internalisation and subsequent degradation of the protease. Eventually, this leads to a substantial downregulation of mature ADAM17. Our results therefore imply that physiological activation of ADAM17 does not rely on its relocalisation, but that PMA-induced PKC activity drastically dysregulates the localisation of ADAM17. PMID:27731361

  6. Role of Mitochondrial Reactive Oxygen Species in the Activation of Cellular Signals, Molecules, and Function

    DEFF Research Database (Denmark)

    Indo, Hiroko P.; Hawkins, Clare L; Nakanishi, Ikuo

    2017-01-01

    -κB) and GATA signaling pathways. We have also reviewed the effects of ROS on the activation of MMP and HIF. There is significant evidence to support the hypothesis that mitochondrial superoxide can initiate signaling pathways following transport into the cytosol. In this study, we provide evidence of TATA...

  7. Ancient cellular structures and modern humans: change of survival strategies before prolonged low solar activity period

    Science.gov (United States)

    Ragulskaya, Mariya; Rudenchik, Evgeniy; Gromozova, Elena; Voychuk, Sergei; Kachur, Tatiana

    The study of biotropic effects of modern space weather carries the information about the rhythms and features of adaptation of early biological systems to the outer space influence. The influence of cosmic rays, ultraviolet waves and geomagnetic field on early life has its signs in modern biosphere processes. These phenomena could be experimentally studied on present-day biological objects. Particularly inorganic polyphosphates, so-called "fossil molecules", attracts special attention as the most ancient molecules which arose in inanimate nature and have been accompanying biological objects at all stages of evolution. Polyphosphates-containing graves of yeast's cells of Saccharomyces cerevisiae strain Y-517, , from the Ukrainian Collection of Microorganisms was studied by daily measurements during 2000-2013 years. The IZMIRAN daily data base of physiological parameters dynamics during 2000-2013 years were analyzed simultaneously (25 people). The analysis showed significant simultaneous changes of the statistical parameters of the studied biological systems in 2004 -2006. The similarity of simultaneous changes of adaptation strategies of human organism and the cell structures of Saccharomyces cerevisiae during the 23-24 cycles of solar activity are discussed. This phenomenon could be due to a replacement of bio-effective parameters of space weather during the change from 23rd to 24th solar activity cycle and nonstandard geophysical peculiarities of the 24th solar activity cycle. It could be suggested that the observed similarity arose as the optimization of evolution selection of the living systems in expectation of probable prolonged period of low solar activity (4-6 cycles of solar activity).

  8. Immune activation alters cellular and humoral responses to yellow fever 17D vaccine.

    Science.gov (United States)

    Muyanja, Enoch; Ssemaganda, Aloysius; Ngauv, Pearline; Cubas, Rafael; Perrin, Helene; Srinivasan, Divya; Canderan, Glenda; Lawson, Benton; Kopycinski, Jakub; Graham, Amanda S; Rowe, Dawne K; Smith, Michaela J; Isern, Sharon; Michael, Scott; Silvestri, Guido; Vanderford, Thomas H; Castro, Erika; Pantaleo, Giuseppe; Singer, Joel; Gillmour, Jill; Kiwanuka, Noah; Nanvubya, Annet; Schmidt, Claudia; Birungi, Josephine; Cox, Josephine; Haddad, Elias K; Kaleebu, Pontiano; Fast, Patricia; Sekaly, Rafick-Pierre; Trautmann, Lydie; Gaucher, Denis

    2014-07-01

    Defining the parameters that modulate vaccine responses in African populations will be imperative to design effective vaccines for protection against HIV, malaria, tuberculosis, and dengue virus infections. This study aimed to evaluate the contribution of the patient-specific immune microenvironment to the response to the licensed yellow fever vaccine 17D (YF-17D) in an African cohort. We compared responses to YF-17D in 50 volunteers in Entebbe, Uganda, and 50 volunteers in Lausanne, Switzerland. We measured the CD8+ T cell and B cell responses induced by YF-17D and correlated them with immune parameters analyzed by flow cytometry prior to vaccination. We showed that YF-17D-induced CD8+ T cell and B cell responses were substantially lower in immunized individuals from Entebbe compared with immunized individuals from Lausanne. The impaired vaccine response in the Entebbe cohort associated with reduced YF-17D replication. Prior to vaccination, we observed higher frequencies of exhausted and activated NK cells, differentiated T and B cell subsets and proinflammatory monocytes, suggesting an activated immune microenvironment in the Entebbe volunteers. Interestingly, activation of CD8+ T cells and B cells as well as proinflammatory monocytes at baseline negatively correlated with YF-17D-neutralizing antibody titers after vaccination. Additionally, memory T and B cell responses in preimmunized volunteers exhibited reduced persistence in the Entebbe cohort but were boosted by a second vaccination. Together, these results demonstrate that an activated immune microenvironment prior to vaccination impedes efficacy of the YF-17D vaccine in an African cohort and suggest that vaccine regimens may need to be boosted in African populations to achieve efficient immunity. Registration is not required for observational studies. This study was funded by Canada's Global Health Research Initiative, Defense Threat Reduction Agency, National Institute of Allergy and Infectious Diseases

  9. (S)Pot on Mitochondria: Cannabinoids Disrupt Cellular Respiration to Limit Neuronal Activity.

    Science.gov (United States)

    Harkany, Tibor; Horvath, Tamas L

    2017-01-10

    Classical views posit G protein-coupled cannabinoid receptor 1s (CB1Rs) at the cell surface with cytosolic Giα-mediated signal transduction. Hebert-Chatelain et al. (2016) instead place CB 1 Rs at mitochondria limiting neuronal respiration by soluble adenylyl cyclase-dependent modulation of complex I activity. Thus, neuronal bioenergetics link to synaptic plasticity and, globally, learning and memory. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Relationship between complement activation, cellular uptake and surface physicochemical aspects of novel PEG-modifed nanocapsules.

    OpenAIRE

    Mosqueira, Vanessa Carla Furtado; Legrand, Philippe; Gulik, Annette; Bourdon, Olivier; Gref, Ruxandra; Labarre, Denis; Barratt, Gillian

    2001-01-01

    ABSTRACT: The aim of our work was to examine the relationship between modi"cations of the surface of nanocapsules (NC) by adsorption or covalent grafting of poly(ethylene oxide) (PEG), and changes in their phospholipid (PL) content on complement activation (C3 cleavage) and on uptake by macrophages. The physicochemical characterization of the NC included an investigation of their properties, such as surface charge, size, hydrophilicity, morphology and homogeneity. This is the "rst ti...

  11. Cellular gravity

    NARCIS (Netherlands)

    F.C. Gruau; J.T. Tromp (John)

    1999-01-01

    textabstractWe consider the problem of establishing gravity in cellular automata. In particular, when cellular automata states can be partitioned into empty, particle, and wall types, with the latter enclosing rectangular areas, we desire rules that will make the particles fall down and pile up on

  12. Relationship between complement activation, cellular uptake and surface physicochemical aspects of novel PEG-modified nanocapsules.

    Science.gov (United States)

    Mosqueira, V C; Legrand, P; Gulik, A; Bourdon, O; Gref, R; Labarre, D; Barratt, G

    2001-11-01

    The aim of our work was to examine the relationship between modifications of the surface of nanocapsules (NC) by adsorption or covalent grafting of poly(ethylene oxide) (PEG), and changes in their phospholipid (PL) content on complement activation (C3 cleavage) and on uptake by macrophages. The physicochemical characterization of the NC included an investigation of their properties, such as surface charge, size, hydrophilicity, morphology and homogeneity. This is the first time that such properties have been correlated with biological interactions for NC, a novel carrier system with a structure more complex than nanospheres. C3 crossed immunoelectrophoresis revealed the reduced activation for NC with longer PEG chain and higher density, although all formulations induced C3 cleavage to a lesser or greater extent. NC bearing PEG covalently bound to the surface were weaker activators of complement than plain PLA [poly(D,L-lactide)] NC or nanospheres (NS). Furthermore, the fluorescent/confocal microscopy of J774A1 cells in contact with NC reveal a dramatically reduced interaction with PEG-bearing NC. However, the way in which PEG was attached (covalent or adsorbed) seemed to affect the mechanism of uptake. Taken together, these results suggest that the low level of protein binding to NC covered with a high density of 20kDa PEG chains is likely to be due to the steric barriers surrounding these particles, which prevents protein adsorption and reduces their interaction with macrophages.

  13. [Cellular mechanism of the generation of spontaneous activity in gastric muscle].

    Science.gov (United States)

    Nakamura, Eri; Kito, Yoshihiko; Fukuta, Hiroyasu; Yanai, Yoshimasa; Hashitani, Hikaru; Yamamoto, Yoshimichi; Suzuki, Hikaru

    2004-03-01

    In gastric smooth muscles, interstitial cells of Cajal (ICC) might be the pacemaker cells of spontaneous activities since ICC are rich in mitochondria and are connected with smooth muscle cells via gap junctions. Several types of ICC are distributed widely in the stomach wall. A group of ICC distributed in the myenteric layer (ICC-MY) were the pacemaker cells of gastrointestinal smooth muscles. Pacemaker potentials were generated in ICC-MY, and the potentials were conducted to circular smooth muscles to trigger slow waves and also conducted to longitudinal muscles to form follower potentials. In circular muscle preparations, interstitial cells distributed within muscle bundles (ICC-IM) produced unitary potentials, which were conducted to circular muscles to form slow potentials by summation. In mutant mice lacking inositol trisphosphate (IP(3)) receptor, slow waves were absent in gastric smooth muscles. The generation of spontaneous activity was impaired by the inhibition of Ca(2+)-release from internal stores through IP(3) receptors, inhibition of mitochondrial Ca(2+)-handling with proton pump inhibitors, and inhibition of ATP-sensitive K(+)-channels at the mitochondrial inner membrane. These results suggested that mitochondrial Ca(2+)-handling causes the generation of spontaneous activity in pacemaker cells. Possible involvement of protein kinase C (PKC) in the Ca(2+) signaling system was also suggested.

  14. Cellular immune responses and phagocytic activity of fishes exposed to pollution of volcano mud.

    Science.gov (United States)

    Risjani, Yenny; Yunianta; Couteau, Jerome; Minier, Christophe

    2014-05-01

    Since May 29, 2006, a mud volcano in the Brantas Delta of the Sidoarjo district has emitted mud that has inundated nearby villages. Pollution in this area has been implicated in detrimental effects on fish health. In fishes, leukocyte and phagocytic cells play a vital role in body defenses. We report for the first time the effect of "LUSI" volcano mud on the immune systems of fish in the Brantas Delta. The aim of this study was to find biomarkers to allow the evaluation of the effects of volcanic mud and anthropogenic pollution on fish health in the Brantas Delta. The study took places at the Brantas Delta, which was polluted by volcano mud, and at reference sites in Karangkates and Pasuruan. Leukocyte numbers were determined using a Neubauer hemocytometer and a light microscope. Differential leukocyte counts were determined using blood smears stained with May Grunwald-Giemsa, providing neutrophil, lymphocyte and monocyte counts. Macrophages were taken from fish kidney, and their phagocytic activity was measured. In vitro analyses revealed that leukocyte and differential leukocyte counts (DLC) were higher in Channa striata and Chanos chanos caught from the polluted area. Macrophage numbers were higher in Oreochromis mossambicus than in the other species, indicating that this species is more sensitive to pollution. In areas close to volcanic mud eruption, all specimens had lower phagocytic activity. Our results show that immune cells were changed and phagocytic activity was reduced in the polluted area indicating cytotoxicity and alteration of the innate immune system in fishes exposed to LUSI volcano mud and anthropogenic pollution. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Estimation of cellular manufacturing cost components using simulation and activity-based costing

    OpenAIRE

    Paul Savory; Robert Williams

    2010-01-01

    It can be difficult estimating all of the cost components that are attributed to a machined part.  This problem is more pronounced when a factory uses group technology manufacturing cells as opposed to a functional or process layout of a job shop.  This paper describes how activity-based costing (ABC) concepts can be integrated into a discrete-event simulation model of a U-shaped manufacturing cell producing a part family with four members.  The simulation model generates detai...

  16. Estimation of cellular manufacturing cost components using simulation and activity-based costing

    OpenAIRE

    Savory, Paul

    2010-01-01

    It can be difficult estimating all of the cost components that are attributed to a machined part. This problem is more pronounced when a factory uses group technology manufacturing cells as opposed to a functional or process layout of a job shop. This paper describes how activity-based costing (ABC) concepts can be integrated into a discrete-event simulation model of a U-shaped manufacturing cell producing a part family with four members. The simulation model generates detailed Bills of Ac...

  17. Use of Cellular Decapping Activators by Positive-Strand RNA Viruses

    Directory of Open Access Journals (Sweden)

    Jennifer Jungfleisch

    2016-12-01

    Full Text Available Positive-strand RNA viruses have evolved multiple strategies to not only circumvent the hostile decay machinery but to trick it into being a priceless collaborator supporting viral RNA translation and replication. In this review, we describe the versatile interaction of positive-strand RNA viruses and the 5′-3′ mRNA decay machinery with a focus on the viral subversion of decapping activators. This highly conserved viral trickery is exemplified with the plant Brome mosaic virus, the animal Flock house virus and the human hepatitis C virus.

  18. 17-AAG inhibits vemurafenib-associated MAP kinase activation and is synergistic with cellular immunotherapy in a murine melanoma model.

    Science.gov (United States)

    Joshi, Sandeep S; Jiang, Shunlin; Unni, Emmanual; Goding, Stephen R; Fan, Tao; Antony, Paul A; Hornyak, Thomas J

    2018-01-01

    Heat shock protein 90 (HSP90) is a molecular chaperone which stabilizes client proteins with important roles in tumor growth. 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90 ATPase activity, occupies the ATP binding site of HSP90 causing a conformational change which destabilizes client proteins and directs them towards proteosomal degradation. Malignant melanomas have active RAF-MEK-ERK signaling which can occur either through an activating mutation in BRAF (BRAFV600E) or through activation of signal transduction upstream of BRAF. Prior work showed that 17-AAG inhibits cell growth in BRAFV600E and BRAF wildtype (BRAFWT) melanomas, although there were conflicting reports about the dependence of BRAFV600E and BRAFWT upon HSP90 activity for stability. Here, we demonstrate that BRAFWT and CRAF are bound by HSP90 in BRAFWT, NRAS mutant melanoma cells. HSP90 inhibition by 17-AAG inhibits ERK signaling and cell growth by destabilizing CRAF but not BRAFWT in the majority of NRAS mutant melanoma cells. The highly-selective BRAFV600E inhibitor, PLX4032 (vemurafenib), inhibits ERK signaling and cell growth in mutant BRAF melanoma cells, but paradoxically enhances signaling in cells with wild-type BRAF. In our study, we examined whether 17-AAG could inhibit PLX4032-enhanced ERK signaling in BRAFWT melanoma cells. As expected, PLX4032 alone enhanced ERK signaling in the BRAFWT melanoma cell lines Mel-Juso, SK-Mel-2, and SK-Mel-30, and inhibited signaling and cell growth in BRAFV600E A375 cells. However, HSP90 inhibition by 17-AAG inhibited PLX4032-enhanced ERK signaling and inhibited cell growth by destabilizing CRAF. Surprisingly, 17-AAG also stimulated melanin production in SK-Mel-30 cells and enhanced TYRP1 and DCT expression without stimulating TYR production in all three BRAFWT cell lines studied as well as in B16F10 mouse melanoma cells. In vivo, the combination of 17-AAG and cellular immunotherapy directed against Tyrp1 enhanced the inhibition of

  19. 17-AAG inhibits vemurafenib-associated MAP kinase activation and is synergistic with cellular immunotherapy in a murine melanoma model

    Science.gov (United States)

    Unni, Emmanual; Goding, Stephen R.; Fan, Tao; Antony, Paul A.; Hornyak, Thomas J.

    2018-01-01

    Heat shock protein 90 (HSP90) is a molecular chaperone which stabilizes client proteins with important roles in tumor growth. 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90 ATPase activity, occupies the ATP binding site of HSP90 causing a conformational change which destabilizes client proteins and directs them towards proteosomal degradation. Malignant melanomas have active RAF-MEK-ERK signaling which can occur either through an activating mutation in BRAF (BRAFV600E) or through activation of signal transduction upstream of BRAF. Prior work showed that 17-AAG inhibits cell growth in BRAFV600E and BRAF wildtype (BRAFWT) melanomas, although there were conflicting reports about the dependence of BRAFV600E and BRAFWT upon HSP90 activity for stability. Here, we demonstrate that BRAFWT and CRAF are bound by HSP90 in BRAFWT, NRAS mutant melanoma cells. HSP90 inhibition by 17-AAG inhibits ERK signaling and cell growth by destabilizing CRAF but not BRAFWT in the majority of NRAS mutant melanoma cells. The highly-selective BRAFV600E inhibitor, PLX4032 (vemurafenib), inhibits ERK signaling and cell growth in mutant BRAF melanoma cells, but paradoxically enhances signaling in cells with wild-type BRAF. In our study, we examined whether 17-AAG could inhibit PLX4032-enhanced ERK signaling in BRAFWT melanoma cells. As expected, PLX4032 alone enhanced ERK signaling in the BRAFWT melanoma cell lines Mel-Juso, SK-Mel-2, and SK-Mel-30, and inhibited signaling and cell growth in BRAFV600E A375 cells. However, HSP90 inhibition by 17-AAG inhibited PLX4032-enhanced ERK signaling and inhibited cell growth by destabilizing CRAF. Surprisingly, 17-AAG also stimulated melanin production in SK-Mel-30 cells and enhanced TYRP1 and DCT expression without stimulating TYR production in all three BRAFWT cell lines studied as well as in B16F10 mouse melanoma cells. In vivo, the combination of 17-AAG and cellular immunotherapy directed against Tyrp1 enhanced the inhibition of

  20. Active subsurface cellular function in the Baltic Sea Basin, IODP Exp 347

    Science.gov (United States)

    Reese, B. K.; Zinke, L. A.; Bird, J. T.; Lloyd, K. G.; Marshall, I.; Amend, J.; Jørgensen, B. B.

    2016-12-01

    The Baltic Sea Basin is a unique depositional setting that has experienced periods of glaciation and deglaciation as a result of global temperature fluctuations over the course of several hundred thousand years. This has resulted in laminated sediments formed during periods with strong permanent salinity stratification. The high sedimentation rates (100-500 cm/1000 y) make this an ideal setting to understand the microbial structure of a deep biosphere community in a high-organic matter environment. The responses of deep sediment microbial communities to variations in conditions during and after deposition are poorly understood. Samples were collected through scientific drilling during the International Ocean Discovery Program (IODP) Expedition 347 on board the Greatship Manisha, September-November 2013. We examined the active microbial community structure using the 16S rRNA gene transcript and active functional genes through metatranscriptome sequencing. Major biogeochemical shifts have been observed in response to the depositional history between the limnic, brackish, and marine phases. The microbial community structure in the BSB is diverse and reflective of the unique changes in the geochemical profile. These data further define the existence life in the deep subsurface and the survival mechanisms required for this extreme environment.

  1. Antiarrhythmic effect of IKr activation in a cellular model of LQT3

    DEFF Research Database (Denmark)

    Diness, Jonas Goldin; Hansen, Rie Schultz; Nissen, Jakob Dahl

    2008-01-01

    . Application of ATX-II (10 nM) was proarrhythmic, causing a profound increase of APD(90) as well as early afterdepolarizations and increased beat-to-beat variability. Two independent I(Kr) activators attenuated the proarrhythmic effects of ATX-II. NS3623 did not affect the late sodium current (I......BACKGROUND: Long QT syndrome type 3 (LQT3) is an inherited cardiac disorder caused by gain-of-function mutations in the cardiac voltage-gated sodium channel, Na(v)1.5. LQT3 is associated with the polymorphic ventricular tachycardia torsades de pointes (TdP), which can lead to syncope and sudden...... cardiac death. The sea anemone toxin ATX-II has been shown to inhibit the inactivation of Na(v)1.5, thereby closely mimicking the underlying cause of LQT3 in patients. OBJECTIVE: The hypothesis for this study was that activation of the I(Kr) current could counteract the proarrhythmic effects of ATX...

  2. Ethanol alters cellular activation and CD14 partitioning in lipid rafts

    International Nuclear Information System (INIS)

    Dai Qun; Zhang Jun; Pruett, Stephen B.

    2005-01-01

    Alcohol consumption interferes with innate immunity. In vivo EtOH administration suppresses cytokine responses induced through Toll-like receptor 4 (TLR4) and inhibits TLR4 signaling. Actually, EtOH exhibits a generalized suppressive effect on signaling and cytokine responses induced by through most TLRs. However, the underlying mechanism remains unknown. RAW264.7 cells were treated with LPS or co-treated with EtOH or with lipid raft-disrupting drugs. TNF-α production, IRAK-1 activation, and CD14 partition were evaluated. EtOH or nystatin, a lipid raft-disrupting drug, suppressed LPS-induced production of TNF-α. The suppressive effect of EtOH on LPS-induced TNF-α production was additive with that of methyl-β-cyclodextrin (MCD), another lipid raft-disrupting drug. EtOH interfered with IRAK-1 activation, an early TLR4 intracellular signaling event. Cell fractionation analyses show that acute EtOH altered LPS-related partition of CD14, a critical component of the LPS receptor complex. These results suggest a novel mechanism of EtOH action that involves interference with lipid raft clustering induced by LPS. This membrane action of EtOH might be one of the mechanisms by which EtOH acts as a generalized suppressor for TLR signaling

  3. Biocompatibility index of antiseptic agents by parallel assessment of antimicrobial activity and cellular cytotoxicity.

    Science.gov (United States)

    Müller, Gerald; Kramer, Axel

    2008-06-01

    To assess the suitability of an antiseptic agent, both the microbicidal activity and the cytotoxic effect must be taken into consideration to derive biocompatible antibacterial agents. We defined the biocompatibility index (BI) by measuring the antibacterial activity against the test organisms Escherichia coli and Staphylococcus aureus and, in parallel, the cytotoxicity on cultured murine fibroblasts. The antiseptic agents tested were benzalkonium chloride (BAC), cetylpyridinium chloride (CPC), chlorhexidine digluconate (CHX), mild silver protein (MSP), octenidine dihydrochloride (OCT), polyhexamethylene biguanide (PHMB), povidone iodine in solution [PVP-I(s)], povidone iodine in ointment [PVP-I(o)], silver nitrate (AgNO(3)), silver (I) sulfadiazine (SSD) and triclosan (TRI). Assays were carried out for 30 min of exposure at 37 degrees C in the presence of cell culture medium containing 10% fetal bovine serum. The resulting dimensionless BI was defined as the ratio of the concentration at which 50% of the murine fibroblasts are damaged and the microbicidal effect producing at least 3 log(10) (99.9%) reduction. The resulting rank ordering of BI for the ratio of fibroblast cytotoxicity to E. coli toxicity was OCT > PHMB > CHX > PVP-I(o) > PVP-I(s) > BAC > CPC > TRI > MSP and that to S. aureus was OCT > PHMB > CHX > CPC > PVP-I(o) > BAC > PVP(s) > TRI > MSP. OCT and PHMB were the most suitable agents with a BI greater than 1. The BI presented may be a useful tool to evaluate antiseptic agents for use in clinical practice.

  4. Sesquiterpene lactones: Mechanism of antineoplastic activity; relationship of cellular glutathione to cytotoxicity; and disposition

    International Nuclear Information System (INIS)

    Grippo, A.A.

    1987-01-01

    Helenalin, a sesquiterpene lactone, inhibited the growth of P388 lymphocytic and L1210 lymphoid leukemia, and Ehrlich ascites and KB carcinoma cells. The L1210 leukemia cells were most sensitive to the cytotoxic effects of helenalin. Helenalin's antineoplastic effects were due to inhibition of DNA synthesis by suppressing the activities of enzymes involved in this biosynthetic pathway; i.e., IMP dehydrogenase, ribonucleoside diphosphate reductase, thioredoxin complex, GSH disulfide oxidoreductase and DNA polymerase α activities. The relationship of reduced glutathione (GSH) to the cytotoxic effects of helanalin was evaluated. L1210 cells, which were more sensitive to helenalin's toxicity, contained lower basal concentrations of GSH. Helenalin decreased the concentration of reduced glutathione in both L1210 and P388 leukemia cells. Concurrent administration of helanalin with agents reported to raise GSH concentrations did not substantially effect GSH levels, nor were survival times of tumor-bearing mice enhanced. Following intraperitoneal administration of 3 H-plenolin, no radioactive drug and/or metabolite was sequestered in the organs of BDF 1 mice. Approximately 50% of 3 H-plenolin and/or its metabolites were eliminated via urine while lesser amounts of radioactive drug and/or metabolites were eliminated in the feces

  5. The rational design of a novel potent analogue of the 5’-AMP-activated protein kinase inhibitor compound C with improved selectivity and cellular activity

    Science.gov (United States)

    Machrouhi, Fouzia; Ouhamou, Nouara; Laderoute, Keith; Calaoagan, Joy; Bukhtiyarova, Marina; Ehrlich, Paula J.; Klon, Anthony E.

    2010-01-01

    We have designed and synthesized analogues of compound C, a non-specific inhibitor of 5’-AMP-activated protein kinase (AMPK), using a computational fragment-based drug design (FBDD) approach. Synthesizing only twenty-seven analogues yielded a compound that was equipotent to compound C in the inhibition of the human AMPK (hAMPK) α2 subunit in the heterotrimeric complex in vitro, exhibited significantly improved selectivity against a subset of relevant kinases, and demonstrated enhanced cellular inhibition of AMPK. PMID:20932747

  6. [Drug vectorization or how to modulate tissular and cellular distribution of biologically active compounds].

    Science.gov (United States)

    Couvreur, P

    2001-07-01

    Drug vectorization has undergone considerable development over the last few years. This review focuses on the intravenous route of administration. Colloid formulations allow a modulation of drug tissue distribution. Using liposomes and nanoparticles with unmodified surfaces, drugs can be targeted to macrophages of the reticulum endothelium system. When the liposomes or nanoparticles are covered with hydrophilic or flexible polymers, the vascular phase can be favored in order, for example, to facilitate selective extravasation at a tumor site. Therapeutic applications of these systems are presented. The development of "intelligent" vectors capable of modulating intracellular distribution of an active compounds is an equally interesting approach, for example pH-sensitive liposomes or nanoparticles decorated with folic acid capable of targeting intracellular cytoplasm.

  7. Digital Morphing Wing: Active Wing Shaping Concept Using Composite Lattice-Based Cellular Structures.

    Science.gov (United States)

    Jenett, Benjamin; Calisch, Sam; Cellucci, Daniel; Cramer, Nick; Gershenfeld, Neil; Swei, Sean; Cheung, Kenneth C

    2017-03-01

    We describe an approach for the discrete and reversible assembly of tunable and actively deformable structures using modular building block parts for robotic applications. The primary technical challenge addressed by this work is the use of this method to design and fabricate low density, highly compliant robotic structures with spatially tuned stiffness. This approach offers a number of potential advantages over more conventional methods for constructing compliant robots. The discrete assembly reduces manufacturing complexity, as relatively simple parts can be batch-produced and joined to make complex structures. Global mechanical properties can be tuned based on sub-part ordering and geometry, because local stiffness and density can be independently set to a wide range of values and varied spatially. The structure's intrinsic modularity can significantly simplify analysis and simulation. Simple analytical models for the behavior of each building block type can be calibrated with empirical testing and synthesized into a highly accurate and computationally efficient model of the full compliant system. As a case study, we describe a modular and reversibly assembled wing that performs continuous span-wise twist deformation. It exhibits high performance aerodynamic characteristics, is lightweight and simple to fabricate and repair. The wing is constructed from discrete lattice elements, wherein the geometric and mechanical attributes of the building blocks determine the global mechanical properties of the wing. We describe the mechanical design and structural performance of the digital morphing wing, including their relationship to wind tunnel tests that suggest the ability to increase roll efficiency compared to a conventional rigid aileron system. We focus here on describing the approach to design, modeling, and construction as a generalizable approach for robotics that require very lightweight, tunable, and actively deformable structures.

  8. Antimicrobial activity and cellular toxicity of nanoparticle-polymyxin B conjugates

    Science.gov (United States)

    Park, Soonhyang; Chibli, Hicham; Wong, Jody; Nadeau, Jay L.

    2011-05-01

    We investigate the antimicrobial activity and cytotoxicity to mammalian cells of conjugates of the peptide antibiotic polymyxin B (PMB) to Au nanoparticles and CdTe quantum dots. Au nanoparticles fully covered with PMB are identical in antimicrobial activity to the free drug alone, whereas partially-conjugated Au particles show decreased effectiveness in proportion to the concentration of Au. CdTe-PMB conjugates are more toxic to Escherichia coli than PMB alone, resulting in a flattening of the steep PMB dose-response curve. The effect is most pronounced at low concentrations of PMB, with a greater effect on the concentration required to reduce growth by half (IC50) than on the concentration needed to inhibit all growth (minimum inhibitory concentration, MIC). The Gram positive organism Staphylococcus aureus is resistant to both PMB and CdTe, showing minimal increased sensitivity when the two are conjugated. Measurement of reactive oxygen species (ROS) generation shows a significant reduction in photo-generated hydroxyl and superoxide radicals with CdTe-PMB as compared with bare CdTe. There is a corresponding reduction in toxicity of QD-PMB versus bare CdTe to mammalian cells, with nearly 100% survival in fibroblasts exposed to bactericidal concentrations of QD-PMB. The situation in bacteria is more complex: photoexcitation of the CdTe particles plays a small role in IC50 but has a significant effect on the MIC, suggesting that at least two different mechanisms are responsible for the antimicrobial action seen. These results show that it is possible to create antimicrobial agents using concentrations of CdTe quantum dots that do not harm mammalian cells.

  9. Antimicrobial activity and cellular toxicity of nanoparticle-polymyxin B conjugates

    International Nuclear Information System (INIS)

    Park, Soonhyang; Chibli, Hicham; Wong, Jody; Nadeau, Jay L

    2011-01-01

    We investigate the antimicrobial activity and cytotoxicity to mammalian cells of conjugates of the peptide antibiotic polymyxin B (PMB) to Au nanoparticles and CdTe quantum dots. Au nanoparticles fully covered with PMB are identical in antimicrobial activity to the free drug alone, whereas partially-conjugated Au particles show decreased effectiveness in proportion to the concentration of Au. CdTe-PMB conjugates are more toxic to Escherichia coli than PMB alone, resulting in a flattening of the steep PMB dose-response curve. The effect is most pronounced at low concentrations of PMB, with a greater effect on the concentration required to reduce growth by half (IC50) than on the concentration needed to inhibit all growth (minimum inhibitory concentration, MIC). The Gram positive organism Staphylococcus aureus is resistant to both PMB and CdTe, showing minimal increased sensitivity when the two are conjugated. Measurement of reactive oxygen species (ROS) generation shows a significant reduction in photo-generated hydroxyl and superoxide radicals with CdTe-PMB as compared with bare CdTe. There is a corresponding reduction in toxicity of QD-PMB versus bare CdTe to mammalian cells, with nearly 100% survival in fibroblasts exposed to bactericidal concentrations of QD-PMB. The situation in bacteria is more complex: photoexcitation of the CdTe particles plays a small role in IC50 but has a significant effect on the MIC, suggesting that at least two different mechanisms are responsible for the antimicrobial action seen. These results show that it is possible to create antimicrobial agents using concentrations of CdTe quantum dots that do not harm mammalian cells.

  10. Antimicrobial activity and cellular toxicity of nanoparticle-polymyxin B conjugates

    Energy Technology Data Exchange (ETDEWEB)

    Park, Soonhyang; Chibli, Hicham; Wong, Jody; Nadeau, Jay L, E-mail: jay.nadeau@mcgill.ca [Department of Biomedical Engineering, McGill University, 3775 University Street, Montreal QC, H3A 2B4 (Canada)

    2011-05-06

    We investigate the antimicrobial activity and cytotoxicity to mammalian cells of conjugates of the peptide antibiotic polymyxin B (PMB) to Au nanoparticles and CdTe quantum dots. Au nanoparticles fully covered with PMB are identical in antimicrobial activity to the free drug alone, whereas partially-conjugated Au particles show decreased effectiveness in proportion to the concentration of Au. CdTe-PMB conjugates are more toxic to Escherichia coli than PMB alone, resulting in a flattening of the steep PMB dose-response curve. The effect is most pronounced at low concentrations of PMB, with a greater effect on the concentration required to reduce growth by half (IC50) than on the concentration needed to inhibit all growth (minimum inhibitory concentration, MIC). The Gram positive organism Staphylococcus aureus is resistant to both PMB and CdTe, showing minimal increased sensitivity when the two are conjugated. Measurement of reactive oxygen species (ROS) generation shows a significant reduction in photo-generated hydroxyl and superoxide radicals with CdTe-PMB as compared with bare CdTe. There is a corresponding reduction in toxicity of QD-PMB versus bare CdTe to mammalian cells, with nearly 100% survival in fibroblasts exposed to bactericidal concentrations of QD-PMB. The situation in bacteria is more complex: photoexcitation of the CdTe particles plays a small role in IC50 but has a significant effect on the MIC, suggesting that at least two different mechanisms are responsible for the antimicrobial action seen. These results show that it is possible to create antimicrobial agents using concentrations of CdTe quantum dots that do not harm mammalian cells.

  11. The cellular responses and antibacterial activities of silver nanoparticles stabilized by different polymers

    Science.gov (United States)

    Lin, Jiang-Jen; Lin, Wen-Chun; Dong, Rui-Xuan; Hsu, Shan-hui

    2012-02-01

    Silver nanoparticles (AgNPs) are known for their excellent antibacterial activities. The possible toxicity, however, is a major concern for their applications. Three types of AgNPs were prepared in this study by chemical processes. Each was stabilized by a polymer surfactant, which was expected to reduce the exposure of cells to AgNPs and therefore their cytotoxicity. The polymer stabilizers included poly(oxyethylene)-segmented imide (POEM), poly(styrene-co-maleic anhydride)-grafting poly(oxyalkylene) (SMA) and poly(vinyl alcohol) (PVA). The cytotoxicity of these chemically produced AgNPs to mouse skin fibroblasts (L929), human hepatocarcinoma cells (HepG2), and mouse monocyte macrophages (J774A1) was compared to that of physically produced AgNPs and gold nanoparticles (AuNPs) as well as the standard reference material RM8011 AuNPs. Results showed that SMA-AgNPs were the least cytotoxic among all materials, but cytotoxicity was still observed at higher silver concentrations (>30 ppm). Macrophages demonstrated the inflammatory response with cell size increase and viability decrease upon exposure to 10 ppm of the chemically produced AgNPs. SMA-AgNPs did not induce hemolysis at a silver concentration below 1.5 ppm. Regarding the antibacterial activity, POEM-AgNPs and SMA-AgNPs at 1 ppm silver content showed 99.9% and 99.3% growth inhibition against E. coli, while PVA-AgNPs at the same silver concentration displayed 79.1% inhibition. Overall, SMA-AgNPs demonstrated better safety in vitro and greater antibacterial effects than POEM-AgNPs and PVA-AgNPs. This study suggested that polymer stabilizers may play an important role in determining the toxicity of AgNPs.

  12. Exogenous coenzyme Q10 modulates MMP-2 activity in MCF-7 cell line as a breast cancer cellular model

    Directory of Open Access Journals (Sweden)

    Mirmiranpour Hossein

    2010-11-01

    Full Text Available Abstract Background/Aims Matrix Metalloproteinases 2 is a key molecule in cellular invasion and metastasis. Mitochondrial ROS has been established as a mediator of MMP activity. Coenzyme Q10 contributes to intracellular ROS regulation. Coenzyme Q10 beneficial effects on cancer are still in controversy but there are indications of Coenzyme Q10 complementing effect on tamoxifen receiving breast cancer patients. Methods In this study we aimed to investigate the correlation of the effects of co-incubation of coenzyme Q10 and N-acetyl-L-cysteine (NAC on intracellular H2O2 content and Matrix Metalloproteinase 2 (MMP-2 activity in MCF-7 cell line. Results and Discussion Our experiment was designed to assess the effect in a time and dose related manner. Gelatin zymography and Flowcytometric measurement of H2O2 by 2'7',-dichlorofluorescin-diacetate probe were employed. The results showed that both coenzyme Q10 and N-acetyl-L-cysteine reduce MMP-2 activity along with the pro-oxidant capacity of the MCF-7 cell in a dose proportionate manner. Conclusions Collectively, the present study highlights the significance of Coenzyme Q10 effect on the cell invasion/metastasis effecter molecules.

  13. Development of a novel LC/MS method to quantitate cellular stearoyl-CoA desaturase activity

    International Nuclear Information System (INIS)

    Dillon, Roslyn; Greig, Michael J.; Bhat, B. Ganesh

    2008-01-01

    Stearoyl-CoA desaturase 1 (SCD1) is an enzyme that catalyzes the rate-limiting step in de novo synthesis of monounsaturated fatty acids-mainly oleate and palmitoleate from stearoyl-CoA and palmitoyl-Co A, respectively. These products are the most abundant monounsaturated fatty acids in membrane phospholipids, triglycerides, cholesterol esters. Reports on mice with a targeted disruption of SCD1 gene (SCD1-/-) exhibit improved glucose tolerance and insulin sensitivity compared to wild-type suggesting SCD1 could be a therapeutic target for diabetes and related metabolic diseases. Measurement of SCD1 activity is technically challenging and traditional cell-based SCD1 assay procedure is labor intensive with low throughput. We describe here a novel medium-throughput LC/MS cell-based assay for determining cellular SCD1 activity, facilitating screening of potential SCD1 inhibitor compounds. Confluent HepG2 cells were grown in 24-well plates and incubated with vehicle or an inhibitor followed by incubation with deuterium labeled saturated fatty acid substrates. Total cell lipids were extracted and the conversion of stearate to oleate was measured by liquid chromatography-mass spectrometry. Sterculate, a known inhibitor of SCD1, inhibited the enzyme activity in a dose dependent manner in this assay with a calculated EC 50 of 247 nM. The medium-throughput method described here is an important step towards identifying an inhibitor of SCD1 to treat diabetes and related metabolic diseases

  14. Chemical constituents of Hericium erinaceum associated with the inhibitory activity against cellular senescence in human umbilical vascular endothelial cells.

    Science.gov (United States)

    Noh, Hyung Jun; Yang, Hyo Hyun; Kim, Geum Soog; Lee, Seung Eun; Lee, Dae Young; Choi, Je Hun; Kim, Seung Yu; Lee, Eun Suk; Ji, Seung Heon; Kang, Ki Sung; Park, Hye-Jin; Kim, Jae-Ryong; Kim, Ki Hyun

    2015-12-01

    Hericium erinaceum is an edible and medicinal mushroom widely used in Korea, Japan, and China. On the search for biologically active compounds supporting the medicinal usage, the MeOH extract of the fruiting bodies of H. erinaceum was investigated for its chemical constituents. Six compounds were isolated and identified as hericenone D (1), (22E,24R)-5α,8α-epidioxyergosta-6,22-dien-3β-ol (2), erinacerin B (3), hericenone E (4), hericenone F (5) and isohericerin (6) by comparing their spectroscopic data with previously reported values. The inhibitory effects on adriamycin-induced cellular senescence in human dermal fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs) of the isolates (1-6) were studied. Among the isolated compounds, ergosterol peroxide (2) reduced senescence associated β-galactosidase (SA-β-gal) activity increased in HUVECs treated with adriamycin. According to experimental data obtained, the active compound may inspire the development of a new pharmacologically useful substance to be used in the treatment and prevention of age-related diseases.

  15. The fungal quorum-sensing molecule farnesol activates innate immune cells but suppresses cellular adaptive immunity.

    Science.gov (United States)

    Leonhardt, Ines; Spielberg, Steffi; Weber, Michael; Albrecht-Eckardt, Daniela; Bläss, Markus; Claus, Ralf; Barz, Dagmar; Scherlach, Kirstin; Hertweck, Christian; Löffler, Jürgen; Hünniger, Kerstin; Kurzai, Oliver

    2015-03-17

    Farnesol, produced by the polymorphic fungus Candida albicans, is the first quorum-sensing molecule discovered in eukaryotes. Its main function is control of C. albicans filamentation, a process closely linked to pathogenesis. In this study, we analyzed the effects of farnesol on innate immune cells known to be important for fungal clearance and protective immunity. Farnesol enhanced the expression of activation markers on monocytes (CD86 and HLA-DR) and neutrophils (CD66b and CD11b) and promoted oxidative burst and the release of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α] and macrophage inflammatory protein 1 alpha [MIP-1α]). However, this activation did not result in enhanced fungal uptake or killing. Furthermore, the differentiation of monocytes to immature dendritic cells (iDC) was significantly affected by farnesol. Several markers important for maturation and antigen presentation like CD1a, CD83, CD86, and CD80 were significantly reduced in the presence of farnesol. Furthermore, farnesol modulated migrational behavior and cytokine release and impaired the ability of DC to induce T cell proliferation. Of major importance was the absence of interleukin 12 (IL-12) induction in iDC generated in the presence of farnesol. Transcriptome analyses revealed a farnesol-induced shift in effector molecule expression and a down-regulation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor during monocytes to iDC differentiation. Taken together, our data unveil the ability of farnesol to act as a virulence factor of C. albicans by influencing innate immune cells to promote inflammation and mitigating the Th1 response, which is essential for fungal clearance. Farnesol is a quorum-sensing molecule which controls morphological plasticity of the pathogenic yeast Candida albicans. As such, it is a major mediator of intraspecies communication. Here, we investigated the impact of farnesol on human innate immune cells known to be

  16. The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation

    Directory of Open Access Journals (Sweden)

    Zarbock Ralf

    2012-03-01

    Full Text Available Abstract Background Surfactant protein C (SP-C is important for the function of pulmonary surfactant. Heterozygous mutations in SFTPC, the gene encoding SP-C, cause sporadic and familial interstitial lung disease (ILD in children and adults. Mutations mapping to the BRICHOS domain located within the SP-C proprotein result in perinuclear aggregation of the proprotein. In this study, we investigated the effects of the mutation A116D in the BRICHOS domain of SP-C on cellular homeostasis. We also evaluated the ability of drugs currently used in ILD therapy to counteract these effects. Methods SP-CA116D was expressed in MLE-12 alveolar epithelial cells. We assessed in vitro the consequences for cellular homeostasis, immune response and effects of azathioprine, hydroxychloroquine, methylprednisolone and cyclophosphamide. Results Stable expression of SP-CA116D in MLE-12 alveolar epithelial cells resulted in increased intracellular accumulation of proSP-C processing intermediates. SP-CA116D expression further led to reduced cell viability and increased levels of the chaperones Hsp90, Hsp70, calreticulin and calnexin. Lipid analysis revealed decreased intracellular levels of phosphatidylcholine (PC and increased lyso-PC levels. Treatment with methylprednisolone or hydroxychloroquine partially restored these lipid alterations. Furthermore, SP-CA116D cells secreted soluble factors into the medium that modulated surface expression of CCR2 or CXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine effect of SP-CA116D on neighboring cells in the alveolar space. Conclusions We show that the A116D mutation leads to impaired processing of proSP-C in alveolar epithelial cells, alters cell viability and lipid composition, and also activates cells of the immune system. In addition, we show that some of the effects of the mutation on cellular homeostasis can be antagonized by application of pharmaceuticals commonly applied in ILD therapy

  17. High Cellular Monocyte Activation in People Living With Human Immunodeficiency Virus on Combination Antiretroviral Therapy and Lifestyle-Matched Controls Is Associated With Greater Inflammation in Cerebrospinal Fluid

    NARCIS (Netherlands)

    Booiman, Thijs; Wit, Ferdinand W.; Maurer, Irma; de Francesco, Davide; Sabin, Caroline A.; Harskamp, Agnes M.; Prins, Maria; Garagnani, Paolo; Pirazzini, Chiara; Franceschi, Claudio; Fuchs, Dietmar; Gisslén, Magnus; Winston, Alan; Reiss, Peter; Kootstra, Neeltje A.; Schouten, J.; Kooij, K. W.; van Zoest, R. A.; Elsenga, B. C.; Janssen, F. R.; Heidenrijk, M.; Zikkenheiner, W.; van der Valk, M.; Booiman, T.; Harskamp-Holwerda, A. M.; Boeser-Nunnink, B.; Maurer, I.; Mangas Ruiz, M. M.; Girigorie, A. F.; Villaudy, J.; Frankin, E.; Pasternak, A.; Berkhout, B.; van der Kuyl, T.; Portegies, P.; Schmand, B. A.; Geurtsen, G. J.; ter Stege, J. A.; Klein Twennaar, M.; Majoie, C. B. L. M.; Caan, M. W. A.; Su, T.; Weijer, K.; Bisschop, P. H. L. T.; Kalsbeek, A.; Wezel, M.; Visser, I.; Ruhé, H. G.; Franceschi, C.; Garagnani, P.

    2017-01-01

    Increased monocyte activation and intestinal damage have been shown to be predictive for the increased morbidity and mortality observed in treated people living with human immunodeficiency virus (PLHIV). A cross-sectional analysis of cellular and soluble markers of monocyte activation, coagulation,

  18. Saccharomyces cerevisiae Ngl3p is an active 3′–5′ exonuclease with a specificity towards poly-A RNA reminiscent of cellular deadenylases

    DEFF Research Database (Denmark)

    Feddersen, Ane; Dedic, Emil; Poulsen, Esben Guldahl

    2012-01-01

    RNAs that yeast Ngl3p is a functional 3′–5′ exonuclease most active at slightly acidic conditions. We further show that the enzyme depends on divalent metal ions for activity and possesses specificity towards poly-A RNA similar to what has been observed for cellular deadenylases. The results suggest that Ngl3p...

  19. Hemin activation of innate cellular response blocks human immunodeficiency virus type-1-induced osteoclastogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Takeda, Kazuyo [Microscopy and Imaging Core Facility, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD (United States); Adhikari, Rewati [Division of Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD (United States); Yamada, Kenneth M. [National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD (United States); Dhawan, Subhash, E-mail: subhash.dhawan@fda.hhs.gov [Division of Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD (United States)

    2015-08-14

    The normal skeletal developmental and homeostatic process termed osteoclastogenesis is exacerbated in numerous pathological conditions and causes excess bone loss. In cancer and HIV-1-infected patients, this disruption of homeostasis results in osteopenia and eventual osteoporesis. Counteracting the factors responsible for these metabolic disorders remains a challenge for preventing or minimizing this co-morbidity associated with these diseases. In this report, we demonstrate that a hemin-induced host protection mechanism not only suppresses HIV-1 associated osteoclastogenesis, but it also exhibits anti-osteoclastogenic activity for non-infected cells. Since the mode of action of hemin is both physiological and pharmacological through induction of heme oxygenase-1 (HO-1), an endogenous host protective response to an FDA-licensed therapeutic used to treat another disease, our study suggests an approach to developing novel, safe and effective therapeutic strategies for treating bone disorders, because hemin administration in humans has previously met required FDA safety standards. - Highlights: • HIV-1 infection induced osteoclastogenesis in primary human macrophages. • Heme oxygenase-1 (HO-1) induction inhibited HIV-1-induced osteoclastogenesis in macrophages. • HO-1 induction suppressed RANKL-enhanced osteoclastogenesis in HIV-1-infected macrophages. • This inverse relationship between HO-1 and HIV-1 pathogenesis may define a novel host defense response against HIV-1 infection.

  20. Studies on cellular distribution of elements in human hepatocellular carcinoma samples by molecular activation analysis

    International Nuclear Information System (INIS)

    Deng Guilong; Chen Chunying; Zhang Peiqun; Zhao Jiujiang; Chai Zhifang

    2005-01-01

    The distribution patterns of 17 elements in the subcellular fractions of nuclei, mitochondria, lysosome, microsome and cytosol of human hepatocellular carcinoma (HCC) and normal liver samples were investigated by using molecular activation analysis (MAA) and differential centrifugation. Their significant difference was checked by the Studient's t-test. These elements exhibit inhomogeneous distributions in each subcellular fraction. Some elements have no significant difference between hepatocellular carcinoma and normal liver samples. However, the concentrations of Br, Ca, Cd and Cs are significantly higher in each component of hepatocarcinoma than in normal liver. The content of Fe in microsome of HCC is significantly lower, almost half of normal liver samples, but higher in other subcellular fractions than in those of normal tissues. The rare earth elements of La and Ce have the patterns similar to Fe. The concentrations of Sb and Zn in nuclei of HCC are obviously lower (P<0.05, P<0.05). The contents of K and Na are higher in cytosol of HCC (P<0.05). The distributions of Ba and Rb show no significant difference between two groups. The relationships of Fe, Cd and K with HCC were also discussed. The levels of some elements in subcellular fractions of tumor were quite different from those of normal liver, which suggested that trace elements might play important roles in the occurrence and development of hepatocellular carcinoma. (authors)

  1. On the Photonic Cellular Interaction and the Electric Activity of Neurons in the Human Brain

    International Nuclear Information System (INIS)

    Salari, V; Tuszynski, J; Bokkon, I; Rahnama, M; Cifra, M

    2011-01-01

    The subject of Ultraweak Photon Emission (UPE) by biological systems is very fascinating, and both evidence of its effects and applications are growing rapidly due to improvements in experimental techniques. Since the relevant equipment should be ultrasensitive with high quantum efficiencies and very low noise levels, the subject of UPE is still hotly debated and some of the interpretations need stronger empirical evidence to be accepted at face value. In this paper we first review different types of interactions between light and living systems based on recent publications. We then discuss the feasibility of UPE production in the human brain. The subject of UPE in the brain is still in early stages of development and needs more accurate experimental methods for proper analysis. In this work we also discuss a possible role of mitochondria in the production of UPE in the neurons of the brain and the plausibility of their effects on microtubules (MTs). MTs have been implicated as playing an important role in the signal and information processing taking place in the mammalian (especially human) brain. Finally, we provide a short discussion about the feasible effects of MTs on electric neural activity in the human brain.

  2. Hemin activation of innate cellular response blocks human immunodeficiency virus type-1-induced osteoclastogenesis

    International Nuclear Information System (INIS)

    Takeda, Kazuyo; Adhikari, Rewati; Yamada, Kenneth M.; Dhawan, Subhash

    2015-01-01

    The normal skeletal developmental and homeostatic process termed osteoclastogenesis is exacerbated in numerous pathological conditions and causes excess bone loss. In cancer and HIV-1-infected patients, this disruption of homeostasis results in osteopenia and eventual osteoporesis. Counteracting the factors responsible for these metabolic disorders remains a challenge for preventing or minimizing this co-morbidity associated with these diseases. In this report, we demonstrate that a hemin-induced host protection mechanism not only suppresses HIV-1 associated osteoclastogenesis, but it also exhibits anti-osteoclastogenic activity for non-infected cells. Since the mode of action of hemin is both physiological and pharmacological through induction of heme oxygenase-1 (HO-1), an endogenous host protective response to an FDA-licensed therapeutic used to treat another disease, our study suggests an approach to developing novel, safe and effective therapeutic strategies for treating bone disorders, because hemin administration in humans has previously met required FDA safety standards. - Highlights: • HIV-1 infection induced osteoclastogenesis in primary human macrophages. • Heme oxygenase-1 (HO-1) induction inhibited HIV-1-induced osteoclastogenesis in macrophages. • HO-1 induction suppressed RANKL-enhanced osteoclastogenesis in HIV-1-infected macrophages. • This inverse relationship between HO-1 and HIV-1 pathogenesis may define a novel host defense response against HIV-1 infection

  3. Protective Activity of N-acetyl-L-cysteine (NAC) against Cellular Oxidative Stress Induced by Radiation

    Energy Technology Data Exchange (ETDEWEB)

    Han, Min; Hyun, Kyung Man; Kim, Jin Kyu [Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of); Nili, Mohammad [Dawnesh Radiation Research Institute, Barcelona (Spain); Aroutiounian, Rouben [Yerevan State University, Yerevan (Armenia)

    2009-10-15

    Oxidative stress occurs due to numerous factors such as irradiation, redox decomposition by ions of hydroperoxides or hydrogen peroxide, and thermal decomposition of free radical initiators including peroxides and hyponitrites. The antioxidant and free-radical scavenger N-acetyl- L-cysteine (NAC) is used extensively as a conditional nutrient. NAC acts as a cysteine donor and maintains or even increases the intracellular levels of glutathione (GSH), a tripeptide which protects cells from toxins such as free-radicals. With regard to the radioprotective effects of NAC, the majority of studies have been performed in vitro. NAC were used to protect the Chinese hamster ovary (CHO) cells from radiationinduced apoptosis by controlling the enzyme that triggers programmed cell death. Some studies have successfully demonstrated sporadic radioprotection following low-level chronic administration of NAC, though the mode and optimal dose of NAC are yet to be fully determined. This study was designed to evaluate the effects of NAC in different doses on the activity levels of GSH and the cell viability in the fish cell line against ionizing radiation.

  4. Preferential replication of FIV in activated CD4+CD25+T cells independent of cellular proliferation

    International Nuclear Information System (INIS)

    Joshi, Anjali; Vahlenkamp, Thomas W.; Garg, Himanshu; Tompkins, Wayne A.F.; Tompkins, Mary B.

    2004-01-01

    Studies attempting to identify reservoirs of HIV-1 latency have documented that the virus persists as both a latent and productive infection in subsets of CD4 + cells. Reports regarding establishment of a stable HIV-1 infection in quiescent T cells in vitro, however, are controversial. In the present study, we investigated the susceptibility of naive and activated CD4 + cell subsets (distinguished by differential expression of CD25) to feline immunodeficiency virus (FIV) infection, their ability to replicate the virus, and potentially act as a reservoir for virus persistence in infected animals. While both CD4 + CD25 + and CD4 + CD25 - cells are susceptible to FIV infection in vitro and in vivo, only CD4 + CD25 + cells produce infectious virions when cultured with interleukin-2 (IL-2). Latently infected CD4 + CD25 - cells produce infectious virions following ConcanvalinA (ConA) stimulation, which correlates with upregulated surface expression of CD25. In contrast to CD4 + CD25 - cells, CD4 + CD25 + cells remain unresponsive to mitogen stimulation and are relatively resistant to apoptosis whether or not infected with FIV. The ability of CD4 + CD25 + cells to replicate FIV efficiently in the presence of IL-2 but remain anergic and unresponsive to apoptotic signaling suggests that these cells may provide a reservoir of productive FIV infection. On the contrary, CD4 + CD25 - cells seem to establish as latent viral reservoirs capable of being reactivated after stimulation

  5. Studies on cellular distribution of elements in human hepatocellular carcinoma samples by molecular activation analysis

    Energy Technology Data Exchange (ETDEWEB)

    Guilong, Deng [Chinese Academy of Sciences, Beijing (China). Inst. of High Energy Physics, Key Laboratory of Nuclear Analytical Techniques; Department of General Surgery, the Second Affiliated Hospital, School of Medicine, Zhejiang Univ., Hangzhou (China); Chunying, Chen; Peiqun, Zhang; Jiujiang, Zhao; Zhifang, Chai [Chinese Academy of Sciences, Beijing (China). Inst. of High Energy Physics, Key Laboratory of Nuclear Analytical Techniques; Yingbin, Liu; Jianwei, Wang; Bin, Xu; Shuyou, Peng [Department of General Surgery, the Second Affiliated Hospital, School of Medicine, Zhejiang Univ., Hangzhou (China)

    2005-07-15

    The distribution patterns of 17 elements in the subcellular fractions of nuclei, mitochondria, lysosome, microsome and cytosol of human hepatocellular carcinoma (HCC) and normal liver samples were investigated by using molecular activation analysis (MAA) and differential centrifugation. Their significant difference was checked by the Studient's t-test. These elements exhibit inhomogeneous distributions in each subcellular fraction. Some elements have no significant difference between hepatocellular carcinoma and normal liver samples. However, the concentrations of Br, Ca, Cd and Cs are significantly higher in each component of hepatocarcinoma than in normal liver. The content of Fe in microsome of HCC is significantly lower, almost half of normal liver samples, but higher in other subcellular fractions than in those of normal tissues. The rare earth elements of La and Ce have the patterns similar to Fe. The concentrations of Sb and Zn in nuclei of HCC are obviously lower (P<0.05, P<0.05). The contents of K and Na are higher in cytosol of HCC (P<0.05). The distributions of Ba and Rb show no significant difference between two groups. The relationships of Fe, Cd and K with HCC were also discussed. The levels of some elements in subcellular fractions of tumor were quite different from those of normal liver, which suggested that trace elements might play important roles in the occurrence and development of hepatocellular carcinoma. (authors)

  6. Insecticidal activity of the metalloprotease AprA occurs through suppression of host cellular and humoral immunity.

    Science.gov (United States)

    Lee, Seung Ah; Jang, Seong Han; Kim, Byung Hyun; Shibata, Toshio; Yoo, Jinwook; Jung, Yunjin; Kawabata, Shun-Ichiro; Lee, Bok Luel

    2018-04-01

    The biochemical characterization of virulence factors from entomopathogenic bacteria is important to understand entomopathogen-insect molecular interactions. Pseudomonas entomophila is a typical entomopathogenic bacterium that harbors virulence factors against several insects. However, the molecular actions of these factors against host innate immune responses are not clearly elucidated. In this study, we observed that bean bugs (Riptortus pedestris) that were injected with P. entomophila were highly susceptible to this bacterium. To determine how P. entomophila counteracts the host innate immunity to survive within the insect, we purified a highly enriched protein with potential host insect-killing activity from the culture supernatant of P. entomophila. Then, a 45-kDa protein was purified to homogeneity and identified as AprA which is an alkaline zinc metalloprotease of the genus Pseudomonas by liquid chromatography mass spectrometry (LC-MS). Purified AprA showed a pronounced killing effect against host insects and suppressed both host cellular and humoral innate immunity. Furthermore, to show that AprA is an important insecticidal protein of P. entomophila, we used an aprA-deficient P. entomophila mutant strain (ΔaprA). When ΔaprA mutant cells were injected to host insects, this mutant exhibited extremely attenuated virulence. In addition, the cytotoxicity against host hemocytes and the antimicrobial peptide-degrading ability of the ΔaprA mutant were greatly decreased. These findings suggest that AprA functions as an important insecticidal protein of P. entomophila via suppression of host cellular and humoral innate immune responses. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Increased cellular levels of spermidine or spermine are required for optimal DNA synthesis in lymphocytes activated by concanavalin A.

    Science.gov (United States)

    Fillingame, R H; Jorstad, C M; Morris, D R

    1975-01-01

    There are large increases in cellular levels of the polyamines spermidine and spermine in lymphocytes induced to transform by concanavalin A. The anti-leukemic agent methylglyoxal bis(guanylhydrazone) (MGBG) blocks synthesis of these polyamines by inhibiting S-adenosylmethionine decarboxylase. Previous results showed that when cells are activated in the presence of MGBG the synthesis and processing of RNA, as well as protein synthesis, proceed as in the absence of the drug. In contrast, the incorporation of [methyl-3H]thymidine into DNA and the rate of entry of the cells into mitosis are inhibited by 60% in the presence of MGBG. Several experiments suggest that MGBG inhibits cell proliferation by directly blocking polyamine synthesis and not by an unrelated pharmacological effect: (1) the inhibitory action of MGBG is reversed by exogenously added spermidine or spermine; (2) inhibition of DNA synthesis by MGBG shows the same dose-response curve as does inhibition of spermidine and spermine synthesis; and (3) if MGBG is added to cells which have been allowed to accumulate their maximum complement of polyamines, there is no inhibition of thymidine incorporation. MGBG-treated and control cultures initiate DNA synthesis at the same time and show the same percentage of labeled cells by autoradiography. Therefore, it appears that in the absence of increased cellular levels of polyamines, lymphocytes progress normally from G0 through G1 and into S-phase. Furthermore, these experiments suggest that the increased levels of spermidine and spermine generally seen in rapidly proliferating eukaryotic systems are necessary for enhanced rates of DNA replication. PMID:1060087

  8. Cellular therapy without cells: extracellular vesicles promote activation of stem cells after irradiation

    International Nuclear Information System (INIS)

    Lange, C.

    2016-01-01

    Mesenchymal stromal cells from the bone marrow (MSC) have been shown to be effective in several cell therapeutic treatments. However, MSC accumulate in lungs after i.v. injection. How do MSC transfer their potential to organs with therapeutic need? We show that released extracellular vesicles (EV) might be playing an active role in this transfer. EV were isolated from MSC supernatant and characterized with flow cytometry, proteomics and next generation sequencing. Our data showed the transfer of RNAs, clustering into several protective gene groups. Besides, we repeatedly detected genomic DNA on vesicles. Using a plant - derived detector gene we showed horizontal DNA transfer via EV. Furthermore, we showed that EV were able to salvage stem/progenitor cells in vitro from radiation suppression. Three selected proteins from proteomics data were examined for stem cell protection after irradiation. EV derived from down-regulated producer MSC showed a substantial loss of protection in irradiated stem cells supporting their relevance for stem cell protection. Finally, we showed that EV after i.v. injection into lethally irradiated animals colocalize within 2-4 hours with hematopoietic stem cells in the bone marrow giving hint to direct protection of stem cells by EV. In conclusion, EV derived from bone marrow MSC were able to transfer several cargo compounds leading potentially to change of the genetic properties. Importantly, EV protect irradiated hematopoietic stem cells, stimulate their recovery and proliferation and rescue lethally irradiated animals long-term. Thus, EV might be an alternative for future cell therapeutic treatment particularly in radiation-based events. (author)

  9. Cellular Trojan horse based polymer nanoreactors with light-sensitive activity.

    Science.gov (United States)

    Baumann, Patric; Spulber, Mariana; Dinu, Ionel Adrian; Palivan, Cornelia G

    2014-08-07

    production, stability, and Trojan horse activity inside cells support our light-sensitive nanoreactors for medical applications which require ROS to be generated with precise time and space control.

  10. Acetyl-CoA carboxylase in Reuber hepatoma cells: variation in enzyme activity, insulin regulation, and cellular lipid content.

    Science.gov (United States)

    Bianchi, A; Evans, J L; Nordlund, A C; Watts, T D; Witters, L A

    1992-01-01

    Reuber hepatoma cells are useful cultured lines for the study of insulin action, lipid and lipoprotein metabolism, and the regulation of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid biosynthesis. During investigations in different clonal lines of these cells, we have uncovered marked intercellular variability in the activity, enzyme content, and insulin regulation of ACC paralleled by differences in cellular neutral lipid (triglyceride) content. Two contrasting clonal lines, Fao and H356A-1, have been studied in detail. Several features distinguish these two lines, including differences in ACC activity and enzyme kinetics, the content of the two major hepatic ACC isozymes (Mr 280,000 and 265,000 Da) and their heteroisozymic complex, the extent of ACC phosphorylation, and the ability of ACC to be activated on stimulation by insulin and insulinomimetic agonists. As studied by Nile Red staining and fluorescence-activated cell sorting, these two lines also display marked differences in neutral lipid content, which correlates with both basal levels of ACC activity and inhibition of ACC by the fatty acid analog, 5-(tetradecyloxy)-2-furoic acid (TOFA). These results emphasize the importance of characterization of any particular clonal line of Reuber cells for studies of enzyme regulation, substrate metabolism, and hormone action. With respect to ACC, studies in contrasting clonal lines of Reuber cells could provide valuable clues to understanding both the complex mechanisms of intracellular ACC regulation in the absence and presence of hormones and its regulatory role(s) in overall hepatic lipid metabolism.

  11. Novel water-soluble polyurethane nanomicelles for cancer chemotherapy: physicochemical characterization and cellular activities

    Directory of Open Access Journals (Sweden)

    Khosroushahi Ahmad

    2012-01-01

    Full Text Available Abstract Background Efficient delivery of anticancer chemotherapies such as paclitaxel (PTX can improve treatment strategy in a variety of tumors such as breast and ovarian cancers. Accordingly, researches on polymeric nanomicelles continue to find suitable delivery systems. However, due to biocompatibility concerns, a few micellar nanoformulations have exquisitely been translated into clinical uses. Here, we report the synthesis of novel water-soluble nanomicelles using bioactive polyurethane (PU polymer and efficient delivery of PTX in the human breast cancer MCF-7 cells. Results The amphiphilic polyurethane was prepared through formation of urethane bounds between hydroxyl groups in poly (tetramethylene ether glycol (PTMEG and dimethylol propionic acid with isocyanate groups in toluene diisocyanate (TDI. The free isocyanate groups were blocked with phenol, while the free carboxyl groups of dimethylol propionic acid were reacted with triethylamine to attain ionic centers in the polymer backbone. These hydrophobic PTMEG blocks displayed self-assembly forming polymeric nanomicelles in water. The PTX loaded PU nanomicelles showed suitable physical stability, negative zeta potential charge (-43 and high loading efficiency (80% with low level of critical micelle concentration (CMC. In vitro drug release profile showed a faster rate of drug liberation at pH 5.4 as compared to that of pH 7.4, implying involvement of a pH-sensitive mechanism for drug release from the nanomicelles. The kinetic of release exquisitely obeyed the Higuchi model, confirming involvement of diffusion and somewhat erosion at pH 5.4. These nanomicelles significantly inhibited the growth and proliferation of the human breast cancer MCF-7 cells, leading them to apoptosis. The real time RT-PCR analysis confirmed the activation of apoptosis as result of liberation of cytochrome c in the cells treated with the PTX loaded PU nanomicelles. The comet assay analysis showed somewhat DNA

  12. Monoacylated Cellular Prion Proteins Reduce Amyloid-β-Induced Activation of Cytoplasmic Phospholipase A2 and Synapse Damage

    Directory of Open Access Journals (Sweden)

    Ewan West

    2015-06-01

    Full Text Available Alzheimer’s disease (AD is a progressive neurodegenerative disease characterized by the accumulation of amyloid-β (Aβ and the loss of synapses. Aggregation of the cellular prion protein (PrPC by Aβ oligomers induced synapse damage in cultured neurons. PrPC is attached to membranes via a glycosylphosphatidylinositol (GPI anchor, the composition of which affects protein targeting and cell signaling. Monoacylated PrPC incorporated into neurons bound “natural Aβ”, sequestering Aβ outside lipid rafts and preventing its accumulation at synapses. The presence of monoacylated PrPC reduced the Aβ-induced activation of cytoplasmic phospholipase A2 (cPLA2 and Aβ-induced synapse damage. This protective effect was stimulus specific, as treated neurons remained sensitive to α-synuclein, a protein associated with synapse damage in Parkinson’s disease. In synaptosomes, the aggregation of PrPC by Aβ oligomers triggered the formation of a signaling complex containing the cPLA2.a process, disrupted by monoacylated PrPC. We propose that monoacylated PrPC acts as a molecular sponge, binding Aβ oligomers at the neuronal perikarya without activating cPLA2 or triggering synapse damage.

  13. Phyto-mediated nanostructured carriers based on dual vegetable actives involved in the prevention of cellular damage.

    Science.gov (United States)

    Istrati, D; Lacatusu, I; Bordei, N; Badea, G; Oprea, O; Stefan, L M; Stan, R; Badea, N; Meghea, A

    2016-07-01

    The growing scientific interest in exploitation of vegetable bioactives has raised a number of questions regarding their imminent presence in pharmaceutical formulations. This study intends to demonstrate that a dual combination between vegetable oil (e.g. thistle oil, safflower oil, sea buckthorn oil) and a carrot extract represents an optimal approach to formulate safe carrier systems that manifest cell regeneration effect and promising antioxidant and anti-inflammatory activity. Inclusion of both natural actives into lipid carriers imparted a strong negative charge on the nanocarrier surface (up to -45mV) and displayed average sizes of 70nm to 140nm. The entrapment efficiency of carrot extract into nanostructured carriers ranged between 78.3 and 88.3%. The in vitro release study has demonstrated that the entrapment of the extract represents a viable way for an equilibrated release of carotenoids. Besides the excellent antioxidant properties (e.g. scavenging up to 98% of the free oxygen radicals), the results of cellular integrity (e.g. cell viability of 133%) recommend these nanocarriers based on dual carrot extract-bioactive oil as a promising trend for the treatment of certain disorders in which oxidative stress plays a prominent role. In addition, the lipid nanocarriers based on safflower oil and sea buckthorn oil demonstrated an anti-inflammatory effect on LPS induced THP-1 macrophages, by inhibiting the secretion of two pro-inflammatory cytokines, IL-6 and TNF-α. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Multivalent adhesion molecule 7 clusters act as signaling platform for host cellular GTPase activation and facilitate epithelial barrier dysfunction.

    Directory of Open Access Journals (Sweden)

    Jenson Lim

    2014-09-01

    Full Text Available Vibrio parahaemolyticus is an emerging bacterial pathogen which colonizes the gastrointestinal tract and can cause severe enteritis and bacteraemia. During infection, V. parahaemolyticus primarily attaches to the small intestine, where it causes extensive tissue damage and compromises epithelial barrier integrity. We have previously described that Multivalent Adhesion Molecule (MAM 7 contributes to initial attachment of V. parahaemolyticus to epithelial cells. Here we show that the bacterial adhesin, through multivalent interactions between surface-induced adhesin clusters and phosphatidic acid lipids in the host cell membrane, induces activation of the small GTPase RhoA and actin rearrangements in host cells. In infection studies with V. parahaemolyticus we further demonstrate that adhesin-triggered activation of the ROCK/LIMK signaling axis is sufficient to redistribute tight junction proteins, leading to a loss of epithelial barrier function. Taken together, these findings show an unprecedented mechanism by which an adhesin acts as assembly platform for a host cellular signaling pathway, which ultimately facilitates breaching of the epithelial barrier by a bacterial pathogen.

  15. Cisplatin as an Anti-Tumor Drug: Cellular Mechanisms of Activity, Drug Resistance and Induced Side Effects

    International Nuclear Information System (INIS)

    Florea, Ana-Maria; Büsselberg, Dietrich

    2011-01-01

    Platinum complexes are clinically used as adjuvant therapy of cancers aiming to induce tumor cell death. Depending on cell type and concentration, cisplatin induces cytotoxicity, e.g., by interference with transcription and/or DNA replication mechanisms. Additionally, cisplatin damages tumors via induction of apoptosis, mediated by the activation of various signal transduction pathways, including calcium signaling, death receptor signaling, and the activation of mitochondrial pathways. Unfortunately, neither cytotoxicity nor apoptosis are exclusively induced in cancer cells, thus, cisplatin might also lead to diverse side-effects such as neuro- and/or renal-toxicity or bone marrow-suppression. Moreover, the binding of cisplatin to proteins and enzymes may modulate its biochemical mechanism of action. While a combination-chemotherapy with cisplatin is a cornerstone for the treatment of multiple cancers, the challenge is that cancer cells could become cisplatin-resistant. Numerous mechanisms of cisplatin resistance were described including changes in cellular uptake, drug efflux, increased detoxification, inhibition of apoptosis and increased DNA repair. To minimize cisplatin resistance, combinatorial therapies were developed and have proven more effective to defeat cancers. Thus, understanding of the biochemical mechanisms triggered by cisplatin in tumor cells may lead to the design of more efficient platinum derivates (or other drugs) and might provide new therapeutic strategies and reduce side effects

  16. Cisplatin as an Anti-Tumor Drug: Cellular Mechanisms of Activity, Drug Resistance and Induced Side Effects

    Energy Technology Data Exchange (ETDEWEB)

    Florea, Ana-Maria [Department of Neuropathology, Heinrich-Heine University, Düsseldorf (Germany); Büsselberg, Dietrich, E-mail: dib2015@qatar-med.cornell.edu [Weil Cornell Medical College in Qatar, Qatar Foundation-Education City, P.O. Box 24144, Doha (Qatar)

    2011-03-15

    Platinum complexes are clinically used as adjuvant therapy of cancers aiming to induce tumor cell death. Depending on cell type and concentration, cisplatin induces cytotoxicity, e.g., by interference with transcription and/or DNA replication mechanisms. Additionally, cisplatin damages tumors via induction of apoptosis, mediated by the activation of various signal transduction pathways, including calcium signaling, death receptor signaling, and the activation of mitochondrial pathways. Unfortunately, neither cytotoxicity nor apoptosis are exclusively induced in cancer cells, thus, cisplatin might also lead to diverse side-effects such as neuro- and/or renal-toxicity or bone marrow-suppression. Moreover, the binding of cisplatin to proteins and enzymes may modulate its biochemical mechanism of action. While a combination-chemotherapy with cisplatin is a cornerstone for the treatment of multiple cancers, the challenge is that cancer cells could become cisplatin-resistant. Numerous mechanisms of cisplatin resistance were described including changes in cellular uptake, drug efflux, increased detoxification, inhibition of apoptosis and increased DNA repair. To minimize cisplatin resistance, combinatorial therapies were developed and have proven more effective to defeat cancers. Thus, understanding of the biochemical mechanisms triggered by cisplatin in tumor cells may lead to the design of more efficient platinum derivates (or other drugs) and might provide new therapeutic strategies and reduce side effects.

  17. Activated α2 -Macroglobulin Induces Mesenchymal Cellular Migration Of Raw264.7 Cells Through Low-Density Lipoprotein Receptor-Related Protein 1.

    Science.gov (United States)

    Ferrer, Darío G; Dato, Virginia Actis; Fincati, Javier R Jaldín; Lorenc, Valeria E; Sánchez, María C; Chiabrando, Gustavo A

    2017-07-01

    Distinct modes of cell migration contribute to diverse types of cell movements. The mesenchymal mode is characterized by a multistep cycle of membrane protrusion, the formation of focal adhesion, and the stabilization at the leading edge associated with the degradation of extracellular matrix (ECM) components and with regulated extracellular proteolysis. Both α 2 -Macroglobulin (α 2 M) and its receptor, low density lipoprotein receptor-related protein 1 (LRP1), play important roles in inflammatory processes, by controlling the extracellular activity of several proteases. The binding of the active form of α 2 M (α 2 M*) to LRP1 can also activate different signaling pathways in macrophages, thus inducing extracellular matrix metalloproteinase-9 (MMP-9) activation and cellular proliferation. In the present study, we investigated whether the α 2 M*/LRP1 interaction induces cellular migration of the macrophage-derived cell line, Raw264.7. By using the wound-scratch migration assay and confocal microscopy, we demonstrate that α 2 M* induces LRP1-mediated mesenchymal cellular migration. This migration exhibits the production of enlarged cellular protrusions, MT1-MMP distribution to these leading edge protrusions, actin polymerization, focal adhesion formation, and increased intracellular LRP1/β1-integrin colocalization. Moreover, the presence of calphostin-C blocked the α 2 M*-stimulated cellular protrusions, suggesting that the PKC activation is involved in the cellular motility of Raw264.7 cells. These findings could constitute a therapeutic target for inflammatory processes with deleterious consequences for human health, such as rheumatoid arthritis, atherosclerosis and cancer. J. Cell. Biochem. 118: 1810-1818, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  18. Regulation of HTLV-1 Tax Stability, Cellular Trafficking and NF-κB Activation by the Ubiquitin-Proteasome Pathway

    Science.gov (United States)

    Lavorgna, Alfonso; Harhaj, Edward William

    2014-01-01

    Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that infects CD4+ T cells and causes adult T-cell leukemia/lymphoma (ATLL) in 3%–5% of infected individuals after a long latent period. HTLV-1 Tax is a trans-activating protein that regulates viral gene expression and also modulates cellular signaling pathways to enhance T-cell proliferation and cell survival. The Tax oncoprotein promotes T-cell transformation, in part via constitutive activation of the NF-κB transcription factor; however, the underlying mechanisms remain unknown. Ubiquitination is a type of post-translational modification that occurs in a three-step enzymatic cascade mediated by E1, E2 and E3 enzymes and regulates protein stability as well as signal transduction, protein trafficking and the DNA damage response. Emerging studies indicate that Tax hijacks the ubiquitin machinery to activate ubiquitin-dependent kinases and downstream NF-κB signaling. Tax interacts with the E2 conjugating enzyme Ubc13 and is conjugated on C-terminal lysine residues with lysine 63-linked polyubiquitin chains. Tax K63-linked polyubiquitination may serve as a platform for signaling complexes since this modification is critical for interactions with NEMO and IKK. In addition to NF-κB signaling, mono- and polyubiquitination of Tax also regulate its subcellular trafficking and stability. Here, we review recent advances in the diverse roles of ubiquitin in Tax function and how Tax usurps the ubiquitin-proteasome pathway to promote oncogenesis. PMID:25341660

  19. Phyto-mediated nanostructured carriers based on dual vegetable actives involved in the prevention of cellular damage

    International Nuclear Information System (INIS)

    Istrati, D.; Lacatusu, I.; Bordei, N.; Badea, G.; Oprea, O.; Stefan, L.M.; Stan, R.; Badea, N.; Meghea, A.

    2016-01-01

    The growing scientific interest in exploitation of vegetable bioactives has raised a number of questions regarding their imminent presence in pharmaceutical formulations. This study intends to demonstrate that a dual combination between vegetable oil (e.g. thistle oil, safflower oil, sea buckthorn oil) and a carrot extract represents an optimal approach to formulate safe carrier systems that manifest cell regeneration effect and promising antioxidant and anti-inflammatory activity. Inclusion of both natural actives into lipid carriers imparted a strong negative charge on the nanocarrier surface (up to − 45 mV) and displayed average sizes of 70 nm to 140 nm. The entrapment efficiency of carrot extract into nanostructured carriers ranged between 78.3 and 88.3%. The in vitro release study has demonstrated that the entrapment of the extract represents a viable way for an equilibrated release of carotenoids. Besides the excellent antioxidant properties (e.g. scavenging up to 98% of the free oxygen radicals), the results of cellular integrity (e.g. cell viability of 133%) recommend these nanocarriers based on dual carrot extract–bioactive oil as a promising trend for the treatment of certain disorders in which oxidative stress plays a prominent role. In addition, the lipid nanocarriers based on safflower oil and sea buckthorn oil demonstrated an anti-inflammatory effect on LPS induced THP-1 macrophages, by inhibiting the secretion of two pro-inflammatory cytokines, IL-6 and TNF-α. - Highlights: • Safety phyto-mediated nanostructured carriers (NLC) based on two kinds of bioactives • Carrot extract incorporation into nanostructured carriers ranged from 78 to 88.3%. • High antioxidant activity of NLC by scavenging up to 98% free oxygen radicals • Extract entrapment represents a viable way for an equilibrated release of carotenoids. • Remarkable regenerative effect of L929 cell, with a proliferation of 133.4%

  20. Phyto-mediated nanostructured carriers based on dual vegetable actives involved in the prevention of cellular damage

    Energy Technology Data Exchange (ETDEWEB)

    Istrati, D.; Lacatusu, I.; Bordei, N.; Badea, G.; Oprea, O. [University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Polizu Street No. 1, 011061 Bucharest (Romania); Stefan, L.M. [National Institute of Research and Development for Biological Sciences, Splaiul Independentei Street No. 296, 060031 Bucharest (Romania); Stan, R. [University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Polizu Street No. 1, 011061 Bucharest (Romania); Badea, N., E-mail: nicoleta.badea@gmail.com [University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Polizu Street No. 1, 011061 Bucharest (Romania); Meghea, A. [University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Polizu Street No. 1, 011061 Bucharest (Romania)

    2016-07-01

    The growing scientific interest in exploitation of vegetable bioactives has raised a number of questions regarding their imminent presence in pharmaceutical formulations. This study intends to demonstrate that a dual combination between vegetable oil (e.g. thistle oil, safflower oil, sea buckthorn oil) and a carrot extract represents an optimal approach to formulate safe carrier systems that manifest cell regeneration effect and promising antioxidant and anti-inflammatory activity. Inclusion of both natural actives into lipid carriers imparted a strong negative charge on the nanocarrier surface (up to − 45 mV) and displayed average sizes of 70 nm to 140 nm. The entrapment efficiency of carrot extract into nanostructured carriers ranged between 78.3 and 88.3%. The in vitro release study has demonstrated that the entrapment of the extract represents a viable way for an equilibrated release of carotenoids. Besides the excellent antioxidant properties (e.g. scavenging up to 98% of the free oxygen radicals), the results of cellular integrity (e.g. cell viability of 133%) recommend these nanocarriers based on dual carrot extract–bioactive oil as a promising trend for the treatment of certain disorders in which oxidative stress plays a prominent role. In addition, the lipid nanocarriers based on safflower oil and sea buckthorn oil demonstrated an anti-inflammatory effect on LPS induced THP-1 macrophages, by inhibiting the secretion of two pro-inflammatory cytokines, IL-6 and TNF-α. - Highlights: • Safety phyto-mediated nanostructured carriers (NLC) based on two kinds of bioactives • Carrot extract incorporation into nanostructured carriers ranged from 78 to 88.3%. • High antioxidant activity of NLC by scavenging up to 98% free oxygen radicals • Extract entrapment represents a viable way for an equilibrated release of carotenoids. • Remarkable regenerative effect of L929 cell, with a proliferation of 133.4%.

  1. Native aggregation as a cause of origin of temporary cellular structures needed for all forms of cellular activity, signaling and transformations

    Directory of Open Access Journals (Sweden)

    Matveev Vladimir V

    2010-06-01

    Full Text Available Abstract According to the hypothesis explored in this paper, native aggregation is genetically controlled (programmed reversible aggregation that occurs when interacting proteins form new temporary structures through highly specific interactions. It is assumed that Anfinsen's dogma may be extended to protein aggregation: composition and amino acid sequence determine not only the secondary and tertiary structure of single protein, but also the structure of protein aggregates (associates. Cell function is considered as a transition between two states (two states model, the resting state and state of activity (this applies to the cell as a whole and to its individual structures. In the resting state, the key proteins are found in the following inactive forms: natively unfolded and globular. When the cell is activated, secondary structures appear in natively unfolded proteins (including unfolded regions in other proteins, and globular proteins begin to melt and their secondary structures become available for interaction with the secondary structures of other proteins. These temporary secondary structures provide a means for highly specific interactions between proteins. As a result, native aggregation creates temporary structures necessary for cell activity. "One of the principal objects of theoretical research in any department of knowledge is to find the point of view from which the subject appears in its greatest simplicity." Josiah Willard Gibbs (1839-1903

  2. Native aggregation as a cause of origin of temporary cellular structures needed for all forms of cellular activity, signaling and transformations.

    Science.gov (United States)

    Matveev, Vladimir V

    2010-06-09

    According to the hypothesis explored in this paper, native aggregation is genetically controlled (programmed) reversible aggregation that occurs when interacting proteins form new temporary structures through highly specific interactions. It is assumed that Anfinsen's dogma may be extended to protein aggregation: composition and amino acid sequence determine not only the secondary and tertiary structure of single protein, but also the structure of protein aggregates (associates). Cell function is considered as a transition between two states (two states model), the resting state and state of activity (this applies to the cell as a whole and to its individual structures). In the resting state, the key proteins are found in the following inactive forms: natively unfolded and globular. When the cell is activated, secondary structures appear in natively unfolded proteins (including unfolded regions in other proteins), and globular proteins begin to melt and their secondary structures become available for interaction with the secondary structures of other proteins. These temporary secondary structures provide a means for highly specific interactions between proteins. As a result, native aggregation creates temporary structures necessary for cell activity."One of the principal objects of theoretical research in any department of knowledge is to find the point of view from which the subject appears in its greatest simplicity."Josiah Willard Gibbs (1839-1903).

  3. Rac1 Regulates the Activity of mTORC1 and mTORC2 and Controls Cellular Size

    Science.gov (United States)

    Saci, Abdelhafid; Cantley, Lewis C.; Carpenter, Christopher L.

    2013-01-01

    SUMMARY Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that exists in two separate complexes, mTORC1 and mTORC2, that function to control cell size and growth in response to growth factors, nutrients, and cellular energy levels. Low molecular weight GTP-binding proteins of the Rheb and Rag families are key regulators of the mTORC1 complex, but regulation of mTORC2 is poorly understood. Here, we report that Rac1, a member of the Rho family of GTPases, is a critical regulator of both mTORC1 and mTORC2 in response to growth-factor stimulation. Deletion of Rac1 in primary cells using an inducible-Cre/Lox approach inhibits basal and growth-factor activation of both mTORC1 and mTORC2. Rac1 appears to bind directly to mTOR and to mediate mTORC1 and mTORC2 localization at specific membranes. Binding of Rac1 to mTOR does not depend on the GTP-bound state of Rac1, but on the integrity of its C-terminal domain. This function of Rac1 provides a means to regulate mTORC1 and mTORC2 simultaneously. PMID:21474067

  4. Structure of modified [epsilon]-polylysine micelles and their application in improving cellular antioxidant activity of curcuminoids

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Hailong; Li, Ji; Shi, Ke; Huang, Qingrong (Rutgers)

    2015-10-15

    The micelle structure of octenyl succinic anhydride modified {var_epsilon}-polylysine (M-EPL), an anti-microbial surfactant prepared from natural peptide {var_epsilon}-polylysine in aqueous solution has been studied using synchrotron small-angle X-ray scattering (SAXS). Our results revealed that M-EPLs formed spherical micelles with individual size of 24-26 {angstrom} in aqueous solution which could further aggregate to form a larger dimension with averaged radius of 268-308 {angstrom}. Furthermore, M-EPL micelle was able to encapsulate curcuminoids, a group of poorly-soluble bioactive compounds from turmeric with poor oral bioavailability, and improve their water solubility. Three loading methods, including solvent evaporation, dialysis, and high-speed homogenization were compared. The results indicated that the dialysis method generated the highest loading capacity and curcuminoids water solubility. The micelle encapsulation was confirmed as there were no free curcuminoid crystals detected in the differential scanning calorimetry analysis. It was also demonstrated that M-EPL encapsulation stabilized curcuminoids against hydrolysis at pH 7.4 and the encapsulated curcuminoids showed elevated cellular antioxidant activity compared with free curcuminoids. This work suggested that M-EPL could be used as new biopolymer micelles for delivering poorly soluble drugs/phytochemicals and improving their bioactivities.

  5. Fabrication of curcumin-loaded bovine serum albumin (BSA)-dextran nanoparticles and the cellular antioxidant activity.

    Science.gov (United States)

    Fan, Yuting; Yi, Jiang; Zhang, Yuzhu; Yokoyama, Wallace

    2018-01-15

    Bovine serum albumin (BSA)-dextran conjugate was prepared with glycation. Self-assembly nanoparticles were synthesized with a green, and facile approach. The effects of dry-heating time on the fabrication and characteristics of BSA-dextran conjugate nanoparticles were examined. Stable nanoparticles (dextran was grafted onto the BSA to provide significant steric hindrance. Particle size decreased with the increase of dry-heating time and the lowest particle size (51.2nm) was obtained after 24h dry-heating. The nanoparticles were stable in a wide pH range (pH 2.0-7.0). The particle size of nanoparticles increased to 115nm after curcumin incorporation and was stable even after one-month storage. TEM results demonstrated that curcumin-loaded nanoparticles displayed a spherical structure and were homogeneously dispersed. Curcumin in BSA-dextran nanoparticle showed better stability, compared to free curcumin. In addition, BSA-dextran nanoparticles can improve the cellular antioxidant activity of curcumin in Caco-2 cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Ebselen impairs cellular oxidative state and induces endoplasmic reticulum stress and activation of crucial mitogen-activated protein kinases in pancreatic tumour AR42J cells.

    Science.gov (United States)

    Santofimia-Castaño, Patricia; Izquierdo-Alvarez, Alicia; Plaza-Davila, María; Martinez-Ruiz, Antonio; Fernandez-Bermejo, Miguel; Mateos-Rodriguez, Jose M; Salido, Gines M; Gonzalez, Antonio

    2018-01-01

    Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is an organoselenium radical scavenger compound, which has strong antioxidant and anti-inflammatory effects. However, evidence suggests that this compound could exert deleterious actions on cell physiology. In this study, we have analyzed the effect of ebselen on rat pancreatic AR42J cells. Cytosolic free-Ca 2+ concentration ([Ca 2+ ] c ), cellular oxidative status, setting of endoplasmic reticulum stress, and phosphorylation of major mitogen-activated protein kinases were analyzed. Our results show that ebselen evoked a concentration-dependent increase in [Ca 2+ ] c . The compound induced an increase in the generation of reactive oxygen species in the mitochondria. We also observed an increase in global cysteine oxidation in the presence of ebselen. In the presence of ebselen an impairment of cholecystokinin-evoked amylase release was noted. Moreover, involvement of the unfolded protein response markers, ER chaperone and signaling regulator GRP78/BiP, eukaryotic translation initiation factor 2α and X-box binding protein 1 was detected. Finally, increases in the phosphorylation of SAPK/JNK, p38 MAPK, and p44/42 MAPK in the presence of ebselen were also observed. Our results provide evidences for an impairment of cellular oxidative state and enzyme secretion, the induction of endoplasmic reticulum stress and the activation of crucial mitogen-activated protein kinases in the presence of ebselen. As a consequence ebselen exerts a potential toxic effect on AR42J cells. © 2017 Wiley Periodicals, Inc.

  7. High Cellular Monocyte Activation in People Living With Human Immunodeficiency Virus on Combination Antiretroviral Therapy and Lifestyle-Matched Controls Is Associated With Greater Inflammation in Cerebrospinal Fluid

    NARCIS (Netherlands)

    Booiman, Thijs; Wit, Ferdinand W N M; Maurer, Irma; De Francesco, Davide; Sabin, Caroline A; Harskamp, Agnes M; Prins, Maria; Garagnani, Paolo; Pirazzini, Chiara; Franceschi, Claudio; Fuchs, Dietmar; Gisslén, Magnus; Winston, Alan; Reiss, Peter; Kootstra, Neeltje A; Kalsbeek, A.

    2017-01-01

    BACKGROUND: Increased monocyte activation and intestinal damage have been shown to be predictive for the increased morbidity and mortality observed in treated people living with human immunodeficiency virus (PLHIV). METHODS: A cross-sectional analysis of cellular and soluble markers of monocyte

  8. Perfluorinated chemicals: Differential toxicity, inhibition of aromatase activity and alteration of cellular lipids in human placental cells

    Energy Technology Data Exchange (ETDEWEB)

    Gorrochategui, Eva; Pérez-Albaladejo, Elisabet [Department of Environmental Chemistry, IDAEA–CSIC, 08034 Barcelona, Catalonia (Spain); Casas, Josefina [Department of Biomedicinal Chemistry, IQAC–CSIC, 08034 Barcelona, Catalonia (Spain); Lacorte, Sílvia, E-mail: slbqam@cid.csic.es [Department of Environmental Chemistry, IDAEA–CSIC, 08034 Barcelona, Catalonia (Spain); Porte, Cinta, E-mail: cinta.porte@cid.csic.es [Department of Environmental Chemistry, IDAEA–CSIC, 08034 Barcelona, Catalonia (Spain)

    2014-06-01

    The cytotoxicity of eight perfluorinated chemicals (PFCs), namely, perfluorobutanoic acid (PFBA), perfluorohexanoic acid (PFHxA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorododecanoic acid (PFDoA), perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHxS) and perfluorooctanesulfonate (PFOS) was assessed in the human placental choriocarcinoma cell line JEG-3. Only the long chain PFCs – PFOS, PFDoA, PFNA, PFOA – showed significant cytotoxicity in JEG-3 cells with EC50 values in the range of 107 to 647 μM. The observed cytotoxicity was to some extent related to a higher uptake of the longer chain PFCs by cells (PFDoA > PFOS ≫ PFNA > PFOA > PFHxA). Moreover, this work evidences a high potential of PFOS, PFOA and PFBS to act as aromatase inhibitors in placental cells with IC50s in the range of 57–80 μM, the inhibitory effect of PFBS being particularly important despite the rather low uptake of the compound by cells. Finally, exposure of JEG-3 cells to a mixture of the eight PFCs (0.6 μM each) led to a relative increase (up to 3.4-fold) of several lipid classes, including phosphatidylcholines (PCs), plasmalogen PC and lyso plasmalogen PC, which suggests an interference of PFCs with membrane lipids. Overall, this work highlights the ability of the PFC mixture to alter cellular lipid pattern at concentrations well below those that generate toxicity, and the potential of the short chain PFBS, often considered a safe substitute of PFOS, to significantly inhibit aromatase activity in placental cells. - Highlights: • Eight perfluorinated chemicals of different chain lengths have been selected. • Long chain ones – PFOS, PFDoA, PFNA, PFOA – were cytotoxic in placenta cells. • The uptake of long chain perfluorinated chemicals by cells was comparatively higher. • PFOS, PFOA and the short chain PFBS significantly inhibited aromatase activity. • A mixture of perfluorinated chemicals significantly altered placenta cell

  9. Activation of p53 by nutlin-3a induces apoptosis and cellular senescence in human glioblastoma multiforme.

    Directory of Open Access Journals (Sweden)

    Ruth Villalonga-Planells

    2011-04-01

    Full Text Available Glioblastoma multiforme (GBM is the most common and aggressive primary brain tumor in adults. Despite concerted efforts to improve current therapies and develop novel clinical approaches, patient survival remains poor. As such, increasing attention has focused on developing new therapeutic strategies that specifically target the apoptotic pathway in order to improve treatment responses. Recently, nutlins, small-molecule antagonists of MDM2, have been developed to inhibit p53-MDM2 interaction and activate p53 signaling in cancer cells. Glioma cell lines and primary cultured glioblastoma cells were treated with nutlin-3a. Nutlin-3a induced p53-dependent G1- and G2-M cell cycle arrest and apoptosis in glioma cell lines with normal TP53 status. In addition, nutlin-arrested glioma cells show morphological features of senescence and persistent induction of p21 protein. Furthermore, senescence induced by nutlin-3a might be depending on mTOR pathway activity. In wild-type TP53 primary cultured cells, exposure to nutlin-3a resulted in variable degrees of apoptosis as well as cellular features of senescence. Nutlin-3a-induced apoptosis and senescence were firmly dependent on the presence of functional p53, as revealed by the fact that glioblastoma cells with knockdown p53 with specific siRNA, or cells with mutated or functionally impaired p53 pathway, were completely insensitive to the drug. Finally, we also found that nutlin-3a increased response of glioma cells to radiation therapy. The results provide a basis for the rational use of MDM2 antagonists as a novel treatment option for glioblastoma patients.

  10. Perfluorinated chemicals: Differential toxicity, inhibition of aromatase activity and alteration of cellular lipids in human placental cells

    International Nuclear Information System (INIS)

    Gorrochategui, Eva; Pérez-Albaladejo, Elisabet; Casas, Josefina; Lacorte, Sílvia; Porte, Cinta

    2014-01-01

    The cytotoxicity of eight perfluorinated chemicals (PFCs), namely, perfluorobutanoic acid (PFBA), perfluorohexanoic acid (PFHxA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorododecanoic acid (PFDoA), perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHxS) and perfluorooctanesulfonate (PFOS) was assessed in the human placental choriocarcinoma cell line JEG-3. Only the long chain PFCs – PFOS, PFDoA, PFNA, PFOA – showed significant cytotoxicity in JEG-3 cells with EC50 values in the range of 107 to 647 μM. The observed cytotoxicity was to some extent related to a higher uptake of the longer chain PFCs by cells (PFDoA > PFOS ≫ PFNA > PFOA > PFHxA). Moreover, this work evidences a high potential of PFOS, PFOA and PFBS to act as aromatase inhibitors in placental cells with IC50s in the range of 57–80 μM, the inhibitory effect of PFBS being particularly important despite the rather low uptake of the compound by cells. Finally, exposure of JEG-3 cells to a mixture of the eight PFCs (0.6 μM each) led to a relative increase (up to 3.4-fold) of several lipid classes, including phosphatidylcholines (PCs), plasmalogen PC and lyso plasmalogen PC, which suggests an interference of PFCs with membrane lipids. Overall, this work highlights the ability of the PFC mixture to alter cellular lipid pattern at concentrations well below those that generate toxicity, and the potential of the short chain PFBS, often considered a safe substitute of PFOS, to significantly inhibit aromatase activity in placental cells. - Highlights: • Eight perfluorinated chemicals of different chain lengths have been selected. • Long chain ones – PFOS, PFDoA, PFNA, PFOA – were cytotoxic in placenta cells. • The uptake of long chain perfluorinated chemicals by cells was comparatively higher. • PFOS, PFOA and the short chain PFBS significantly inhibited aromatase activity. • A mixture of perfluorinated chemicals significantly altered placenta cell

  11. Nogo-receptor gene activity: cellular localization and developmental regulation of mRNA in mice and humans.

    Science.gov (United States)

    Josephson, Anna; Trifunovski, Alexandra; Widmer, Hans Ruedi; Widenfalk, Johan; Olson, Lars; Spenger, Christian

    2002-11-18

    Nogo (reticulon-4) is a myelin-associated protein that is expressed in three different splice variants, Nogo-A, Nogo-B, and Nogo-C. Nogo-A inhibits neurite regeneration in the central nervous system. Messenger RNA encoding Nogo is expressed in oligodendrocytes and central and peripheral neurons, but not in astrocytes or Schwann cells. Nogo is a transmembraneous protein; the extracellular domain is termed Nogo-66, and a Nogo-66-receptor (Nogo-R) has been identified. We performed in situ hybridization in human and mouse nervous tissues to map the cellular distribution of Nogo-R gene activity patterns in fetal and adult human spinal cord and sensory ganglia, adult human brain, and the nervous systems of developing and adult mice. In the human fetus Nogo-R was transcribed in the ventral horn of the spinal cord and in dorsal root ganglia. In adult human tissues Nogo-R gene activity was found in neocortex, hippocampus, amygdala, and a subset of large and medium-sized neurons of the dorsal root ganglia. Nogo-R mRNA was not expressed in the adult human spinal cord at detectable levels. In the fetal mouse, Nogo-R was diffusely expressed in brain, brainstem, trigeminal ganglion, spinal cord, and dorsal root ganglia at all stages. In the adult mouse strong Nogo-R mRNA expression was found in neurons in neocortex, hippocampus, amygdala, habenula, thalamic nuclei, brainstem, the granular cell layer of cerebellum, and the mitral cell layer of the olfactory bulb. Neurons in the adult mouse striatum, the medial septal nucleus, and spinal cord did not express Nogo-R mRNA at detectable levels. In summary, Nogo-66-R mRNA expression in humans and mice was observed in neurons of the developing nervous system Expression was downregulated in the adult spinal cord of both species, and specific expression patterns were seen in the adult brain. Copyright 2002 Wiley-Liss, Inc.

  12. Improved cellular activity of antisense peptide nucleic acids by conjugation to a cationic peptide-lipid (CatLip) domain

    DEFF Research Database (Denmark)

    Koppelhus, Uffe; Shiraishi, Takehiko; Zachar, Vladimir

    2008-01-01

    Conjugation to cationic cell penetrating peptides (such as Tat, Penetratin, or oligo arginines) efficiently improves the cellular uptake of large hydrophilic molecules such as oligonucleotides and peptide nucleic acids, but the cellular uptake is predominantly via an unproductive endosomal pathway...... for future in vivo applications. We find that simply conjugating a lipid domain (fatty acid) to the cationic peptide (a CatLip conjugate) increases the biological effect of the corresponding PNA (CatLip) conjugates in a luciferase cellular antisense assay up to 2 orders of magnitude. The effect increases...... with increasing length of the fatty acid (C8-C16) but in parallel also results in increased cellular toxicity, with decanoic acid being optimal. Furthermore, the relative enhancement is significantly higher for Tat peptide compared to oligoarginine. Confocal microscopy and chloroquine enhancement indicates...

  13. Restriction on an energy-dense diet improves markers of metabolic health and cellular aging in mice through decreasing hepatic mTOR activity.

    Science.gov (United States)

    Schloesser, Anke; Campbell, Graeme; Glüer, Claus-Christian; Rimbach, Gerald; Huebbe, Patricia

    2015-02-01

    Dietary restriction (DR) on a normal low-fat diet improves metabolic health and may prolong life span. However, it is still uncertain whether restriction of an energy-dense, high-fat diet would also be beneficial and mitigate age-related processes. In the present study, we determined biomarkers of metabolic health, energy metabolism, and cellular aging in obesity-prone mice subjected to 30% DR on a high-fat diet for 6 months. Dietary-restricted mice had significantly lower body weights, less adipose tissue, lower energy expenditure, and altered substrate oxidation compared to their ad libitum-fed counterparts. Hepatic major urinary proteins (Mup) expression, which is linked to glucose and energy metabolism, and biomarkers of metabolic health, including insulin, glucose, cholesterol, and leptin/adiponectin ratio, were likewise reduced in high-fat, dietary-restricted mice. Hallmarks of cellular senescence such as Lamp2a and Hsc70 that mediate chaperone-mediated autophagy were induced and mechanistic target of rapamycin (mTOR) signaling mitigated upon high-fat DR. In contrast to DR applied in low-fat diets, anti-oxidant gene expression, proteasome activity, as well as 5'-adenosine monophosphate-activated protein kinase (AMPK) activation were not changed, suggesting that high-fat DR may attenuate some processes associated with cellular aging without the induction of cellular stress response or energy deprivation.

  14. Cellular MR Imaging

    Directory of Open Access Journals (Sweden)

    Michel Modo

    2005-07-01

    Full Text Available Cellular MR imaging is a young field that aims to visualize targeted cells in living organisms. In order to provide a different signal intensity of the targeted cell, they are either labeled with MR contrast agents in vivo or prelabeled in vitro. Either (ultrasmall superparamagnetic iron oxide [(USPIO] particles or (polymeric paramagnetic chelates can be used for this purpose. For in vivo cellular labeling, Gd3+- and Mn2+- chelates have mainly been used for targeted hepatobiliary imaging, and (USPIO-based cellular imaging has been focused on imaging of macrophage activity. Several of these magneto-pharmaceuticals have been FDA-approved or are in late-phase clinical trials. As for prelabeling of cells in vitro, a challenge has been to induce a sufficient uptake of contrast agents into nonphagocytic cells, without affecting normal cellular function. It appears that this issue has now largely been resolved, leading to an active research on monitoring the cellular biodistribution in vivo following transplantation or transfusion of these cells, including cell migration and trafficking. New applications of cellular MR imaging will be directed, for instance, towards our understanding of hematopoietic (immune cell trafficking and of novel guided (stem cell-based therapies aimed to be translated to the clinic in the future.

  15. Dermal quercetin lipid nanocapsules: Influence of the formulation on antioxidant activity and cellular protection against hydrogen peroxide.

    Science.gov (United States)

    Hatahet, T; Morille, M; Shamseddin, A; Aubert-Pouëssel, A; Devoisselle, J M; Bégu, S

    2017-02-25

    Quercetin is a plant flavonoid with strong antioxidant and antiinflammatory properties interesting for skin protection. However, its poor water solubility limits its penetration and so its efficiency on skin. For this purpose, quercetin lipid nanocapsules were formulated implementing phase inversion technique wherein several modifications were introduced to enhance quercetin loading. Quercetin lipid nanocapsules were formulated with two particle size range, (50nm and 20nm) allowing a drug loading of 18.6 and 32mM respectively. The successful encapsulation of quercetin within lipid nanocapsules increased its apparent water solubility by more than 5000 fold (from 0.5μg/ml to about 5mg/ml). The physicochemical properties of these formulations such as surface charge, stability and morphology were characterized. Lipid nanocapsules had spherical shape and were stable for 28days at 25°C. Quercetin release from lipid nanocapsules was studied and revealed a prolonged release kinetics during 24h. Using DPPH assay, we demonstrated that the formulation process of lipid nanocapsules did not modify the antioxidant activity of quercetin in vitro (92.3%). With the goal of a future dermal application, quercetin lipid nanocapsules were applied to THP-1 monocytes and proved the cellular safety of the formulation up to 2μg/ml of quercetin. Finally, formulated quercetin was as efficient as the crude form in the protection of THP-1 cells from oxidative stress by exogenous hydrogen peroxide. With its lipophilic nature and occlusive effect on skin, lipid nanocapsules present a promising strategy to deliver quercetin to skin tissue and can be of value for other poorly water soluble drug candidates. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Cellular metabolism

    International Nuclear Information System (INIS)

    Hildebrand, C.E.; Walters, R.A.

    1977-01-01

    Progress is reported on the following research projects: chromatin structure; the use of circular synthetic polydeoxynucleotides as substrates for the study of DNA repair enzymes; human cellular kinetic response following exposure to DNA-interactive compounds; histone phosphorylation and chromatin structure in cell proliferation; photoaddition products induced in chromatin by uv light; pollutants and genetic information transfer; altered RNA metabolism as a function of cadmium accumulation and intracellular distribution in cultured cells; and thymidylate chromophore destruction by water free radicals

  17. Effects of wearing bio-active material coated fabric against γ-irradiation-induced cellular damaged in Sprague-Dawley rats

    International Nuclear Information System (INIS)

    Kang, Jung Ae; Kim, Hye Rim; Yoon, Sun Hye; Nam, Sang Hyun; Park, Sang Hyun; Jang, Beom Su; Go, Kyung Chan; Yang, Gwang Wung; Rho, Young Hwan; Park, Hyo Suk

    2016-01-01

    Ionizing radiation causes cellular damage and death through the direct damage and/or indirectly the production of ROS, which induces oxidative stress. This study was designed to evaluate the in vivo radioprotective effects of a bio-active material coated fabric (BMCF) against γ-irradiation-induced cellular damage in Sprague-Dawley (SD) rats. Healthy male SD rats wore bio-active material coated (concentrations in 10% and 30%) fabric for 7 days after 3 Gy of γ-irradiation. Radioprotective effects were evaluated by performing various biochemical assays including spleen and thymus index, WBC count, hepatic damage marker enzymes [aspartate transaminase (AST) and alanine transaminase (ALT)] in plasma, liver antioxidant enzymes, and mitochondrial activity in muscle. Exposure to γ-irradiation resulted in hepatocellular and immune systemic damage. Gamma-irradiation induced decreases in antioxidant enzymes. However, wearing the BMCF-30% decreased significantly AST and ALT activities in plasma. Furthermore, wearing the BMCF-30% increased SOD (superoxide dismutase) and mitochondrial activity. These results suggest that wearing BMCF offers effective radioprotection against γ-irradiation-induced cellular damage in SD rats

  18. Effects of wearing bio-active material coated fabric against γ-irradiation-induced cellular damaged in Sprague-Dawley rats

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Jung Ae; Kim, Hye Rim; Yoon, Sun Hye; Nam, Sang Hyun; Park, Sang Hyun; Jang, Beom Su [Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of); Go, Kyung Chan; Yang, Gwang Wung; Rho, Young Hwan; Park, Hyo Suk [Research and Development Center, VENTEX Co. Ltd., Seoul (Korea, Republic of)

    2016-09-15

    Ionizing radiation causes cellular damage and death through the direct damage and/or indirectly the production of ROS, which induces oxidative stress. This study was designed to evaluate the in vivo radioprotective effects of a bio-active material coated fabric (BMCF) against γ-irradiation-induced cellular damage in Sprague-Dawley (SD) rats. Healthy male SD rats wore bio-active material coated (concentrations in 10% and 30%) fabric for 7 days after 3 Gy of γ-irradiation. Radioprotective effects were evaluated by performing various biochemical assays including spleen and thymus index, WBC count, hepatic damage marker enzymes [aspartate transaminase (AST) and alanine transaminase (ALT)] in plasma, liver antioxidant enzymes, and mitochondrial activity in muscle. Exposure to γ-irradiation resulted in hepatocellular and immune systemic damage. Gamma-irradiation induced decreases in antioxidant enzymes. However, wearing the BMCF-30% decreased significantly AST and ALT activities in plasma. Furthermore, wearing the BMCF-30% increased SOD (superoxide dismutase) and mitochondrial activity. These results suggest that wearing BMCF offers effective radioprotection against γ-irradiation-induced cellular damage in SD rats.

  19. Macrophages From Irradiated Tumors Express Higher Levels of iNOS, Arginase-I and COX-2, and Promote Tumor Growth

    International Nuclear Information System (INIS)

    Tsai, C.-S.; Chen, F.-H.; Wang, C.-C.; Huang, H.-L.; Jung, Shih-Ming; Wu, C.-J.; Lee, C.-C.; McBride, William H.; Chiang, C.-S.; Hong, J.-H.

    2007-01-01

    Purpose: To investigate the effects of single and fractionated doses of radiation on tumors and tumor-associated macrophages (TAMs), and to elucidate the potential of TAMs to influence tumor growth. Methods and Materials: A murine prostate cell line, TRAMP-C1, was grown in C57Bl/6J mice to 4-mm tumor diameter and irradiated with either 25 Gy in a single dose, or 60 Gy in 15 fractions. The tumors were removed at the indicated times and assessed for a variety of markers related to TAM content, activation status, and function. Results: In tumors receiving a single radiation dose, arginase (Arg-I), and cycloxygenase-2 (COX-2) mRNA expression increased as a small transient wave within 24 h and a larger persistent wave starting after 3 days. Inducible nitric oxide synthase (iNOS) mRNA was elevated only after 3 days and continued to increase up to 3 weeks. After fractionated irradiation, Arg-1 and COX-2 mRNA levels increased within 5 days, whereas iNOS was increased only after 10 fractions of irradiation had been given. Increased levels of Arg-I, COX-2, and, to a lesser extent, iNOS protein were found to associate with TAMs 1-2 weeks after tumor irradiation. Function of TAMs were compared by mixing them with TRAMP-C1 cells and injecting them into mice; TRAMP-C1 cells mixed with TAMs from irradiated tumors appeared earlier and grew significantly faster than those mixed with TAMs from unirradiated tumors or TRAMP-C1 alone. Conclusions: Tumor-associated macrophages in the postirradiated tumor microenvironment express higher levels of Arg-1, COX-2, and iNOS, and promote early tumor growth in vivo

  20. Continuous DC-CIK infusions restore CD8+ cellular immunity, physical activity and improve clinical efficacy in advanced cancer patients unresponsive to conventional treatments.

    Science.gov (United States)

    Zhao, Yan-Jie; Jiang, Ni; Song, Qing-Kun; Wu, Jiang-Ping; Song, Yu-Guang; Zhang, Hong-Mei; Chen, Feng; Zhou, Lei; Wang, Xiao-Li; Zhou, Xin-Na; Yang, Hua-Bing; Ren, Jun; Lyerly, Herbert Kim

    2015-01-01

    There are few choices for treatment of advanced cancer patients who do not respond to or tolerate conventional anti-cancer treatments. Therefore this study aimed to deploy the benefits and clinical efficacy of continuous dendritic cell-cytokine induced killer cell infusions in such patients. A total of 381 infusions (from 67 advanced cases recruited) were included in this study. All patients underwent peripheral blood mononuclear cell apheresis for the following cellular therapy and dendritic cells-cytokine induced killer cells were expanded in vitro. Peripheral blood T lymphocyte subsets were quantified through flow cytometry to address the cellular immunity status. Clinical efficacy and physical activities were evaluated by RECIST criteria and Eastern Cooperative Oncology Group scores respectively. Logistic regression model was used to estimate the association between cellular infusions and clinical benefits. An average of 5.7±2.94x10(9) induced cells were infused each time and patients were exposed to 6 infusions. Cellular immunity was improved in that cytotoxic CD8+CD28+T lymphocytes were increased by 74% and suppressive CD8+CD28-T lymphocytes were elevated by 16% (p<0.05). Continuous infusion of dendritic cells-cytokine induced killer cells was associated with improvement of both patient status and cellular immunity. A median of six infusions were capable of reducing risk of progression by 70% (95%CI 0.10-0.91). Every elevation of one ECOG score corresponded to a 3.90-fold higher progression risk (p<0.05) and 1% increase of CD8+CD28- T cell proportion reflecting a 5% higher risk of progression (p<0.05). In advanced cancer patients, continuous dendritic cell-cytokine induced killer cell infusions are capable of recovering cellular immunity, improving patient status and quality of life in those who are unresponsive to conventional cancer treatment.

  1. Mapping whole-brain activity with cellular resolution by light-sheet microscopy and high-throughput image analysis (Conference Presentation)

    Science.gov (United States)

    Silvestri, Ludovico; Rudinskiy, Nikita; Paciscopi, Marco; Müllenbroich, Marie Caroline; Costantini, Irene; Sacconi, Leonardo; Frasconi, Paolo; Hyman, Bradley T.; Pavone, Francesco S.

    2016-03-01

    Mapping neuronal activity patterns across the whole brain with cellular resolution is a challenging task for state-of-the-art imaging methods. Indeed, despite a number of technological efforts, quantitative cellular-resolution activation maps of the whole brain have not yet been obtained. Many techniques are limited by coarse resolution or by a narrow field of view. High-throughput imaging methods, such as light sheet microscopy, can be used to image large specimens with high resolution and in reasonable times. However, the bottleneck is then moved from image acquisition to image analysis, since many TeraBytes of data have to be processed to extract meaningful information. Here, we present a full experimental pipeline to quantify neuronal activity in the entire mouse brain with cellular resolution, based on a combination of genetics, optics and computer science. We used a transgenic mouse strain (Arc-dVenus mouse) in which neurons which have been active in the last hours before brain fixation are fluorescently labelled. Samples were cleared with CLARITY and imaged with a custom-made confocal light sheet microscope. To perform an automatic localization of fluorescent cells on the large images produced, we used a novel computational approach called semantic deconvolution. The combined approach presented here allows quantifying the amount of Arc-expressing neurons throughout the whole mouse brain. When applied to cohorts of mice subject to different stimuli and/or environmental conditions, this method helps finding correlations in activity between different neuronal populations, opening the possibility to infer a sort of brain-wide 'functional connectivity' with cellular resolution.

  2. [Arginase inhibitor nor-NOHA induces apoptosis and inhibits invasion and migration of HepG2 cells].

    Science.gov (United States)

    Li, Xiangnan; Zhu, Fangyu; He, Yongsong; Luo, Fang

    2017-04-01

    Objective To investigate the cell inhibitory effect of arginase inhibitor nor-NOHA on HepG2 hepatocellular carcinoma cells and related mechanism. Methods CCK-8 assay was used to detect the cell proliferation and flow cytometry to detect the apoptosis of HepG2 cells treated with (0, 0.5, 1.0, 2.0, 3.0) ng/μL nor-NOHA. The protein levels of arginase 1 (Arg1), P53, matrix metalloproteinase-2 (MMP-2), E-cadherin (ECD) were determined by Western blotting. Real time quantitative PCR was employed to examine the changes in the mRNA level of inducible nitric oxide synthase (iNOS). Griess assay was used to measure the concentration of nitric oxide (NO) in HepG2 cells. Transwell TM assay and wound-healing assay were performed to evaluate the changes of the cell invasion and migration ability, respectively. Results nor-NOHA inhibited the proliferation and induced the apoptosis of HepG2 cells. It also decreased the expression levels of Arg1 and MMP-2, increased the expression levels of P53 and ECD as well as the production of NO; in addition, nor-NOHA inhibited the invasion and migration of HepG2 cells. Conclusion Nor-NOHA can induce cell apoptosis and inhibit the ability of invasion and migration of HepG2 cells by inhibiting Arg1, which is related with the increase of iNOS expression and the high concentration of NO.

  3. The cellular transcription factor CREB corresponds to activating transcription factor 47 (ATF-47) and forms complexes with a group of polypeptides related to ATF-43.

    OpenAIRE

    Hurst, H C; Masson, N; Jones, N C; Lee, K A

    1990-01-01

    Promoter elements containing the sequence motif CGTCA are important for a variety of inducible responses at the transcriptional level. Multiple cellular factors specifically bind to these elements and are encoded by a multigene family. Among these factors, polypeptides termed activating transcription factor 43 (ATF-43) and ATF-47 have been purified from HeLa cells and a factor referred to as cyclic AMP response element-binding protein (CREB) has been isolated from PC12 cells and rat brain. We...

  4. The Na{sup +}/K{sup +} -pump in rat peritoneal mast cells: Some aspects of regulatio of activity and cellular fusion

    Energy Technology Data Exchange (ETDEWEB)

    Knudsen, T. [Odense Univ., Dept. of Pharmacology, Inst. of Medical Biology, The Faculty of Health Scineces (Denmark)

    1995-12-31

    The mast cell contains potent mediators of inflammation which are released after IgE-directed and non-IgE-directed stimulation of the cell. This highly specialized cell is therefore ascribed a role in the pathogenesis of disease states in which the inflammatory response plays a role for the development of the clinical symptoms. Thus, besides being of interest in basic research, studies of the cellular processes leading to release of inflammatory mediators from the mast cell also also have important clinical implications. The aim of the present work has been to document the existence of the Na{sup +}/K{sup +}-pump in rat peritoneal mast cells, to investigate the regulation of the pump activity and to explore whether modulation of the pump activity interferes with the cellular stimulus/secretion coupling mechanism. The Na{sup +}/K{sup +}-pump activity following stimulation of the mast cell was also investigated. The pump activity was assessed as the ouabain-sensitive cellular potassium uptake with {sup 86}Rb{sup +} as a tracer for potassium. The histamine release from the mast cell following IgE-directed and non-IgE-directed stimulation of the cell was used as a parameter of cellular degranulation. Histamine was measured by spectrofluorometry. Besides describing aspects of the function and regulation of the Na{sup +}/K{sup +}-pump in the rat peritoneal mast cell, this thesis points to the potential role of sodium transport mechanisms in mast cell physiology. Pharmacological manipulations of such transport mechanisms might in the future add to the treatment of allergic diseases. (au) 253 refs.

  5. A cellular system for quantitation of vitamin K cycle activity: structure-activity effects on vitamin K antagonism by warfarin metabolites

    Science.gov (United States)

    Haque, Jamil A.; McDonald, Matthew G.; Kulman, John D.

    2014-01-01

    Warfarin and other 4-hydroxycoumarins inhibit vitamin K epoxide reductase (VKOR) by depleting reduced vitamin K that is required for posttranslational modification of vitamin K–dependent clotting factors. In vitro prediction of the in vivo potency of vitamin K antagonists is complicated by the complex multicomponent nature of the vitamin K cycle. Here we describe a sensitive assay that enables quantitative analysis of γ-glutamyl carboxylation and its antagonism in live cells. We engineered a human embryonic kidney (HEK) 293–derived cell line (HEK 293-C3) to express a chimeric protein (F9CH) comprising the Gla domain of factor IX fused to the transmembrane and cytoplasmic regions of proline-rich Gla protein 2. Maximal γ-glutamyl carboxylation of F9CH required vitamin K supplementation, and was dose-dependently inhibited by racemic warfarin at a physiologically relevant concentration. Cellular γ-glutamyl carboxylation also exhibited differential VKOR inhibition by warfarin enantiomers (S > R) consistent with their in vivo potencies. We further analyzed the structure-activity relationship for inhibition of γ-glutamyl carboxylation by warfarin metabolites, observing tolerance to phenolic substitution at the C-5 and especially C-6, but not C-7 or C-8, positions on the 4-hydroxycoumarin nucleus. After correction for in vivo concentration and protein binding, 10-hydroxywarfarin and warfarin alcohols were predicted to be the most potent inhibitory metabolites in vivo. PMID:24297869

  6. Histogram analysis parameters of apparent diffusion coefficient reflect tumor cellularity and proliferation activity in head and neck squamous cell carcinoma.

    Science.gov (United States)

    Surov, Alexey; Meyer, Hans Jonas; Winter, Karsten; Richter, Cindy; Hoehn, Anna-Kathrin

    2018-05-04

    Our purpose was to analyze associations between apparent diffusion coefficient (ADC) histogram analysis parameters and histopathologicalfeatures in head and neck squamous cell carcinoma (HNSCC). The study involved 32 patients with primary HNSCC. For every tumor, the following histogram analysis parameters were calculated: ADCmean, ADCmax, ADC min , ADC median , ADC mode , P10, P25, P75, P90, kurtosis, skewness, and entropy. Furthermore, proliferation index KI 67, cell count, total and average nucleic areas were estimated. Spearman's correlation coefficient (p) was used to analyze associations between investigated parameters. In overall sample, all ADC values showed moderate inverse correlations with KI 67. All ADC values except ADCmax correlated inversely with tumor cellularity. Slightly correlations were identified between total/average nucleic area and ADC mean , ADC min , ADC median , and P25. In G1/2 tumors, only ADCmode correlated well with Ki67. No statistically significant correlations between ADC parameters and cellularity were found. In G3 tumors, Ki 67 correlated with all ADC parameters except ADCmode. Cell count correlated well with all ADC parameters except ADCmax. Total nucleic area correlated inversely with ADC mean , ADC min , ADC median , P25, and P90. ADC histogram parameters reflect proliferation potential and cellularity in HNSCC. The associations between histopathology and imaging depend on tumor grading.

  7. A perillyl alcohol-conjugated analog of 3-bromopyruvate without cellular uptake dependency on monocarboxylate transporter 1 and with activity in 3-BP-resistant tumor cells.

    Science.gov (United States)

    Chen, Thomas C; Yu, Jiali; Nouri Nigjeh, Eslam; Wang, Weijun; Myint, Phyo Thazin; Zandi, Ebrahim; Hofman, Florence M; Schönthal, Axel H

    2017-08-01

    The anticancer agent 3-bromopyruvate (3-BP) is viewed as a glycolytic inhibitor that preferentially kills glycolytic cancer cells through energy depletion. However, its cytotoxic activity is dependent on cellular drug import through transmembrane monocarboxylate transporter 1 (MCT-1), which restricts its anticancer potential to MCT-1-positive tumor cells. We created and characterized an MCT-1-independent analog of 3-BP, called NEO218. NEO218 was synthesized by covalently conjugating 3-BP to perillyl alcohol (POH), a natural monoterpene. The responses of various tumor cell lines to treatment with either compound were characterized in the presence or absence of supplemental pyruvate or antioxidants N-acetyl-cysteine (NAC) and glutathione (GSH). Drug effects on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) enzyme activity were investigated by mass spectrometric analysis. The development of 3-BP resistance was investigated in MCT-1-positive HCT116 colon carcinoma cells in vitro. Our results show that NEO218: (i) pyruvylated GAPDH on all 4 of its cysteine residues and shut down enzymatic activity; (ii) severely lowered cellular ATP content below life-sustaining levels, and (iii) triggered rapid necrosis. Intriguingly, supplemental antioxidants effectively prevented cytotoxic activity of NEO218 as well as 3-BP, but supplemental pyruvate powerfully protected cells only from 3-BP, not from NEO218. Unlike 3-BP, NEO218 exerted its potent cytotoxic activity irrespective of cellular MCT-1 status. Treatment of HCT116 cells with 3-BP resulted in prompt development of resistance, based on the emergence of MCT-1-negative cells. This was not the case with NEO218, and highly 3-BP-resistant cells remained exquisitely sensitive to NEO218. Thus, our study identifies a mechanism by which tumor cells develop rapid resistance to 3-BP, and presents NEO218 as a superior agent not subject to this cellular defense. Furthermore, our results offer alternative interpretations of previously

  8. Dual inhibition of γ-oryzanol on cellular melanogenesis: inhibition of tyrosinase activity and reduction of melanogenic gene expression by a protein kinase A-dependent mechanism.

    Science.gov (United States)

    Jun, Hee-jin; Lee, Ji Hae; Cho, Bo-Ram; Seo, Woo-Duck; Kang, Hang-Won; Kim, Dong-Woo; Cho, Kang-Jin; Lee, Sung-Joon

    2012-10-26

    The in vitro effects on melanogenesis of γ-oryzanol (1), a rice bran-derived phytosterol, were investigated. The melanin content in B16F1 cells was significantly and dose-dependently reduced (-13% and -28% at 3 and 30 μM, respectively). Tyrosinase enzyme activity was inhibited by 1 both in a cell-free assay and when analyzed based on the measurement of cellular tyrosinase activity. Transcriptome analysis was performed to investigate the biological pathways altered by 1, and it was found that gene expression involving protein kinase A (PKA) signaling was markedly altered. Subsequent analyses revealed that 1 stimulation in B16 cells reduced cytosolic cAMP concentrations, PKA activity (-13% for cAMP levels and -40% for PKA activity), and phosphorylation of the cAMP-response element binding protein (-57%), which, in turn, downregulated the expression of microphthalmia-associated transcription factor (MITF; -59% for mRNA and -64% for protein), a key melanogenic gene transcription factor. Accordingly, tyrosinase-related protein 1 (TRP-1; -69% for mRNA and -82% for protein) and dopachrome tautomerase (-51% for mRNA and -92% for protein) in 1-stimulated B16F1 cells were also downregulated. These results suggest that 1 has dual inhibitory activities for cellular melanogenesis by inhibiting tyrosinase enzyme activity and reducing MITF and target genes in the PKA-dependent pathway.

  9. Ectopic AP4 expression induces cellular senescence via activation of p53 in long-term confluent retinal pigment epithelial cells.

    Science.gov (United States)

    Wang, Yiping; Wong, Matthew Man-Kin; Zhang, Xiaojian; Chiu, Sung-Kay

    2015-11-15

    When cells are grown to confluence, cell-cell contact inhibition occurs and drives the cells to enter reversible quiescence rather than senescence. Confluent retinal pigment epithelial (RPE) cells exhibiting contact inhibition was used as a model in this study to examine the role of overexpression of transcription factor AP4, a highly expressed transcription factor in many types of cancer, in these cells during long-term culture. We generated stable inducible RPE cell clones expressing AP4 or AP4 without the DNA binding domain (DN-AP4) and observed that, when cultured for 24 days, RPE cells with a high level of AP4 exhibit a large, flattened morphology and even cease proliferating; these changes were not observed in DN-AP4-expressing cells or non-induced cells. In addition, AP4-expressing cells exhibited senescence-associated β-galactosidase activity and the senescence-associated secretory phenotype. We demonstrated that the induced cellular senescence was mediated by enhanced p53 expression and that AP4 regulates the p53 gene by binding directly to two of the three E-boxes present on the promoter of the p53 gene. Moreover, we showed that serum is essential for AP4 in inducing p53-associated cellular senescence. Collectively, we showed that overexpression of AP4 mediates cellular senescence involving in activation of p53 in long-term post-confluent RPE cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Suitability of the cellular viability technique as a control tool of the chlorine dosage on the activated sludge of a biological process affected by bulking

    International Nuclear Information System (INIS)

    Montaya Martinez, T.; Zornoza Zornoza, A.; Granell Munoz, P.; Fayos, G.; Fajarddo, V.; Zorrilla, F.; Alonso Molina, J. L.; Morenilla Martinez, J. J.; Bernacer Bonora, I.; Martinez Francisco, F. J.

    2009-01-01

    This work demonstrates the suitability of the cellular viability technique as a control tool of the chlorine dosage on the activated sludge of a biological process affected by the overabundance of the filamentous bacteria (Thiothrix-021N). This technique was used to establish the chlorine dosage according to the observed damages on cellular membranes of both, floc-forming bacteria as well as filamentous bacteria. To identify the filamentous bacteria responsible for the macro-structural alteration of the flocs, several criteria were, met, including morphologic characteristics as well as conventional microbiological stains: Gram, Neisser and polyhydroxy alkanoates. FISH was used to confirm the obtained results, providing a definitive identification of the filamentous bacteria responsible for the alteration. (Author) 11 refs

  11. Human T lymphotropic virus type-1 p30II alters cellular gene expression to selectively enhance signaling pathways that activate T lymphocytes

    Directory of Open Access Journals (Sweden)

    Feuer Gerold

    2004-11-01

    Full Text Available Abstract Background Human T-lymphotropic virus type-1 (HTLV-1 is a deltaretrovirus that causes adult T-cell leukemia/lymphoma and is implicated in a variety of lymphocyte-mediated disorders. HTLV-1 contains both regulatory and accessory genes in four pX open reading frames. pX ORF-II encodes two proteins, p13II and p30II, which are incompletely defined in the virus life cycle or HTLV-1 pathogenesis. Proviral clones of the virus with pX ORF-II mutations diminish the ability of the virus to maintain viral loads in vivo. Exogenous expression of p30II differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein/p300 and represses tax/rex RNA nuclear export. Results Herein, we further characterized the role of p30II in regulation of cellular gene expression, using stable p30II expression system employing lentiviral vectors to test cellular gene expression with Affymetrix U133A arrays, representing ~33,000 human genes. Reporter assays in Jurkat T cells and RT-PCR in Jurkat and primary CD4+ T-lymphocytes were used to confirm selected gene expression patterns. Our data reveals alterations of interrelated pathways of cell proliferation, T-cell signaling, apoptosis and cell cycle in p30II expressing Jurkat T cells. In all categories, p30II appeared to be an overall repressor of cellular gene expression, while selectively increasing the expression of certain key regulatory genes. Conclusions We are the first to demonstrate that p30II, while repressing the expression of many genes, selectively activates key gene pathways involved in T-cell signaling/activation. Collectively, our data suggests that this complex retrovirus, associated with lymphoproliferative diseases, relies upon accessory gene products to modify cellular environment to promote clonal expansion of the virus genome and thus maintain proviral loads in vivo.

  12. The ROS-mediated activation of IL-6/STAT3 signaling pathway is involved in the 27-hydroxycholesterol-induced cellular senescence in nerve cells.

    Science.gov (United States)

    Liu, Jiao; Liu, Yun; Chen, Juan; Hu, Chunyan; Teng, Mengying; Jiao, Kailin; Shen, Zhaoxia; Zhu, Dongmei; Yue, Jia; Li, Zhong; Li, Yuan

    2017-12-01

    The oxysterol 27-hydroxycholesterol (27HC) is a selective estrogen receptor modulator (SERMs), which like endogenous estrogen 17β-estradiol (E 2 ) induces the proliferation of ER-positive breast cancer cells in vitro. Interestingly, the observation that 27HC induces adverse effects in neural system, distinguishing it from E 2 . It has been suggested that high levels of circulating cholesterol increase the entry of 27HC into the brain, which may induce learning and memory impairment. Based on this evidence, 27HC may be associated with neurodegenerative processes and interrupted cholesterol homeostasis in the brain. However, the biological events that participate in this process remain largely elusive. In the present study, we demonstrated that 27HC induced apparent cellular senescence in nerve cells. Senescence-associated β-galactosidase (SA-β-Gal) assay revealed that 27HC induced senescence in both BV2 cells and PC12 cells. Furthermore, we demonstrated that 27HC promoted the accumulation of cellular reactive oxygen species (ROS) in nerve cells and subsequently activation of IL-6/STAT3 signaling pathway. Notably, treatment with the ROS scavenger N-acetylcysteine (NAC) markedly blocked 27HC-induced ROS production and activation of IL-6/STAT3 signaling pathway. Either blocking the generation of ROS or inhibition of IL-6/STAT3 both attenuated 27HC-induced cellular senescence. In sum, these findings not only suggested a mechanism whereby 27HC induced cellular senescence in nerve cells, but also helped to recognize the 27HC as a novel harmful factor in neurodegenerative diseases. Copyright © 2017. Published by Elsevier Ltd.

  13. Relation of murine thoracic aortic structural and cellular changes with aging to passive and active mechanical properties.

    Science.gov (United States)

    Wheeler, Jason B; Mukherjee, Rupak; Stroud, Robert E; Jones, Jeffrey A; Ikonomidis, John S

    2015-02-25

    Maintenance of the structure and mechanical properties of the thoracic aorta contributes to aortic function and is dependent on the composition of the extracellular matrix and the cellular content within the aortic wall. Age-related alterations in the aorta include changes in cellular content and composition of the extracellular matrix; however, the precise roles of these age-related changes in altering aortic mechanical function are not well understood. Thoracic aortic rings from the descending segment were harvested from C57BL/6 mice aged 6 and 21 months. Thoracic aortic diameter and wall thickness were higher in the old mice. Cellular density was reduced in the medial layer of aortas from the old mice; concomitantly, collagen content was higher in old mice, but elastin content was similar between young and old mice. Stress relaxation, an index of compliance, was reduced in aortas from old mice and correlated with collagen fraction. Contractility of the aortic rings following potassium stimulation was reduced in old versus young mice. Furthermore, collagen gel contraction by aortic smooth muscle cells was reduced with age. These results demonstrate that numerous age-related structural changes occurred in the thoracic aorta and were related to alterations in mechanical properties. Aortic contractility decreased with age, likely because of a reduction in medial cell number in addition to a smooth muscle contractile deficit. Together, these unique findings provide evidence that the age-related changes in structure and mechanical function coalesce to provide an aortic substrate that may be predisposed to aortopathies. © 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

  14. Macrófagos e inducción de arginasa como mecanismo de evasión de parásitos Macrophages and arginase induction as a mechanism for parasite escape

    Directory of Open Access Journals (Sweden)

    Cinthia C Stempin

    2007-12-01

    make possible their survival and replication in the host. Some parasites modulate the production of several toxic molecules synthesized by the immune system. Several parasites are highly sensitive to nitric oxide (ON and their derivatives. ON is produced in macrophages (MΦ after stimulation with microbial products or cytokines. In the past, M Φ were defined as inflammatory cells (classically activated MΦ, able to produce inflammatory mediators, to act like antigens presenting cells and to kill intracellular pathogens. Nevertheless, activated MΦ involve a more heterogeneous group of cells with different biological markers that can carry out different immunological functions. Alternatively activated MΦ fail to produce ON due to the arginase induction and consequently they have diminished their capacity to kill intracellular pathogens. It has been reported the induction of arginase by different parasites; therefore this mechanism could favor their survival in the host. In our group, we studied the participation of arginase in a model of Trypanosoma cruzi infection and the intracellular signals involved in the replication of this parasite in MΦ. The data obtained from our works would allow the understanding of some mechanisms by which cells can be programmed to favor the establishment of chronic parasitic infections.

  15. Unique mitochondrial localization of arginase 1 and 2 in hepatocytes of air-breathing walking catfish, Clarias batrachus and their differential expression patterns under hyper-ammonia stress.

    Science.gov (United States)

    Banerjee, Bodhisattwa; Koner, Debaprasad; Lal, Priyanka; Saha, Nirmalendu

    2017-07-30

    Arginase (ARG) catalyzes the final step of ornithine-urea cycle (OUC) leading to a conversion of L-arginine to L-ornithine and urea. Several isoforms of ARG have been reported in vertebrates, out of which the two predominant isoforms are the cytosolic ARG1 and the mitochondrial ARG2. The air-breathing walking catfish (Clarias batrachus) is frequently being challenged by different environmental insults such as hyper-ammonia, dehydration and osmotic stresses in their natural habitats throughout the year. The present study investigated the active presence of ARG1 and ARG2 isoforms in hepatocytes along with unique localization of both the isoforms inside the mitochondria, and also their specific expression patterns under hyper-ammonia stress (5mM NH 4 Cl) in isolated hepatocytes of walking catfish. Initially, full length sequences of both arg1 and arg2 genes were obtained by RACE-PCR. Studies on molecular characterization demonstrated the presence of all the conserved amino acids required for stability and activity of binuclear metal center in both the isoforms. Phylogenetic analysis of the amino acid sequences of ARG isoforms showed a differentiation of the ARG1 and ARG2 into two distinct clusters with their respective isoforms from other species. Most interestingly, both the isoforms of ARG in hepatocytes were found to be localized inside the mitochondria as evidenced by the presence of mitochondrial target peptide (mTP) in N-terminal of the derived amino acid sequences, and exclusive localization of ARG activity in the mitochondrial fraction. This was additionally confirmed by Western blot analysis of ARGs in mitochondrial and cytosolic fractions, and by immunocytochemical analysis in isolated hepatocytes. Although the possible reasons associated with the presence of both the isoforms of ARGs inside the mitochondria is not clearly understood, perhaps this mitochondrial localization of ARG is functionally advantageous in this catfish for the synthesis of N

  16. An Asynchronous Recurrent Network of Cellular Automaton-Based Neurons and Its Reproduction of Spiking Neural Network Activities.

    Science.gov (United States)

    Matsubara, Takashi; Torikai, Hiroyuki

    2016-04-01

    Modeling and implementation approaches for the reproduction of input-output relationships in biological nervous tissues contribute to the development of engineering and clinical applications. However, because of high nonlinearity, the traditional modeling and implementation approaches encounter difficulties in terms of generalization ability (i.e., performance when reproducing an unknown data set) and computational resources (i.e., computation time and circuit elements). To overcome these difficulties, asynchronous cellular automaton-based neuron (ACAN) models, which are described as special kinds of cellular automata that can be implemented as small asynchronous sequential logic circuits have been proposed. This paper presents a novel type of such ACAN and a theoretical analysis of its excitability. This paper also presents a novel network of such neurons, which can mimic input-output relationships of biological and nonlinear ordinary differential equation model neural networks. Numerical analyses confirm that the presented network has a higher generalization ability than other major modeling and implementation approaches. In addition, Field-Programmable Gate Array-implementations confirm that the presented network requires lower computational resources.

  17. A high-throughput cellular assay to quantify the p53-degradation activity of E6 from different human papillomavirus types.

    Science.gov (United States)

    Gagnon, David; Archambault, Jacques

    2015-01-01

    A subset of human papillomaviruses (HPVs), known as the high-risk types, are the causative agents of cervical cancer and other malignancies of the anogenital region and oral mucosa. The capacity of these viruses to induce cancer and to immortalize cells in culture relies in part on a critical function of their E6 oncoprotein, that of promoting the poly-ubiquitination of the cellular tumor suppressor protein p53 and its subsequent degradation by the proteasome. Here, we describe a cellular assay to measure the p53-degradation activity of E6 from different HPV types. This assay is based on a translational fusion of p53 to Renilla luciferase (Rluc-p53) that remains sensitive to degradation by high-risk E6 and whose steady-state levels can be accurately measured in standard luciferase assays. The p53-degradation activity of any E6 protein can be tested and quantified in transiently transfected cells by determining the amount of E6-expression vector required to reduce by half the levels of RLuc-p53 luciferase activity (50 % effective concentration [EC50]). The high-throughput and quantitative nature of this assay makes it particularly useful to compare the p53-degradation activities of E6 from several HPV types in parallel.

  18. Cellular dosimetry

    International Nuclear Information System (INIS)

    Humm, J.L.; Chin, L.M.

    1989-01-01

    Radiation dose is a useful predictive parameter for describing radiation toxicity in conventional radiotherapy. Traditionally, in vitro radiation biology dose-effect relations are expressed in the form of cell survival curves, a semilog plot of cell survival versus dose. However, the characteristic linear or linear quadratic survival curve shape, for high- and low-LET radiations respectively, is only strictly valid when the radiation dose is uniform across the entire target population. With an external beam of 60 Co gamma rays or x-rays, a uniform field may be readily achievable. When radionuclides are incorporated into a cell milieu, several new problems emerge which can result in a departure from uniformity in energy deposition throughout a cell population. This nonuniformity can have very important consequences for the shape of the survival curve. Cases in which perturbations of source uniformity may arise include: 1. Elemental sources may equilibrate in the cell medium with partition coefficients between the extracellular, cytosol, and nuclear compartments. The effect of preferential cell internalization or binding to cell membrane of some radionuclides can increase or decrease the slope of the survival curve. 2. Radionuclides bound to antibodies, hormones, metabolite precursors, etc., may result in a source localization pattern characteristic of the carrier agent, i.e., the sources may bind to cell surface receptors or antigens, be internalized, bind to secreted antigen concentrated around a fraction of the cell population, or become directly incorporated into the cell DNA. We propose to relate the distribution of energy deposition in cell nuclei to biological correlates of cellular inactivation. The probability of each cell's survival is weighted by its individual radiation burden, and the summation of these probabilities for the cell population can be used to predict the number or fraction of cell survivors

  19. Poloxamer-Based Thermoreversible Gel for Topical Delivery of Emodin: Influence of P407 and P188 on Solubility of Emodin and Its Application in Cellular Activity Screening

    Directory of Open Access Journals (Sweden)

    Eunmi Ban

    2017-02-01

    Full Text Available Emodin is a component in a Chinese herb, Rheum officinale Baill, traditionally used for diabetes and anticancer. Its poor solubility is one of the major challenges to pharmaceutical scientists. We previously reported on thermoreversible gel formulations based on poloxamer for the topical delivery of emodin. The present study was to understand the effect of poloxamer type on emodin solubility and its application in cellular activity screening. Various gel formulations composed of poloxamer 407 (P407, poloxamer 188 (P188 and PEG400 were prepared and evaluated. Major evaluation parameters were the gelation temperature (Tgel and solubility of emodin. The emodin solubility increased with increasing poloxamer concentration and the Tgel was modulated by the proper combination of P407. In particular, this study showed that the amount of P407 in thermoreversible poloxamer gel (PG was the dominant factor in enhancing solubility and P188 was effective at fixing gelation temperature in the desired range. A thermoreversible emodin PG was selected as the proper composition with the liquid state at room temperature and gel state at body temperature. The gel showed the solubility enhancement of emodin at least 100-fold compared to 10% ethanol or water. The thermoreversible formulation was applied for in vitro cellular activity screening in the human dermal fibroblast cell line and DLD-1 colon cancer cell line after dilution with cell culture media. The thermoreversible gel formulation remained as a clear solution in the microplate, which allowed reliable cellular activity screening. In contrast, emodin solution in ethanol or DMSO showed precipitation at the corresponding emodin concentration, complicating data interpretation. In conclusion, the gel formulation is proposed as a useful prototype topical formulation for testing emodin in vivo as well as in vitro.

  20. Cellular inhibitor of apoptosis protein 2 (cIAP2) controls human colonic epithelial restitution, migration and Rac1 activation

    DEFF Research Database (Denmark)

    Seidelin, JB; Larsen, Sylvester; Linnemann, D

    2015-01-01

    epithelial cells (IECs) was increased at the wound edge after 24 h (P 2 was induced in vitro in regenerating Caco2 IECs after wound infliction (P ...Identification of pathways involved in wound healing is important for understanding the pathogenesis of various intestinal diseases. Cellular inhibitor of apoptosis protein 2 (cIAP2) regulates proliferation and migration in nonepithelial cells and is expressed in human colonocytes. The aim...... of the study was to investigate the role of cIAP2 for wound healing in the normal human colon. Wound tissue was generated by taking rectosigmoidal biopsies across an experimental ulcer in healthy subjects after 5, 24, and 48 h. In experimental ulcers, the expression of cIAP2 in regenerating intestinal...

  1. Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting.

    Science.gov (United States)

    Juzwa, W; Duber, A; Myszka, K; Białas, W; Czaczyk, K

    2016-09-01

    In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells.

  2. The MAP kinase-activated protein kinase Rck2p regulates cellular responses to cell wall stresses, filamentation and virulence in the human fungal pathogen Candida albicans.

    Science.gov (United States)

    Li, Xichuan; Du, Wei; Zhao, Jingwen; Zhang, Lilin; Zhu, Zhiyan; Jiang, Linghuo

    2010-06-01

    Rck2p is the Hog1p-MAP kinase-activated protein kinase required for the attenuation of protein synthesis in response to an osmotic challenge in Saccharomyces cerevisiae. Rck2p also regulates rapamycin sensitivity in both S. cerevisiae and Candida albicans. In this study, we demonstrate that the deletion of CaRCK2 renders C. albicans cells sensitive to, and CaRck2p translocates from the cytosol to the nucleus in response to, cell wall stresses caused by Congo red, Calcoflor White, elevated heat and zymolyase. However, the kinase activity of CaRck2p is not required for the cellular response to these cell wall stresses. Furthermore, transcripts of cell wall protein-encoding genes CaBGL2, CaHWP1 and CaXOG1 are reduced in C. albicans cells lacking CaRCK2. The deletion of CaRCK2 also reduces the in vitro filamentation of C. albicans and its virulence in a mouse model of systemic candidasis. The kinase activity of CaRck2p is required for the virulence, but not for the in vitro filamentation, in C. albicans. Therefore, Rck2p regulates cellular responses to cell wall stresses, filamentation and virulence in the human fungal pathogen C. albicans.

  3. Cellularized Cellular Solids via Freeze-Casting.

    Science.gov (United States)

    Christoph, Sarah; Kwiatoszynski, Julien; Coradin, Thibaud; Fernandes, Francisco M

    2016-02-01

    The elaboration of metabolically active cell-containing materials is a decisive step toward the successful application of cell based technologies. The present work unveils a new process allowing to simultaneously encapsulate living cells and shaping cell-containing materials into solid-state macroporous foams with precisely controlled morphology. Our strategy is based on freeze casting, an ice templating materials processing technique that has recently emerged for the structuration of colloids into macroporous materials. Our results indicate that it is possible to combine the precise structuration of the materials with cellular metabolic activity for the model organism Saccharomyces cerevisiae. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Expression of human papilloma virus type 16 E5 protein in amelanotic melanoma cells regulates endo-cellular pH and restores tyrosinase activity

    Directory of Open Access Journals (Sweden)

    Coccia Raffaella

    2009-01-01

    Full Text Available Abstract Background Melanin synthesis, the elective trait of melanocytes, is regulated by tyrosinase activity. In tyrosinase-positive amelanotic melanomas this rate limiting enzyme is inactive because of acidic endo-melanosomal pH. The E5 oncogene of the Human Papillomavirus Type 16 is a small transmembrane protein with a weak transforming activity and a role during the early steps of viral infections. E5 has been shown to interact with 16 kDa subunit C of the trans-membrane Vacuolar ATPase proton pump ultimately resulting in its functional suppressions. However, the cellular effects of such an interaction are still under debate. With this work we intended to explore whether the HPV16 E5 oncoprotein does indeed interact with the vacuolar ATPase proton pump once expressed in intact human cells and whether this interaction has functional consequences on cell metabolism and phenotype. Methods The expression of the HPV16-E5 oncoproteins was induced in two Tyrosinase-positive amelanotic melanomas (the cell lines FRM and M14 by a retroviral expression construct. Modulation of the intracellular pH was measured with Acridine orange and fluorescence microscopy. Expression of tyrosinase and its activity was followed by RT-PCR, Western Blot and enzyme assay. The anchorage-independence growth and the metabolic activity of E5 expressing cells were also monitored. Results We provide evidence that in the E5 expressing cells interaction between E5 and V-ATPase determines an increase of endo-cellular pH. The cellular alkalinisation in turn leads to the post-translational activation of tyrosinase, melanin synthesis and phenotype modulation. These effects are associated with an increased activation of tyrosine analogue anti-blastic drugs. Conclusion Once expressed within intact human cells the HPV16-E5 oncoprotein does actually interact with the vacuolar V-ATPase proton pump and this interaction induces a number of functional effects. In amelanotic melanomas these

  5. Cellular defense of the avian respiratory system: effects of Pasteurella multocida on respiratory burst activity of avian respiratory tract phagocytes.

    Science.gov (United States)

    Ochs, D L; Toth, T E; Pyle, R H; Siegel, P B

    1988-12-01

    The respiratory tract of healthy chickens contain few free-residing phagocytic cells. Intratracheal inoculation with Pasteurella multocida stimulated a significant (P less than 0.05) migration of cells to the lungs and air sacs of White Rock chickens within 2 hours after inoculation. We found the maximal number of avian respiratory tract phagocytes (22.9 +/- 14.0 x 10(6] at 8 hours after inoculation. Flow cytometric analysis of these cells revealed 2 populations on the basis of cell-size and cellular granularity. One of these was similar in size and granularity to those of blood heterophils. Only this population was capable of generating oxidative metabolites in response to phorbol myristate acetate. The ability of the heterophils to produce hydrogen peroxide, measured as the oxidation of intracellularly loaded 2',7'-dichlorofluorescein, decreased with time after inoculation. These results suggest that the migration of heterophils, which are capable of high levels of oxidative metabolism, to the lungs and air sacs may be an important defense mechanism of poultry against bacterial infections of the respiratory tract.

  6. Antibody-dependent cellular cytotoxicity and neutralizing activity in sera of HIV-1-infected mothers and their children.

    Science.gov (United States)

    Broliden, K; Sievers, E; Tovo, P A; Moschese, V; Scarlatti, G; Broliden, P A; Fundaro, C; Rossi, P

    1993-01-01

    The prognostic and protective role of antibodies mediating cellular cytotoxicity (ADCC) and neutralization was evaluated in sera of HIV-1-infected mothers and their consecutively followed children. The presence and titres of ADCC mediating and/or neutralizing antibodies in maternal sera did not predict HIV-1 infection in their respective children. No significant difference in the sera from the children was seen when comparing the presence of neutralizing antibodies between the uninfected and infected children. Stratification of the infected group according to clinical status revealed differences. Only one of 24 AIDS patients had a high neutralizing titre against IIIB. Four patients had a very low titre and the remaining had no detectable neutralizing antibodies at all. In contrast, 10/17 infected non-AIDS children had neutralizing antibodies. Similarly, no significant difference was seen when comparing the presence of ADCC-mediating antibodies between the uninfected and the infected group of children. However, a significantly higher frequency of ADCC was seen in the seropositive non-AIDS children compared with the AIDS children. This study clearly shows that the presence of antibodies mediating ADCC and neutralization in infected children, 0-2 years old, is associated with a better clinical status and delayed disease progression. PMID:8324904

  7. Identification of cellular and viral factors related to anti-hepatitis C virus activity of cyclophilin inhibitor.

    Science.gov (United States)

    Goto, Kaku; Watashi, Koichi; Inoue, Daisuke; Hijikata, Makoto; Shimotohno, Kunitada

    2009-10-01

    We have so far reported that an immunosuppressant cyclosporin A (CsA), a well-known cyclophilin (CyP) inhibitor (CPI), strongly suppressed hepatitis C virus (HCV) replication in cell culture, and that CyPB was a cellular cofactor for viral replication. To further investigate antiviral mechanisms of CPI, we here developed cells carrying CsA-resistant HCV replicons, by culturing the HCV subgenomic replicon cells for 4 weeks in the presence of CsA with G418. Transfection of total RNA from the isolated CsA-resistant cells to naïve Huh7 cells conferred CsA resistance, suggesting that the replicon RNA itself was responsible for the resistant phenotype. Of the identified amino acid mutations, D320E in NS5A conferred the CsA resistance. The replicon carrying the D320E mutation was sensitive to interferon-alpha, but was resistant to CsA and other CPIs including NIM811 and sanglifehrin A. Knockdown of individual CyP subtypes revealed CyP40, in addition to CyPA and CyPB, contributed to viral replication, and CsA-resistant replicons acquired independence from CyPA for efficient replication. These data provide important evidence on the mechanisms underlying the regulation of HCV replication by CyP and for designing novel and specific anti-HCV strategies with CPIs.

  8. Tat-dependent repression of human immunodeficiency virus type 1 long terminal repeat promoter activity by fusion of cellular transcription factors

    International Nuclear Information System (INIS)

    Zhao Cunyou; Chen Yali; Park, Jiyoung; Kim, Jae Bum; Tang Hong

    2004-01-01

    Transcription initiation from HIV-1 long terminal repeat (LTR) promoter requires the virally encoded transactivator, Tat, and several cellular co-factors to accomplish the Tat-dependent processive transcription elongation. Individual cellular transcription activators, LBP-1b and Oct-1, on the other hand, have been shown to inhibit LTR promoter activities probably via competitive binding against TFIID to the TATA-box in LTR promoter. To explore the genetic interference strategies against the viral replication, we took advantage of the existence of the bipartite DNA binding domains and the repression domains of LBP-1b and Oct-1 factors to generate a chimeric transcription repressor. Our results indicated that the fusion protein of LBP-1b and Oct-1 exhibited higher DNA binding affinity to the viral promoter than the individual factors, and little interference with the host cell gene expression due to its anticipated rare cognate DNA sites in the host cell genome. Moreover, the chimera exerted increased Tat-dependent repression of transcription initiation at the LTR promoter both in vitro and in vivo compared to LBP-1b, Oct-1 or combination of LBP-1b and Oct-1. These results might provide the lead in generating a therapeutic reagent useful to suppress HIV-1 replication

  9. Decreased Fc receptor expression on innate immune cells is associated with impaired antibody-mediated cellular phagocytic activity in chronically HIV-1 infected individuals.

    Science.gov (United States)

    Dugast, Anne-Sophie; Tonelli, Andrew; Berger, Christoph T; Ackerman, Margaret E; Sciaranghella, Gaia; Liu, Qingquan; Sips, Magdalena; Toth, Ildiko; Piechocka-Trocha, Alicja; Ghebremichael, Musie; Alter, Galit

    2011-07-05

    In addition to neutralization, antibodies mediate other antiviral activities including antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), as well as complement deposition. While it is established that progressive HIV infection is associated with reduced ADCC and ADCP, the underlying mechanism for this loss of function is unknown. Here we report considerable changes in FcR expression over the course of HIV infection on both mDCs and monocytes, including elevated FcγRI expression in acute HIV infection and reduced expression of FcγRII and FcγRIIIa in chronic HIV infection. Furthermore, selective blockade of FcγRII alone was associated with a loss in ADCP activity, suggesting that FcγRII plays a central role in modulating ADCP. Overall, HIV infection is associated with a number of changes in FcR expression on phagocytic cells that are associated with changes in their ability to respond to antibody-opsonized targets, potentially contributing to a failure in viral clearance in progressive HIV-1 infection. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Decreased Fc-Receptor expression on innate immune cells is associated with impaired antibody mediated cellular phagocytic activity in chronically HIV-1 infected individuals

    Science.gov (United States)

    Dugast, Anne-Sophie; Tonelli, Andrew; Berger, Christoph T.; Ackerman, Margaret E.; Sciaranghella, Gaia; Liu, Qingquan; Sips, Magdalena; Toth, Ildiko; Piechocka-Trocha, Alicja; Ghebremichael, Musie; Alter, Galit

    2011-01-01

    In addition to neutralization, antibodies mediate other antiviral activities including antibody-dependent cellular-phagocytosis (ADCP), antibody dependent cellular-cytotoxicity (ADCC), as well as complement deposition. While it is established that progressive HIV infection is associated with reduced ADCC and ADCP, the underlying mechanism for this loss of function is unknown. Here we report considerable changes in FcR expression over the course of HIV infection on both mDCs and monocytes, including elevated FcγRI expression in acute HIV infection and reduced expression of FcγRII and FcγRIIIa in chronic HIV infection. Furthermore, selective blockade of FcγRII alone was associated with a loss in ADCP activity, suggesting that FcγRII plays a central role in modulating ADCP. Overall, HIV infection is associated with a number of changes in FcR expression on phagocytic cells that are associated with changes in their ability to respond to antibody-opsonized targets, potentially contributing to a failure in viral clearance in progressive HIV-1 infection. PMID:21565376

  11. Cellular uptake of {sup 99m}TcN-NOET in human leukaemic HL-60 cells is related to calcium channel activation and cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Guillermet, Stephanie; Vuillez, Jean-Philippe; Caravel, Jean-Pierre; Marti-Batlle, Daniele; Fagret, Daniel [Universite de Grenoble, Radiopharmaceutiques Biocliniques, La Tronche (France); Fontaine, Eric [Universite de Grenoble, Laboratoire de Bioenergetique Fondamentale et Appliquee, Grenoble (France); Pasqualini, Roberto [Cis Bio International Schering SA, Gif-sur-Yvette (France)

    2006-01-01

    A major goal of nuclear oncology is the development of new radiolabelled tracers as proliferation markers. Intracellular calcium waves play a fundamental role in the course of the cell cycle. These waves occur in non-excitable tumour cells via store-operated calcium channels (SOCCs). Bis(N-ethoxy, N-ethyldithiocarbamato) nitrido technetium (V)-99m ({sup 99m}TcN-NOET) has been shown to interact with L-type voltage-operated calcium channels (VOCCs) in cultured cardiomyocytes. Considering the analogy between VOCCs and SOCCs, we sought to determine whether {sup 99m}TcN-NOET also binds to activated SOCCs in tumour cells in order to clarify the potential value of this tracer as a proliferation marker. Uptake kinetics of {sup 99m}TcN-NOET were measured in human leukaemic HL-60 cells over 60 min and the effect of several calcium channel modulators on 1-min tracer uptake was studied. The uptake kinetics of {sup 99m}TcN-NOET were compared both with the variations of cytosolic free calcium concentration measured by indo-1/AM and with the variations in the SG{sub 2}M cellular proliferation index. All calcium channel inhibitors significantly decreased the cellular uptake of {sup 99m}TcN-NOET whereas the activator thapsigargin induced a significant 10% increase. In parallel, SOCC activation by thapsigargin, as measured using the indo-1/AM probe, was inhibited by nicardipine. These results indicate that the uptake of {sup 99m}TcN-NOET is related to the activation of SOCCs. Finally, a correlation was observed between the tracer uptake and variations in the proliferation index SG{sub 2}M. The uptake of {sup 99m}TcN-NOET seems to be related to SOCC activation and to cell proliferation in HL-60 cells. These results indicate that {sup 99m}TcN-NOET might be a marker of cell proliferation. (orig.)

  12. Cellular uptake of 99mTcN-NOET in human leukaemic HL-60 cells is related to calcium channel activation and cell proliferation

    International Nuclear Information System (INIS)

    Guillermet, Stephanie; Vuillez, Jean-Philippe; Caravel, Jean-Pierre; Marti-Batlle, Daniele; Fagret, Daniel; Fontaine, Eric; Pasqualini, Roberto

    2006-01-01

    A major goal of nuclear oncology is the development of new radiolabelled tracers as proliferation markers. Intracellular calcium waves play a fundamental role in the course of the cell cycle. These waves occur in non-excitable tumour cells via store-operated calcium channels (SOCCs). Bis(N-ethoxy, N-ethyldithiocarbamato) nitrido technetium (V)-99m ( 99m TcN-NOET) has been shown to interact with L-type voltage-operated calcium channels (VOCCs) in cultured cardiomyocytes. Considering the analogy between VOCCs and SOCCs, we sought to determine whether 99m TcN-NOET also binds to activated SOCCs in tumour cells in order to clarify the potential value of this tracer as a proliferation marker. Uptake kinetics of 99m TcN-NOET were measured in human leukaemic HL-60 cells over 60 min and the effect of several calcium channel modulators on 1-min tracer uptake was studied. The uptake kinetics of 99m TcN-NOET were compared both with the variations of cytosolic free calcium concentration measured by indo-1/AM and with the variations in the SG 2 M cellular proliferation index. All calcium channel inhibitors significantly decreased the cellular uptake of 99m TcN-NOET whereas the activator thapsigargin induced a significant 10% increase. In parallel, SOCC activation by thapsigargin, as measured using the indo-1/AM probe, was inhibited by nicardipine. These results indicate that the uptake of 99m TcN-NOET is related to the activation of SOCCs. Finally, a correlation was observed between the tracer uptake and variations in the proliferation index SG 2 M. The uptake of 99m TcN-NOET seems to be related to SOCC activation and to cell proliferation in HL-60 cells. These results indicate that 99m TcN-NOET might be a marker of cell proliferation. (orig.)

  13. Cellular localization of peptide hydrolases in chicken embryo tissues and influence of gamma irradiation on their activity

    Energy Technology Data Exchange (ETDEWEB)

    Khristov, D; Marinopolski, G

    1975-01-01

    Studied was the influence of chicken embryo irradiation at 600 R and 1000 R gamma rays on the activity of tissue peptide hydrolases in mitochondrial-lysosomal, microsomal and supernatant (cell hyaloplasm) cell fractions. The investigation was performed 50 to 168 hours post irradiation. The wole tissue (of the whole embryo) was examined following irradiation of 4-day-old embryos whose liver, muscle and brain tissues were post irradiation examined on day 12 and 16 of incubation. Prior to treatment, the tissues were threfold rinsed with sucrose solution to eliminate proeinase inhibitors. Lysosome membranes were destroyed by adding 0.5 % desoxycholate. It was found that: Peptide hydrolase activity of mitochondrial-lysosomal cell fractions of tissues of whole 6-day chicken embryos is 4-5 times as high as that of cell hyaloplasm. Peptide hydrolase activity of mitochondrial-lysosomal fractions of liver tissues decreases on day 18 and 19 post incubation, while the same fraction of muscle and brain tissues shows high activity. Peptide hydrolase activity of microsomal fraction and of cell hyaloplasm rises during embryonal development and exceeds the activity of liver tissue mitochondrial fraction. Peptide hydrolase activity of mitochondrial-lysosomal fraction of tissue of whole 6-day-old embryos 50 hours post irradiation is higher than the activity of non-irradiated embryos. Later the activity of this fraction diminishes and on the 168 hr post irradiation it drops below the normal. Microsomal fraction and cell hyaloplasm activity likewise show deviation from the norm. Peptide hydrolase activity of mitochondrial-lysosomal fraction of liver, muscle and brain tissue of 14 and 18-day-old embryos is higher than the control 50 hours post irradiation and then declines. The activity of mitochondrial-lysosomal fraction of embryo brain tissue changes most strikingly on irradiation, while other brain cell fractions change less compared with liver and muscle fractions.

  14. Cellular localization of peptide hydrolases in chicken embryo tissues and influence of gamma irradiation on their activity

    International Nuclear Information System (INIS)

    Khristov, D.; Marinopolski, G.

    1975-01-01

    Studied was the influence of chicken embryo irradiation at 600 R and 1000 R gamma rays on the activity of tissue peptide hydrolases in mitochondrial-lysosomal, microsomal and supernatant (cell hyaloplasm) cell fractions. The investigation was performed 50 to 168 hours post irradiation. The wole tissue (of the whole embryo) was examined following irradiation of 4-day-old embryos whose liver, muscle and brain tissues were post irradiation examined on day 12 and 16 of incubation. Prior to treatment, the tissues were threfold rinsed with sucrose solution to eliminate proeinase inhibitors. Lysosome membranes were destroyed by adding 0.5 % desoxycholate. It was found that: Peptide hydrolase activity of mitochondrial-lysosomal cell fractions of tissues of whole 6-day chicken embryos is 4-5 times as high as that of cell hyaloplasm. Peptide hydrolase activity of mitochondrial-lysosomal fractions of liver tissues decreases on day 18 and 19 post incubation, while the same fraction of muscle and brain tissues shows high activity. Peptide hydrolase activity of microsomal fraction and of cell hyaloplasm rises during embryonal development and exceeds the activity of liver tissue mitochondrial fraction. Peptide hydrolase activity of mitochondrial-lysosomal fraction of tissue of whole 6-day-old embryos 50 hours post irradiation is higher than the activity of non-irradiated embryos. Later the activity of this fraction diminishes and on the 168 hr post irradiation it drops below the normal. Microsomal fraction and cell hyaloplasm activity likewise show deviation from the norm. Peptide hydrolase activity of mitochondrial-lysosomal fraction of liver, muscle and brain tissue of 14 and 18-day-old embryos is higher than the control 50 hours post irradiation and then declines. The activity of mitochondrial-lysosomal fraction of embryo brain tissue changes most strikingly on irradiation, while other brain cell fractions change less compared with liver and muscle fractions

  15. Inhibition of cAMP-Activated Intestinal Chloride Secretion by Diclofenac: Cellular Mechanism and Potential Application in Cholera

    OpenAIRE

    Pongkorpsakol, Pawin; Pathomthongtaweechai, Nutthapoom; Srimanote, Potjanee; Soodvilai, Sunhapas; Chatsudthipong, Varanuj; Muanprasat, Chatchai

    2014-01-01

    Cyclic AMP-activated intestinal Cl- secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl- secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl- secretion in human intestinal epithelial (T84) cells with IC50 of ∼ 20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell...

  16. Inhibition of cAMP-activated intestinal chloride secretion by diclofenac: cellular mechanism and potential application in cholera.

    Science.gov (United States)

    Pongkorpsakol, Pawin; Pathomthongtaweechai, Nutthapoom; Srimanote, Potjanee; Soodvilai, Sunhapas; Chatsudthipong, Varanuj; Muanprasat, Chatchai

    2014-09-01

    Cyclic AMP-activated intestinal Cl- secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl- secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl- secretion in human intestinal epithelial (T84) cells with IC50 of ∼ 20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell monolayers to diclofenac, either in apical or basolateral solutions, produced similar degree of inhibitions. Analyses of the apical Cl- current showed that diclofenac reversibly inhibited CFTR Cl- channel activity (IC50 ∼ 10 µM) via mechanisms not involving either changes in intracellular cAMP levels or CFTR channel inactivation by AMP-activated protein kinase and protein phosphatase. Of interest, diclofenac had no effect on Na(+)-K(+) ATPases and Na(+)-K(+)-Cl- cotransporters, but inhibited cAMP-activated basolateral K(+) channels with IC50 of ∼ 3 µM. In addition, diclofenac suppressed Ca(2+)-activated Cl- channels, inwardly rectifying Cl- channels, and Ca(2+)-activated basolateral K(+) channels. Furthermore, diclofenac (up to 200 µM; 24 h of treatment) had no effect on cell viability and barrier function in T84 cells. Importantly, cholera toxin (CT)-induced Cl- secretion across T84 cell monolayers was effectively suppressed by diclofenac. Intraperitoneal administration of diclofenac (30 mg/kg) reduced both CT and Vibrio cholerae-induced intestinal fluid secretion by ∼ 70% without affecting intestinal fluid absorption in mice. Collectively, our results indicate that diclofenac inhibits both cAMP-activated and Ca(2+)-activated Cl- secretion by inhibiting both apical Cl- channels and basolateral K+ channels in intestinal epithelial cells. Diclofenac may be useful in the treatment of cholera and other types of secretory diarrheas resulting from intestinal

  17. Inhibition of cAMP-activated intestinal chloride secretion by diclofenac: cellular mechanism and potential application in cholera.

    Directory of Open Access Journals (Sweden)

    Pawin Pongkorpsakol

    2014-09-01

    Full Text Available Cyclic AMP-activated intestinal Cl- secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl- secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl- secretion in human intestinal epithelial (T84 cells with IC50 of ∼ 20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell monolayers to diclofenac, either in apical or basolateral solutions, produced similar degree of inhibitions. Analyses of the apical Cl- current showed that diclofenac reversibly inhibited CFTR Cl- channel activity (IC50 ∼ 10 µM via mechanisms not involving either changes in intracellular cAMP levels or CFTR channel inactivation by AMP-activated protein kinase and protein phosphatase. Of interest, diclofenac had no effect on Na(+-K(+ ATPases and Na(+-K(+-Cl- cotransporters, but inhibited cAMP-activated basolateral K(+ channels with IC50 of ∼ 3 µM. In addition, diclofenac suppressed Ca(2+-activated Cl- channels, inwardly rectifying Cl- channels, and Ca(2+-activated basolateral K(+ channels. Furthermore, diclofenac (up to 200 µM; 24 h of treatment had no effect on cell viability and barrier function in T84 cells. Importantly, cholera toxin (CT-induced Cl- secretion across T84 cell monolayers was effectively suppressed by diclofenac. Intraperitoneal administration of diclofenac (30 mg/kg reduced both CT and Vibrio cholerae-induced intestinal fluid secretion by ∼ 70% without affecting intestinal fluid absorption in mice. Collectively, our results indicate that diclofenac inhibits both cAMP-activated and Ca(2+-activated Cl- secretion by inhibiting both apical Cl- channels and basolateral K+ channels in intestinal epithelial cells. Diclofenac may be useful in the treatment of cholera and other types of secretory diarrheas resulting from intestinal

  18. Wavefront cellular learning automata.

    Science.gov (United States)

    Moradabadi, Behnaz; Meybodi, Mohammad Reza

    2018-02-01

    This paper proposes a new cellular learning automaton, called a wavefront cellular learning automaton (WCLA). The proposed WCLA has a set of learning automata mapped to a connected structure and uses this structure to propagate the state changes of the learning automata over the structure using waves. In the WCLA, after one learning automaton chooses its action, if this chosen action is different from the previous action, it can send a wave to its neighbors and activate them. Each neighbor receiving the wave is activated and must choose a new action. This structure for the WCLA is necessary in many dynamic areas such as social networks, computer networks, grid computing, and web mining. In this paper, we introduce the WCLA framework as an optimization tool with diffusion capability, study its behavior over time using ordinary differential equation solutions, and present its accuracy using expediency analysis. To show the superiority of the proposed WCLA, we compare the proposed method with some other types of cellular learning automata using two benchmark problems.

  19. Wavefront cellular learning automata

    Science.gov (United States)

    Moradabadi, Behnaz; Meybodi, Mohammad Reza

    2018-02-01

    This paper proposes a new cellular learning automaton, called a wavefront cellular learning automaton (WCLA). The proposed WCLA has a set of learning automata mapped to a connected structure and uses this structure to propagate the state changes of the learning automata over the structure using waves. In the WCLA, after one learning automaton chooses its action, if this chosen action is different from the previous action, it can send a wave to its neighbors and activate them. Each neighbor receiving the wave is activated and must choose a new action. This structure for the WCLA is necessary in many dynamic areas such as social networks, computer networks, grid computing, and web mining. In this paper, we introduce the WCLA framework as an optimization tool with diffusion capability, study its behavior over time using ordinary differential equation solutions, and present its accuracy using expediency analysis. To show the superiority of the proposed WCLA, we compare the proposed method with some other types of cellular learning automata using two benchmark problems.

  20. Tissue level, activation and cellular localisation of TGF-β1 and association with survival in gastric cancer patients

    NARCIS (Netherlands)

    Hawinkels, L.J.A.C.; Verspaget, H.W.; Duijn, W. van; Zon, J.M. van der; Zuidwijk, K.; Kubben, F.J.G.M.; Verheijen, J.H.; Hommes, D.W.; Lamers, C.B.H.W.; Sier, C.F.M.

    2007-01-01

    Transforming growth factor-β1 (TGF-β1), a tumour suppressing as well as tumour-promoting cytokine, is stored as an extracellular matrix-bound latent complex. We examined TGF-β1 activation and localisation of TGF-β1 activity in gastric cancer. Gastric tumours showed increased stromal and epithelial

  1. Discovery of novel high potent and cellular active ADC type PTP1B inhibitors with selectivity over TC-PTP via modification interacting with C site.

    Science.gov (United States)

    Du, Yongli; Zhang, Yanhui; Ling, Hao; Li, Qunyi; Shen, Jingkang

    2018-01-20

    PTP1B serving as a key negative regulator of insulin signaling is a novel target for type 2 diabetes and obesity. Modification at ring B of N-{4-[(3-Phenyl-ureido)-methyl]-phenyl}-methane-sulfonamide template to interact with residues Arg47 and Lys41 in the C site of PTP1B by molecular docking aided design resulted in the discovery of a series of novel high potent and selective inhibitors of PTP1B. The structure activity relationship interacting with the C site of PTP1B was well illustrated. Compounds 8 and 18 were shown to be the high potent and most promising PTP1B inhibitors with cellular activity and great selectivity over the highly homologous TCPTP and other PTPs. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  2. Semen modulated secretory activity of oviductal epithelial cells is linked to cellular proteostasis network remodeling: Proteomic insights into the early phase of interaction in the oviduct in vivo.

    Science.gov (United States)

    Steinberger, Birgit; Yu, Hans; Brodmann, Theodor; Milovanovic, Daniela; Reichart, Ursula; Besenfelder, Urban; Artemenko, Konstantin; Razzazi-Fazeli, Ebrahim; Brem, Gottfried; Mayrhofer, Corina

    2017-06-23

    The oviductal epithelium is crucial for the integrity of the female organ. Previously we got evidence that the surface proteome of oviductal epithelial cells (Oecs) is promptly altered in response to insemination and thus suggested that this early phase plays a notable regulatory role in maintaining cellular function. This study further aimed to assess the effect of semen on the cellular and molecular mechanisms in rabbit Oecs. A quantitative gel-based proteomic approach was applied to analyze changes at three time points (0h, 1h, 2h) after intrauterine insemination (IUI) compared to time matched controls. Within two hours the abundance of 22 protein species was evidently altered in the intracellular fraction. Functional analysis revealed that the proteins were primarily involved in proteostasis as well as metabolic processes. The analysis of phosphoproteins specified a role of mitogen-activated protein kinase (MAPK) signaling molecules. Concurrently, semen increased oviduct-specific glycoprotein (OVGP1) secretion. A correlation between OVGP1 abundance and microtubule-associated proteins 1A/1B-light chain 3 lipidation was observed. The localization and changes in abundance of selected proteins were corroborated by antibody-based methods. These results clearly show that the early phase of interaction acts as a trigger for cellular adaptation to meet an altered demand in the female organ. The oviductal epithelium and its secreted proteins exert a pivotal role in reproductive processes, including the final maturation of male gametes. Thereby, the regulation and subsequently the functionality of the oviductal epithelial cell layer are important factors for the establishment of the appropriate milieu in the female reproductive tract. Notably, male gametes themselves have been shown to be an extrinsic modulatory factor of the oviductal epithelium. Accordingly a comprehensive knowledge about the underlying cellular and molecular mechanisms in the epithelial cells is of

  3. Molecular and Cellular Signaling

    CERN Document Server

    Beckerman, Martin

    2005-01-01

    A small number of signaling pathways, no more than a dozen or so, form a control layer that is responsible for all signaling in and between cells of the human body. The signaling proteins belonging to the control layer determine what kinds of cells are made during development and how they function during adult life. Malfunctions in the proteins belonging to the control layer are responsible for a host of human diseases ranging from neurological disorders to cancers. Most drugs target components in the control layer, and difficulties in drug design are intimately related to the architecture of the control layer. Molecular and Cellular Signaling provides an introduction to molecular and cellular signaling in biological systems with an emphasis on the underlying physical principles. The text is aimed at upper-level undergraduates, graduate students and individuals in medicine and pharmacology interested in broadening their understanding of how cells regulate and coordinate their core activities and how diseases ...

  4. Design and cellular kinetics of dansyl-labeled CADA derivatives with anti-HIV and CD4 receptor down-modulating activity.

    Science.gov (United States)

    Vermeire, Kurt; Lisco, Andrea; Grivel, Jean-Charles; Scarbrough, Emily; Dey, Kaka; Duffy, Noah; Margolis, Leonid; Bell, Thomas W; Schols, Dominique

    2007-08-15

    A new class of anti-retrovirals, cyclotriazadisulfonamide (CADA) and its derivatives, specifically down-regulate CD4, the main receptor of HIV, and prevent HIV infection in vitro. In this work, several CADA derivatives, chemically labeled with a fluorescent dansyl group, were evaluated for their biological features and cellular uptake kinetics. We identified a derivative KKD-016 with antiviral and CD4 down-modulating capabilities similar to those of the parental compound CADA. By using flow cytometry, we demonstrated that the dose-dependent cellular uptake of this derivative correlated with CD4 down-modulation. The uptake and activity of the dansyl-labeled compounds were not dependent on the level of expression of CD4 at the cell surface. Removal of the CADA compounds from the cell culture medium resulted in their release from the cells followed by a complete restoration of CD4 expression. The inability of several fluorescent CADA derivatives to down-modulate CD4 was not associated with their lower cellular uptake and was not reversed by facilitating their cell penetration by a surfactant. These results prove the successful integration of the dansyl fluorophore into the chemical structure of a CD4 down-modulating anti-HIV compound, and show the feasibility of tracking a receptor and its down-modulator simultaneously. These fluorescent CADA analogs with reversible CD4 down-regulating potency can now be applied in further studies on receptor modulation, and in the exploration of their potentials as preventive and therapeutic anti-HIV drugs.

  5. The Effect of a p38 Mitogen-Activated Protein Kinase Inhibitor on Cellular Senescence of Cultivated Human Corneal Endothelial Cells.

    Science.gov (United States)

    Hongo, Akane; Okumura, Naoki; Nakahara, Makiko; Kay, EunDuck P; Koizumi, Noriko

    2017-07-01

    We have begun a clinical trial of a cell-based therapy for corneal endothelial dysfunction in Japan. The purpose of this study was to investigate the usefulness of a p38 MAPK inhibitor for prevention cellular senescence in cultivated human corneal endothelial cells (HCECs). HCECs of 10 donor corneas were divided and cultured with or without SB203580 (a p38 MAPK inhibitor). Cell density and morphology were evaluated by phase-contrast microscopy. Expression of function-related proteins was examined by immunofluorescent staining. Cellular senescence was evaluated by SA-β-gal staining and Western blotting for p16 and p21. Senescence-associated factors were evaluated by membrane blotting array, quantitative PCR, and ELISA. Phase-contrast microscopy showed a significantly higher cell density for HCECs cultured with SB203580 than without SB203580 (2623 ± 657 cells/mm2 and 1752 ± 628 cells/mm2, respectively). The HCECs cultured with SB203580 maintained a hexagonal morphology and expressed ZO-1, N-cadherin, and Na+/K+-ATPase in the plasma membrane, whereas the control HCECs showed an altered staining pattern for these marker proteins. HCECs cultured without SB203580 showed high positive SA-β-gal staining, a low nuclear/cytoplasm ratio, and expression of p16 and p21. IL-6, IL-8, CCL2, and CXCL1 were observed at high levels in low cell density HCECs cultured without SB203580. Activation of p38 MAPK signaling due to culture stress might be a causative factor that induces cellular senescence; therefore, the use of p38 MAPK inhibitor to counteract senescence may achieve sufficient numbers of HCECs for tissue engineering therapy for corneal endothelial dysfunction.

  6. Involvement of stress-activated protein kinase in the cellular response to 1-beta-D-arabinofuranosylcytosine and other DNA-damaging agents.

    Science.gov (United States)

    Saleem, A; Datta, R; Yuan, Z M; Kharbanda, S; Kufe, D

    1995-12-01

    The cellular response to 1-beta-D-arabinofuranosylcytosine (ara-C) includes activation of Jun/AP-1, induction of c-jun transcription, and programmed cell death. The stress-activated protein (SAP) kinases stimulate the transactivation function of c-jun by amino terminal phosphorylation. The present work demonstrates that ara-C activates p54 SAP kinase. The finding that SAP kinase is also activated by alkylating agents (mitomycin C and cisplatinum) and the topoisomerase I inhibitor 9-amino-camptothecin supports DNA damage as an initial signal in this cascade. The results demonstrate that ara-C also induces binding of SAP kinase to the SH2/SH3-containing adapter protein Grb2. SAP kinase binds to the SH3 domains of Grb2, while interaction of the p85 alpha-subunit of phosphatidylinositol 3-kinase complex. The results also demonstrate that ara-C treatment is associated with inhibition of lipid and serine kinase activities of PI 3-kinase. The potential significance of the ara-C-induced interaction between SAP kinase and PI 3-kinase is further supported by the demonstration that Wortmannin, an inhibitor of PI 3-kinase, stimulates SAP kinase activity. The finding that Wortmannin treatment is also associated with internucleosomal DNA fragmentation may support a potential link between PI 3-kinase and regulation of both SAP kinase and programmed cell death.

  7. Bufalin-loaded mPEG-PLGA-PLL-cRGD nanoparticles: preparation, cellular uptake, tissue distribution, and anticancer activity

    Directory of Open Access Journals (Sweden)

    Duan YR

    2012-07-01

    Full Text Available Peihao Yin,1,* Yan Wang,1,* YanYan Qiu,1 LiLi Hou,1 Xuan Liu,1 Jianmin Qin,1 Yourong Duan,2 Peifeng Liu,2 Ming Qiu,3 Qi Li11Department of Clinical Oncology, Putuo Hospital and Interventional Cancer Institute of Integrative Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China; 2Shanghai Cancer Institute, Jiaotong University, Shanghai, China; 3Department of General Surgery, Changzheng Hospital, Second Military Medical University, Shanghai, China *These authors contributed equally to this workBackground: Recent studies have shown that bufalin has a good antitumor effect but has high toxicity, poor water solubility, a short half-life, a narrow therapeutic window, and a toxic dose that is close to the therapeutic dose, which all limit its clinical application. This study aimed to determine the targeting efficacy of nanoparticles (NPs made of methoxy polyethylene glycol (mPEG, polylactic-co-glycolic acid (PLGA, poly-L-lysine (PLL, and cyclic arginine-glycine-aspartic acid (cRGD loaded with bufalin, ie, bufalin-loaded mPEG-PLGA-PLL-cRGD nanoparticles (BNPs, in SW620 colon cancer-bearing mice.Methods: BNPs showed uniform size. The size, shape, zeta potential, drug loading, encapsulation efficiency, and release of these nanoparticles were studied in vitro. The tumor targeting, cellular uptake, and growth-inhibitory effect of BNPs in vivo were tested.Results: BNPs were of uniform size with an average particle size of 164 ± 84 nm and zeta potential of 2.77 mV. The encapsulation efficiency was 81.7% ± 0.89%, and the drug load was 3.92% ± 0.16%. The results of in vitro cytotoxicity studies showed that although the blank NPs were nontoxic, they enhanced the cytotoxicity of bufalin in BNPs. Drug release experiments showed that the release of the drug was prolonged and sustained. The results of confocal laser scanning microscopy indicated that BNPs could effectively bind to human umbilical vein endothelial cells. In the SW620

  8. Kurarinol induces hepatocellular carcinoma cell apoptosis through suppressing cellular signal transducer and activator of transcription 3 signaling

    International Nuclear Information System (INIS)

    Shu, Guangwen; Yang, Jing; Zhao, Wenhao; Xu, Chan; Hong, Zongguo; Mei, Zhinan; Yang, Xinzhou

    2014-01-01

    Kurarinol is a flavonoid isolated from roots of the medical plant Sophora flavescens. However, its cytotoxic activity against hepatocellular carcinoma (HCC) cells and toxic effects on mammalians remain largely unexplored. Here, the pro-apoptotic activities of kurarinol on HCC cells and its toxic impacts on tumor-bearing mice were evaluated. The molecular mechanisms underlying kurarinol-induced HCC cell apoptosis were also investigated. We found that kurarinol dose-dependently provoked HepG2, Huh-7 and H22 HCC cell apoptosis. In addition, kurarinol gave rise to a considerable decrease in the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) in HCC cells. Suppression of STAT3 signaling is involved in kurarinol-induced HCC cell apoptosis. In vivo studies showed that kurarinol injection substantially induced transplanted H22 cell apoptosis with low toxic impacts on tumor-bearing mice. Similarly, the transcriptional activity of STAT3 in transplanted tumor tissues was significantly suppressed after kurarinol treatment. Collectively, our current research demonstrated that kurarinol has the capacity of inducing HCC cell apoptosis both in vitro and in vivo with undetectable toxic impacts on the host. Suppressing STAT3 signaling is implicated in kurarinol-mediated HCC cell apoptosis. - Highlights: • Kurarinol induces hepatocellular carcinoma (HCC) cell apoptosis. • Kurarinol induces HCC cell apoptosis via inhibiting STAT3. • Kurarinol exhibits low toxic effects on tumor-bearing animals

  9. siRNA - Mediated LRP/LR knock-down reduces cellular viability of malignant melanoma cells through the activation of apoptotic caspases.

    Science.gov (United States)

    Rebelo, Thalia M; Vania, Leila; Ferreira, Eloise; Weiss, Stefan F T

    2018-07-01

    The 37 kDa/67 kDa laminin receptor (LRP/LR) is over-expressed in tumor cells and has been implicated in several tumourigenic processes such as metastasis and telomerase activation, however, more importantly the focus of the present study is on the maintenance of cellular viability and the evasion of apoptosis. The aim of the study was to investigate the role of LRP/LR on the cellular viability of early (A375) and late stage (A375SM) malignant melanoma cells. Flow cytometry and western blot analysis revealed that A375SM cells contain more cell-surface and total LRP/LR levels in comparison to the A375 cells, respectively. In order to determine the effect of LRP/LR on cell viability and apoptosis, LRP was down-regulated via siRNA technology. MTT assays revealed that LRP knock-down led to significant reductions in the viability of A375 and A375SM cells. Confocal microscopy indicated nuclear morphological changes suggestive of apoptotic induction in both cell lines and Annexin-V FITC/PI assays confirmed this observation. Additionally, caspase-3 activity assays revealed that apoptosis was induced in both cell lines after siRNA-mediated down-regulation of LRP. Caspase-8 and -9 activity assays suggested that post LRP knock-down; A375 cells undergo apoptosis solely via the extrinsic pathway, while A375SM cells undergo apoptosis via the intrinsic pathway. siRNAs mediated LRP knock-down might represent a powerful alternative therapeutic strategy for the treatment of malignant melanoma through the induction of apoptosis. Copyright © 2018. Published by Elsevier Inc.

  10. Stimulation of toll-like receptor 2 with bleomycin results in cellular activation and secretion of pro-inflammatory cytokines and chemokines

    International Nuclear Information System (INIS)

    Razonable, Raymund R.; Henault, Martin; Paya, Carlos V.

    2006-01-01

    The clinical use of bleomycin results in systemic and pulmonary inflammatory syndromes that are mediated by the production of cytokines and chemokines. In this study, we demonstrate that cell activation is initiated upon the recognition of bleomycin as a pathogen-associated molecular pattern by toll-like receptor (TLR) 2. The THP1 human monocytic cell line, which constitutively expresses high levels of TLR2, secretes interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α during bleomycin exposure. The TLR2-dependent nature of cell activation and cytokine secretion is supported by (1) the inability of TLR2-deficient human embryonic kidney (HEK) 293 cells to exhibit nuclear factor-kappa B (NF-κB) activation and secrete IL-8 in response to bleomycin; (2) the acquired ability of HEK293 to exhibit NF-κB activation and secrete IL-8 upon experimental expression of TLR2; and (3) the inhibition of cell activation in TLR2-expressing HEK293 and THP1 by anti-TLR2 monoclonal antibody. Collectively, these observations identify TLR2 activation as a critical event that triggers NF-κB activation and secretion of cytokines and chemokines during bleomycin exposure. Our in vitro findings could serve as a molecular mechanism underlying the pro-inflammatory toxicity associated with bleomycin. Whether bleomycin engages with other cellular receptors that results in activation of alternate signaling pathways and whether the TLR2-agonist activity of bleomycin contribute to its anti-neoplastic property deserve further study

  11. Sorafenib targets the mitochondrial electron transport chain complexes and ATP synthase to activate the PINK1-Parkin pathway and modulate cellular drug response.

    Science.gov (United States)

    Zhang, Conggang; Liu, Zeyu; Bunker, Eric; Ramirez, Adrian; Lee, Schuyler; Peng, Yinghua; Tan, Aik-Choon; Eckhardt, S Gail; Chapnick, Douglas A; Liu, Xuedong

    2017-09-08

    Sorafenib (Nexavar) is a broad-spectrum multikinase inhibitor that proves effective in treating advanced renal-cell carcinoma and liver cancer. Despite its well-characterized mechanism of action on several established cancer-related protein kinases, sorafenib causes variable responses among human tumors, although the cause for this variation is unknown. In an unbiased screening of an oncology drug library, we found that sorafenib activates recruitment of the ubiquitin E3 ligase Parkin to damaged mitochondria. We show that sorafenib inhibits the activity of both complex II/III of the electron transport chain and ATP synthase. Dual inhibition of these complexes, but not inhibition of each individual complex, stabilizes the serine-threonine protein kinase PINK1 on the mitochondrial outer membrane and activates Parkin. Unlike the protonophore carbonyl cyanide m -chlorophenylhydrazone, which activates the mitophagy response, sorafenib treatment triggers PINK1/Parkin-dependent cellular apoptosis, which is attenuated upon Bcl-2 overexpression. In summary, our results reveal a new mechanism of action for sorafenib as a mitocan and suggest that high Parkin activity levels could make tumor cells more sensitive to sorafenib's actions, providing one possible explanation why Parkin may be a tumor suppressor gene. These insights could be useful in developing new rationally designed combination therapies with sorafenib. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Natural cytolytic activity in mice with natural or induced cellular defects. I. Differential ability of in vitro interleukin-2 addition to augment natural cytolytic function

    International Nuclear Information System (INIS)

    Ades, E.W.; Hinson, A.; Butler, L.D.

    1986-01-01

    The ability of in vitro addition of recombinant interleukin 2 (rIL-2) to differentially enhance natural cytotoxicity was assessed using cells from mice with natural and induced cellular defects. In vivo treatment with most immunosuppressive or cytoreductive agents, anti-asialo-GM1 antibody, or gamma irradiation dramatically reduced in vitro cytotoxicity against natural killer (NK) sensitive targets by direct reduction in either percentage specific lysis or lytic units per spleen. In most cases, in vitro addition of rIL-2 (at concentrations causing augmented NK function in cells from naive Balb/C mice) enhanced cytotoxic activity of cells from treatment groups to a normal value but not within the rIL-2-enhanced range of nontreated animals. Additionally, cytotoxic activity of cells from animals treated with certain drugs or gamma irradiation could be augmented by rIL-2 when measured by percentage lysis but not lytic units per spleen. In vivo treatment with cyclosporin A did not affect natural cytotoxic activity and addition of rIL-2 augmented the NK activity in a similar fashion to the profile of naive cells. In experiments using cells from beige (C57Bl/6-bg) mice which have a natural defect in NK activity against YAC-1 targets, addition of rIL-2 (at concentrations causing augmented natural cytotoxic function in cells from C57Bl/6 mice) could not effectively enhance in vitro natural cytotoxic function

  13. Natural cytolytic activity in mice with natural or induced cellular defects. I. Differential ability of in vitro interleukin-2 addition to augment natural cytolytic function

    Energy Technology Data Exchange (ETDEWEB)

    Ades, E.W.; Hinson, A.; Butler, L.D.

    1986-08-01

    The ability of in vitro addition of recombinant interleukin 2 (rIL-2) to differentially enhance natural cytotoxicity was assessed using cells from mice with natural and induced cellular defects. In vivo treatment with most immunosuppressive or cytoreductive agents, anti-asialo-GM1 antibody, or gamma irradiation dramatically reduced in vitro cytotoxicity against natural killer (NK) sensitive targets by direct reduction in either percentage specific lysis or lytic units per spleen. In most cases, in vitro addition of rIL-2 (at concentrations causing augmented NK function in cells from naive Balb/C mice) enhanced cytotoxic activity of cells from treatment groups to a normal value but not within the rIL-2-enhanced range of nontreated animals. Additionally, cytotoxic activity of cells from animals treated with certain drugs or gamma irradiation could be augmented by rIL-2 when measured by percentage lysis but not lytic units per spleen. In vivo treatment with cyclosporin A did not affect natural cytotoxic activity and addition of rIL-2 augmented the NK activity in a similar fashion to the profile of naive cells. In experiments using cells from beige (C57Bl/6-bg) mice which have a natural defect in NK activity against YAC-1 targets, addition of rIL-2 (at concentrations causing augmented natural cytotoxic function in cells from C57Bl/6 mice) could not effectively enhance in vitro natural cytotoxic function.

  14. Algorithm for cellular reprogramming.

    Science.gov (United States)

    Ronquist, Scott; Patterson, Geoff; Muir, Lindsey A; Lindsly, Stephen; Chen, Haiming; Brown, Markus; Wicha, Max S; Bloch, Anthony; Brockett, Roger; Rajapakse, Indika

    2017-11-07

    The day we understand the time evolution of subcellular events at a level of detail comparable to physical systems governed by Newton's laws of motion seems far away. Even so, quantitative approaches to cellular dynamics add to our understanding of cell biology. With data-guided frameworks we can develop better predictions about, and methods for, control over specific biological processes and system-wide cell behavior. Here we describe an approach for optimizing the use of transcription factors (TFs) in cellular reprogramming, based on a device commonly used in optimal control. We construct an approximate model for the natural evolution of a cell-cycle-synchronized population of human fibroblasts, based on data obtained by sampling the expression of 22,083 genes at several time points during the cell cycle. To arrive at a model of moderate complexity, we cluster gene expression based on division of the genome into topologically associating domains (TADs) and then model the dynamics of TAD expression levels. Based on this dynamical model and additional data, such as known TF binding sites and activity, we develop a methodology for identifying the top TF candidates for a specific cellular reprogramming task. Our data-guided methodology identifies a number of TFs previously validated for reprogramming and/or natural differentiation and predicts some potentially useful combinations of TFs. Our findings highlight the immense potential of dynamical models, mathematics, and data-guided methodologies for improving strategies for control over biological processes. Copyright © 2017 the Author(s). Published by PNAS.

  15. Nanomolar Cellular Antisense Activity of Peptide Nucleic Acid (PNA) Cholic Acid ("Umbrella") and Cholesterol Conjugates Delivered by Cationic Lipids

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Nielsen, Peter E

    2012-01-01

    of cholesterol and cholic acid ("umbrella") derivatives of splice correction antisense PNA oligomers. While the conjugates alone were practically inactive up to 1 µM, their activity was dramatically improved when delivered by a cationic lipid transfection agent (LipofectAMINE2000). In particular, PNAs...

  16. Telomerase activity is spontaneously increased in lymphocytes from patients with atopic dermatitis and correlates with cellular proliferation

    DEFF Research Database (Denmark)

    Wu, Kehuai; Volke, Anne Rehné; Lund, Marianne

    1999-01-01

    blood mononuclear cells (PBMC) were isolated from 15 patients with AD and 13 healthy donors. Cells were stimulated with purified protein derivative (PPD) of tuberculin (10 microg/ml), interleukin 2 (IL-2) (100 U/ml), anti-CD3 monoclonal antibody (anti-CD3) (1 microg/ml), anti-CD3 plus IL-2......-thymidine incorporation. We found that telomerase activity in non-stimulated PBMC from patients with AD was significantly up-regulated without any stimulation during the 72 h of in vitro incubation. The most potent stimulator of telomerase activity was SEA, followed by anti-CD3 plus IL-2, anti-CD3 alone, and PPD. IL-2...

  17. Cellular cooperation in lymphocyte activation. III. B-cell helper effect in the enhancement of T-cell response.

    Science.gov (United States)

    Kasahara, T; Kin, K; Itoh, Y; Kawai, T; Kano, Y; Shioiri-Nakano, K

    1979-01-01

    T and B cells were purified from human tonsil and peripheral blood by the removal of phagocytic cells, followed by filtration through a nylon fiber column (NC) and E-rosette formation. Purified T and B cells contained less than 1% of other cell types. The responses of T cells to concanavalin A (Con A) and soluble protein A were greatly enhanced in the presence of autologous B cells. Participation of B cells in T-cell enhancement was confirmed by the following observations: (a) purified B copulation, which was separated further from adherent B cells, retained its enhancing activity. (b) Another adherent cell-free B-cell preparation, which was purified from the NC-passed fraction, and (c) no T lymphoid but some B lymphoid cell lines, elicited strong T-cell enhancement. It was also found that the enhancing capacity of B cells required no metabolic activity, but rather an intact cell form and direct cell-to-cell contact with responding cells. The stimulatory determinants on B cells were resistant to trypsin and neuraminidase treatment. In this paper a hypothesis will be presented that at least two signals are prerequisite for the effective activation of T cells.

  18. Elevated Levels of Endocannabinoids in Chronic Hepatitis C May Modulate Cellular Immune Response and Hepatic Stellate Cell Activation

    Directory of Open Access Journals (Sweden)

    Eleonora Patsenker

    2015-03-01

    Full Text Available The endocannabinoid (EC system is implicated in many chronic liver diseases, including hepatitis C viral (HCV infection. Cannabis consumption is associated with fibrosis progression in patients with chronic hepatitis C (CHC, however, the role of ECs in the development of CHC has never been explored. To study this question, anandamide (AEA and 2-arachidonoyl glycerol (2-AG were quantified in samples of HCV patients and healthy controls by gas and liquid chromatography mass spectrometry. Fatty acid amide hydrolase (FAAH and monoaclyglycerol lipase (MAGL activity was assessed by [3H]AEA and [3H]2-AG hydrolysis, respectively. Gene expression and cytokine release were assayed by TaqMan PCR and ELISpot, respectively. AEA and 2-AG levels were increased in plasma of HCV patients, but not in liver tissues. Hepatic FAAH and MAGL activity was not changed. In peripheral blood mononuclear cells (PBMC, ECs inhibited IFN-γ, TNF-α, and IL-2 secretion. Inhibition of IL-2 by endogenous AEA was stronger in PBMC from HCV patients. In hepatocytes, 2-AG induced the expression of IL-6, -17A, -32 and COX-2, and enhanced activation of hepatic stellate cells (HSC co-cultivated with PBMC from subjects with CHC. In conclusion, ECs are increased in plasma of patients with CHC and might reveal immunosuppressive and profibrogenic effects.

  19. Cellular demise and inflammatory microglial activation during beta-amyloid toxicity are governed by Wnt1 and canonical signaling pathways.

    Science.gov (United States)

    Chong, Zhao Zhong; Li, Faqi; Maiese, Kenneth

    2007-06-01

    Initially described as a modulator of embryogenesis for a number of organ systems, Wnt1 has recently been linked to the development of several neurodegenerative disorders, none being of greater significance than Alzheimer's disease. We therefore examined the ability of Wnt1 to oversee vital pathways responsible for cell survival during beta-amyloid (Abeta1-42) exposure. Here we show that Wnt1 is critical for protection in the SH-SY5Y neuronal cell line against genomic DNA degradation, membrane phosphatidylserine (PS) exposure, and microglial activation, since these neuroprotective attributes of Wnt1 are lost during gene silencing of Wnt1 protein expression. Intimately tied to Wnt1 protection is the presence and activation of Akt1. Pharmacological inhibition of the PI 3-K pathway or gene silencing of Akt1 expression can abrogate the protective capacity of Wnt1. Closely aligned with Wnt1 and Akt1 are the integrated canonical pathways of synthase kinase-3beta (GSK-3beta) and beta-catenin. Through Akt1 dependent pathways, Wnt1 phosphorylates GSK-3beta and maintains beta-catenin integrity to insure its translocation from the cytoplasm to the nucleus to block apoptosis. Our work outlines a highly novel role for Wnt1 and its integration with Akt1, GSK-3beta, and beta-catenin to foster neuronal cell survival and repress inflammatory microglial activation that can identify new avenues of therapy against neurodegenerative disorders.

  20. Cellular renewal and improvement of local cell effector activity in peritoneal cavity in response to infectious stimuli.

    Directory of Open Access Journals (Sweden)

    Alexandra dos Anjos Cassado

    Full Text Available The peritoneal cavity (PerC is a singular compartment where many cell populations reside and interact. Despite the widely adopted experimental approach of intraperitoneal (i.p. inoculation, little is known about the behavior of the different cell populations within the PerC. To evaluate the dynamics of peritoneal macrophage (MØ subsets, namely small peritoneal MØ (SPM and large peritoneal MØ (LPM, in response to infectious stimuli, C57BL/6 mice were injected i.p. with zymosan or Trypanosoma cruzi. These conditions resulted in the marked modification of the PerC myelo-monocytic compartment characterized by the disappearance of LPM and the accumulation of SPM and monocytes. In parallel, adherent cells isolated from stimulated PerC displayed reduced staining for β-galactosidase, a biomarker for senescence. Further, the adherent cells showed increased nitric oxide (NO and higher frequency of IL-12-producing cells in response to subsequent LPS and IFN-γ stimulation. Among myelo-monocytic cells, SPM rather than LPM or monocytes, appear to be the central effectors of the activated PerC; they display higher phagocytic activity and are the main source of IL-12. Thus, our data provide a first demonstration of the consequences of the dynamics between peritoneal MØ subpopulations by showing that substitution of LPM by a robust SPM and monocytes in response to infectious stimuli greatly improves PerC effector activity.

  1. Exposure of Daphnia magna to trichloroethylene (TCE) and vinyl chloride (VC): evaluation of gene transcription, cellular activity, and life-history parameters.

    Science.gov (United States)

    Houde, Magali; Douville, Mélanie; Gagnon, Pierre; Sproull, Jim; Cloutier, François

    2015-06-01

    Trichloroethylene (TCE) is a ubiquitous contaminant classified as a human carcinogen. Vinyl chloride (VC) is primarily used to manufacture polyvinyl chloride and can also be a degradation product of TCE. Very few data exist on the toxicity of TCE and VC in aquatic organisms particularly at environmentally relevant concentrations. The aim of this study was to evaluate the sub-lethal effects (10 day exposure; 0.1; 1; 10 µg/L) of TCE and VC in Daphnia magna at the gene, cellular, and life-history levels. Results indicated impacts of VC on the regulation of genes related to glutathione-S-transferase (GST), juvenile hormone esterase (JHE), and the vitelline outer layer membrane protein (VMO1). On the cellular level, exposure to 0.1, 1, and 10 µg/L of VC significantly increased the activity of JHE in D. magna and TCE increased the activity of chitinase (at 1 and 10 µg/L). Results for life-history parameters indicated a possible tendency of TCE to affect the number of molts at the individual level in D. magna (p=0.051). Measurement of VG-like proteins using the alkali-labile phosphates (ALP) assay did not show differences between TCE treated organisms and controls. However, semi-quantitative measurement using gradient gel electrophoresis (213-218 kDa) indicated significant decrease in VG-like protein levels following exposure to TCE at all three concentrations. Overall, results indicate effects of TCE and VC on genes and proteins related to metabolism, reproduction, and growth in D. magna. Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.

  2. Cellular hyper-excitability caused by mutations that alter the activation process of voltage-gated sodium channels

    Directory of Open Access Journals (Sweden)

    Mohamed-Yassine eAMAROUCH

    2015-02-01

    Full Text Available Voltage-gated sodium channels (Nav are widely expressed as macro-molecular complexes in both excitable and non-excitable tissues. In excitable tissues, the upstroke of the action potential is the result of the passage of a large and rapid influx of sodium ions through these channels. NaV dysfunction has been associated with an increasingly wide range of neurological, muscular and cardiac disorders. The purpose of this review is to summarize the recently identified sodium channel mutations that are linked to hyper-excitability phenotypes and associated with the alteration of the activation process of voltage gated sodium channels. Indeed, several clinical manifestations that demonstrate an alteration of tissue excitability were recently shown to be strongly associated with the presence of mutations that affect the activation process of the voltage-gated sodium channels. These emerging genotype-phenotype correlations have expanded the clinical spectrum of sodium channelopathies to include disorders which feature a hyper-excitability phenotype that may or may not be associated with a cardiomyopathy. The p.I141V mutation in SCN4A and SCN5A, as well as its homologous p.I136V mutation in SCN9A, are interesting examples of mutations that have been linked to inherited hyperexcitability myotonia, exercise-induced polymorphic ventricular arrhythmias and erythromelalgia, respectively. Regardless of which sodium channel isoform is investigated, the substitution of the isoleucine to valine in the locus 141 induces similar modifications in the biophysical properties of the voltage-gated sodium channels by shifting the voltage-dependence of steady state activation towards more negative potentials.

  3. Quantification of cellular NEMO content and its impact on NF-κB activation by genotoxic stress.

    Directory of Open Access Journals (Sweden)

    Byounghoon Hwang

    Full Text Available NF-κB essential modulator, NEMO, plays a key role in canonical NF-κB signaling induced by a variety of stimuli, including cytokines and genotoxic agents. To dissect the different biochemical and functional roles of NEMO in NF-κB signaling, various mutant forms of NEMO have been previously analyzed. However, transient or stable overexpression of wild-type NEMO can significantly inhibit NF-κB activation, thereby confounding the analysis of NEMO mutant phenotypes. What levels of NEMO overexpression lead to such an artifact and what levels are tolerated with no significant impact on NEMO function in NF-κB activation are currently unknown. Here we purified full-length recombinant human NEMO protein and used it as a standard to quantify the average number of NEMO molecules per cell in a 1.3E2 NEMO-deficient murine pre-B cell clone stably reconstituted with full-length human NEMO (C5. We determined that the C5 cell clone has an average of 4 x 10(5 molecules of NEMO per cell. Stable reconstitution of 1.3E2 cells with different numbers of NEMO molecules per cell has demonstrated that a 10-fold range of NEMO expression (0.6-6x10(5 molecules per cell yields statistically equivalent NF-κB activation in response to the DNA damaging agent etoposide. Using the C5 cell line, we also quantified the number of NEMO molecules per cell in several commonly employed human cell lines. These results establish baseline numbers of endogenous NEMO per cell and highlight surprisingly normal functionality of NEMO in the DNA damage pathway over a wide range of expression levels that can provide a guideline for future NEMO reconstitution studies.

  4. Cellular stress induces cancer stem-like cells through expression of DNAJB8 by activation of heat shock factor 1.

    Science.gov (United States)

    Kusumoto, Hiroki; Hirohashi, Yoshihiko; Nishizawa, Satoshi; Yamashita, Masamichi; Yasuda, Kazuyo; Murai, Aiko; Takaya, Akari; Mori, Takashi; Kubo, Terufumi; Nakatsugawa, Munehide; Kanaseki, Takayuki; Tsukahara, Tomohide; Kondo, Toru; Sato, Noriyuki; Hara, Isao; Torigoe, Toshihiko

    2018-03-01

    In a previous study, we found that DNAJB8, a heat shock protein (HSP) 40 family member is expressed in kidney cancer stem-like cells (CSC)/cancer-initiating cells (CIC) and that it has a role in the maintenance of kidney CSC/CIC. Heat shock factor (HSF) 1 is a key transcription factor for responses to stress including heat shock, and it induces HSP family expression through activation by phosphorylation. In the present study, we therefore examined whether heat shock (HS) induces CSC/CIC. We treated the human kidney cancer cell line ACHN with HS, and found that HS increased side population (SP) cells. Western blot analysis and qRT-PCR showed that HS increased the expression of DNAJB8 and SOX2. Gene knockdown experiments using siRNAs showed that the increase in SOX2 expression and SP cell ratio depends on DNAJB8 and that the increase in DNAJB8 and SOX2 depend on HSF1. Furthermore, treatment with a mammalian target of rapamycin (mTOR) inhibitor, temsirolimus, decreased the expression of DNAJB8 and SOX2 and the ratio of SP cells. Taken together, the results indicate that heat shock induces DNAJB8 by activation of HSF1 and induces cancer stem-like cells. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  5. Impact of metal binding on the antitumor activity and cellular imaging of a metal chelator cationic imidazopyridine derivative.

    Science.gov (United States)

    Roy, Mithun; Chakravarthi, Balabhadrapatruni V S K; Jayabaskaran, Chelliah; Karande, Anjali A; Chakravarty, Akhil R

    2011-05-14

    A new water soluble cationic imidazopyridine species, viz. (1E)-1-((pyridin-2-yl)methyleneamino)-3-(3-(pyridin-2-yl)imidazo[1,5-a]pyridin-2(3H)-yl)propan-2-ol (1), as a metal chelator is prepared as its PF(6) salt and characterized. Compound 1 shows fluorescence at 438 nm on excitation at 342 nm in Tris-HCl buffer giving a fluorescence quantum yield (φ) of 0.105 and a life-time of 5.4 ns. Compound 1, as an avid DNA minor groove binder, shows pUC19 DNA cleavage activity in UV-A light of 365 nm forming singlet oxygen species in a type-II pathway. The photonuclease potential of 1 gets enhanced in the presence of Fe(2+), Cu(2+) or Zn(2+). Compound 1 itself displays anticancer activity in HeLa, HepG2 and Jurkat cells with an enhancement on addition of the metal ions. Photodynamic effect of 1 at 365 nm also gets enhanced in the presence of Fe(2+) and Zn(2+). Fluorescence-based cell cycle analysis shows a significant dead cell population in the sub-G1 phase of the cell cycle suggesting apoptosis via ROS generation. A significant change in the nuclear morphology is observed from Hoechst 33258 and an acridine orange/ethidium bromide (AO/EB) dual nuclear staining suggesting apoptosis in cells when treated with 1 alone or in the presence of the metal ions. Apoptosis is found to be caspase-dependent. Fluorescence imaging to monitor the distribution of 1 in cells shows that 1 in the presence of metal ions accumulates predominantly in the cytoplasm. Enhanced uptake of 1 into the cells within 12 h is observed in the presence of Fe(2+) and Zn(2+).

  6. Shared activity patterns arising at genetic susceptibility loci reveal underlying genomic and cellular architecture of human disease.

    Science.gov (United States)

    Baillie, J Kenneth; Bretherick, Andrew; Haley, Christopher S; Clohisey, Sara; Gray, Alan; Neyton, Lucile P A; Barrett, Jeffrey; Stahl, Eli A; Tenesa, Albert; Andersson, Robin; Brown, J Ben; Faulkner, Geoffrey J; Lizio, Marina; Schaefer, Ulf; Daub, Carsten; Itoh, Masayoshi; Kondo, Naoto; Lassmann, Timo; Kawai, Jun; Mole, Damian; Bajic, Vladimir B; Heutink, Peter; Rehli, Michael; Kawaji, Hideya; Sandelin, Albin; Suzuki, Harukazu; Satsangi, Jack; Wells, Christine A; Hacohen, Nir; Freeman, Thomas C; Hayashizaki, Yoshihide; Carninci, Piero; Forrest, Alistair R R; Hume, David A

    2018-03-01

    Genetic variants underlying complex traits, including disease susceptibility, are enriched within the transcriptional regulatory elements, promoters and enhancers. There is emerging evidence that regulatory elements associated with particular traits or diseases share similar patterns of transcriptional activity. Accordingly, shared transcriptional activity (coexpression) may help prioritise loci associated with a given trait, and help to identify underlying biological processes. Using cap analysis of gene expression (CAGE) profiles of promoter- and enhancer-derived RNAs across 1824 human samples, we have analysed coexpression of RNAs originating from trait-associated regulatory regions using a novel quantitative method (network density analysis; NDA). For most traits studied, phenotype-associated variants in regulatory regions were linked to tightly-coexpressed networks that are likely to share important functional characteristics. Coexpression provides a new signal, independent of phenotype association, to enable fine mapping of causative variants. The NDA coexpression approach identifies new genetic variants associated with specific traits, including an association between the regulation of the OCT1 cation transporter and genetic variants underlying circulating cholesterol levels. NDA strongly implicates particular cell types and tissues in disease pathogenesis. For example, distinct groupings of disease-associated regulatory regions implicate two distinct biological processes in the pathogenesis of ulcerative colitis; a further two separate processes are implicated in Crohn's disease. Thus, our functional analysis of genetic predisposition to disease defines new distinct disease endotypes. We predict that patients with a preponderance of susceptibility variants in each group are likely to respond differently to pharmacological therapy. Together, these findings enable a deeper biological understanding of the causal basis of complex traits.

  7. Phenolic Acids Profiles and Cellular Antioxidant Activity in Tortillas Produced from Mexican Maize Landrace Processed by Nixtamalization and Lime Extrusion Cooking.

    Science.gov (United States)

    Gaxiola-Cuevas, Nallely; Mora-Rochín, Saraid; Cuevas-Rodriguez, Edith Oliva; León-López, Liliana; Reyes-Moreno, Cuauhtémoc; Montoya-Rodríguez, Alvaro; Milán-Carrillo, Jorge

    2017-09-01

    Phenolic acids profiles, chemical antioxidant activities (ABTS and ORAC), as well as cellular antioxidant activity (CAA) of tortilla of Mexican native maize landraces elaborated from nixtamalization and lime cooking extrusion processes were studied. Both cooking procedures decreased total phenolics, chemicals antioxidant activity when compared to raw grains. Extruded tortillas retained 79.6-83.5%, 74.1-77.6% and 79.8-80.5% of total phenolics, ABTS and ORAC values, respectively, compared to 47.8-49.8%, 41.3-42.3% and 43.7-44.4% assayed in traditional tortillas, respectively. Approximately 72.5-88.2% of ferulic acid in raw grains and their tortillas were in the bound form. Regarding of the CAA initially found in raw grains, the retained percentage for traditional and extruded tortillas ranged from 47.4 to 48.7% and 72.8 to 77.5%, respectively. These results suggest that Mexican maize landrace used in this study could be considered for the elaboration of nixtamalized and extruded food products with nutraceutical potential.

  8. Cellular size as a means of tracking mTOR activity and cell fate of CD4+ T cells upon antigen recognition.

    Directory of Open Access Journals (Sweden)

    Kristen N Pollizzi

    Full Text Available mTOR is a central integrator of metabolic and immunological stimuli, dictating immune cell activation, proliferation and differentiation. In this study, we demonstrate that within a clonal population of activated T cells, there exist both mTORhi and mTORlo cells exhibiting highly divergent metabolic and immunologic functions. By taking advantage of the role of mTOR activation in controlling cellular size, we demonstrate that upon antigen recognition, mTORhi CD4+ T cells are destined to become highly glycolytic effector cells. Conversely, mTORlo T cells preferentially develop into long-lived cells that express high levels of Bcl-2, CD25, and CD62L. Furthermore, mTORlo T cells have a greater propensity to differentiate into suppressive Foxp3+ T regulatory cells, and this paradigm was also observed in human CD4+ T cells. Overall, these studies provide the opportunity to track the development of effector and memory T cells from naïve precursors, as well as facilitate the interrogation of immunologic and metabolic programs that inform these fates.

  9. Noninvasive Evaluation of Cellular Proliferative Activity in Brain Neurogenic Regions in Rats under Depression and Treatment by Enhanced [18F]FLT-PET Imaging.

    Science.gov (United States)

    Tamura, Yasuhisa; Takahashi, Kayo; Takata, Kumi; Eguchi, Asami; Yamato, Masanori; Kume, Satoshi; Nakano, Masayuki; Watanabe, Yasuyoshi; Kataoka, Yosky

    2016-08-03

    Neural stem cells in two neurogenic regions, the subventricular zone and the subgranular zone (SGZ) of the hippocampal dentate gyrus, can divide and produce new neurons throughout life. Hippocampal neurogenesis is related to emotions, including depression/anxiety, and the therapeutic effects of antidepressants, as well as learning and memory. The establishment of in vivo imaging for proliferative activity of neural stem cells in the SGZ might be used to diagnose depression and to monitor the therapeutic efficacy of antidepressants. Positron emission tomography (PET) imaging with 3'-deoxy-3'-[(18)F]fluoro-l-thymidine ([(18)F]FLT) has been studied to allow visualization of proliferative activity in two neurogenic regions of adult mammals; however, the PET imaging has not been widely used because of lower accumulation of [(18)F]FLT, which does not allow quantitative assessment of the decline in cellular proliferative activity in the SGZ under the condition of depression. We report the establishment of an enhanced PET imaging method with [(18)F]FLT combined with probenecid, an inhibitor of drug transporters at the blood-brain barrier, which can allow the quantitative visualization of neurogenic activity in rats. Enhanced PET imaging allowed us to evaluate reduced cell proliferation in the SGZ of rats with corticosterone-induced depression, and further the recovery of proliferative activity in rats under treatment with antidepressants. This enhanced [(18)F]FLT-PET imaging technique with probenecid can be used to assess the dynamic alteration of neurogenic activity in the adult mammalian brain and may also provide a means for objective diagnosis of depression and monitoring of the therapeutic effect of antidepressant treatment. Adult hippocampal neurogenesis may play a role in major depression and antidepressant therapy. Establishment of in vivo imaging for hippocampal neurogenic activity may be useful to diagnose depression and monitor the therapeutic efficacy of

  10. Activation of cellular apoptosis in the caecal epithelium is associated with increased oxidative reactions in lactating goats after feeding a high-concentrate diet.

    Science.gov (United States)

    Tao, Shiyu; Tian, Jing; Cong, Rihua; Sun, Lili; Duanmu, Yongqian; Dong, Haibo; Ni, Yingdong; Zhao, Ruqian

    2015-03-01

    What is the central question of this study? What are the ultrastructural changes of the caecal mucosa and the status of epithelial cellular apoptosis and oxidative reactions in lactating goats after prolonged feeding with a high-concentrate diet? What is the main finding and its importance? High-concentrate diet results in ultrastructural damage to the caprine caecal epithelium. Increased oxidative and decreased antioxidative reactions are involved in the process of activating epithelial apoptosis in the caecal epithelium of goats fed a high-concentrate diet. Our results provide new insight into the relationship between abnormal fermentation in the hindgut and damage to the intestinal mucosal barrier. The effect of feeding a high-concentrate diet (HC) to lactating ruminants on their hindgut epithelial structure remains unknown. In this study, 12 lactating goats were randomly assigned to either HC (65% of dry matter as concentrate; n = 6) or a low-concentrate diet (LC; 35% of dry matter as concentrate; n = 6). After 10 weeks, the epithelial ultrastructure and cell apoptotic status in the caecal mucosa were determined by transmission electron microscopy and TUNEL, respectively. The results showed that the level of free lipopolysaccharide (P epithelium, as evidenced by more TUNEL-positive apoptotic cells. Western blot analysis showed that there was no significant difference in activated caspase-3, Bax protein expression in caecal epithelial mucosa between HC- and LC-fed goats (P > 0.05). However, the level of malondialdehyde content in the caecal epithelium from HC-fed goats was markedly higher than that in LC-fed goats (P < 0.05), whereas the level of glutathione peroxidase and the superoxide dismutase activity were significantly decreased. Gene expressions of cytokines, including interleukin-1β, interleukin-6, interleukin-10, tumour necrosis factor-α and interferon-γ, as well as myeloperoxidase activity in the caecal mucosa did not show any significant

  11. Structural, molecular and cellular functions of MSH2 and MSH6 during DNA mismatch repair, damage signaling and other noncanonical activities

    Energy Technology Data Exchange (ETDEWEB)

    Edelbrock, Michael A., E-mail: Edelbrock@findlay.edu [The University of Findlay, 1000 North Main Street, Findlay, OH 45840 (United States); Kaliyaperumal, Saravanan, E-mail: Saravanan.Kaliyaperumal@hms.harvard.edu [Division of Comparative Medicine and Pathology, New England Primate Research Center, One Pine Hill Drive, Southborough, MA 01772 (United States); Williams, Kandace J., E-mail: Kandace.williams@utoledo.edu [University of Toledo College of Medicine and Life Sciences, Department of Biochemistry and Cancer Biology, 3000 Transverse Dr., Toledo, OH 43614 (United States)

    2013-03-15

    The field of DNA mismatch repair (MMR) has rapidly expanded after the discovery of the MutHLS repair system in bacteria. By the mid 1990s yeast and human homologues to bacterial MutL and MutS had been identified and their contribution to hereditary non-polyposis colorectal cancer (HNPCC; Lynch syndrome) was under intense investigation. The human MutS homologue 6 protein (hMSH6), was first reported in 1995 as a G:T binding partner (GTBP) of hMSH2, forming the hMutSα mismatch-binding complex. Signal transduction from each DNA-bound hMutSα complex is accomplished by the hMutLα heterodimer (hMLH1 and hPMS2). Molecular mechanisms and cellular regulation of individual MMR proteins are now areas of intensive research. This review will focus on molecular mechanisms associated with mismatch binding, as well as emerging evidence that MutSα, and in particular, MSH6, is a key protein in MMR-dependent DNA damage response and communication with other DNA repair pathways within the cell. MSH6 is unstable in the absence of MSH2, however it is the DNA lesion-binding partner of this heterodimer. MSH6, but not MSH2, has a conserved Phe-X-Glu motif that recognizes and binds several different DNA structural distortions, initiating different cellular responses. hMSH6 also contains the nuclear localization sequences required to shuttle hMutSα into the nucleus. For example, upon binding to O{sup 6}meG:T, MSH6 triggers a DNA damage response that involves altered phosphorylation within the N-terminal disordered domain of this unique protein. While many investigations have focused on MMR as a post-replication DNA repair mechanism, MMR proteins are expressed and active in all phases of the cell cycle. There is much more to be discovered about regulatory cellular roles that require the presence of MutSα and, in particular, MSH6.

  12. Structural, molecular and cellular functions of MSH2 and MSH6 during DNA mismatch repair, damage signaling and other noncanonical activities

    International Nuclear Information System (INIS)

    Edelbrock, Michael A.; Kaliyaperumal, Saravanan; Williams, Kandace J.

    2013-01-01

    The field of DNA mismatch repair (MMR) has rapidly expanded after the discovery of the MutHLS repair system in bacteria. By the mid 1990s yeast and human homologues to bacterial MutL and MutS had been identified and their contribution to hereditary non-polyposis colorectal cancer (HNPCC; Lynch syndrome) was under intense investigation. The human MutS homologue 6 protein (hMSH6), was first reported in 1995 as a G:T binding partner (GTBP) of hMSH2, forming the hMutSα mismatch-binding complex. Signal transduction from each DNA-bound hMutSα complex is accomplished by the hMutLα heterodimer (hMLH1 and hPMS2). Molecular mechanisms and cellular regulation of individual MMR proteins are now areas of intensive research. This review will focus on molecular mechanisms associated with mismatch binding, as well as emerging evidence that MutSα, and in particular, MSH6, is a key protein in MMR-dependent DNA damage response and communication with other DNA repair pathways within the cell. MSH6 is unstable in the absence of MSH2, however it is the DNA lesion-binding partner of this heterodimer. MSH6, but not MSH2, has a conserved Phe-X-Glu motif that recognizes and binds several different DNA structural distortions, initiating different cellular responses. hMSH6 also contains the nuclear localization sequences required to shuttle hMutSα into the nucleus. For example, upon binding to O 6 meG:T, MSH6 triggers a DNA damage response that involves altered phosphorylation within the N-terminal disordered domain of this unique protein. While many investigations have focused on MMR as a post-replication DNA repair mechanism, MMR proteins are expressed and active in all phases of the cell cycle. There is much more to be discovered about regulatory cellular roles that require the presence of MutSα and, in particular, MSH6

  13. Positive correlation between decreased cellular uptake, NADPH-glutathione reductase activity and adriamycin resistance in Ehrlich ascites tumor lines.

    Science.gov (United States)

    Scheulen, M E; Hoensch, H; Kappus, H; Seeber, S; Schmidt, C G

    1987-01-01

    From a wild type strain of Ehrlich ascites tumor (EATWT) sublines resistant to daunorubicin (EATDNM), etoposide (EATETO), and cisplatinum (EATCIS) have been developed in vivo. Increase in survival and cure rate caused by adriamycin (doxorubicin) have been determined in female NMRI mice which were inoculated i.p. with EAT cells. Adriamycin concentrations causing 50% inhibition of 3H-thymidine (ICT) and 3H-uridine incorporation (ICU) and intracellular adriamycin steady-state concentrations (SSC) were measured in vitro. Adriamycin resistance increased and SSC decreased in the following sequence: EATWT - EATCIS - EATDNM - EATETO. When ICT and ICU were corrected for intracellular adriamycin concentrations in consideration of the different SSC (ICTc, ICUc), ICTc and ICUc still varied up to the 3.2 fold in EATCIS, EATDNM and EATETO in comparison to EATWT. Thus, in addition to different SSC other factors must be responsible for adriamycin resistance. Therefore, enzymes which may play a role in the cytotoxicity related to adriamycin metabolism (NADPH-cytochrome P-450 reductase, NADPH-glutathione reductase, NADP-glucose-6-phosphate dehydrogenase, NADP-isocitrate dehydrogenase) were measured. In contrast to the other parameters determined, NADPH-glutathione reductase was significantly (p less than 0.01) increased up to the 3.2 fold parallel to adriamycin resistance as determined by increase in life span, cure rate, ICTc, and ICUc, respectively. It is concluded that high activities of NADPH-glutathione reductase may contribute to an increase in adriamycin resistance of malignant tumors.

  14. Numerical modelling of local deposition patients, activity distributions and cellular hit probabilities of inhaled radon progenies in human airways

    International Nuclear Information System (INIS)

    Farkas, A.; Balashazy, I.; Szoeke, I.

    2003-01-01

    The general objective of our research is modelling the biophysical processes of the effects of inhaled radon progenies. This effort is related to the rejection or support of the linear no threshold (LNT) dose-effect hypothesis, which seems to be one of the most challenging tasks of current radiation protection. Our approximation and results may also serve as a useful tool for lung cancer models. In this study, deposition patterns, activity distributions and alpha-hit probabilities of inhaled radon progenies in the large airways of the human tracheobronchial tree are computed. The airflow fields and related particle deposition patterns strongly depend on the shape of airway geometry and breathing pattern. Computed deposition patterns of attached an unattached radon progenies are strongly inhomogeneous creating hot spots at the carinal regions and downstream of the inner sides of the daughter airways. The results suggest that in the vicinity of the carinal regions the multiple hit probabilities are quite high even at low average doses and increase exponentially in the low-dose range. Thus, even the so-called low doses may present high doses for large clusters of cells. The cell transformation probabilities are much higher in these regions and this phenomenon cannot be modeled with average burdens. (authors)

  15. Arginase-1 expressing microglia in close proximity to motor neurons were increased early in disease progression in canine degenerative myelopathy, a model of amyotrophic lateral sclerosis.

    Science.gov (United States)

    Toedebusch, Christine M; Snyder, John C; Jones, Maria R; Garcia, Virginia B; Johnson, Gayle C; Villalón, Eric L; Coates, Joan R; Garcia, Michael L

    2018-04-01

    Toxicity within superoxide dismutase-1 (SOD1)-associated familial amyotrophic lateral sclerosis (ALS) is non-cell autonomous with direct contribution from microglia. Microglia exhibit variable expression of neuroprotective and neurotoxic molecules throughout disease progression. The mechanisms regulating microglial phenotype within ALS are not well understood. This work presents a first study to examine the specific microglial phenotypic response in close association to motor neurons in a naturally occurring disease model of ALS, canine degenerative myelopathy (DM). Microglia closely associated with motor neurons were increased in all stages of DM progression, although only DM Late reached statistical significance. Furthermore, the number of arginase-1 expressing microglia per motor neuron were significantly increased in early stages of DM, whereas the number of inducible nitric oxide synthase (iNOS)-expressing microglia per motor neuron was indistinguishable from aged controls at all stages of disease. Fractalkine, a chemotactic molecule for microglia, was expressed in motor neurons, and the fractalkine receptor was specifically localized to microglia. However, we found no correlation between microglial response and lumbar spinal cord fractalkine levels. Taken together, these data suggest that arginase-1-expressing microglia are recruited to the motor neuron early in DM disease through a fractalkine-independent mechanism. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. Arginine Deficiency Causes Runting in the Suckling Period by Selectively Activating the Stress Kinase GCN2

    NARCIS (Netherlands)

    Marion, Vincent; Sankaranarayanan, Selvakumari; de Theije, Chiel; van Dijk, Paul; Lindsey, Patrick; Lamers, Marinus C.; Harding, Heather P.; Ron, David; Lamers, Wouter H.; Koehler, S. Eleonore

    2011-01-01

    Suckling "F/A2" mice, which overexpress arginase-I in their enterocytes, develop a syndrome (hypoargininemia, reduced hair and muscle growth, impaired B-cell maturation) that resembles IGF1 deficiency. The syndrome may result from an impaired function of the GH-IGF1 axis, activation of the

  17. [L-arginine metabolism enzyme activities in rat liver subcellular fractions under condition of protein deprivation].

    Science.gov (United States)

    Kopyl'chuk, G P; Buchkovskaia, I M

    2014-01-01

    The features of arginase and NO-synthase pathways of arginine's metabolism have been studied in rat liver subcellular fractions under condition of protein deprivation. During the experimental period (28 days) albino male rats were kept on semi synthetic casein diet AIN-93. The protein deprivation conditions were designed as total absence of protein in the diet and consumption of the diet partially deprived with 1/2 of the casein amount compared to in the regular diet. Daily diet consumption was regulated according to the pair feeding approach. It has been shown that the changes of enzyme activities, involved in L-arginine metabolism, were characterized by 1.4-1.7 fold decrease in arginase activity, accompanied with unchanged NO-synthase activity in cytosol. In mitochondrial fraction the unchanged arginase activity was accompanied by 3-5 fold increase of NO-synthase activity. At the terminal stages of the experiment the monodirectional dynamics in the studied activities have been observed in the mitochondrial and cytosolfractions in both experimental groups. In the studied subcellular fractions arginase activity decreased (2.4-2.7 fold with no protein in the diet and 1.5 fold with partly supplied protein) and was accompanied by NO-synthase activity increase by 3.8 fold in cytosole fraction, by 7.2 fold in mitochondrial fraction in the group with no protein in the diet and by 2.2 and 3.5 fold in the group partialy supplied with protein respectively. The observed tendency is presumably caused by the switch of L-arginine metabolism from arginase into oxidizing NO-synthase parthway.

  18. Concomitant administration of fluoxetine and amantadine modulates the activity of peritoneal macrophages of rats subjected to a forced swimming test.

    Science.gov (United States)

    Roman, Adam; Rogóz, Zofia; Kubera, Marta; Nawrat, Dominika; Nalepa, Irena

    2009-01-01

    Recent studies show that administration of a non-competitive NMDA receptor antagonist, amantadine (AMA), potentiates the action of antidepressant drugs. Since antidepressants may modulate functioning of the immune system and activation of a pro-inflammatory response in depressive disorders is frequently reported, the aim of the present study was to examine whether a combined administration of AMA and the antidepressant, fluoxetine (FLU), to rats subsequently subjected to a forced swimming test (FST) modifies the parameters of macrophage activity, directly related to their immunomodulatory functions, i.e., arginase (ARG) activity and synthesis of nitric oxide (NO). We found that 10 mg/kg AMA and 10 mg/kg FLU, ineffective in FST for antidepressant-like activity when administered alone, increased the ARG/NO ratio in macrophages when administered concomitantly. This effect was accompanied by a decrease of cellular adherence. Concurrently, the basal metabolic activity of the cells measured with reduction of resazurin, and intracellular host defense as assessed by a synthesis of superoxide anion, were not affected by such antidepressive treatment. Our data indicate that co-administration of AMA and FLU decreases the pro-inflammatory properties of macrophages and causes a redirection of immune response toward anti-inflammatory activity, as one can anticipate in the case of an effective antidepressive treatment.

  19. The cellular transcription factor CREB corresponds to activating transcription factor 47 (ATF-47) and forms complexes with a group of polypeptides related to ATF-43.

    Science.gov (United States)

    Hurst, H C; Masson, N; Jones, N C; Lee, K A

    1990-12-01

    Promoter elements containing the sequence motif CGTCA are important for a variety of inducible responses at the transcriptional level. Multiple cellular factors specifically bind to these elements and are encoded by a multigene family. Among these factors, polypeptides termed activating transcription factor 43 (ATF-43) and ATF-47 have been purified from HeLa cells and a factor referred to as cyclic AMP response element-binding protein (CREB) has been isolated from PC12 cells and rat brain. We demonstrated that CREB and ATF-47 are identical and that CREB and ATF-43 form protein-protein complexes. We also found that the cis requirements for stable DNA binding by ATF-43 and CREB are different. Using antibodies to ATF-43 we have identified a group of polypeptides (ATF-43) in the size range from 40 to 43 kDa. ATF-43 polypeptides are related by their reactivity with anti-ATF-43, DNA-binding specificity, complex formation with CREB, heat stability, and phosphorylation by protein kinase A. Certain cell types vary in their ATF-43 complement, suggesting that CREB activity is modulated in a cell-type-specific manner through interaction with ATF-43. ATF-43 polypeptides do not appear simply to correspond to the gene products of the ATF multigene family, suggesting that the size of the ATF family at the protein level is even larger than predicted from cDNA-cloning studies.

  20. Simultaneous development of antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) activity in irradiated mice reconstituted with bone marrow cells

    International Nuclear Information System (INIS)

    Sihvola, M.; Hurme, M.

    1987-01-01

    Spleen cells from irradiated, bone marrow-reconstituted mice were tested for their ability to mediate antibody-dependent cellular cytotoxicity against P815 target (ADCC-P815), ADCC against sheep red blood cells (ADCC-SRBC), and natural killer (NK) activity judged as YAC-1 lysis at different times after bone marrow reconstitution. Donor-derived ADCC-P815 effectors were found to appear in the spleens 10-12 days after bone marrow reconstitution simultaneously with the appearance of donor-derived NK cells. NK cells recently derived from bone marrow are known to express the Thy-1 antigen; the phenotype of the ''early'' ADCC-P815 effectors was found to be the same as that of NK cells, i.e., Thy-1+, asialo-GM1+. These data suggest that ADCC-P815 effector cells belong to the NK cell population. ADCC-SRBC, in contrast to ADCC-P815 and NK activity, was already high on Day 7 after bone marrow reconstitution. However, it was mediated partly by recipient-derived effectors. ADCC-SRBC effectors were characterized to be different from ADCC-P815 effectors

  1. Antiglycopeptide Mouse Monoclonal Antibody LpMab-21 Exerts Antitumor Activity Against Human Podoplanin Through Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity.

    Science.gov (United States)

    Kato, Yukinari; Kunita, Akiko; Fukayama, Masashi; Abe, Shinji; Nishioka, Yasuhiko; Uchida, Hiroaki; Tahara, Hideaki; Yamada, Shinji; Yanaka, Miyuki; Nakamura, Takuro; Saidoh, Noriko; Yoshida, Kanae; Fujii, Yuki; Honma, Ryusuke; Takagi, Michiaki; Ogasawara, Satoshi; Murata, Takeshi; Kaneko, Mika K

    2017-02-01

    The interaction between podoplanin (PDPN) and C-type lectin-like receptor 2 (CLEC-2) is involved in tumor malignancy. We have established many monoclonal antibodies (mAbs) against human podoplanin using the cancer-specific mAb (CasMab) technology. LpMab-21, one of the mouse antipodoplanin mAbs, is of the IgG 2a subclass, and its minimum epitope was determined to be Thr76-Arg79 of the human podoplanin. Importantly, sialic acid is linked to Thr76; therefore, LpMab-21 is an antiglycopeptide mAb (GpMab). In this study, we investigated whether LpMab-21 shows antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cell lines in vitro and also studied its antitumor activities using a xenograft model. LpMab-21 showed high ADCC and CDC activities against not only podoplanin-expressing Chinese hamster ovary cells but also LN319 glioblastoma cells and PC-10 lung cancer cells, both of which endogenously express podoplanin. Furthermore, LpMab-21 decreased tumor growth in vivo, indicating that LpMab-21 could be useful for antibody therapy against human podoplanin-expressing cancers.

  2. Cellular corepressor TLE2 inhibits replication-and-transcription- activator-mediated transactivation and lytic reactivation of Kaposi's sarcoma-associated herpesvirus.

    Science.gov (United States)

    He, Zhiheng; Liu, Yunhua; Liang, Deguang; Wang, Zhuo; Robertson, Erle S; Lan, Ke

    2010-02-01

    Replication and transcription activator (RTA) encoded by open reading frame 50 (ORF50) of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential and sufficient to initiate lytic reactivation. RTA activates its target genes through direct binding with high affinity to its responsive elements or by interaction with cellular factors, such as RBP-Jkappa, Ap-1, C/EBP-alpha, and Oct-1. In this study, we identified transducin-like enhancer of split 2 (TLE2) as a novel RTA binding protein by using yeast two-hybrid screening of a human spleen cDNA library. The interaction between TLE2 and RTA was confirmed by glutathione S-transferase (GST) binding and coimmunoprecipitation assays. Immunofluorescence analysis showed that TLE2 and RTA were colocalized in the same nuclear compartment in KSHV-infected cells. This interaction recruited TLE2 to RTA bound to its recognition sites on DNA and repressed RTA auto-activation and transactivation activity. Moreover, TLE2 also inhibited the induction of lytic replication and virion production driven by RTA. We further showed that the Q (Gln-rich), SP (Ser-Pro-rich), and WDR (Trp-Asp repeat) domains of TLE2 and the Pro-rich domain of RTA were essential for this interaction. RBP-Jkappa has been shown previously to bind to the same Pro-rich domain of RTA, and this binding can be subject to competition by TLE2. In addition, TLE2 can form a complex with RTA to access the cognate DNA sequence of the RTA-responsive element at different promoters. Intriguingly, the transcription level of TLE2 could be upregulated by RTA during the lytic reactivation process. In conclusion, we identified a new RTA binding protein, TLE2, and demonstrated that TLE2 inhibited replication and transactivation mediated by RTA. This provides another potentially important mechanism for maintenance of KSHV viral latency through interaction with a host protein.

  3. Ex-vivo expanded human NK cells express activating receptors that mediate cytotoxicity of allogeneic and autologous cancer cell lines by direct recognition and antibody directed cellular cytotoxicity

    Directory of Open Access Journals (Sweden)

    Campana Dario

    2010-10-01

    Full Text Available Abstract Background The possibility that autologous NK cells could serve as an effective treatment modality for solid tumors has long been considered. However, implementation is hampered by (i the small number of NK cells in peripheral blood, (ii the difficulties associated with large-scale production of GMP compliant cytolytic NK cells, (iii the need to activate the NK cells in order to induce NK cell mediated killing and (iv the constraints imposed by autologous inhibitory receptor-ligand interactions. To address these issues, we determined (i if large numbers of NK cells could be expanded from PBMC and GMP compliant cell fractions derived by elutriation, (ii their ability to kill allogeneic and autologous tumor targets by direct cytotoxitiy and by antibody-mediated cellular cytotoxicity and (iii defined NK cell specific receptor-ligand interactions that mediate tumor target cell killing. Methods Human NK cells were expanded during 14 days. Expansion efficiency, NK receptor repertoire before and after expansion, expression of NK specific ligands, cytolytic activity against allogeneic and autologous tumor targets, with and without the addition of chimeric EGFR monoclonal antibody, were investigated. Results Cell expansion shifted the NK cell receptor repertoire towards activation and resulted in cytotoxicity against various allogeneic tumor cell lines and autologous gastric cancer cells, while sparing normal PBMC. Blocking studies confirmed that autologous cytotoxicity is established through multiple activating receptor-ligand interactions. Importantly, expanded NK cells also mediated ADCC in an autologous and allogeneic setting by antibodies that are currently being used to treat patients with select solid tumors. Conclusion These data demonstrate that large numbers of cytolytic NK cells can be generated from PBMC and lymphocyte-enriched fractions obtained by GMP compliant counter current elutriation from PBMC, establishing the preclinical

  4. Subjective Positive and Negative Sleep Variables Differentially Affect Cellular Immune Activity in a Breast Cancer Survivor: A Time-series Analysis Approach

    Directory of Open Access Journals (Sweden)

    Magdalena Singer

    2018-01-01

    Full Text Available This study on a breast cancer survivor suffering from cancer-related fatigue (CaRF and depression investigated the bidirectional relationship between cellular immune activity and subjective sleep. The 49-year-old patient (breast cancer diagnosis 5 years before the study, currently in remission collected her full urine output for 28 days in 12-h intervals (8:00 p.m. to 8:00 a.m. and 8:00 a.m. to 8:00 p.m.. These urine samples were used to determine urinary neopterin (cellular immune activation marker and creatinine concentrations via high-pressure liquid chromatography (HPLC. Each morning, the patient answered questions on five sleep variables: sleep quality (SQ, sleep recreational value (SRV, total sleep time (TST, total wake time (TWT, and awakenings during sleep period (ADS. For the purpose of this study, the time series of the nighttime urinary neopterin levels and the five sleep variables were determined. Using centered moving average (CMA smoothing and cross-correlational analysis, this study showed that increases in the positive sleep variables SQ and SRV were followed by urinary neopterin concentration decreases after 96–120 h (SQ, lag 4: r = −0.411; p = 0.044; SRV: lag 4: r = −0.472; p = 0.021 and 120–144 h (SRV, lag 5: r = −0.464; p = 0.026. Increases in the negative sleep variable TWT, by contrast, were followed by increases in urinary neopterin concentrations 72–96 h later (lag 3: r = 0.522; p = 0.009. No systematic effects in the other direction, i.e., from urinary neopterin levels to sleep, were observed in this study. Although preliminary, the findings of this study highlight the benefit of carefully investigating temporal delays and directions of effects when studying the dynamic relationship between sleep and immune variables in the natural context of everyday life.

  5. Diclofenac Inhibits Tumor Growth in a Murine Model of Pancreatic Cancer by Modulation of VEGF Levels and Arginase Activity

    OpenAIRE

    Mayorek, Nina; Naftali-Shani, Nili; Grunewald, Myriam

    2010-01-01

    BACKGROUND: Diclofenac is one of the oldest anti-inflammatory drugs in use. In addition to its inhibition of cyclooxygenases (COX), diclofenac potently inhibits phospholipase A(2) (PLA(2)), thus yielding a broad anti-inflammatory effect. Since inflammation is an important factor in the development of pancreatic tumors we explored the potential of diclofenac to inhibit tumor growth in mice inoculated with PANCO2 cells orthotopically. METHODOLOGY/PRINCIPAL FINDINGS: We found that diclofenac tre...

  6. Cellular mechanics and motility

    Science.gov (United States)

    Hénon, Sylvie; Sykes, Cécile

    2015-10-01

    cross-linked or branched networks. It is a highly dynamical system in which filaments are able to elongate or slide one on the other with the contribution of very active cellular proteins like molecular motors. The versatile properties of this cytoskeleton ensure the diversity of mechanical behaviors to explain cell rigidity as well as cell motility.

  7. Inhibition of S-adenosylmethionine decarboxylase and diamine oxidase activities by analogues of methylglyoxal bis(guanylhydrazone) and their cellular uptake during lymphocyte activation.

    Science.gov (United States)

    Jänne, J; Morris, D R

    1984-03-15

    Several congeners of methylglyoxal bis(guanylhydrazone) were tested for their ability to inhibit eukaryotic putrescine-activated S-adenosylmethionine decarboxylase (EC 4.1.1.50) and intestinal diamine oxidase (EC 1.4.3.6). All the compounds tested, namely methylglyoxal bis(guanylhydrazone), ethylglyoxal bis(guanylhydrazone), dimethylglyoxal bis(guanylhydrazone) and the di-N"-methyl derivative of methylglyoxal bis(guanylhydrazone), were strong inhibitors of both yeast and mouse liver adenosylmethionine decarboxylase activity in vitro. The enzyme from both sources was most powerfully inhibited by ethylglyoxal bis(guanylhydrazone). All the diguanidines likewise inhibited diamine oxidase activity in vitro. The maximum intracellular concentrations of the ethyl and dimethylated analogues achieved in activated lymphocytes were only about one-fifth of that of the parent compound. However, both derivatives appeared to utilize the polyamine-carrier system, as indicated by competition experiments with spermidine.

  8. ChLpMab-23: Cancer-Specific Human-Mouse Chimeric Anti-Podoplanin Antibody Exhibits Antitumor Activity via Antibody-Dependent Cellular Cytotoxicity.

    Science.gov (United States)

    Kaneko, Mika K; Nakamura, Takuro; Kunita, Akiko; Fukayama, Masashi; Abe, Shinji; Nishioka, Yasuhiko; Yamada, Shinji; Yanaka, Miyuki; Saidoh, Noriko; Yoshida, Kanae; Fujii, Yuki; Ogasawara, Satoshi; Kato, Yukinari

    2017-06-01

    Podoplanin is expressed in many cancers, including oral cancers and brain tumors. The interaction between podoplanin and its receptor C-type lectin-like receptor 2 (CLEC-2) has been reported to be involved in cancer metastasis and tumor malignancy. We previously established many monoclonal antibodies (mAbs) against human podoplanin using the cancer-specific mAb (CasMab) technology. LpMab-23 (IgG 1 , kappa), one of the mouse anti-podoplanin mAbs, was shown to be a CasMab. However, we have not shown the usefulness of LpMab-23 for antibody therapy against podoplanin-expressing cancers. In this study, we first determined the minimum epitope of LpMab-23 and revealed that Gly54-Leu64 peptide, especially Gly54, Thr55, Ser56, Glu57, Asp58, Arg59, Tyr60, and Leu64 of podoplanin, is a critical epitope of LpMab-23. We further produced human-mouse chimeric LpMab-23 (chLpMab-23) and investigated whether chLpMab-23 exerts antibody-dependent cellular cytotoxicity (ADCC) and antitumor activity. In flow cytometry, chLpMab-23 showed high sensitivity against a podoplanin-expressing glioblastoma cell line, LN319, and an oral cancer cell line, HSC-2. chLpMab-23 also showed ADCC activity against podoplanin-expressing CHO cells (CHO/podoplanin). In xenograft models with HSC-2 and CHO/podoplanin, chLpMab-23 exerts antitumor activity using human natural killer cells, indicating that chLpMab-23 could be useful for antibody therapy against podoplanin-expressing cancers.

  9. Silencing of ribosomal protein S9 elicits a multitude of cellular responses inhibiting the growth of cancer cells subsequent to p53 activation.

    Directory of Open Access Journals (Sweden)

    Mikael S Lindström

    Full Text Available BACKGROUND: Disruption of the nucleolus often leads to activation of the p53 tumor suppressor pathway through inhibition of MDM2 that is mediated by a limited set of ribosomal proteins including RPL11 and RPL5. The effects of ribosomal protein loss in cultured mammalian cells have not been thoroughly investigated. Here we characterize the cellular stress response caused by depletion of ribosomal protein S9 (RPS9. METHODOLOGY/PRINCIPAL FINDINGS: Depletion of RPS9 impaired production of 18S ribosomal RNA and induced p53 activity. It promoted p53-dependent morphological differentiation of U343MGa Cl2:6 glioma cells as evidenced by intensified expression of glial fibrillary acidic protein and profound changes in cell shape. U2OS osteosarcoma cells displayed a limited senescence response with increased expression of DNA damage response markers, whereas HeLa cervical carcinoma cells underwent cell death by apoptosis. Knockdown of RPL11 impaired p53-dependent phenotypes in the different RPS9 depleted cell cultures. Importantly, knockdown of RPS9 or RPL11 also markedly inhibited cell proliferation through p53-independent mechanisms. RPL11 binding to MDM2 was retained despite decreased levels of RPL11 protein following nucleolar stress. In these settings, RPL11 was critical for maintaining p53 protein stability but was not strictly required for p53 protein synthesis. CONCLUSIONS: p53 plays an important role in the initial restriction of cell proliferation that occurs in response to decreased level of RPS9. Our results do not exclude the possibility that other nucleolar stress sensing molecules act upstream or in parallel to RPL11 to activate p53. Inhibiting the expression of certain ribosomal proteins, such as RPS9, could be one efficient way to reinitiate differentiation processes or to induce senescence or apoptosis in rapidly proliferating tumor cells.

  10. P2X7 Cell Death Receptor Activation and Mitochondrial Impairment in Oxaliplatin-Induced Apoptosis and Neuronal Injury: Cellular Mechanisms and In Vivo Approach.

    Directory of Open Access Journals (Sweden)

    France Massicot

    Full Text Available Limited information is available regarding the cellular mechanisms of oxaliplatin-induced painful neuropathy during exposure of patients to this drug. We therefore determined oxidative stress in cultured cells and evaluated its occurrence in C57BL/6 mice. Using both cultured neuroblastoma (SH-SY5Y and macrophage (RAW 264.7 cell lines and also brain tissues of oxaliplatin-treated mice, we investigated whether oxaliplatin (OXA induces oxidative stress and apoptosis. Cultured cells were treated with 2-200 µM OXA for 24 h. The effects of pharmacological inhibitors of oxidative stress or inflammation (N-acetyl cysteine, ibuprofen, acetaminophen were also tested. Inhibitors were added 30 min before OXA treatment and then in combination with OXA for 24 h. In SH-SY5Y cells, OXA caused a significant dose-dependent decrease in viability, a large increase in ROS and NO production, lipid peroxidation and mitochondrial impairment as assessed by a drop in mitochondrial membrane potential, which are deleterious for the cell. An increase in levels of negatively charged phospholipids such as cardiolipin but also phosphatidylserine and phosphatidylinositol, was also observed. Additionally, OXA caused concentration-dependent P2X7 receptor activation, increased chromatin condensation and caspase-3 activation associated with TNF-α and IL-6 release. The majority of these toxic effects were equally observed in Raw 264.7 which also presented high levels of PGE2. Pretreatment of SH-SY5Y cells with pharmacological inhibitors significantly reduced or blocked all the neurotoxic OXA effects. In OXA-treated mice (28 mg/kg cumulated dose significant cold hyperalgesia and oxidative stress in the tested brain areas were shown. Our study suggests that targeting P2X7 receptor activation and mitochondrial impairment might be a potential therapeutic strategy against OXA-induced neuropathic pain.

  11. Interferon-β induces cellular senescence in cutaneous human papilloma virus-transformed human keratinocytes by affecting p53 transactivating activity.

    Directory of Open Access Journals (Sweden)

    Maria V Chiantore

    Full Text Available Interferon (IFN-β inhibits cell proliferation and affects cell cycle in keratinocytes transformed by both mucosal high risk Human Papilloma Virus (HPV and cutaneous HPV E6 and E7 proteins. In particular, upon longer IFN-β treatments, cutaneous HPV38 expressing cells undergo senescence. IFN-β appears to induce senescence by upregulating the expression of the tumor suppressor PML, a well known IFN-induced gene. Indeed, experiments in gene silencing via specific siRNAs have shown that PML is essential in the execution of the senescence programme and that both p53 and p21 pathways are involved. IFN-β treatment leads to a modulation of p53 phosphorylation and acetylation status and a reduction in the expression of the p53 dominant negative ΔNp73. These effects allow the recovery of p53 transactivating activity of target genes involved in the control of cell proliferation. Taken together, these studies suggest that signaling through the IFN pathway might play an important role in cellular senescence. This additional understanding of IFN antitumor action and mechanisms influencing tumor responsiveness or resistance appears useful in aiding further promising development of biomolecular strategies in the IFN therapy of cancer.

  12. Histogram Analysis of Diffusion Weighted Imaging at 3T is Useful for Prediction of Lymphatic Metastatic Spread, Proliferative Activity, and Cellularity in Thyroid Cancer.

    Science.gov (United States)

    Schob, Stefan; Meyer, Hans Jonas; Dieckow, Julia; Pervinder, Bhogal; Pazaitis, Nikolaos; Höhn, Anne Kathrin; Garnov, Nikita; Horvath-Rizea, Diana; Hoffmann, Karl-Titus; Surov, Alexey

    2017-04-12

    Pre-surgical diffusion weighted imaging (DWI) is increasingly important in the context of thyroid cancer for identification of the optimal treatment strategy. It has exemplarily been shown that DWI at 3T can distinguish undifferentiated from well-differentiated thyroid carcinoma, which has decisive implications for the magnitude of surgery. This study used DWI histogram analysis of whole tumor apparent diffusion coefficient (ADC) maps. The primary aim was to discriminate thyroid carcinomas which had already gained the capacity to metastasize lymphatically from those not yet being able to spread via the lymphatic system. The secondary aim was to reflect prognostically important tumor-biological features like cellularity and proliferative activity with ADC histogram analysis. Fifteen patients with follicular-cell derived thyroid cancer were enrolled. Lymph node status, extent of infiltration of surrounding tissue, and Ki-67 and p53 expression were assessed in these patients. DWI was obtained in a 3T system using b values of 0, 400, and 800 s/mm². Whole tumor ADC volumes were analyzed using a histogram-based approach. Several ADC parameters showed significant correlations with immunohistopathological parameters. Most importantly, ADC histogram skewness and ADC histogram kurtosis were able to differentiate between nodal negative and nodal positive thyroid carcinoma. histogram analysis of whole ADC tumor volumes has the potential to provide valuable information on tumor biology in thyroid carcinoma. However, further studies are warranted.

  13. Review of quantitative phase-digital holographic microscopy: promising novel imaging technique to resolve neuronal network activity and identify cellular biomarkers of psychiatric disorders

    KAUST Repository

    Marquet, Pierre

    2014-09-22

    Quantitative phase microscopy (QPM) has recently emerged as a new powerful quantitative imaging technique well suited to noninvasively explore a transparent specimen with a nanometric axial sensitivity. In this review, we expose the recent developments of quantitative phase-digital holographic microscopy (QP-DHM). Quantitative phase-digital holographic microscopy (QP-DHM) represents an important and efficient quantitative phase method to explore cell structure and dynamics. In a second part, the most relevant QPM applications in the field of cell biology are summarized. A particular emphasis is placed on the original biological information, which can be derived from the quantitative phase signal. In a third part, recent applications obtained, with QP-DHM in the field of cellular neuroscience, namely the possibility to optically resolve neuronal network activity and spine dynamics, are presented. Furthermore, potential applications of QPM related to psychiatry through the identification of new and original cell biomarkers that, when combined with a range of other biomarkers, could significantly contribute to the determination of high risk developmental trajectories for psychiatric disorders, are discussed.

  14. Viral capsid assembly as a model for protein aggregation diseases: Active processes catalyzed by cellular assembly machines comprising novel drug targets.

    Science.gov (United States)

    Marreiros, Rita; Müller-Schiffmann, Andreas; Bader, Verian; Selvarajah, Suganya; Dey, Debendranath; Lingappa, Vishwanath R; Korth, Carsten

    2015-09-02

    Viruses can be conceptualized as self-replicating multiprotein assemblies, containing coding nucleic acids. Viruses have evolved to exploit host cellular components including enzymes to ensure their replicative life cycle. New findings indicate that also viral capsid proteins recruit host factors to accelerate their assembly. These assembly machines are RNA-containing multiprotein complexes whose composition is governed by allosteric sites. In the event of viral infection, the assembly machines are recruited to support the virus over the host and are modified to achieve that goal. Stress granules and processing bodies may represent collections of such assembly machines, readily visible by microscopy but biochemically labile and difficult to isolate by fractionation. We hypothesize that the assembly of protein multimers such as encountered in neurodegenerative or other protein conformational diseases, is also catalyzed by assembly machines. In the case of viral infection, the assembly machines have been modified by the virus to meet the virus' need for rapid capsid assembly rather than host homeostasis. In the case of the neurodegenerative diseases, it is the monomers and/or low n oligomers of the so-called aggregated proteins that are substrates of assembly machines. Examples for substrates are amyloid β peptide (Aβ) and tau in Alzheimer's disease, α-synuclein in Parkinson's disease, prions in the prion diseases, Disrupted-in-schizophrenia 1 (DISC1) in subsets of chronic mental illnesses, and others. A likely continuum between virus capsid assembly and cell-to-cell transmissibility of aggregated proteins is remarkable. Protein aggregation diseases may represent dysfunction and dysregulation of these assembly machines analogous to the aberrations induced by viral infection in which cellular homeostasis is pathologically reprogrammed. In this view, as for viral infection, reset of assembly machines to normal homeostasis should be the goal of protein aggregation

  15. Assessment of cellular estrogenic activity based on estrogen receptor-mediated reduction of soluble-form catechol-O-methyltransferase (COMT expression in an ELISA-based system.

    Directory of Open Access Journals (Sweden)

    Philip Wing-Lok Ho

    cellular components, a cell-based COMT assay provides useful initial screening to supplement the current assessments of xenoestrogens for potential estrogenic activity.

  16. Repaglinide at a cellular level

    DEFF Research Database (Denmark)

    Krogsgaard Thomsen, M; Bokvist, K; Høy, M

    2002-01-01

    To investigate the hormonal and cellular selectivity of the prandial glucose regulators, we have undertaken a series of experiments, in which we characterised the effects of repaglinide and nateglinide on ATP-sensitive potassium ion (KATP) channel activity, membrane potential and exocytosis in ra...

  17. Radiation, nitric oxide and cellular death

    International Nuclear Information System (INIS)

    Dubner, D.; Perez, M.R. Del; Michelin, S.C.; Gisone, P.A.

    1997-01-01

    The mechanisms of radiation induced cellular death constitute an objective of research ever since the first biological effects of radiation were first observed. The explosion of information produced in the last 20 years calls for a careful analysis due to the apparent contradictory data related to the cellular system studied and the range of doses used. This review focuses on the role of the active oxygen species, in particular the nitric oxides, in its relevance as potential mediator of radiation induced cellular death

  18. Inhibition of S-adenosylmethionine decarboxylase and diamine oxidase activities by analogues of methylglyoxal bis(guanylhydrazone) and their cellular uptake during lymphocyte activation.

    OpenAIRE

    Jänne, J; Morris, D R

    1984-01-01

    Several congeners of methylglyoxal bis(guanylhydrazone) were tested for their ability to inhibit eukaryotic putrescine-activated S-adenosylmethionine decarboxylase (EC 4.1.1.50) and intestinal diamine oxidase (EC 1.4.3.6). All the compounds tested, namely methylglyoxal bis(guanylhydrazone), ethylglyoxal bis(guanylhydrazone), dimethylglyoxal bis(guanylhydrazone) and the di-N"-methyl derivative of methylglyoxal bis(guanylhydrazone), were strong inhibitors of both yeast and mouse liver adenosylm...

  19. Structure–activity relationships of the human prothrombin kringle-2 peptide derivative NSA9: anti-proliferative activity and cellular internalization

    OpenAIRE

    Hwang, Hyun Sook; Kim, Dong Won; Kim, Soung Soo

    2006-01-01

    The human prothrombin kringle-2 protein inhibits angiogenesis and LLC (Lewis lung carcinoma) growth and metastasis in mice. Additionally, the NSA9 peptide (NSAVQLVEN) derived from human prothrombin kringle-2 has been reported to inhibit the proliferation of BCE (bovine capillary endothelial) cells and CAM (chorioallantoic membrane) angiogenesis. In the present study, we examined the structure–activity relationships of the NSA9 peptide in inhibiting the proliferation of endothelial cells lines...

  20. Improvement in antioxidant activity, angiotensin-converting enzyme inhibitory activity and in vitro cellular properties of fermented pepino milk by Lactobacillus strains containing the glutamate decarboxylase gene.

    Science.gov (United States)

    Chiu, Tsai-Hsin; Tsai, Shwu-Jene; Wu, Tsung-Yen; Fu, Szu-Chieh; Hwang, Yi-Ting

    2013-03-15

    The purpose of this study was to evaluate the functional potential of fermented pepino extract (PE) milk by Lactobacillus strains containing the glutamate decarboxylase (GAD) gene. Three Lactobacillus strains were selected, including L. brevis BCRC 12310, L. casei BCRC 14082 and L. salivarius subsp. salivarius BCRC 14759. The contents of free amino acids, total phenolics content, total carotenoids and the associated functional and antioxidant abilities were analyzed, including angiotensin-converting enzyme (ACE) inhibition activity, 1,1-diphenyl-2-picylhydrazyl (DPPH) radical-scavenging ability and oxygen radical absorbance capacity (ORAC). Cell proliferation of fermented PE milk was also evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Compared to the unfermented PE, fermented PE milk from Lactobacillus strains with the GAD gene showed higher levels of total phenolics, γ-aminobutyric acid, ACE inhibitory activity, DPPH, and ORAC. The viability of human promyelocytic leukemia cells (HL-60) determined by the MTT method decreased significantly when the cells were incubated with the PE and the fermented PE milk extracts. The consumption of fermented PE milk from Lactobacillus strains with the GAD gene is expected to benefit health. Further application as a health food is worthy of investigation. © 2012 Society of Chemical Industry. © 2012 Society of Chemical Industry.

  1. Interactive effects of CO2 and trace metals on the proteasome activity and cellular stress response of marine bivalves Crassostrea virginica and Mercenaria mercenaria

    International Nuclear Information System (INIS)

    Götze, Sandra; Matoo, Omera B.; Beniash, Elia; Saborowski, Reinhard; Sokolova, Inna M.

    2014-01-01

    the oyster proteasome were slightly inhibited by Cd exposure in normocapnia but this inhibition was reversed at elevated P CO 2 . Cu exposure inhibited the chymotrypsin-like activity of the oyster proteasome regardless of the exposure P CO 2 . The effects of metal exposure on the proteasome activity were less pronounced in clams, likely due to the lower metal accumulation. However, the general trends (i.e. an increase during Cd exposure, inhibition during exposure to Cu, and overall stimulatory effects of elevated P CO 2 ) were similar to those found in oysters. Levels of mRNA for ubiquitin and tumor suppressor p53 were suppressed by metal exposures in normocapnia in both species but this effect was alleviated or reversed at elevated P CO 2 . Cellular energy status of oysters was maintained at all metal and CO 2 exposures, while in clams the simultaneous exposure to Cu and moderate hypercapnia (∼800 μatm P CO 2 ) led to a decline in glycogen, ATP and ADP levels and an increase in AMP indicating energy deficiency. These data suggest that environmental CO