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Sample records for deaminase cd levels

  1. The Binding Site of Human Adenosine Deaminase for Cd26/Dipeptidyl Peptidase IV

    PubMed Central

    Richard, Eva; Arredondo-Vega, Francisco X.; Santisteban, Ines; Kelly, Susan J.; Patel, Dhavalkumar D.; Hershfield, Michael S.

    2000-01-01

    Human, but not murine, adenosine deaminase (ADA) forms a complex with the cell membrane protein CD26/dipeptidyl peptidase IV. CD26-bound ADA has been postulated to regulate extracellular adenosine levels and to modulate the costimulatory function of CD26 on T lymphocytes. Absence of ADA–CD26 binding has been implicated in causing severe combined immunodeficiency due to ADA deficiency. Using human–mouse ADA hybrids and ADA point mutants, we have localized the amino acids critical for CD26 binding to the helical segment 126–143. Arg142 in human ADA and Gln142 in mouse ADA largely determine the capacity to bind CD26. Recombinant human ADA bearing the R142Q mutation had normal catalytic activity per molecule, but markedly impaired binding to a CD26+ ADA-deficient human T cell line. Reduced CD26 binding was also found with ADA from red cells and T cells of a healthy individual whose only expressed ADA has the R142Q mutation. Conversely, ADA with the E217K active site mutation, the only ADA expressed by a severely immunodeficient patient, showed normal CD26 binding. These findings argue that ADA binding to CD26 is not essential for immune function in humans. PMID:11067872

  2. The effect of native and ACC deaminase-containing Azospirillum brasilense Cd1843 on the rooting of carnation cuttings.

    PubMed

    Li, Qiaosi; Saleh-Lakha, Saleema; Glick, Bernard R

    2005-06-01

    Carnation cuttings treated with non-transformed and 1-aminocyclopropane (ACC) deaminase-containing Azospirillum brasilense Cd1843 produced significantly more roots than untreated controls and fewer roots than cuttings treated with 0.1% indolebutyric acid (IBA). The roots produced by cuttings treated with ACC deaminase-containing Azospirillum brasilense Cd1843 were the longest roots resulting from any of the treatments, followed by non-transformed Azospirillum brasilense Cd1843, 0.1% IBA, and treatment with water. The results are interpreted in terms of a previously proposed model of bacterial promotion of plant growth by ACC deaminase and indoleacetic acid, and may have implications for the use of plant growth-promoting bacteria in the flower industry. PMID:16121231

  3. Feed-Forward Inhibition of CD73 and Upregulation of Adenosine Deaminase Contribute to the Loss of Adenosine Neuromodulation in Postinflammatory Ileitis

    PubMed Central

    Magalhães-Cardoso, Maria Teresa; Ferreirinha, Fátima; Dias, Ana Sofia; Pelletier, Julie

    2014-01-01

    Purinergic signalling is remarkably plastic during gastrointestinal inflammation. Thus, selective drugs targeting the “purinome” may be helpful for inflammatory gastrointestinal diseases. The myenteric neuromuscular transmission of healthy individuals is fine-tuned and controlled by adenosine acting on A2A excitatory receptors. Here, we investigated the neuromodulatory role of adenosine in TNBS-inflamed longitudinal muscle-myenteric plexus of the rat ileum. Seven-day postinflammation ileitis lacks adenosine neuromodulation, which may contribute to acceleration of gastrointestinal transit. The loss of adenosine neuromodulation results from deficient accumulation of the nucleoside at the myenteric synapse despite the fact that the increases in ATP release were observed. Disparity between ATP outflow and adenosine deficit in postinflammatory ileitis is ascribed to feed-forward inhibition of ecto-5′-nucleotidase/CD73 by high extracellular ATP and/or ADP. Redistribution of NTPDase2, but not of NTPDase3, from ganglion cell bodies to myenteric nerve terminals leads to preferential ADP accumulation from released ATP, thus contributing to the prolonged inhibition of muscle-bound ecto-5′-nucleotidase/CD73 and to the delay of adenosine formation at the inflamed neuromuscular synapse. On the other hand, depression of endogenous adenosine accumulation may also occur due to enhancement of adenosine deaminase activity. Both membrane-bound and soluble forms of ecto-5′-nucleotidase/CD73 and adenosine deaminase were detected in the inflamed myenteric plexus. These findings provide novel therapeutic targets for inflammatory gut motility disorders. PMID:25210228

  4. Diagnostic value of sputum adenosine deaminase (ADA) level in pulmonary tuberculosis

    PubMed Central

    Binesh, Fariba; Jalali, Hadi; Zare, Mohammad Reza; Behravan, Farhad; Tafti, Arefeh Dehghani; Behnaz, Fatemah; Tabatabaee, Mohammad; Shahcheraghi, Seyed Hossein

    2016-01-01

    Introduction Tuberculosis is still a considerable health problem in many countries. Rapid diagnosis of this disease is important, and adenosine deaminase (ADA) has been used as a diagnostic test. The aim of this study was to assess the diagnostic value of ADA in the sputum of patients with pulmonary tuberculosis. Methods The current study included 40 patients with pulmonary tuberculosis (culture positive, smear ±) and 42 patients with non tuberculosis pulmonary diseases (culture negative). ADA was measured on all of the samples. Results The median value of ADA in non-tuberculosis patients was 2.94 (4.2) U/L and 4.01 (6.54) U/L in tuberculosis patients, but this difference was not statistically significant (p=0.100). The cut-off point of 3.1 U/L had a sensitivity of 61% and a specificity of 53%, the cut-off point of 2.81 U/L had a sensitivity of 64% and a specificity of 50% and the cut-off point of 2.78 U/L had a sensitivity of 65% and a specificity of 48%. The positive predictive values for cut-off points of 3.1, 2.81 and 2.78 U/L were 55.7%, 57.44% and 69.23%, respectively. The negative predictive values for the abovementioned cut-off points were 56.75%, 57.14% and 55.88%, respectively. Conclusion Our results showed that sputum ADA test is neither specific nor sensitive. Because of its low sensitivity and specificity, determination of sputum ADA for the diagnosis of pulmonary tuberculosis is not recommended. PMID:27482515

  5. Aqueous seed extract of Syzygium cumini inhibits the dipeptidyl peptidase IV and adenosine deaminase activities, but it does not change the CD26 expression in lymphocytes in vitro.

    PubMed

    Bellé, Luziane Potrich; Bitencourt, Paula Eliete Rodrigues; Abdalla, Faida Husein; Bona, Karine Santos de; Peres, Alessandra; Maders, Liési Diones Konzen; Moretto, Maria Beatriz

    2013-03-01

    Syzygium cumini (Sc) have been intensively studied in the last years due its beneficial effects including anti-diabetic and anti-inflammatory potential. Thus, the aim of this study was to evaluate the effect of aqueous seed extract of Sc (ASc) in the activity of enzymes involved in lymphocyte functions. To perform this study, we isolated lymphocytes from healthy donors. Lymphocytes were exposed to 10, 30, and 100 mg/mL of ASc during 4 and 6 h and adenosine deaminase (ADA), dipeptidyl peptidase IV (DPP-IV), and acetylcholinesterase (AChE) activities as well as CD26 expression and cellular viability were evaluated. ASc inhibited the ADA and DPP-IV activities without alteration in the CD26 expression (DPP-IV protein). No alterations were observed in the AChE activity or in the cell viability. These results indicate that the inhibition of the DPP-IV and ADA activities was dependent on the time of exposition to ASc. We suggest that ASc exhibits immunomodulatory properties probably via the pathway of DPP-IV-ADA complex, contributing to the understanding of these proceedings in the purinergic signaling. PMID:22798209

  6. Alterations in the cholinesterase and adenosine deaminase activities and inflammation biomarker levels in patients with multiple sclerosis.

    PubMed

    Polachini, C R N; Spanevello, R M; Casali, E A; Zanini, D; Pereira, L B; Martins, C C; Baldissareli, J; Cardoso, A M; Duarte, M F; da Costa, P; Prado, A L C; Schetinger, M R C; Morsch, V M

    2014-04-25

    Multiple sclerosis (MS) is one of the main chronic inflammatory diseases of the CNS that cause functional disability in young adults. It has unknown etiology characterized by the infiltration of lymphocytes and macrophages into the brain. The aim of this study was to evaluate the acetylcholinesterase (AChE) activity in lymphocytes and whole blood, as well as butyrylcholinesterase (BChE) and adenosine deaminase (ADA) activities in serum. We also checked the levels of nucleotides, nucleosides, biomarkers of inflammation such as cytokines (interleukin (IL)-1, IL-6, interferon (IFN)-γ, tumor necrosis factor-alpha (TNF-α) and IL-10) and C-reactive protein (CRP) in serum from 29 patients with the relapsing-remitting form of MS (RRMS) and 29 healthy subjects as the control group. Results showed that AChE in lymphocytes and whole blood as well as BChE, and ADA activities in serum were significantly increased in RRMS patients when compared to the control group (P<0.05). In addition, we observed a decrease in ATP levels and a significant increase in the levels of ADP, AMP, adenosine and inosine in serum from RRMS patients in relation to the healthy subjects (P<0.05). Results also demonstrated an increase in the IFN-γ, TNF-α, IL-1, IL-6 and CRP (P<0.05) and a significant decrease in the IL-10 (P<0.0001) in RRMS patients when compared to control. Our results suggest that alterations in the biomarkers of inflammation and hydrolysis of nucleotides and nucleosides may contribute to the understanding of the neurological dysfunction of RRMS patients.

  7. Assessing the effects of heavy metals in ACC deaminase and IAA production on plant growth-promoting bacteria.

    PubMed

    Carlos, Mendoza-Hernández José; Stefani, Perea-Vélez Yazmin; Janette, Arriola-Morales; Melani, Martínez-Simón Sara; Gabriela, Pérez-Osorio

    2016-01-01

    This study poses a methodology in order to simultaneously quantify ACC deaminase and IAA levels in the same culture medium. Ten bacterial strains isolated from plant rhizosphere naturally settled in mining residues were chosen. These bacterial strains were characterized as PGPB, and all of them showed at least three characteristics (indole-3 acetic acid and siderophore production, ACC deaminase enzyme activity, and inorganic phosphate solubilization). Taxonomic identification showed that the strains belong to Enterobacter, Serratia, Klebsiella, and Escherichia genera. Similarly, both the ACC deaminase enzyme activity and the IAA synthesis in the presence of Cu, As, Pb, Ni, Cd, and Mn were measured. The results showed that both the ACC deaminase enzyme activity and the IAA synthesis were higher with the Pb, As, and Cu treatments than with the Escherichia N16, Enterobacter K131, Enterobacter N9, and Serratia K120 control treatments. On the other hand, Ni, Cd, and Mn negatively affected both the ACC deaminase enzyme activity and the IAA production on every bacterium except on the Klebsiella Mc173 strain. Serratia K120 bacterium got a positive correlation between ACC deaminase and IAA in the presence of every heavy metal, and it also promoted Helianthus annuus plant growth, showing a potential use in phytoremediation systems.

  8. Assessing the effects of heavy metals in ACC deaminase and IAA production on plant growth-promoting bacteria.

    PubMed

    Carlos, Mendoza-Hernández José; Stefani, Perea-Vélez Yazmin; Janette, Arriola-Morales; Melani, Martínez-Simón Sara; Gabriela, Pérez-Osorio

    2016-01-01

    This study poses a methodology in order to simultaneously quantify ACC deaminase and IAA levels in the same culture medium. Ten bacterial strains isolated from plant rhizosphere naturally settled in mining residues were chosen. These bacterial strains were characterized as PGPB, and all of them showed at least three characteristics (indole-3 acetic acid and siderophore production, ACC deaminase enzyme activity, and inorganic phosphate solubilization). Taxonomic identification showed that the strains belong to Enterobacter, Serratia, Klebsiella, and Escherichia genera. Similarly, both the ACC deaminase enzyme activity and the IAA synthesis in the presence of Cu, As, Pb, Ni, Cd, and Mn were measured. The results showed that both the ACC deaminase enzyme activity and the IAA synthesis were higher with the Pb, As, and Cu treatments than with the Escherichia N16, Enterobacter K131, Enterobacter N9, and Serratia K120 control treatments. On the other hand, Ni, Cd, and Mn negatively affected both the ACC deaminase enzyme activity and the IAA production on every bacterium except on the Klebsiella Mc173 strain. Serratia K120 bacterium got a positive correlation between ACC deaminase and IAA in the presence of every heavy metal, and it also promoted Helianthus annuus plant growth, showing a potential use in phytoremediation systems. PMID:27296962

  9. Assignment of the DPP4 gene encoding adenosine deaminase binding protein (CD26/dipeptidylpeptidase IV) to 2q23

    SciTech Connect

    Mathew, S.; Morrison, M.E.; Murty, V.V.V.S.

    1994-07-01

    FISH was performed on chromosome preparations obtained from phytohemagglutinin-stimulated human blood lymphocytes. cDNA encoding ADAbp was isolated from the SK-RC-28 human renal cell carcinomas cell line using PCR technique and was cloned in pSVK3 plasmid for use as a probe. The PCR primers were constructed from the known nucleotide sequence of CD26, and the cDNA product was extracted from nucleotides 1 to 2344. The vector containing the probe was labeled by nick-translation with biotin-11-dUTP. Hybridization to chromosome spreads, washings, detection with FITC-conjugated avidin, selection and photography of metaphases, analysis of signals, and banding were performed according to the described method.

  10. Increased expression with differential subcellular location of cytidine deaminase APOBEC3G in human CD4(+) T-cell activation and dendritic cell maturation.

    PubMed

    Oliva, Harold; Pacheco, Rodrigo; Martinez-Navio, José M; Rodríguez-García, Marta; Naranjo-Gómez, Mar; Climent, Núria; Prado, Carolina; Gil, Cristina; Plana, Montserrat; García, Felipe; Miró, José M; Franco, Rafael; Borras, Francesc E; Navaratnam, Naveenan; Gatell, José M; Gallart, Teresa

    2016-08-01

    APOBEC3G (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G; A3G) is an innate defense protein showing activity against retroviruses and retrotransposons. Activated CD4(+) T cells are highly permissive for HIV-1 replication, whereas resting CD4(+) T cells are refractory. Dendritic cells (DCs), especially mature DCs, are also refractory. We investigated whether these differences could be related to a differential A3G expression and/or subcellular distribution. We found that A3G mRNA and protein expression is very low in resting CD4(+) T cells and immature DCs, but increases strongly following T-cell activation and DC maturation. The Apo-7 anti-A3G monoclonal antibody (mAb), which was specifically developed, confirmed these differences at the protein level and disclosed that A3G is mainly cytoplasmic in resting CD4(+) T cells and immature DCs. Nevertheless, A3G translocates to the nucleus in activated-proliferating CD4(+) T cells, yet remaining cytoplasmic in matured DCs, a finding confirmed by immunoblotting analysis of cytoplasmic and nuclear fractions. Apo-7 mAb was able to immunoprecipitate endogenous A3G allowing to detect complexes with numerous proteins in activated-proliferating but not in resting CD4(+) T cells. The results show for the first time the nuclear translocation of A3G in activated-proliferating CD4(+) T cells.

  11. A 30-year-old female Behçet's disease patient with recurrent pleural and pericardial effusion and elevated adenosine deaminase levels: case report.

    PubMed

    Choi, Joon Young; Kim, Sung-Hwan; Kwok, Seung-Ki; Jung, Jung Im; Lee, Kyo-Young; Kim, Tae-Jung; Kang, Ji Young

    2016-07-01

    Behçet's disease is a systemic disease which may involve various organs. We describe a case of a patient diagnosed as pleuropericardial involvement of Behçet's disease. A 30-year-old woman visited our clinic presented with left pleuritic chest pain for s days. She had been diagnosed as Behçet's disease and admitted to our clinic due to pericardial and pleural effusion repeatedly in past two years. In the previous studies, effusion analysis revealed to be lympho-dominant exudate with high adenosine deaminase level. Acid-fast bacilli (AFB) culture and polymerase chain reaction (PCR) for mycobacterial tuberculosis (M.TB) were negative in the pericardial tissue, and pathologic finding showed mild endothelitis with micro-thrombi formation in the lumen. The patient had been treated with antituberculous medication for a year. In the current admission, chest computed tomography (CT) again showed left pleural effusion without other significant lesion. Pleural fluid analysis was similar with the previous study. Video-assisted thoracoscopic pleural biopsy was performed to obtain the definite diagnosis. Pathology confirmed the diagnosis as pleuropericardial involvement of Behçet's disease, and we treated the patient with oral steroid in the out-patient department. Pleuropericardial involvement of Behçet's disease may mimic TB pleurisy or pericarditis due to high adenosine deaminase (ADA) level in effusion analysis. Clinicians should keep in mind that Behçet's disease may manifest as pleural or pericardial effusion, and pathologic confirmation could be helpful for the definite diagnosis. PMID:27499994

  12. A 30-year-old female Behçet’s disease patient with recurrent pleural and pericardial effusion and elevated adenosine deaminase levels: case report

    PubMed Central

    Choi, Joon Young; Kim, Sung-Hwan; Kwok, Seung-Ki; Jung, Jung Im; Lee, Kyo-Young; Kim, Tae-Jung

    2016-01-01

    Behçet’s disease is a systemic disease which may involve various organs. We describe a case of a patient diagnosed as pleuropericardial involvement of Behçet’s disease. A 30-year-old woman visited our clinic presented with left pleuritic chest pain for s days. She had been diagnosed as Behçet’s disease and admitted to our clinic due to pericardial and pleural effusion repeatedly in past two years. In the previous studies, effusion analysis revealed to be lympho-dominant exudate with high adenosine deaminase level. Acid-fast bacilli (AFB) culture and polymerase chain reaction (PCR) for mycobacterial tuberculosis (M.TB) were negative in the pericardial tissue, and pathologic finding showed mild endothelitis with micro-thrombi formation in the lumen. The patient had been treated with antituberculous medication for a year. In the current admission, chest computed tomography (CT) again showed left pleural effusion without other significant lesion. Pleural fluid analysis was similar with the previous study. Video-assisted thoracoscopic pleural biopsy was performed to obtain the definite diagnosis. Pathology confirmed the diagnosis as pleuropericardial involvement of Behçet’s disease, and we treated the patient with oral steroid in the out-patient department. Pleuropericardial involvement of Behçet’s disease may mimic TB pleurisy or pericarditis due to high adenosine deaminase (ADA) level in effusion analysis. Clinicians should keep in mind that Behçet’s disease may manifest as pleural or pericardial effusion, and pathologic confirmation could be helpful for the definite diagnosis. PMID:27499994

  13. Genetics Home Reference: adenosine deaminase 2 deficiency

    MedlinePlus

    ... Health Conditions adenosine deaminase 2 deficiency adenosine deaminase 2 deficiency Enable Javascript to view the expand/collapse ... PDF Open All Close All Description Adenosine deaminase 2 (ADA2) deficiency is a disorder characterized by abnormal ...

  14. Adenosine deaminase isoenzyme levels in patients with human T-cell lymphotropic virus type 1 and human immunodeficiency virus type 1 infections.

    PubMed Central

    Tsuboi, I; Sagawa, K; Shichijo, S; Yokoyama, M M; Ou, D W; Wiederhold, M D

    1995-01-01

    In serum, the enzyme adenosine deaminase (ADA) is known to be divided into two isoenzymes, ADA1 and ADA2, which have different molecular weights and kinetic properties. The present study investigated ADA isoenzyme levels in the sera of patients infected with retroviruses associated with adult T-cell leukemia (ATL), human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy (HAM), and AIDS, ADA isoenzyme activities were found to be significantly (P < 0.001) higher in the sera of patients with ATL, HAM, and AIDS than in the sera of healthy controls. In the case of the ADA subtypes in the sera of patients with ATL, ADA1 activity was significantly (P < 0.001) elevated in patients with the acute and lymphoma types of ATL compared with that in patients with the chronic and smoldering types of ATL. ADA2 activity was significantly elevated in the sera of patients with the acute, lymphoma, and chronic types of ATL (P < 0.001) compared with that in patients with smoldering ATL and HTLV-1 carriers. In the case of patients with human immunodeficiency virus type 1 (HIV-1) infection, ADA1 and ADA2 activities in the sera of patients with AIDS and HIV-1 antibody-positive individuals were significantly (P < 0.001) higher than those in the sera of HIV-1 antibody-negative individuals. A significant elevation in ADA2 activity was also seen in the sera of AIDS patients (P < 0.01) compared with that in the sera of HIV-1 antibody-positive individuals. These results suggest that the magnitude of elevation of ADA isoenzyme levels in serum correlates well with the clinical conditions of the patients with these diseases. Measurement of the activities of ADA isoenzymes may therefore provide an additional parameter for distinguishing the subtypes of ATL and may prove to be useful as prognostic and therapeutic monitors in diseases associated with HTLV-1 and HIV-1 infections. PMID:8548545

  15. [Adenosine deaminase in experimental trypanosomiasis: future implications].

    PubMed

    Pérez-Aguilar, Mary Carmen; Rondón-Mercado, Rocío

    2015-09-01

    The adenosine deaminase represents a control point in the regulation of extracellular adenosine levels, thus playing a critical role in the modulation of purinergic responses to certain pathophysiological events. Several studies have shown that serum and plasma enzyme levels are elevated in some diseases caused by microorganisms, which may represent a compensatory mechanism due to the elevated levels of adenosine and the release of inflammatory mediators. Recent research indicates that adenosine deaminase activity decreases and affects hematological parameters of infected animals with Trypanosoma evansi, so that such alterations could have implications in the pathogenesis of the disease. In addition, the enzyme has been detected in this parasite; allowing the inference that it could be associated with the vital functions of the same, similar to what occurs in mammals. This knowledge may be useful in the association of chemotherapy with specific inhibitors of the enzyme in future studies.

  16. Treatment of severe combined immunodeficiency disease (SCID) due to adenosine deaminase deficiency with CD34+ selected autologous peripheral blood cells transduced with a human ADA gene. Amendment to clinical research project, Project 90-C-195, January 10, 1992.

    PubMed

    Blaese, R M; Culver, K W; Chang, L; Anderson, W F; Mullen, C; Nienhuis, A; Carter, C; Dunbar, C; Leitman, S; Berger, M

    1993-08-01

    Significant increases in lymphocyte adenosine deaminase activity, T cell numbers and immune function have been achieved in the two children with SCID thus far treated with autologous T cells genetically-corrected by retroviral-mediated insertion of a normal ADA gene. Although the data obtained to date demonstrate that the use of ADA gene corrected peripheral T cells appears to be an effective treatment for ADA(-)SCID, it is theoretically preferable to try to develop a treatment for these children that will result in stem cell gene correction. The genetic correction of T cell progenitors with long-term immune reconstituting ability would be more desirable because repeated infusions of genetically altered cells should not be necessary and the generation of a more complete repertoire of T cell specificities might also be possible. Furthermore, the present treatment protocol involves indefinite continuation of enzyme replacement treatment with PEG-ADA. The demonstration of ADA gene expression in the progeny of transduced stem cells may simplify the decision concerning cessation of this very costly enzyme treatment (approximately $250,000/yr./patient). Recent evidence suggests that a small fraction of bone marrow or peripheral blood mononuclear cells bearing the CD34 antigen contains hematopoietic stem cells with both lymphoid and myeloid reconstituting ability. We propose in this amendment to supplement the infusion of human ADA gene-transduced autologous T cells in children with ADA(-)SCID with autologous peripheral blood CD34+ cells transduced with a second, readily distinguishable ADA vector.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Purine metabolism in adenosine deaminase deficiency.

    PubMed Central

    Mills, G C; Schmalstieg, F C; Trimmer, K B; Goldman, A S; Goldblum, R M

    1976-01-01

    Purine and pyrimidine metabolites were measured in erythrocytes, plasma, and urine of a 5-month-old infant with adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) deficiency. Adenosine and adenine were measured using newly devised ion exchange separation techniques and a sensitive fluorescence assay. Plasma adenosine levels were increased, whereas adenosine was normal in erythrocytes and not detectable in urine. Increased amounts of adenine were found in erythrocytes and urine as well as in the plasma. Erythrocyte adenosine 5'-monophosphate and adenosine diphosphate concentrations were normal, but adenosine triphosphate content was greatly elevated. Because of the possibility of pyrimidine starvation, pyrimidine nucleotides (pyrimidine coenzymes) in erythrocytes and orotic acid in urine were measured. Pyrimidine nucleotide concentrations were normal, while orotic acid was not detected. These studies suggest that the immune deficiency associated with adenosine deaminase deficiency may be related to increased amounts of adenine, adenosine, or adenine nucleotides. PMID:1066699

  18. Levels of expression of complement regulatory proteins CD46, CD55 and CD59 on resting and activated human peripheral blood leucocytes

    PubMed Central

    Christmas, Stephen E; de la Mata Espinosa, Claudia T; Halliday, Deborah; Buxton, Cheryl A; Cummerson, Joanne A; Johnson, Peter M

    2006-01-01

    The cell surface complement regulatory (CReg) proteins CD46, CD55 and CD59 are widely expressed on human lymphoid and non-lymphoid cells. This study aimed to compare systematically levels of CReg expression by different leucocyte subsets and to determine whether levels were increased following activation in vitro. Levels of each CReg protein were similar on freshly isolated monocytes and all major lymphocyte subsets, except that CD4+ cells expressed significantly less CD46 than CD8+ cells (P < 0·05) while the reverse was observed for CD55 (P < 0·02). CD56+ cells, predominantly natural killer cells, expressed significantly lower levels of CD59 than T cells (P < 0·02). CD45RO+ cells had higher levels of surface CD46 and CD59, but lower levels of CD55, than CD45RO– cells (P < 0·02); CD25+ cells also expressed significantly less CD55 than CD25− cells (P < 0·002). Neutrophils expressed higher levels of CD59, but lower levels of CD55, than monocytes. Following activation with phytohaemagglutinin, CD46 was up-regulated on all leucocyte subsets with the exception of CD56+ cells. Both CD55 and CD59 were also markedly up-regulated on monocytes, and CD55 expression was greater on CD8+ than CD4+ cells following activation (P < 0·02). Lipopolysaccharide treatment did not significantly alter B-cell expression of CReg proteins whereas CD55 and CD59, but not CD46, were significantly up-regulated on monocytes (P < 0·02). These observations that CReg proteins are up-regulated on certain activated leucocyte subsets indicate that levels would be increased following immune responses in vivo. This could enhance both protection against local complement activation at inflammatory sites and also the immunoregulatory properties of these leucocytes. PMID:16999828

  19. Levels of expression of complement regulatory proteins CD46, CD55 and CD59 on resting and activated human peripheral blood leucocytes.

    PubMed

    Christmas, Stephen E; de la Mata Espinosa, Claudia T; Halliday, Deborah; Buxton, Cheryl A; Cummerson, Joanne A; Johnson, Peter M

    2006-12-01

    The cell surface complement regulatory (CReg) proteins CD46, CD55 and CD59 are widely expressed on human lymphoid and non-lymphoid cells. This study aimed to compare systematically levels of CReg expression by different leucocyte subsets and to determine whether levels were increased following activation in vitro. Levels of each CReg protein were similar on freshly isolated monocytes and all major lymphocyte subsets, except that CD4(+) cells expressed significantly less CD46 than CD8(+) cells (P < 0.05) while the reverse was observed for CD55 (P < 0.02). CD56(+) cells, predominantly natural killer cells, expressed significantly lower levels of CD59 than T cells (P < 0.02). CD45RO(+) cells had higher levels of surface CD46 and CD59, but lower levels of CD55, than CD45RO(-) cells (P < 0.02); CD25(+) cells also expressed significantly less CD55 than CD25(-) cells (P < 0.002). Neutrophils expressed higher levels of CD59, but lower levels of CD55, than monocytes. Following activation with phytohaemagglutinin, CD46 was up-regulated on all leucocyte subsets with the exception of CD56(+) cells. Both CD55 and CD59 were also markedly up-regulated on monocytes, and CD55 expression was greater on CD8(+) than CD4(+) cells following activation (P < 0.02). Lipopolysaccharide treatment did not significantly alter B-cell expression of CReg proteins whereas CD55 and CD59, but not CD46, were significantly up-regulated on monocytes (P < 0.02). These observations that CReg proteins are up-regulated on certain activated leucocyte subsets indicate that levels would be increased following immune responses in vivo. This could enhance both protection against local complement activation at inflammatory sites and also the immunoregulatory properties of these leucocytes.

  20. Action of troxacitabine on cells transduced with human cytidine deaminase cDNA.

    PubMed

    Boivin, Anne-Julie; Gourdeau, Henriette; Momparler, Richard L

    2004-01-01

    Troxacitabine (beta-L-Dioxolane-cytidine; Troxatyl) is a beta-L-nucleoside analog, which has shown preclinical antitumor activity in human xenograft tumor models and antileukemic response in patients with relapsed myeloid leukemia. Troxacitabine is activated by cellular kinases and incorporated into DNA, inhibiting its replication. In contrast to other cytosine nucleoside analogs, troxacitabine is resistant to inactivation by cytidine deaminase (CD). In this study we have investigated the effects of increased intracellular levels of CD on the antineoplastic action of troxacitabine and the related antileukemic drug, cytosine arabinoside (ARA-C). Retroviral transduction of the human CD gene in A549 lung carcinoma cells (A549-CD cells) markedly increased the expression of this gene. The A549-CD cells were more resistant to the cytotoxic action of ARA-C than the wild type A549 cells as determined by clonogenic assays. In contrast, the CD-transduced cells were as or more sensitive to the cytotoxic action of troxacitabine than the wild type cells. These results suggest that troxacitabine may be an effective antineoplastic agent against tumors with high levels of CD that show drug resistance to cytosine nucleoside analogs.

  1. ACTIVATION OF A CRYPTIC D-SERINE DEAMINASE (DSD) GENE FROM PSEUDOMONAS CEPACIA 17616

    EPA Science Inventory

    D-serine inhibits growth of P. cepacia 17616; however, resistant mutants able to express an ordinarily cryptic D-serine deaminase (dsd) gene were isolated readily. The resistant strains formed high levels of a D-serine deaminase active on D-threonine as well as D-serine. IS eleme...

  2. The current structural and functional understanding of APOBEC deaminases.

    PubMed

    Bransteitter, Ronda; Prochnow, Courtney; Chen, Xiaojiang S

    2009-10-01

    The apolipoprotein B mRNA-editing enzyme catalytic polypeptide (APOBEC) family of cytidine deaminases has emerged as an intensively studied field as a result of their important biological functions. These enzymes are involved in lipid metabolism, antibody diversification, and the inhibition of retrotransposons, retroviruses, and some DNA viruses. The APOBEC proteins function in these roles by deaminating single-stranded (ss) DNA or RNA. There are two high-resolution crystal structures available for the APOBEC family, Apo2 and the C-terminal catalytic domain (CD2) of Apo3G or Apo3G-CD2 [Holden et al. (Nature 456:121-124, 2008); Prochnow et al. (Nature 445:447-451, 2007)]. Additionally, the structure of Apo3G-CD2 has also been determined using NMR [Chen et al. (Nature 452:116-119, 2008); Furukawa et al. (EMBO J 28:440-451, 2009); Harjes et al. (J Mol Biol, 2009)]. A detailed structural analysis of the APOBEC proteins and a comparison to other zinc-coordinating deaminases can facilitate our understanding of how APOBEC proteins bind nucleic acids, recognize substrates, and form oligomers. Here, we review the recent development of structural and functional studies that apply to Apo3G as well as the APOBEC deaminase family. PMID:19547914

  3. Metabolism of 5-fluorocytosine to 5-fluorouracil in human colorectal tumor cells transduced with the cytosine deaminase gene: significant antitumor effects when only a small percentage of tumor cells express cytosine deaminase.

    PubMed Central

    Huber, B E; Austin, E A; Richards, C A; Davis, S T; Good, S S

    1994-01-01

    The gene encoding cytosine deaminase (CD) has been expressed in the human colorectal carcinoma cell line WiDr. Metabolism studies confirm that tumor cells expressing CD convert the very nontoxic prodrug 5-fluorocytosine (5FCyt) to 5-fluorouracil (5FUra) and 5FUra metabolites. Tumor xenografts composed of CD-expressing cells can selectively generate tumor levels of > 400 microM 5FUra when the host mouse is dosed with nontoxic levels of 5FCyt. The selective metabolic conversion of 5FCyt to 5FUra in CD-expressing tumor cells results in the inhibition of thymidylate synthase and incorporation of 5FUra into RNA. 5FUra is also liberated into the surrounding environment when CD-expressing tumor cells are treated with 5FCyt. The liberated 5FUra is able to kill neighboring, non-CD-expressing tumor cells in vitro and in vivo. Most importantly, when only 2% of the tumor mass contains CD-expressing cells (98% non-CD-expressing cells), significant regressions in all tumors are observed when the host mouse is dosed with nontoxic levels of 5FCyt. PMID:8058798

  4. Levels of human platelet-derived soluble CD40 ligand depend on haplotypes of CD40LG-CD40-ITGA2.

    PubMed

    Aloui, Chaker; Prigent, Antoine; Tariket, Sofiane; Sut, Caroline; Fagan, Jocelyne; Cognasse, Fabrice; Chakroun, Tahar; Garraud, Olivier; Laradi, Sandrine

    2016-01-01

    Increased circulating soluble CD40 ligand (sCD40L) is commonly associated with inflammatory disorders. We aimed to investigate whether gene polymorphisms in CD40LG, CD40 and ITGA2 are associated with a propensity to secrete sCD40L; thus, we examined this issue at the level of human platelets, the principal source of sCD40L. We performed single polymorphism and haplotype analyses to test for the effect of twelve polymorphisms across the CD40LG, CD40 and ITGA2 genes in blood donors. ITGA2 presented a positive association with rs1126643, with a significant modification in sCD40L secretion (carriers of C allele, P =  0.02), unlike the investigated CD40LG and CD40 polymorphisms. One CD40LG haplotype (TGGC) showing rs975379 (C/T), rs3092952 (A/G), rs3092933 (A/G) and rs3092929 (A/C) was associated with increased sCD40L levels (1.906 μg/L (95% CI: 1.060 to 2.751); P = 0.000009). The sCD40L level was associated with the inter-chromosomal CD40LG/CD40/ITGA2 haplotype (ATC), displaying rs3092952 (A/G), rs1883832 (C/T) and rs1126643 (C/T), with increased sCD40L levels (P = 0.0135). Our results help to decipher the genetic role of CD40LG, CD40 and ITGA2 with regard to sCD40L levels found in platelet components. Given the crucial role of sCD40L, this haplotype study in a transfusion model may be helpful to further determine the role of haplotypes in inflammatory clinical settings. PMID:27094978

  5. Levels of human platelet-derived soluble CD40 ligand depend on haplotypes of CD40LG-CD40-ITGA2

    PubMed Central

    Aloui, Chaker; Prigent, Antoine; Tariket, Sofiane; Sut, Caroline; Fagan, Jocelyne; Cognasse, Fabrice; Chakroun, Tahar; Garraud, Olivier; Laradi, Sandrine

    2016-01-01

    Increased circulating soluble CD40 ligand (sCD40L) is commonly associated with inflammatory disorders. We aimed to investigate whether gene polymorphisms in CD40LG, CD40 and ITGA2 are associated with a propensity to secrete sCD40L; thus, we examined this issue at the level of human platelets, the principal source of sCD40L. We performed single polymorphism and haplotype analyses to test for the effect of twelve polymorphisms across the CD40LG, CD40 and ITGA2 genes in blood donors. ITGA2 presented a positive association with rs1126643, with a significant modification in sCD40L secretion (carriers of C allele, P = 0.02), unlike the investigated CD40LG and CD40 polymorphisms. One CD40LG haplotype (TGGC) showing rs975379 (C/T), rs3092952 (A/G), rs3092933 (A/G) and rs3092929 (A/C) was associated with increased sCD40L levels (1.906 μg/L (95% CI: 1.060 to 2.751); P = 0.000009). The sCD40L level was associated with the inter-chromosomal CD40LG/CD40/ITGA2 haplotype (ATC), displaying rs3092952 (A/G), rs1883832 (C/T) and rs1126643 (C/T), with increased sCD40L levels (P = 0.0135). Our results help to decipher the genetic role of CD40LG, CD40 and ITGA2 with regard to sCD40L levels found in platelet components. Given the crucial role of sCD40L, this haplotype study in a transfusion model may be helpful to further determine the role of haplotypes in inflammatory clinical settings. PMID:27094978

  6. Guanine deaminase functions as dihydropterin deaminase in the biosynthesis of aurodrosopterin, a minor red eye pigment of Drosophila.

    PubMed

    Kim, Jaekwang; Park, Sang Ick; Ahn, Chiyoung; Kim, Heuijong; Yim, Jeongbin

    2009-08-28

    Dihydropterin deaminase, which catalyzes the conversion of 7,8-dihydropterin to 7,8-dihydrolumazine, was purified 5850-fold to apparent homogeneity from Drosophila melanogaster. Its molecular mass was estimated to be 48 kDa by gel filtration and SDS-PAGE, indicating that it is a monomer under native conditions. The pI value, temperature, and optimal pH of the enzyme were 5.5, 40 degrees C, and 7.5, respectively. Interestingly the enzyme had much higher activity for guanine than for 7,8-dihydropterin. The specificity constant (k(cat)/K(m)) for guanine (8.6 x 10(6) m(-1).s(-1)) was 860-fold higher than that for 7,8-dihydropterin (1.0 x 10(4) m(-1).s(-1)). The structural gene of the enzyme was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis as CG18143, located at region 82A1 on chromosome 3R. The cloned and expressed CG18143 exhibited both 7,8-dihydropterin and guanine deaminase activities. Flies with mutations in CG18143, SUPor-P/Df(3R)A321R1 transheterozygotes, had severely decreased activities in both deaminases compared with the wild type. Among several red eye pigments, the level of aurodrosopterin was specifically decreased in the mutant, and the amount of xanthine and uric acid also decreased considerably to 76 and 59% of the amounts in the wild type, respectively. In conclusion, dihydropterin deaminase encoded by CG18143 plays a role in the biosynthesis of aurodrosopterin by providing one of its precursors, 7,8-dihydrolumazine, from 7,8-dihydropterin. Dihydropterin deaminase also functions as guanine deaminase, an important enzyme for purine metabolism. PMID:19567870

  7. Effects of elevated CO(2) levels on root morphological traits and Cd uptakes of two Lolium species under Cd stress.

    PubMed

    Jia, Yan; Tang, Shi-rong; Ju, Xue-hai; Shu, Li-na; Tu, Shu-xing; Feng, Ren-wei; Giusti, Lorenzino

    2011-04-01

    This study was conducted to investigate the combined effects of elevated CO(2) levels and cadmium (Cd) on the root morphological traits and Cd accumulation in Lolium multiflorum Lam. and Lolium perenne L. exposed to two CO(2) levels (360 and 1 000 μl/L) and three Cd levels (0, 4, and 16 mg/L) under hydroponic conditions. The results show that elevated levels of CO(2) increased shoot biomass more, compared to root biomass, but decreased Cd concentrations in all plant tissues. Cd exposure caused toxicity to both Lolium species, as shown by the restrictions of the root morphological parameters including root length, surface area, volume, and tip numbers. These parameters were significantly higher under elevated levels of CO(2) than under ambient CO(2), especially for the number of fine roots. The increases in magnitudes of those parameters triggered by elevated levels of CO(2) under Cd stress were more than those under non-Cd stress, suggesting an ameliorated Cd stress under elevated levels of CO(2). The total Cd uptake per pot, calculated on the basis of biomass, was significantly greater under elevated levels of CO(2) than under ambient CO(2). Ameliorated Cd toxicity, decreased Cd concentration, and altered root morphological traits in both Lolium species under elevated levels of CO(2) may have implications in food safety and phytoremediation.

  8. Effects of elevated CO2 levels on root morphological traits and Cd uptakes of two Lolium species under Cd stress*

    PubMed Central

    Jia, Yan; Tang, Shi-rong; Ju, Xue-hai; Shu, Li-na; Tu, Shu-xing; Feng, Ren-wei; Giusti, Lorenzino

    2011-01-01

    This study was conducted to investigate the combined effects of elevated CO2 levels and cadmium (Cd) on the root morphological traits and Cd accumulation in Lolium multiflorum Lam. and Lolium perenne L. exposed to two CO2 levels (360 and 1000 μl/L) and three Cd levels (0, 4, and 16 mg/L) under hydroponic conditions. The results show that elevated levels of CO2 increased shoot biomass more, compared to root biomass, but decreased Cd concentrations in all plant tissues. Cd exposure caused toxicity to both Lolium species, as shown by the restrictions of the root morphological parameters including root length, surface area, volume, and tip numbers. These parameters were significantly higher under elevated levels of CO2 than under ambient CO2, especially for the number of fine roots. The increases in magnitudes of those parameters triggered by elevated levels of CO2 under Cd stress were more than those under non-Cd stress, suggesting an ameliorated Cd stress under elevated levels of CO2. The total Cd uptake per pot, calculated on the basis of biomass, was significantly greater under elevated levels of CO2 than under ambient CO2. Ameliorated Cd toxicity, decreased Cd concentration, and altered root morphological traits in both Lolium species under elevated levels of CO2 may have implications in food safety and phytoremediation. PMID:21462388

  9. Study of deep level defects of n+-CdS/p-CdTe solar cells

    NASA Astrophysics Data System (ADS)

    Kharangarh, Poonam Rani

    Among various photovoltaic materials, polycrystalline cadmium telluride thin film is now the most promising material, due to its low production cost excellent stability and reliability. Current-voltage and capacitance-voltage measurements of CdTe photovoltaic devices at different temperatures can provide valuable information about non-idealities in the n-p semiconductor junction. There are certain limitations which limit the efficiency of CdTe solar cells. There is no real distinction between defects and impurities in CdTe solar cells as both act as beneficial dopants or detrimental traps unlike Si where intentional shallow dopants and traps are distinctly different. Therefore, the role of defect states on CdTe solar cell performance, the effect of processing on defect states, and simple and effective characterization techniques must be investigated and identified. In this research the thin film n+-CdS/p-CdTe solar cells made with evaporated Cu as a primary back contact, are characterized by using the temperature dependence of the reverse bias diode current (J-V-T) to determine the energy levels of deep defects. The results of the J-V-T measurements on solar cells made at NJIT show that while modest amounts of Cu enhance cell performance, an excessive high temperature annealing step degrades device quality and reduces efficiency. This work addresses the error that can be introduced during defect energy level estimation if the temperature dependence of the carrier capture cross-section is neglected. Therefore, the location of traps is derived using a Shockley-Read-Hall recombination model with modified assumptions. A Cu-related deep level defect with activation energy of 0.57eV is observed for Cu evaporated back contact cells and an intrinsic defect with activation energy 0.89eV is found. Frequency dispersion in Capacitance-Voltage measurements confirms the presence of Cu-related deep level traps for cells with a Cu evaporated back contact, whereas no such defects

  10. ACC deaminase activity in avirulent Agrobacterium tumefaciens D3.

    PubMed

    Hao, Youai; Charles, Trevor C; Glick, Bernard R

    2011-04-01

    Some plant-growth-promoting bacteria encode the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which breaks down ACC, the direct precursor of ethylene biosynthesis in all higher plants, into ammonia and α-ketobutyrate and, as a result, reduces stress ethylene levels in plants caused by a wide range of biotic and abiotic stresses. It was previously shown that ACC deaminase can inhibit crown gall development induced by Agrobacterium tumefaciens and can partially protect plants from this disease. Agrobacterium tumefaciens D3 has been previously reported to contain a putative ACC deaminase structural gene (acdS) and a regulatory gene (acdR = lrpL). In the present study, it was found that A. tumefaciens D3 is an avirulent strain. ACC deaminase activity and its regulation were also characterized. Under gnotobiotic conditions, wild-type A. tumefaciens D3 was shown to be able to promote plant root elongation, while the acdS and lrpL double mutant strain A. tumefaciens D3-1 lost that ability. When co-inoculated with the virulent strain, A. tumefaciens C58, in wounded castor bean plants, both the wild-type A. tumefaciens D3 and the mutant A. tumefaciens D3-1 were found to be able to significantly inhibit crown gall development induced by A. tumefaciens C58. PMID:21491979

  11. Adenosine-5'-phosphate deaminase. A novel herbicide target.

    PubMed Central

    Dancer, J E; Hughes, R G; Lindell, S D

    1997-01-01

    The isolation of carbocyclic coformycin as the herbicidally active component from a fermentation of Saccharothrix species was described previously (B.D. Bush, G.V. Fitchett, D.A. Gates, D. Langley [1993] Phytochemistry 32: 737-739). Here we report that the primary mode of action of carbocyclic coformycin has been identified as inhibition of the enzyme AMP deaminase (EC 3.5.4.6) following phosphorylation at the 5' hydroxyl on the carbocyclic ring in vivo. When pea (Pisum sativum L. var Onward) seedlings are treated with carbocyclic coformycin, there is a very rapid and dramatic increase in ATP levels, indicating a perturbation in purine metabolism. Investigation of the enzymes of purine metabolism showed a decrease in the extractable activity of AMP deaminase that correlates with a strong, noncovalent association of the phosphorylated natural product with the protein. The 5'-phosphate analog of the carbocyclic coformycin was synthesized and shown to be a potent, tight binding inhibitor of AMP deaminase isolated from pea seedlings. Through the use of a synthetic radiolabeled marker, rapid conversion of carbocyclic coformycin to the 5'-phosphate analog could be demonstrated in vivo. It is proposed that inhibition of AMP deaminase leads to the death of the plant through perturbation of the intracellular ATP pool. PMID:9159944

  12. CD26 Expression on T Helper Populations and sCD26 Serum Levels in Patients with Rheumatoid Arthritis

    PubMed Central

    Cordero, Oscar J.; Varela-Calviño, Rubén; López-González, Tania; Calviño-Sampedro, Cristina; Viñuela, Juan E.; Mouriño, Coral; Hernández-Rodríguez, Íñigo; Rodríguez-López, Marina; Aspe de la Iglesia, Bruno; Pego, José María

    2015-01-01

    We studied dipeptidyl peptidase IV (DPP-IV, CD26) expression in different T helper cells and serum soluble DPP-IV/sCD26 levels in rheumatoid arthritis (RA) patients, correlated these with disease activity score (DAS), and examined how they were affected by different therapies, conventional or biological (anti-TNF, anti-CD20 and anti-IL6R or Ig-CTLA4). The percentage of CD4+CD45R0+CD26- cells was greatly reduced in patients (up to 50%) when compared with healthy subjects. Three other subsets of CD4 cells, including a CD26high Th1-associated population, changed variably with therapies. Data from these subsets (frequency and staining density) significantly correlated with DAS28 or DAS28 components but different in each group of patients undergoing the different therapies. Th17 and Th22 subsets were implicated in RA as independent CCR4+ and CCR4- populations each, with distinct CD26 expression, and were targeted with varying efficiency by each therapy. Serum DPP-IV activity rather than sCD26 levels was lower in RA patients compared to healthy donors. DPP-IV and sCD26 serum levels were found related to specific T cell subsets but not to disease activity. We conclude that, according to their CD26 expression, different cell subsets could serve to monitor RA course, and an uncharacterized T helper CD26- subset, not targeted by therapies, should be monitored for early diagnosis. PMID:26177310

  13. Serum adenosine deaminase activity in cutaneous anthrax

    PubMed Central

    Sunnetcioglu, Mahmut; Karadas, Sevdegul; Aslan, Mehmet; Ceylan, Mehmet Resat; Demir, Halit; Oncu, Mehmet Resit; Karahocagil, Mustafa Kasım; Sunnetcioglu, Aysel; Aypak, Cenk

    2014-01-01

    Background Adenosine deaminase (ADA) activity has been discovered in several inflammatory conditions; however, there are no data associated with cutaneous anthrax. The aim of this study was to investigate serum ADA activity in patients with cutaneous anthrax. Material/Methods Sixteen patients with cutaneous anthrax and 17 healthy controls were enrolled. We measured ADA activity; peripheral blood leukocyte, lymphocyte, neutrophil, and monocyte counts; erythrocyte sedimentation rate; and C reactive protein levels. Results Serum ADA activity was significantly higher in patients with cutaneous anthrax than in the controls (p<0.001). A positive correlation was observed between ADA activity and lymphocyte counts (r=0.589, p=0.021) in the patient group. Conclusions This study suggests that serum ADA could be used as a biochemical marker in cutaneous anthrax. PMID:24997584

  14. Regulation of glucosamine-6-phosphate deaminase synthesis in yeast.

    PubMed

    Singh, B; Datta, A

    1979-02-19

    A basal level of glucosamine-6-phosphate deaminase is detected in yeast cells grown on glucose. However, a burst of enzyme production occurs in the presence of N-acetylglucosamine in pathogenic Candida albicans and non-pathogenic Saccharomyces cervisiae. The enzyme synthesis stops and its concentration in the cells declines rapidly as soon as N-acetylglucosamine is removed from the medium. Experiments with RNA- and protein-synthesis inhibitors indicate that the appearance of new enzyme activity is dependent on concomitant new protein synthesis and the inducer operates at a transcriptional level. However, inhibition of DNA synthesis either by hydroxyurea or by mitomycin-C does not impair the synthesis of glucosamine-6-phosphate deaminase. PMID:369615

  15. Role of Therapeutic Plasma Exchange in Treatment of Tumefactive Multiple Sclerosis-Associated Low CD4 and CD8 Levels

    PubMed Central

    Lew, Kristen; Mewada, Nishith; Ramanujam, Sahana; Hassanzadeh, Bahareh; Donahue, John E.; Peddareddygari, Leema Reddy; Moser, Robert; Kososky, Charles; Grewal, Raji P.

    2016-01-01

    We report a 35-year-old healthy male who developed central nervous system inflammatory demyelinating disease consistent with tumefactive multiple sclerosis. About 2 weeks after onset of symptoms and prior to initiation of therapy, the patient had lymphopenia and low CD4 and CD8 levels. His lymphocyte count was 400 cells/µl (850–3,900 cells/µl), CD4 was 193 cells/µl (490–1,740 cells/µl) and CD8 was 103 cells/µl (180–1,170 cells/µl). He was treated with intravenous methylprednisolone followed by therapeutic plasma exchange, the levels of CD4 and CD8 normalized, and ultimately, he recovered completely. PMID:27721782

  16. Genetics Home Reference: adenosine monophosphate deaminase deficiency

    MedlinePlus

    Skip to main content Your Guide to Understanding Genetic Conditions Enable Javascript for addthis links to activate. ... Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Home Health Conditions adenosine monophosphate deaminase deficiency adenosine ...

  17. Accumulation and localization of cadmium in potato (Solanum tuberosum) under different soil Cd levels.

    PubMed

    Chen, Zhifan; Zhao, Ye; Gu, Lei; Wang, Shuifeng; Li, Yongliang; Dong, Fangli

    2014-06-01

    Phytoavailability and uptake mechanism of Cd in edible plant tissues grown on metal polluted agricultural soils has become a growing concern worldwide. Uptake, transport, accumulation and localization of cadmium in potato organs under different soil Cd levels were investigated using inductively-coupled plasma mass spectrometry and energy dispersive X-ray microanalysis. Results indicated that Cd contents in potato organs increased with increasing soil Cd concentrations, and the order of Cd contents in different organs was leaves > stems/roots > tubers. Root-to-stem Cd translocation coefficients ranged from 0.89 to 1.81. Cd localization in potato tissues suggested that leaves and stems should be the main compartment of Cd storage and uptake. Although low concentrations of Cd migrated from the root to tuber, Cd accumulation in the tuber exceeded the standard for food security. Therefore, the planting of potato plants in farmland containing Cd should be closely evaluated due to its potential to present health risks.

  18. High Soluble CD14 Levels at Primary HIV-1 Infection Predict More Rapid Disease Progression.

    PubMed

    Krastinova, Evguenia; Lecuroux, Camille; Leroy, Carole; Seng, Remonie; Cabie, Andre; Rami, Agathe; Venet, Alain; Meyer, Laurence; Goujard, Cecile

    2015-09-15

    The soluble CD14 (sCD14) level was found associated with mortality during the chronic phase of human immunodeficiency virus (HIV) infection. Here we assessed its prognostic value in 138 patients with primary HIV infection. Higher sCD14 levels were associated with death, from myocardial infarction, but this was based on 3 deaths only. Among 68 untreated patients, those with higher sCD14 levels had more rapid spontaneous CD4 cell decline during the first 18 months following primary infection. This association persisted after adjustment for age, the CD4 cell count, and HIV viral load at diagnosis.

  19. Nucleoside deaminase: an enzymatic marker for stress erythropoiesis in the mouse

    PubMed Central

    Rothman, Ivan K.; Zanjani, Esmail D.; Gordon, Albert S.; Silber, Robert

    1970-01-01

    The level of nucleoside deaminase was determined in extracts of mouse tissues obtained during a period of accelerated erythropoiesis induced by hypoxia, hemorrhage, or the injection of phenylhydrazine. Under these conditions a striking (10- to 100-fold) elevation of the enzyme activity occurred in the spleen. Similar results were obtained with the injection of purified erythropoietin. In control animals, only a trace of nucleoside deaminase activity was detected in the blood. During the reticulocyte response which followed erythropoietic stimulation, there was a sharp increase in the blood level of nucleoside deaminase, which rose up to 120 times that of control animals. By differential centrifugation, the enzyme was localized to the reticulocyte-rich fraction. Erythrocyte nucleoside deaminase remained elevated even after the reticulocyte count had fallen to normal in the phenylhydrazine-treated mice or to zero after the cessation of hypoxia. There was a very gradual decline in the enzyme activity in the blood which fell to the barely detectable control levels about 45 days after the initial reticulocyte response, a time period which corresponds to the survival of the mouse red blood cell. The persistence of high levels of nucleoside deaminase for the full life span of a generation of erythrocytes formed during stress, viewed in contrast to the virtual absence of the enzyme from normal erythrocytes of all ages, represents an enzymatic difference between the normal red blood cell and the cell produced under conditions of accelerated erythropoiesis. PMID:5475986

  20. Perspective of plant growth promoting rhizobacteria (PGPR) containing ACC deaminase in stress agriculture.

    PubMed

    Saleem, Muhammad; Arshad, Muhammad; Hussain, Sarfraz; Bhatti, Ahmad Saeed

    2007-10-01

    Ethylene is a gaseous plant growth hormone produced endogenously by almost all plants. It is also produced in soil through a variety of biotic and abiotic mechanisms, and plays a key role in inducing multifarious physiological changes in plants at molecular level. Apart from being a plant growth regulator, ethylene has also been established as a stress hormone. Under stress conditions like those generated by salinity, drought, waterlogging, heavy metals and pathogenicity, the endogenous production of ethylene is accelerated substantially which adversely affects the root growth and consequently the growth of the plant as a whole. Certain plant growth promoting rhizobacteria (PGPR) contain a vital enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which regulates ethylene production by metabolizing ACC (an immediate precursor of ethylene biosynthesis in higher plants) into alpha-ketobutyrate and ammonia. Inoculation with PGPR containing ACC deaminase activity could be helpful in sustaining plant growth and development under stress conditions by reducing stress-induced ethylene production. Lately, efforts have been made to introduce ACC deaminase genes into plants to regulate ethylene level in the plants for optimum growth, particularly under stressed conditions. In this review, the primary focus is on giving account of all aspects of PGPR containing ACC deaminase regarding alleviation of impact of both biotic and abiotic stresses onto plants and of recent trends in terms of introduction of ACC deaminase genes into plant and microbial species.

  1. Multi-level 3D implementation of thermo-pneumatic pumping on centrifugal microfluidic CD platforms.

    PubMed

    Thio, Tzer Hwai Gilbert; Ibrahim, Fatimah; Al-Faqheri, Wisam; Soin, Norhayati; Abdul Kahar, Maria Kahar Bador; Madou, Marc

    2013-01-01

    Thermo-pneumatic (TP) pumping is a method employing the principle of expanding heated air to transfer fluids back towards the CD center on the centrifugal microfluidic CD platform. While the TP features are easy to fabricate as no moving parts are involved, it consumes extra real estate on the CD, and because heating is involved, it introduces unnecessary heating to the fluids on the CD. To overcome these limitations, we introduce a multi-level 3D approach and implement forced convection heating. In a multi-level 3D CD, the TP features are relocated to a separate top level, while the microfluidic process remains on a lower bottom level. This allows for heat shielding of the fluids in the microfluidic process level, and also improve usage of space on the CD. To aid in future implementations of TP pumping on a multi-level 3D CD, studies on the effect of heat source setting, and the effect of positioning the TP feature (it distance from the CD center) on CD surface heating are also presented. In this work, we successfully demonstrate a multi-level 3D approach to implement TP pumping on the microfluidic CD platform.

  2. ACC deaminase genes are conserved among Mesorhizobium species able to nodulate the same host plant.

    PubMed

    Nascimento, Francisco X; Brígido, Clarisse; Glick, Bernard R; Oliveira, Solange

    2012-11-01

    Rhizobia strains expressing the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase have been reported to display an augmented symbiotic performance as a consequence of lowering the plant ethylene levels that inhibit the nodulation process. Genes encoding ACC deaminase (acdS) have been studied in Rhizobium spp.; however, not much is known about the presence of acdS genes in Mesorhizobium spp. The aim of this study was to assess the prevalence and phylogeny of acdS genes in Mesorhizobium strains including a collection of chickpea-nodulating mesorhizobia from Portugal. ACC deaminase genes were detected in 10 of 12 mesorhizobia type strains as well as in 18 of 18 chickpea Mesorhizobium isolates studied in this work. No ACC deaminase activity was detected in any Mesorhizobium strain tested under free-living conditions. Despite the lack of ACC deaminase activity, it was possible to demonstrate that in Mesorhizobium ciceri UPM-Ca7(T) , the acdS gene is transcribed under symbiotic conditions. Phylogenetic analysis indicates that strains belonging to different species of Mesorhizobium, but nodulating the same host plant, have similar acdS genes, suggesting that acdS genes are horizontally acquired by transfer of the symbiosis island. This data, together with analysis of the symbiosis islands from completely sequenced Mesorhizobium genomes, suggest the presence of the acdS gene in a Mesorhizobium common ancestor that possessed this gene in a unique symbiosis island.

  3. Molecular epidemiology and diagnosis of PBG deaminase gene defects in acute intermittent porphyria.

    PubMed Central

    Puy, H; Deybach, J C; Lamoril, J; Robreau, A M; Da Silva, V; Gouya, L; Grandchamp, B; Nordmann, Y

    1997-01-01

    Acute intermittent porphyria (AIP) is the major autosomal dominant form of acute hepatic porphyrias. The disease is due to mutations in the gene encoding for porphobilinogen (PBG) deaminase and is characterized by life-threatening neurovisceral attacks, often precipitated by drugs, fasting, cyclical hormonal changes, or infectious diseases. This report describes a prospective study on the molecular epidemiology of PBG deaminase gene defects in AIP. It uses a sensitive, reliable, and easy-to-handle method for routine AIP molecular diagnosis and family study based on an exon-by-exon denaturing gradient gel electrophoresis (DGGE) strategy followed by direct sequencing. Fifteen genomic DNA fragments, including all the coding sequence and covering 3.35 kb of the PBG deaminase gene, were investigated in 405 subjects from 121 unrelated French Caucasian AIP families who had not been screened previously at the DNA level. PBG deaminase gene mutations were identified in 109 families, but only 78 were of different type, and each of them had a prevalence rate < 5%. Among these mutations, 33 had not been published previously. Sixty percent of these 78 mutations were located in only three exons (exons 10, 12, and 14), 44% were missense, 18% were splice defect, 19% were frameshift, and 16% were nonsense. In addition, two de novo mutational events were characterized. The evaluation of the efficiency of the standard PBG deaminase enzymatic screening method for gene-carrier detection indicated 95% of concordancy with the molecular-based diagnosis. Images Figure 1 Figure 2 PMID:9199558

  4. Complexes between nuclear factor-κB p65 and signal transducer and activator of transcription 3 are key actors in inducing activation-induced cytidine deaminase expression and immunoglobulin A production in CD40L plus interleukin-10-treated human blood B cells.

    PubMed

    Lafarge, S; Hamzeh-Cognasse, H; Richard, Y; Pozzetto, B; Cogné, M; Cognasse, F; Garraud, O

    2011-11-01

    The signal transducer and activator of transcription 3 (STAT3) transcription factor pathway plays an important role in many biological phenomena. STAT3 transcription is triggered by cytokine-associated signals. Here, we use isolated human B cells to analyse the role of STAT3 in interleukin (IL)-10 induced terminal B cell differentiation and in immunoglobulin (Ig)A production as a characteristic readout of IL-10 signalling. We identified optimal conditions for inducing in-vitro IgA production by purified blood naive B cells using IL-10 and soluble CD40L. We show that soluble CD40L consistently induces the phosphorylation of nuclear factor (NF)-κB p65 but not of STAT3, while IL-10 induces the phosphorylation of STAT3 but not of NF-κB p65. Interestingly, while soluble CD40L and IL-10 were synergistic in driving the terminal maturation of B cells into IgA-producing plasma cells, they did not co-operate earlier in the pathway with regard to the transcription factors NF-κB p65 or STAT3. Blocking either NF-κB p65 or STAT3 profoundly altered the production of IgA and mRNA for activation-induced cytidine deaminase (AID), an enzyme strictly necessary for Ig heavy chain recombination. Finally, the STAT3 pathway was directly activated by IL-10, while IL-6, the main cytokine otherwise known for activating the STAT3 pathway, did not appear to be involved in IL-10-induced-STAT3 activation. Our results suggest that STAT3 and NF-κB pathways co-operate in IgA production, with soluble CD40L rapidly activating the NF-κB pathway, probably rendering STAT3 probably more reactive to IL-10 signalling. This novel role for STAT3 in B cell development reveals a potential therapeutic or vaccine target for eliciting IgA humoral responses at mucosal interfaces.

  5. Yeast Cytosine Deaminase Mutants with Increased Thermostability Impart Sensitivity to 5-Fluorocytosine

    PubMed Central

    Stolworthy, Tiffany S.; Korkegian, Aaron M.; Willmon, Candice L.; Ardiani, Andressa; Cundiff, Jennifer; Stoddard, Barry L.; Black, Margaret E.

    2008-01-01

    SUMMARY Prodrug gene therapy (PGT) is a treatment strategy in which tumor cells are transfected with a 'suicide' gene that encodes a metabolic enzyme capable of converting a nontoxic prodrug into a potent cytotoxin. One of the most promising PGT enzymes is cytosine deaminase (CD), a microbial salvage enzyme that converts cytosine to uracil. CD also converts 5-fluorocytosine (5FC) to 5-fluorouracil (5FU), an inhibitor of DNA synthesis and RNA function. Over 150 studies of cytosine deaminase-mediated PGT applications have been reported since 2000, all using wild-type enzymes. However, various forms of cytosine deaminase are limited by inefficient turnover of 5FC and/or limited thermostability. In a previous study we stabilized and extended the half-life of yeast cytosine deaminase (yCD) by repacking of its hydrophobic core at several positions distant from the active site. Here we report that random mutagenesis of residues selected based on alignment with similar enzymes, followed by selection for enhanced sensitization to 5FC, also produces an enzyme variant (yCD-D92E) with elevated Tm values and increased activity half-life. The new mutation is located at the enzyme's dimer interface, indicating that independent mutational pathways can lead to an increase in the temperature that induces protein unfolding and aggregation in thermal denaturation experiments measured by circular dichroism spectroscopy, and an increase in the half-life of enzyme activity at physiological temperature, as well as more subtle effect on enzyme kinetics. Each independently derived set of mutations significantly improves the enzyme's performance in PGT assays both in cell culture and in animal models. PMID:18291415

  6. Regulation of Activation Induced Deaminase (AID) by Estrogen.

    PubMed

    Pauklin, Siim

    2016-01-01

    Regulation of Activation Induced Deaminase (AID) by the hormone estrogen has important implications for understanding adaptive immune responses as well as the involvement of AID in autoimmune diseases and tumorigenesis. This chapter describes the general laboratory techniques for analyzing AID expression and activity induced by estrogen, focusing on the isolation and preparation of cells for hormone treatment and the subsequent analysis of AID responsiveness to estrogen at the RNA level and for determining the regulation of AID activity via estrogen by analyzing Ig switch circle transcripts and mutations in switch region loci.

  7. Rescue of the Orphan Enzyme Isoguanine Deaminase

    SciTech Connect

    D Hitchcock; A Fedorov; E Fedorov; L Dangott; S Almo; F Raushel

    2011-12-31

    Cytosine deaminase (CDA) from Escherichia coli was shown to catalyze the deamination of isoguanine (2-oxoadenine) to xanthine. Isoguanine is an oxidation product of adenine in DNA that is mutagenic to the cell. The isoguanine deaminase activity in E. coli was partially purified by ammonium sulfate fractionation, gel filtration, and anion exchange chromatography. The active protein was identified by peptide mass fingerprint analysis as cytosine deaminase. The kinetic constants for the deamination of isoguanine at pH 7.7 are as follows: k{sub cat} = 49 s{sup -1}, K{sub m} = 72 {micro}M, and k{sub cat}/K{sub m} = 6.7 x 10{sup 5} M{sup -1} s{sup -1}. The kinetic constants for the deamination of cytosine are as follows: k{sub cat} = 45 s{sup -1}, K{sub m} = 302 {micro}M, and k{sub cat}/K{sub m} = 1.5 x 10{sup 5} M{sup -1} s{sup -1}. Under these reaction conditions, isoguanine is the better substrate for cytosine deaminase. The three-dimensional structure of CDA was determined with isoguanine in the active site.

  8. Anti-CD20 antibody promotes cancer escape via enrichment of tumor-evoked regulatory B cells expressing low levels of CD20 and CD137L.

    PubMed

    Bodogai, Monica; Lee Chang, Catalina; Wejksza, Katarzyna; Lai, Jinping; Merino, Maria; Wersto, Robert P; Gress, Ronald E; Chan, Andrew C; Hesdorffer, Charles; Biragyn, Arya

    2013-04-01

    The possible therapeutic benefits of B-cell depletion in combating tumoral immune escape have been debated. In support of this concept, metastasis of highly aggressive 4T1 breast cancer cells in mice can be abrogated by inactivation of tumor-evoked regulatory B cells (tBreg). Here, we report the unexpected finding that B-cell depletion by CD20 antibody will greatly enhance cancer progression and metastasis. Both murine and human tBregs express low levels of CD20 and, as such, anti-CD20 mostly enriches for these cells. In the 4T1 model of murine breast cancer, this effect of enriching for tBregs suggests that B-cell depletion by anti-CD20 may not be beneficial at all in some cancers. In contrast, we show that in vivo-targeted stimulation of B cells with CXCL13-coupled CpG oligonucleotides (CpG-ODN) can block cancer metastasis by inhibiting CD20(Low) tBregs. Mechanistic investigations suggested that CpG-ODN upregulates low surface levels of 4-1BBL on tBregs to elicit granzyme B-expressing cytolytic CD8(+) T cells, offering some explanative power for the effect. These findings underscore the immunotherapeutic importance of tBreg inactivation as a strategy to enhance cancer therapy by targeting both the regulatory and activating arms of the immune system in vivo. PMID:23365136

  9. Cytosine deaminase adenoviral vector and 5-fluorocytosine selectively reduce breast cancer cells 1 million-fold when they contaminate hematopoietic cells: a potential purging method for autologous transplantation.

    PubMed

    Garcia-Sanchez, F; Pizzorno, G; Fu, S Q; Nanakorn, T; Krause, D S; Liang, J; Adams, E; Leffert, J J; Yin, L H; Cooperberg, M R; Hanania, E; Wang, W L; Won, J H; Peng, X Y; Cote, R; Brown, R; Burtness, B; Giles, R; Crystal, R; Deisseroth, A B

    1998-07-15

    Ad.CMV-CD is a replication incompetent adenoviral vector carrying a cytomegalovirus (CMV)-driven transcription unit of the cytosine deaminase (CD) gene. The CD transcription unit in this vector catalyzes the deamination of the nontoxic pro-drug, 5-fluorocytosine (5-FC), thus converting it to the cytotoxic drug 5-fluorouracil (5-FU). This adenoviral vector prodrug activation system has been proposed for use in selectively sensitizing breast cancer cells, which may contaminate collections of autologous stem cells products from breast cancer patients, to the toxic effects of 5-FC, without damaging the reconstitutive capability of the normal hematopoietic cells. This system could conceivably kill even the nondividing breast cancer cells, because the levels of 5-FU generated by this system are 10 to 30 times that associated with systemic administration of 5-FU. The incorporation of 5-FU into mRNA at these high levels is sufficient to disrupt mRNA processing and protein synthesis so that even nondividing cells die of protein starvation. To test if the CD adenoviral vector sensitizes breast cancer cells to 5-FC, we exposed primary explants of normal human mammary epithelial cells (HMECs) and the established breast cancer cell (BCC) lines MCF-7 and MDA-MB-453 to the Ad.CMV-CD for 90 minutes. This produced a 100-fold sensitization of these epithelial cells to the effects of 48 hours of exposure to 5-FC. We next tested the selectivity of this system for BCC. When peripheral blood mononuclear cells (PBMCs), collected from cancer patients during the recovery phase from conventional dose chemotherapy-induced myelosuppression, were exposed to the Ad.CMV-CD for 90 minutes in serum-free conditions, little or no detectable conversion of 5-FC into 5-FU was seen even after 48 hours of exposure to high doses of 5-FC. In contrast, 70% of 5-FC was converted into the cytotoxic agent 5-FU when MCF-7 breast cancer cells (BCCs) were exposed to the same Ad.CMV-CD vector followed by 5-FC for

  10. Proapoptotic CD95L levels in normal human serum and sera of breast cancer patients.

    PubMed

    Olimón-Andalón, Vicente; Aguilar-Lemarroy, Adriana; Ratkovich-González, Sarah; Uribe-López, Aida; Mariscal-Ramírez, Ignacio; Delgadillo-Cristerna, Raúl; Ortiz-Lazareno, Pablo; Hernández-Flores, Georgina; de Celis, Ruth; Bravo-Cuellar, Alejandro; Jave-Suárez, Luis F

    2015-05-01

    The CD95 pathway is a critical apoptotic pathway used by immune cells to avoid cancer development. CD95 ligand (CD95L) is found in several forms, as a cell membrane-associated form, a soluble metalloprotease-cleaved form, and a soluble but membrane-bound CD95L released on cell-derived exosomes. In this study, we used a cell-based assay to evaluate the activity of proapoptotic CD95L in sera from healthy individuals and breast cancer patients. We confirmed that our cell-based assay using Jurkat cells was sensitive to the presence of proapoptotic CD95L in serum, and apoptosis induction by mechanisms other than CD95 was discriminated using apoptosis-resistant Jurkat subclones. Our results indicated a proapoptotic potential of normal serum that involved CD95L. Sera from breast cancer patients exhibited significantly decreased apoptosis induction, due to increased CD95 receptor levels compared with healthy women. Apoptotic potential tended to decrease as the Breast Imaging Reporting and Data System grade increased, and we observed restoration of proapoptotic potential after tumor removal. The CD95L in serum responsible for apoptotic induction was associated with high-molecular-weight particles, perhaps with exosomes. The sera of healthy individuals generally contain a proapoptotic environment, and this property is mainly maintained by the presence of CD95L. Furthermore, measurement of CD95L-mediated apoptosis induction by sera could be a useful parameter to be evaluated during cancer development and therapeutic response.

  11. Improving wafer level CD uniformity for logic applications utilizing mask level metrology and process

    NASA Astrophysics Data System (ADS)

    Cohen, Avi; Trautzsch, Thomas; Buttgereit, Ute; Graitzer, Erez; Hanuka, Ori

    2013-09-01

    Critical Dimension Uniformity (CDU) is one of the key parameters necessary to assure good performance and reliable functionality of any integrated circuit (IC). The extension of 193nm based lithography usage combined with design rule shrinkage makes process control, in particular the wafer level CDU control, an extremely important and challenging task in IC manufacturing. In this study the WLCD-CDC closed loop solution offered by Carl Zeiss SMS was examined. This solution aims to improve the wafer level intra-field CDU without the need to run wafer prints and extensive wafer CD metrology. It combines two stand-alone tools: The WLCD tool which measures CD based on aerial imaging technology while applying the exact scanner-used illumination conditions to the photomask and the CDC tool which utilizes an ultra-short femto-second laser to write intra-volume shading elements (Shade-In Elements™) inside the photomask bulk material. The CDC process changes the dose going through the photomask down to the wafer, hence the wafer level intra-field CDU improves. The objective of this study was to evaluate how CDC process is affecting the CD for different type of features and pattern density which are typical for logic and system on chip (SOC) devices. The main findings show that the linearity and proximity behavior is maintained by the CDC process and CDU and CDC Ratio (CDCR) show a linear behavior for the different feature types. Finally, it was demonstrated that the CDU errors of the targeted (critical) feature have been effectively eliminated. In addition, the CDU of all other features have been significantly improved as well.

  12. Polyethylene Glycol-Conjugated Adenosine Deaminase (ADA) Therapy Provides Temporary Immune Reconstitution to a Child with Delayed-Onset ADA Deficiency

    PubMed Central

    Lainka, Elke; Hershfield, Michael S.; Santisteban, Ines; Bali, Pawan; Seibt, Annette; Neubert, Jennifer; Friedrich, Wilhelm; Niehues, Tim

    2005-01-01

    We describe the effects of polyethylene glycol-conjugated adenosine deaminase (ADA) replacement therapy on lymphocyte counts, activation, apoptosis, proliferation, and cytokine secretion in a 14-month-old girl with “delayed-onset” ADA deficiency and marked immunodysregulation. Pretreatment lymphopenia affected T cells (CD4, 150/μl; CD8, 459/μl), B cells (16/μl), and NK cells (55/μl). T cells were uniformly activated and largely apoptotic (CD4, 59%; CD8, 82%); and T-cell-dependent cytokine levels in plasma were elevated, including the levels of interleukin 2 (IL-2; 26 pg/ml), IL-4 (81 pg/ml), IL-5 (46 pg/ml), gamma interferon (1,430 pg/ml), tumor necrosis factor alpha (210 pg/ml), and IL-10 (168 pg/ml). Mitogen-stimulated peripheral blood mononuclear cells show reduced IL-2 secretion and proliferation. During the first 5 months of therapy there was clinical improvement and partial immune reconstitution, with nearly normal lymphocyte subset numbers, reduced T-cell activation and CD4-cell apoptosis, and decreased plasma cytokine levels. In parallel, IL-2 secretion and the lymphocyte mitogenic response improved. Between 4 and 7 months, immunoglobulin G antibodies to bovine ADA developed and resulted in the complete reversal of immune recovery. PMID:16002636

  13. Amelioration of high salinity stress damage by plant growth-promoting bacterial endophytes that contain ACC deaminase.

    PubMed

    Ali, Shimaila; Charles, Trevor C; Glick, Bernard R

    2014-07-01

    Plant growth and productivity is negatively affected by soil salinity. However, it is predicted that plant growth-promoting bacterial (PGPB) endophytes that contain 1-aminocyclopropane-1-carboxylate (ACC) deaminase (E.C. 4.1.99.4) can facilitate plant growth and development in the presence of a number of different stresses. In present study, the ability of ACC deaminase containing PGPB endophytes Pseudomonas fluorescens YsS6, Pseudomonas migulae 8R6, and their ACC deaminase deficient mutants to promote tomato plant growth in the absence of salt and under two different levels of salt stress (165 mM and 185 mM) was assessed. It was evidence that wild-type bacterial endophytes (P. fluorescens YsS6 and P. migulae 8R6) promoted tomato plant growth significantly even in the absence of stress (salinity). Plants pretreated with wild-type ACC deaminase containing endophytic strains were healthier and grew to a much larger size under high salinity stress compared to plants pretreated with the ACC deaminase deficient mutants or no bacterial treatment (control). The plants pretreated with ACC deaminase containing bacterial endophytes exhibit higher fresh and dry biomass, higher chlorophyll contents, and a greater number of flowers and buds than the other treatments. Since the only difference between wild-type and mutant bacterial endophytes was ACC deaminase activity, it is concluded that this enzyme is directly responsible for the different behavior of tomato plants in response to salt stress. The use of PGPB endophytes with ACC deaminase activity has the potential to facilitate plant growth on land that is not normally suitable for the majority of crops due to their high salt contents.

  14. Characterization of the Porphobilinogen Deaminase Deficiency in Acute Intermittent Porphyria

    PubMed Central

    Anderson, Peter M.; Reddy, Raman M.; Anderson, Karl E.; Desnick, Robert J.

    1981-01-01

    The molecular pathology of the porphobilinogen (PBG)-deaminase deficiency in heterozygotes for acute intermittent porphyria (AIP) was investigated by means of biochemical and immunologic techniques. The stable enzyme-substrate intermediates (A, B, C, D, and E) of PBG-deaminase were separated by anion-exchange chromatography of erythrocyte lysates from heterozygotes for AIP and normal individuals. In normal lysates, the intermediates eluted in a characteristic pattern with decreasing amounts of activity (A > B > C > D > E), the combined A and B intermediates representing >75% of total recovered activity. In contrast, two different profiles were observed in lysates from heterozygotes for AIP. In most heterozygotes, the elution profile was similar to that of normal individuals, but each intermediate was reduced ∼50%. A second profile in which the C intermediate had disproportionately higher activity than the A or B intermediates was observed in asymptomatic heterozygotes with high urinary levels of PBG (>5 μg/ml) as well as in heterozygotes during acute attacks. These findings suggested that the C intermediate (the dipyrrole-enzyme intermediate) may be rate limiting in the stepwise conversion of the monopyrrole, PBG, to the linear tetrapyrrole, hydroxymethylbilane. To investigate further the nature of the enzymatic defect in AIP, sensitive immunotitration and immunoelectrophoretic assays were developed with the aid of a rabbit anti-human PBG-deaminase IgG preparation produced against the homogeneous enzyme. Equal amounts of erythrocyte lysate activity from 32 heterozygotes for AIP from 22 unrelated families and 35 normal individuals were immunoelectrophoresed. There were no detectable differences in the amounts of cross-reactive immunologic material (CRIM) in lysates from the normal individuals and 25 heterozygotes from 21 of the 22 unrelated families with AIP. In contrast, when equal enzymatic activities were coimmunoelectrophoresed, all seven heterozygotes from

  15. Increased CD28 serum levels are not associated with specific clinical activity in systemic lupus erythematosus.

    PubMed

    Brambila-Tapia, Aniel J L; Gámez-Nava, Jorge I; Salazar-Páramo, Mario; Munoz-Valle, José F; González-López, Laura; Llamas-Covarrubias, Mara A; Gutiérrez-Urena, Sergio R; Vázquez-Del Mercado, Mónica; Dávalos-Rodríguez, Ingrid P

    2011-10-01

    CD28 is one of the main activator receptors involved in systemic lupus erythematosus (SLE) pathogenesis, and its expression and serum levels are significantly higher in patients with SLE and other autoimmune diseases than in healthy controls (HC). However, it is unknown whether this increase is associated with specific organ damage. Therefore, our objective was to measure the CD28 levels in serum from SLE and HC groups to confirm the CD28 serum levels increase, as reported previously, and to determine whether this increase was associated with specific organ activity and the SLE Disease Activity Index (SLEDAI). Forty SLE patients and 40 matched HC were included, and the age, disease duration, SLEDAI and Mexican SLEDAI were recorded for the SLE group. CD28 serum levels were measured by ELISA. There was a statistically significant increase in the CD28 serum levels of SLE patients compared to controls (p = 0.039); however, we did not find any significant correlation with disease activity indices or organ involvement, although we found a significant but low correlation with C3. Our results and a review of the literature suggest that the increase in CD28 serum levels may be the result of CD28 gene overexpression, which could be related to the decrease in CD28+ T cells, T-cell hyporesponsiveness and immune impairment that occurs in SLE.

  16. The Effect of Acute Exercise upon Adenosin Deaminase Oxidant and Antioxidant Activity

    ERIC Educational Resources Information Center

    Kafkas, M. Emin; Karabulut, Aysun Bay; Sahin, Armagan; Otlu, Onder; Savas, Seyfi; Aytac, Aylin

    2012-01-01

    The purpose of this study was to determine the changes of MDA, glutation (GSH), Adenozine deaminase (ADA) and superoxidase dismutaze (SOD) levels with exercise training in obese middle-aged women (body mass index, MMI [greater than or equal to] 30.0). Twelve obese middle-aged women participated in this study. The descriptive statistics of some of…

  17. Oncolytic Herpes simplex virus expressing yeast cytosine deaminase: relationship between viral replication, transgene expression, prodrug bioactivation

    PubMed Central

    Yamada, Suguru; Kuroda, Toshihiko; Fuchs, Bryan C.; He, Xiaoying; Supko, Jeffrey G.; Schmitt, Anthony; McGinn, Christopher M.; Lanuti, Michael; Tanabe, Kenneth K.

    2011-01-01

    Yeast cytosine deaminase (yCD) is a well-characterized prodrug/enzyme system that converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), and has been combined with oncolytic viruses. However, in vivo studies of the interactions between 5-FC bioactivation and viral replication have not been previously reported, nor have the kinetics of transgene expression and the pharmacokinetics of 5-FC and 5-FU. We constructed a replication-conditional HSV-1 expressing yCD and examined cytotoxicity when 5-FC was initiated at different times after viral infection, and observed that earlier 5-FC administration led to greater cytotoxicity than later 5-FC administration in vitro and in vivo. Twelve days of 5-FC administration was superior to 6 days in animal models, but dosing beyond 12 days did not further enhance efficacy. Consistent with the dosing schedule results, both viral genomic DNA copy number and viral titers were observed to peak on Day 3 after viral injection and gradually decrease thereafter. The virus is replication-conditional and was detected in tumors for as long as 2 weeks after viral injection. The maximum relative extent of yCD conversion of 5-FC to 5-FU in tumors was observed on Day 6 after viral injection and it decreased progressively thereafter. The observation that 5-FU generation within tumors did not lead to appreciable levels of systemic 5-FU (<10 ng/ml) is important and has not been previously reported. The approaches used in these studies of the relationship between the viral replication kinetics, transgene expression, prodrug administration and anti-tumor efficacy are useful in the design of clinical trials of armed, oncolytic viruses. PMID:22076044

  18. Association of high post-transplant soluble CD30 serum levels with chronic allograft nephropathy.

    PubMed

    Grenzi, Patricia C; Campos, Érika F; Tedesco-Silva, Hélio; Felipe, Claudia R; Franco, Marcello F; Soares, Maria Fernanda; Medina-Pestana, José Osmar; Gerbase-Delima, Maria

    2013-12-01

    The purpose of this study was to evaluate the association of post-transplant soluble CD30 (sCD30) levels, isolated or in combination with of anti-HLA class II antibodies and of serum creatinine levels, with kidney graft loss due to chronic allograft nephropathy (CAN), and type of lesions in graft biopsies for cause. The study comprised 511 first kidney graft recipients, transplanted at a single center, with a graft functioning for at least 2.8 years. A single blood sample was collected from each patient. sCD30 levels were determined by ELISA, and HLA antibodies by Luminex assay. The minimum follow-up after testing was 9.3 years. High sCD30 levels, set at sCD30 ≥ 34.15 ng/mL, the presence of HLA class II antibodies, and serum creatinine ≥ 1.9 mg/dL were independently associated with CAN-graft loss (P values <0.0001, 0.05, <0.0001, respectively), and the combined hazard ratio for CAN-graft loss was 20.2. Analyses of 166 biopsies for cause showed that high sCD30 levels and creatinine were independently associated with interstitial lesions. Post-transplant sCD30 serum levels, especially in conjunction with information regarding HLA class II antibodies and serum creatinine levels, provide valuable information regarding graft outcome and could be useful for the management of kidney transplant recipients.

  19. High levels of the soluble form of CD30 molecule in rheumatoid arthritis (RA) are expression of CD30+ T cell involvement in the inflamed joints.

    PubMed

    Gerli, R; Muscat, C; Bistoni, O; Falini, B; Tomassini, C; Agea, E; Tognellini, R; Biagini, P; Bertotto, A

    1995-12-01

    The CD30 is a surface molecule expressed by Th2-type lymphokine-producing T cells upon activation. CD30-expressing activated T cells release a soluble form of the molecule, which can be detectable both in vitro and in vivo. In the present study, high levels of soluble CD30 were found in peripheral blood and synovial fluid from patients with RA. However, CD30+ CD3+ cells, either CD4+ or CD8+, were significantly present in synovial fluid, but not in peripheral blood, of RA patients. Serum values of soluble CD30 were higher in active than inactive RA patients and directly correlated with rheumatoid factor serum titres. These data strongly support an involvement of CD30+ T cells in the immune processes of rheumatoid synovitis, and may suggest a relationship between Th2-type cytokine-secreting T cells and the pathological response in RA. PMID:8536371

  20. Cell surface Glut1 levels distinguish human CD4 and CD8 T lymphocyte subsets with distinct effector functions

    PubMed Central

    Cretenet, Gaspard; Clerc, Isabelle; Matias, Maria; Loisel, Severine; Craveiro, Marco; Oburoglu, Leal; Kinet, Sandrina; Mongellaz, Cédric; Dardalhon, Valérie; Taylor, Naomi

    2016-01-01

    CD4 and CD8 T lymphocyte activation requires the generation of sufficient energy to support new biosynthetic demands. Following T cell receptor (TCR) engagement, these requirements are met by an increased glycolysis, due, at least in part, to induction of the Glut1 glucose transporter. As Glut1 is upregulated on tumor cells in response to hypoxia, we assessed whether surface Glut1 levels regulate the antigen responsiveness of human T lymphocytes in both hypoxic and atmospheric oxygen conditions. Notably, Glut1 upregulation in response to TCR stimulation was significantly higher in T lymphocytes activated under hypoxic as compared to atmospheric oxygen conditions. Furthermore, TCR-stimulated human T lymphocytes sorted on the basis of Glut1-Lo and Glut1-Hi profiles maintained distinct characteristics, irrespective of the oxygen tension. While T cells activated in hypoxia divided less than those activated in atmospheric oxygen, Glut1-Hi lymphocytes exhibited increased effector phenotype acquisition, augmented proliferation, and an inverted CD4/CD8 ratio in both oxygen conditions. Moreover, Glut1-Hi T lymphocytes exhibited a significantly enhanced ability to produce IFN-γ and this secretion potential was completely dependent on continued glycolysis. Thus, Glut1 surface levels identify human T lymphocytes with distinct effector functions in both hypoxic and atmospheric oxygen tensions. PMID:27067254

  1. Degradation of the cancer genomic DNA deaminase APOBEC3B by SIV Vif.

    PubMed

    Land, Allison M; Wang, Jiayi; Law, Emily K; Aberle, Ryan; Kirmaier, Andrea; Krupp, Annabel; Johnson, Welkin E; Harris, Reuben S

    2015-11-24

    APOBEC3B is a newly identified source of mutation in many cancers, including breast, head/neck, lung, bladder, cervical, and ovarian. APOBEC3B is a member of the APOBEC3 family of enzymes that deaminate DNA cytosine to produce the pro-mutagenic lesion, uracil. Several APOBEC3 family members function to restrict virus replication. For instance, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H combine to restrict HIV-1 in human lymphocytes. HIV-1 counteracts these APOBEC3s with the viral protein Vif, which targets the relevant APOBEC3s for proteasomal degradation. While APOBEC3B does not restrict HIV-1 and is not targeted by HIV-1 Vif in CD4-positive T cells, we asked whether related lentiviral Vif proteins could degrade APOBEC3B. Interestingly, several SIV Vif proteins are capable of promoting APOBEC3B degradation, with SIVmac239 Vif proving the most potent. This likely occurs through the canonical polyubiquitination mechanism as APOBEC3B protein levels are restored by MG132 treatment and by altering a conserved E3 ligase-binding motif. We further show that SIVmac239 Vif can prevent APOBEC3B mediated geno/cytotoxicity and degrade endogenous APOBEC3B in several cancer cell lines. Our data indicate that the APOBEC3B degradation potential of SIV Vif is an effective tool for neutralizing the cancer genomic DNA deaminase APOBEC3B. Further optimization of this natural APOBEC3 antagonist may benefit cancer therapy.

  2. Levels of CD56+TIM-3- effector CD8 T cells distinguish HIV natural virus suppressors from patients receiving antiretroviral therapy.

    PubMed

    Poonia, Bhawna; Pauza, C David

    2014-01-01

    Prolonged antiretroviral therapy (ART) with effective HIV suppression and reconstitution of CD4 T cells, fails to restore CD8 T cell lytic effector function that is needed to eradicate the viral reservoir. Better understanding of the phenotype and function of circulating CD8 cells in HIV patients will contribute to new targeted therapies directed at increasing CD8 T cell lytic effector function and destruction of the viral reservoir. We show that CD8 T cells from ART treated patients had sharply reduced expression of CD56 (neural cell adhesion molecule-1), a marker associated with cytolytic function whereas elite patients who control HIV in the absence of ART had CD56+ CD8 T cell levels similar to uninfected controls. The CD56+ CD8 T cells had higher perforin upregulation as well as degranulation following stimulation with HIV gag peptides compared with CD56 negative CD8 T cells. Elite patients had the highest frequencies of perforin producing CD56+ CD8 T cells among all HIV+ groups. In patients receiving ART we noted high levels of the exhaustion marker TIM-3 on CD56+ CD8 T cells, implying that defective effector function was related to immune exhaustion. CD56+ CD8 T cells from elite or treated HIV patients responded to PMA plus ionomycin stimulation, and expressed transcription factors T-bet and EOMES at levels similar to uninfected controls. Consequently, the lytic effector defect in chronic HIV disease is due to immune exhaustion and quantitative loss of CD56+ CD8 T cells and this defect is not repaired in patients where viremia is suppressed and CD4 T cells are recovered after ART. Reconstituting the cytotoxic CD56+ subset of CD8+ T cells through new interventions might improve the lytic effector capacity and contribute to reducing the viral reservoir. Our initial studies indicate that IL-15 treatment partly reverses the CD56 defect, implying that myeloid cell defects could be targeted for immune therapy during chronic HIV disease.

  3. Push pull microfluidics on a multi-level 3D CD.

    PubMed

    Thio, Tzer Hwai Gilbert; Ibrahim, Fatimah; Al-Faqheri, Wisam; Moebius, Jacob; Khalid, Noor Sakinah; Soin, Norhayati; Kahar, Maria Kahar Bador Abdul; Madou, Marc

    2013-08-21

    A technique known as thermo-pneumatic (TP) pumping is used to pump fluids on a microfluidic compact disc (CD) back towards the CD center against the centrifugal force that pushes liquids from the center to the perimeter of the disc. Trapped air expands in a TP air chamber during heating, and this creates positive pressure on liquids located in chambers connected to that chamber. While the TP air chamber and connecting channels are easy to fabricate in a one-level CD manufacturing technique, this approach provides only one way pumping between two chambers, is real-estate hungry and leads to unnecessary heating of liquids in close proximity to the TP chamber. In this paper, we present a novel TP push and pull pumping method which allows for pumping of liquid in any direction between two connected liquid chambers. To ensure that implementation of TP push and pull pumping also addresses the issue of space and heating challenges, a multi-level 3D CD design is developed, and localized forced convection heating, rather than infra-red (IR) is applied. On a multi-level 3D CD, the TP features are placed on a top level separate from the rest of the microfluidic processes that are implemented on a lower separate level. This approach allows for heat shielding of the microfluidic process level, and efficient usage of space on the CD for centrifugal handling of liquids. The use of localized forced convection heating, rather than infra-red (IR) or laser heating in earlier implementations allows not only for TP pumping of liquids while the CD is spinning but also makes heat insulation for TP pumping and other fluidic functions easier. To aid in future implementations of TP push and pull pumping on a multi-level 3D CD, study on CD surface heating is also presented. In this contribution, we also demonstrate an advanced application of pull pumping through the implementation of valve-less switch pumping.

  4. The relation between clinical features of adult T-cell leukemia/lymphoma and the serum levels of soluble CD25 and CD30.

    PubMed

    Takemoto, Shigeki; Pornkuna, Ratiorn; Hidaka, Michihiro; Kawano, Fumio

    2016-07-01

    Adult T-cell leukemia (ATL) is an aggressive neoplasm of mature T cells. We aimed to ascertain the relationships between soluble CD25 (sIL-2R) and CD30 (sCD30) levels and nodal or extra-nodal involvement of ATL. Our study subjects were ATL patients whose sIL-2R and sCD30 levels were measured before initial therapy (n=32). Their sCD30 levels correlated significantly with the number of ATL cells in peripheral blood (PB) (ρ=0.456; P=0.009), while sIL-2R levels correlated significantly with the number of nodal lesions (ρ=0.660; P=0.001). We then also assessed the relationships of pulmonary lesions with the number of ATL cells in PB, lactate dehydrogenase levels, sIL-2R levels, and sCD30 levels in 24 patients whose pleural effusions and hilar lymphadenopathy were investigated before initial therapy. The results suggested that a high number of ATL cells in PB may be associated with pulmonary lesions. It is known that metalloproteinases shed and cleave cytokine receptors such as CD25 and CD30 from the cell surface as well as E-cadherin and extracellular matrix. It seems that serum levels of sIL-2R and sCD30 indicate the activation of metalloproteinases associated with ATL involvement in vivo.

  5. Reduced expressions of Fas and Bcl-2 proteins in CD14+ monocytes and normal CD14 soluble levels in juvenile systemic lupus erythematosus.

    PubMed

    Liphaus, B L; Kiss, M H B; Carrasco, S; Goldenstein-Schainberg, C

    2013-08-01

    In order to evaluate Fas and Bcl-2 expressions in CD14+ monocytes, to measure soluble CD14 serum levels and to analyze the relationships with lupus nephritis and disease activity, we enrolled 41 patients with juvenile systemic lupus erythematosus (JSLE) and 27 healthy volunteers. Disease activity was determined by SLEDAI score. Peripheral monocytes were stained for CD14, Fas and Bcl-2 molecules, and cellular expressions were determined by flow cytometry. Soluble CD14 levels were measured by a quantitative ELISA kit. JSLE patients, those with active disease and those with nephritis, presented significantly reduced expressions of Fas and Bcl-2 proteins in CD14+ monocytes compared with healthy controls. Significant inverse correlations between percentages of CD14+Fas+ cells, SLEDAI score and anti-dsDNA antibodies were observed. JSLE patients had soluble CD14 levels similar to controls, although sCD14 levels positively correlated with ESR, but not with SLEDAI score. JSLE patients with nephritis also presented sCD14 levels similar to controls. In conclusion, the reduced expressions of Fas and Bcl-2 proteins in CD14+ monocytes from JSLE patients depict that monocyte apoptotic mechanisms may be important in lupus pathogenesis.

  6. Stabilization of Aspergillus parasiticus cytosine deaminase by immobilization on calcium alginate beads improved enzyme operational stability.

    PubMed

    Zanna, H; Nok, A J; Ibrahim, S; Inuwa, H M

    2013-12-01

    Cytosine deaminase (CD) from Aspergillus parasiticus, which has half-life of 1.10 h at 37°C, was stabilized by immobilization on calcium alginate beads. The immobilized CD had pH and temperature optimum of 5 and 50°C respectively. The immobilized enzyme also stoichiometrically deaminated Cytosine and 5-fluorocytosine (5-FC) with the apparent K(M) values of 0.60 mM and 0.65 mM respectively, displaying activation energy of 10.72 KJ/mol. The immobilization of native CD on calcium alginate beads gave the highest yield of apparent enzymatic activity of 51.60% of the original activity and the enzymatic activity was lost exponentially at 37°C over 12 h with a half-life of 5.80 h. Hence, the operational stability of native CD can be improved by immobilization on calcium alginate beads.

  7. Elevated levels of soluble CD14 in serum of patients with systemic lupus erythematosus.

    PubMed Central

    Nockher, W A; Wigand, R; Schoeppe, W; Scherberich, J E

    1994-01-01

    A soluble form of CD14 (sCD14) was assessed with an ELISA assay in the serum of the following three clinical groups: 35 patients with an inactive phase of systemic lupus erythematosus (SLE), 17 patients with SLE relapses, and 65 normal healthy volunteers. Increased levels of sCD14 were observed in all patients suffering from SLE compared with normal controls. In addition, patients with active SLE revealed higher serum concentrations of sCD14 (median 6.9 mg/l) than patients under remission (4.1 mg/l; P < 0.0001). Serum values of sCD14 correlated neither with the number of peripheral blood monocytes bearing the CD14 membrane antigen, nor with serum concentrations of IL-1 beta. Serum sCD14 was compared with other clinical parameters used to monitor the clinical course of patients with SLE, among them complement C3, anti-dsDNA antibodies and soluble IL-2 receptor (sIL-2R). A good correlation emerged between sCD14 and C3 as well as sIL-2R concentrations, but sCD14 and anti-dsDNA titres disclosed no significant correlation in both groups of patients with SLE. Serial studies in patients with severe SLE showed that serum sCD14 closely parallels the clinical course as defined by an activity score. Our data suggest that serum sCD14 represents a promising parameter to monitor disease activity in patients with SLE. PMID:7512005

  8. The Level of Viral Antigen Presented by Hepatocytes Influences CD8 T-Cell Function▿

    PubMed Central

    Gehring, Adam J.; Sun, Dianxing; Kennedy, Patrick T. F.; Nolte-'t Hoen, Esther; Lim, Seng Gee; Wasser, Shanthi; Selden, Clare; Maini, Mala K.; Davis, Dan M.; Nassal, Michael; Bertoletti, Antonio

    2007-01-01

    CD8 T cells exert their antiviral function through cytokines and lysis of infected cells. Because hepatocytes are susceptible to noncytolytic mechanisms of viral clearance, CD8 T-cell antiviral efficiency against hepatotropic viruses has been linked to their capacity to produce gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). On the other hand, intrahepatic cytokine production triggers the recruitment of mononuclear cells, which sustain acute and chronic liver damage. Using virus-specific CD8 T cells and human hepatocytes, we analyzed the modulation of virus-specific CD8 T-cell function after recognition peptide-pulsed or virally infected hepatocytes. We observed that hepatocyte antigen presentation was generally inefficient, and the quantity of viral antigen strongly influenced CD8 T-cell antiviral function. High levels of hepatitis B virus production induced robust IFN-γ and TNF-α production in virus-specific CD8 T cells, while limiting amounts of viral antigen, both in hepatocyte-like cells and naturally infected human hepatocytes, preferentially stimulated CD8 T-cell degranulation. Our data document a mechanism where virus-specific CD8 T-cell function is influenced by the quantity of virus produced within hepatocytes. PMID:17202217

  9. Pachyman treatment improves CD4+CD25+ Treg counts and serum interleukin 4 and interferon γ levels in a mouse model of Kawasaki disease.

    PubMed

    Chu, Mao-Ping; Wang, Dan; Zhang, Ying-Ying; Li, Bao-Qing; Zhou, Ai-Hua; Chen, Xi-Wen; Qian, Yan

    2012-05-01

    The aim of this study was to explore the effect of pachyman, a mushroom extract, on CD4+CD25+ regulatory T cells (Tregs), serum interleukin 4 (IL-4) and interferon γ (IFN-γ) levels in a mouse model of Kawasaki disease. Lactobacillus casei cell wall extract was diluted to 1 mg/ml in PBS and administered to mice by intraperitoneal injection to establish a model of Kawasaki disease. Sixty female mice were used in this study, 40 of which were randomly assigned to a model (normal saline by gavage, n=20) or experimental group (200 mg/kg/day pachyman by gavage, n=20). The remaining 20 mice were disease and treatment-free, and were used as the control group. Compared to the control mice, mice in the model group exhibited a significantly lower percentage of CD4+CD25+ Tregs and significantly higher serum IL-4 and IFN-γ levels (P<0.05). However, CD4+CD25+ Tregs significantly increased and IL-4 and IFN-γ levels significantly decreased in experimental mice following pachyman treatment (P<0.05). Further analysis showed a negative correlation between CD4+CD25+ Tregs and IL-4/IFN-γ levels (P<0.05). In conclusion, pachyman improves immune function in a mouse model of Kawasaki disease by upregulating CD4+CD25+ Tregs, which may inhibit the cytokine secretion of Th1 and Th2 cells. PMID:22367425

  10. Are increased frequency of macrophage-like and natural killer (NK) cells, together with high levels of NKT and CD4+CD25high T cells balancing activated CD8+ T cells, the key to control Chagas’ disease morbidity?

    PubMed Central

    Vitelli-Avelar, D M; Sathler-Avelar, R; Massara, R L; Borges, J D; Lage, P S; Lana, M; Teixeira-Carvalho, A; Dias, J C P; Elói-Santos, S M; Martins-Filho, O A

    2006-01-01

    The immunological response during early human Trypanosoma cruzi infection is not completely understood, despite its role in driving the development of distinct clinical manifestations of chronic infection. Herein we report the results of a descriptive flow cytometric immunophenotyping investigation of major and minor peripheral blood leucocyte subpopulations in T. cruzi-infected children, characterizing the early stages of the indeterminate clinical form of Chagas’ disease. Our results indicated significant alterations by comparison with uninfected children, including increased values of pre-natural killer (NK)-cells (CD3– CD16+ CD56–), and higher values of proinflammatory monocytes (CD14+ CD16+ HLA-DR++). The higher values of activated B lymphocytes (CD19+ CD23+) contrasted with impaired T cell activation, indicated by lower values of CD4+ CD38+ and CD4+ HLA-DR+ lymphocytes, a lower frequency of CD8+ CD38+ and CD8+ HLA-DR+ cells; a decreased frequency of CD4+ CD25HIGH regulatory T cells was also observed. These findings reinforce the hypothesis that simultaneous activation of innate and adaptive immunity mechanisms in addition to suppression of adaptive cellular immune response occur during early events of Chagas’ disease. Comparative cross-sectional analysis of these immunophenotypes with those exhibited by patients with late chronic indeterminate and cardiac forms of disease suggested that a shift toward high values of macrophage-like cells extended to basal levels of proinflammatory monocytes as well as high values of mature NK cells, NKT and regulatory T cells, may account for limited tissue damage during chronic infection favouring the establishment/maintenance of a lifelong indeterminate clinical form of the disease. On the other hand, development of an adaptive cell-mediated inflammatory immunoprofile characterized by high levels of activated CD8+ cells and basal levels of mature NK cells, NKT and CD4+ CD25HIGH cells might lead to late chronic

  11. Plasma CXCL10, sCD163 and sCD14 Levels Have Distinct Associations with Antiretroviral Treatment and Cardiovascular Disease Risk Factors

    PubMed Central

    Castley, Alison; Williams, Leah; James, Ian; Guelfi, George; Berry, Cassandra; Nolan, David

    2016-01-01

    We investigate the associations of three established plasma biomarkers in the context of HIV and treatment-related variables including a comprehensive cardiovascular disease risk assessment, within a large ambulatory HIV cohort. Patients were recruited in 2010 to form the Royal Perth Hospital HIV/CVD risk cohort. Plasma sCD14, sCD163 and CXCL10 levels were measured in 475 consecutive patients with documented CVD risk (age, ethnicity, gender, smoking, blood pressure, BMI, fasting metabolic profile) and HIV treatment history including immunological/virological outcomes. The biomarkers assessed showed distinct associations with virological response: CXCL10 strongly correlated with HIV-1 RNA (p<0.001), sCD163 was significantly reduced among ‘aviraemic’ patients only (p = 0.02), while sCD14 was unaffected by virological status under 10,000 copies/mL (p>0.2). Associations between higher sCD163 and protease inhibitor therapy (p = 0.05) and lower sCD14 with integrase inhibitor therapy (p = 0.02) were observed. Levels of sCD163 were also associated with CVD risk factors (age, ethnicity, HDL, BMI), with a favourable influence of Framingham score <10% (p = 0.04). Soluble CD14 levels were higher among smokers (p = 0.002), with no effect of other CVD risk factors, except age (p = 0.045). Our findings confirm CXCL10, sCD163 and sCD14 have distinct associations with different aspects of HIV infection and treatment. Levels of CXCL10 correlated with routinely monitored variables, sCD163 levels reflect a deeper level of virological suppression and influence of CVD risk factors, while sCD14 levels were not associated with routinely monitored variables, with evidence of specific effects of smoking and integrase inhibitor therapy warranting further investigation. PMID:27355513

  12. Plasma CXCL10, sCD163 and sCD14 Levels Have Distinct Associations with Antiretroviral Treatment and Cardiovascular Disease Risk Factors.

    PubMed

    Castley, Alison; Williams, Leah; James, Ian; Guelfi, George; Berry, Cassandra; Nolan, David

    2016-01-01

    We investigate the associations of three established plasma biomarkers in the context of HIV and treatment-related variables including a comprehensive cardiovascular disease risk assessment, within a large ambulatory HIV cohort. Patients were recruited in 2010 to form the Royal Perth Hospital HIV/CVD risk cohort. Plasma sCD14, sCD163 and CXCL10 levels were measured in 475 consecutive patients with documented CVD risk (age, ethnicity, gender, smoking, blood pressure, BMI, fasting metabolic profile) and HIV treatment history including immunological/virological outcomes. The biomarkers assessed showed distinct associations with virological response: CXCL10 strongly correlated with HIV-1 RNA (p<0.001), sCD163 was significantly reduced among 'aviraemic' patients only (p = 0.02), while sCD14 was unaffected by virological status under 10,000 copies/mL (p>0.2). Associations between higher sCD163 and protease inhibitor therapy (p = 0.05) and lower sCD14 with integrase inhibitor therapy (p = 0.02) were observed. Levels of sCD163 were also associated with CVD risk factors (age, ethnicity, HDL, BMI), with a favourable influence of Framingham score <10% (p = 0.04). Soluble CD14 levels were higher among smokers (p = 0.002), with no effect of other CVD risk factors, except age (p = 0.045). Our findings confirm CXCL10, sCD163 and sCD14 have distinct associations with different aspects of HIV infection and treatment. Levels of CXCL10 correlated with routinely monitored variables, sCD163 levels reflect a deeper level of virological suppression and influence of CVD risk factors, while sCD14 levels were not associated with routinely monitored variables, with evidence of specific effects of smoking and integrase inhibitor therapy warranting further investigation.

  13. Dynamic Change of CD34 Level during the Survival Process of Narrow Pedicle Flap

    PubMed Central

    Wu, Lijun; Zhao, Tianlan; Yu, Daojiang; Chen, Qi; Han, Wenya; Yu, Wenyuan; Sun, Wei

    2015-01-01

    Objective To evaluate the dynamic change of CD34 level during the survival process of narrow pedicle flaps. Methods Twenty-five white pigs were randomly and equally divided into 5 experimental groups. Five different type of narrow pedicle with different length-to-width ratio were employed, and each type of narrow pedicle was covered with 5 different size random flaps and which was classified into A, B, C, D and E for 5 groups. Group A was control group. Each type narrow pedicle with 5 different skin flaps were implanted onto the back of the pigs along the midline of back with a reverse direction. A 0.3 cm×0.3 cm full thickness skin flap in the middle of distal segment was collected and on 3rd, 5th, 7th and 14th days of post-operation. The expression of CD34 was measured by immunohistochemistry and enzyme-linked immunosorbent (ELISA). Results Histological examination showed that with the increasing of length-to-width ratio of the narrow pedicle skin flaps, the expression of CD34 increased in the skin flaps. Increased level of CD34 was found on 3rd day post-operation, and the peak expression was found on 7th day. Persistent high level of CD34 was found until 14th day. Conclusion Increased CD34 level in the distal skin flap, there is the association between CD34 level and ischemia injury. Moreover, CD34 expression plays an important role during the repair processes of pedicle flaps. PMID:26561392

  14. Phytochelatins do not correlate with the level of Cd accumulation in Chlamydomonas spp.

    PubMed

    Nishikawa, Kahoko; Onodera, Ai; Tominaga, Noriko

    2006-06-01

    Chlamydomonas acidophila KT-1 and Chlamydomonas acidophila DVB238 exhibit a strong heavy metal tolerance, but C. acidophila DVB238 can accumulate a much higher amount of Cadmium (Cd) than C. acidophila KT-1. Phytochelatins (PCs) are known to play an important role in the detoxification of several toxic heavy metals, but the relationship between PCs and Cd accumulation is not clear. PC metabolism and Cd accumulation were investigated by using three Chlamydomonas strains including Chlamydomonas reinhardtii C-9 as a standard alga. The results showed that the PC content did not correlate closely with the level of Cd accumulation, maintenance of a high GSH level seeming to be more important for Cd accumulation. The ultrastructure of C. acidophila KT-1 was extremely disrupted by a great increase in starch granules, which resulted in a moribund state, but hyper-accumulator C. acidophila DVB238 did not exhibit an increase in starch granules in its cells, in spite of Cd accumulation in its chloroplasts, cytosol and vacuoles. These results indicated that C. acidophila DVB238 probably has a developed detoxification system preventing such as destruction of the cells due to Cd toxicity.

  15. Elevated Plasma Soluble CD14 Levels Correlate with the Monocyte Response Status During Hantaan Virus Infection in Humans.

    PubMed

    Tang, Kang; Zhang, Chunmei; Zhang, Yusi; Zhang, Yun; Zhuang, Ran; Jin, Boquan; Ma, Ying

    2015-10-01

    Hantaan virus (HTNV) infection can cause severe hemorrhagic fever with renal syndrome (HFRS) in humans. CD14, a pattern recognition receptor recognizing lipopolysaccharide, is highly expressed on monocytes and can be shed as soluble CD14 (sCD14) upon monocyte activation. To understand the role of sCD14 in HFRS, the sCD14 plasma concentrations from 45 HFRS patients were quantified, and the relationships between the plasma sCD14 level and the monocyte response status and clinical parameters were analyzed. The plasma sCD14 levels were significantly higher in the HFRS patients and they correlated with monocyte expansion and activation, which were characterized by increased blood monocyte counts, the proportion of CD14(++)CD16(+) intermediate monocytes, as well as elevated plasma tumor necrosis factor-α (TNF-α) and soluble CD163 (sCD163) levels. Additionally, the high plasma sCD14 levels positively correlated with white blood cell counts and blood urea nitrogen levels and negatively correlated with platelet counts in the HFRS patients. Taken together, our data indicate that elevated plasma sCD14 levels are associated with the monocyte response status during HTNV infection in humans.

  16. Expression of fas protein on CD4+T cells irradiated by low level He-Ne

    NASA Astrophysics Data System (ADS)

    Nie, Fan; Zhu, Jing; Zhang, Hui-Guo

    2005-07-01

    Objective: To investigate the influence on the Expression of Fas protein on CD4+ T cells irradiated by low level He-Ne laser in the cases of psoriasis. Methods:the expression of CD4+ T Fas protein was determined in the casee of psoriasis(n=5) pre and post-low level laser irradiation(30 min、60min and 120min)by flow cytometry as compared withthe control(n=5). Results:In the cases of psoriasis,the expression of CD4+T FAS protein 21.4+/-3.1% was increased significantly than that of control group 16.8+/-2.1% pre-irradiation, p<0.05in the control,there is no difference between pre and post- irradiation,p>0.05in the cases , the expression of CD4+T Fas protein wae positively corelated to the irradiation times, when the energy density arrived to 22.92J/cm2(60 minutes)and 45.84J/cm2(120minutes), the expression of CD4+ T Fas protein was increased significantly as compared with pre-irradiation,p<0.05.Conclusion: The expression of CD4+T Fas protein may be increased by low level He-Ne laser irradiation ,the uncontrolled status of apoptosis could be corrected.

  17. Defect Levels of Indium-doped CdMnTe Crystals

    SciTech Connect

    K Kim; A Bolotinikov; G Camarda; R Gul; A Hossain; G Yang; Y Cui; R James

    2011-12-31

    Using photoluminescence (PL) and current deep-level transient spectroscopy (I-DLTS), we investigated the electronic defects of indium-doped detector-grade CdMnTe:In (CMT:In) crystals grown by the vertical Bridgman method. We similarly analyzed CdZnTe:In (CZT:In) and undoped CdMnTe (CMT) crystals grown under the amount of same level of excess Te and/or indium doping level to detail the fundamental properties of the electronic defect structure more readily. Extended defects, existing in all the samples, were revealed by synchrotron white beam x-ray diffraction topography and scanning electron microscopy. The electronic structure of CMT is very similar to that of CZT, with shallow traps, A-centers, Cd vacancies, deep levels, and Te antisites. The 1.1-eV deep level, revealed by PL in earlier studies of CZT and CdTe, were attributed to dislocation-induced defects. In our I-DLTS measurements, the 1.1-eV traps showed different activation energies with applied bias voltage and an exponential dependence on the trap-filling time, which are typical characteristics of dislocation-induced defects. We propose a new defect-trap model for indium-doped CMT crystals.

  18. Adenosine deaminase in disorders of purine metabolism and in immune deficiency

    SciTech Connect

    Tritsch, G.L.

    1985-01-01

    This book consists of five parts and a section of poster papers. Some of the selection titles are: Adenosine Deaminase Impairment and Ribonucleotide Reductase in Human Cells; Adenosine Deaminase and Malignant Cells; Inhibition of Adenosine Deaminase to Increase the Antitumor Activity of Adenine Nucleoside Analogues; and Molecular Biology of the Adenosine Deaminase Gene and Messenger RNA.

  19. AID/APOBEC deaminases and cancer

    PubMed Central

    Rebhandl, Stefan; Huemer, Michael; Greil, Richard; Geisberger, Roland

    2015-01-01

    Mutations are the basis for evolution and the development of genetic diseases. Especially in cancer, somatic mutations in oncogenes and tumor suppressor genes alongside the occurrence of passenger mutations have been observed by recent deep-sequencing approaches. While mutations have long been considered random events induced by DNA-replication errors or by DNA damaging agents, genome sequencing led to the discovery of non-random mutation signatures in many human cancer. Common non-random mutations comprise DNA strand-biased mutation showers and mutations restricted to certain DNA motifs, which recently have become attributed to the activity of the AID/APOBEC family of DNA deaminases. Hence, APOBEC enzymes, which have evolved as key players in natural and adaptive immunity, have been proposed to contribute to cancer development and clonal evolution of cancer by inducing collateral genomic damage due to their DNA deaminating activity. This review focuses on how mutagenic events through AID/APOBEC deaminases may contribute to cancer development. PMID:26097867

  20. Prenyltransferases regulate CD20 protein levels and influence anti-CD20 monoclonal antibody-mediated activation of complement-dependent cytotoxicity.

    PubMed

    Winiarska, Magdalena; Nowis, Dominika; Bil, Jacek; Glodkowska-Mrowka, Eliza; Muchowicz, Angelika; Wanczyk, Malgorzata; Bojarczuk, Kamil; Dwojak, Michal; Firczuk, Malgorzata; Wilczek, Ewa; Wachowska, Malgorzata; Roszczenko, Katarzyna; Miaczynska, Marta; Chlebowska, Justyna; Basak, Grzegorz Wladyslaw; Golab, Jakub

    2012-09-14

    Anti-CD20 monoclonal antibodies (mAbs) are successfully used in the management of non-Hodgkin lymphomas and chronic lymphocytic leukemia. We have reported previously that statins induce conformational changes in CD20 molecules and impair rituximab-mediated complement-dependent cytotoxicity. Here we investigated in more detail the influence of farnesyltransferase inhibitors (FTIs) on CD20 expression and antitumor activity of anti-CD20 mAbs. Among all FTIs studied, L-744,832 had the most significant influence on CD20 levels. It significantly increased rituximab-mediated complement-dependent cytotoxicity against primary tumor cells isolated from patients with non-Hodgkin lymphomas or chronic lymphocytic leukemia and increased CD20 expression in the majority of primary lymphoma/leukemia cells. Incubation of Raji cells with L-744,832 led to up-regulation of CD20 at mRNA and protein levels. Chromatin immunoprecipitation assay revealed that inhibition of farnesyltransferase activity was associated with increased binding of PU.1 and Oct-2 to the CD20 promoter sequences. These studies indicate that CD20 expression can be modulated by FTIs. The combination of FTIs with anti-CD20 mAbs is a promising therapeutic approach, and its efficacy should be examined in patients with B-cell tumors.

  1. Soluble CD14 Levels, Interleukin-6, and Mortality Among Prevalent Hemodialysis Patients

    PubMed Central

    Raj, Dominic SC; Carrero, Juan J; Shah, Vallbh O; Qureshi, Abdul R; Barany, Peter; Heimburger, Olof; Lindholm, Bengt; Ferguson, Jennet; Moseley, Pope L; Stenvinkel, Peter

    2009-01-01

    Background: CD14 is a pattern-recognition receptor that plays a central immunomodulatory role in pro-inflammatory signaling in response to a variety of ligands, including endotoxin. CD14 protein is present in two forms soluble (sCD14) and membrane-bound. Hereby we studied the implications of elevated sCD14 in hemodialysis patients. We hypothesized that sCD14 elevation may link to cytokine activation and protein-energy wasting, predisposing to increased mortality risk. Study Design: Prospective observational study of prevalent hemodialysis patients. Setting & Participants: 211 prevalent hemodialysis patients, median age of 66 years, 29 months of vintage dialysis time followed-up for mortality for a median of 31 months. Predictors: Tertiles of baseline circulating sCD14 corresponding to <2.84, 2.85-3.62, and >3.63 μg/mL, respectively. Outcome: The major outcome of interest was all-cause mortality. Measurements: sCD14 and endotoxin, together with other markers of inflammation and protein-energy wasting. Results: The median value of sCD14 was 3.2 μg/mL (25th to 75th percentile, 2.7 to 3.9). sCD14 correlated positively with C-reactive protein, interleukin-6, endotoxin and long pentraxin-3, while negatively with serum albumin, muscle mass and handgrip strength. Patients with elevated sCD14 had lower body mass index and increased prevalence of muscle atrophy. Patients within the highest sCD14 tertile had a crude morality Hazard ratio of 1.94 (95% CI 1.13-3.32), that persisted after adjustment for multiple confounders (Hazard ratio 3.11 [95% CI 1.49-6.46]). Among patients with persistent inflammation, the presence of concurrent elevation of sCD14 levels gradually increased the mortality risk, but this effect was less than multiplicative and failed to show a statistical interaction. Limitations: Those inherent to an observational study. Conclusions: sCD14 is associated with inflammation and protein-energy wasting in hemodialysis patients. It is a strong and independent

  2. Levels of circulating CD45dimCD34+VEGFR2+ progenitor cells correlate with outcome in metastatic renal cell carcinoma patients treated with tyrosine kinase inhibitors

    PubMed Central

    Farace, F; Gross-Goupil, M; Tournay, E; Taylor, M; Vimond, N; Jacques, N; Billiot, F; Mauguen, A; Hill, C; Escudier, B

    2011-01-01

    Background: Predicting the efficacy of antiangiogenic therapy would be of clinical value in patients (pts) with metastatic renal cell carcinoma (mRCC). We tested the hypothesis that circulating endothelial cell (CEC), bone marrow-derived CD45dimCD34+VEGFR2+ progenitor cell or plasma angiogenic factor levels are associated with clinical outcome in mRCC pts undergoing treatment with tyrosine kinase inhibitors (TKI). Methods: Fifty-five mRCC pts were prospectively monitored at baseline (day 1) and day 14 during treatment (46 pts received sunitinib and 9 pts received sorafenib). Circulating endothelial cells (CD45−CD31+CD146+7-amino-actinomycin (7AAD)− cells) were measured in 1 ml whole blood using four-color flow cytometry (FCM). Circulating CD45dimCD34+VEGFR2+7AAD− progenitor cells were measured in progenitor-enriched fractions by four-color FCM. Plasma VEGF, sVEGFR2, SDF-1α and sVCAM-1 levels were determined by ELISA. Correlations between baseline CEC, CD45dimCD34+VEGFR2+7AAD− progenitor cells, plasma factors, as well as day 1–day 14 changes in CEC, CD45dimCD34+VEGFR2+7AAD− progenitor, plasma factor levels, and response to TKI, progression-free survival (PFS) and overall survival (OS) were examined. Results: No significant correlation between markers and response to TKI was observed. No association between baseline CEC, plasma VEGF, sVEGFR-2, SDF-1α, sVCAM-1 levels with PFS and OS was observed. However, baseline CD45dimCD34+VEGFR2+7AAD− progenitor cell levels were associated with PFS (P=0.01) and OS (P=0.006). Changes in this population and in SDF-1α levels between day 1 and day 14 were associated with PFS (P=0.03, P=0.002). Changes in VEGF and SDF-1α levels were associated with OS (P=0.02, P=0.007). Conclusion: Monitoring CD45dimCD34+VEGFR2+ progenitor cells, plasma VEGF and SDF-1α levels could be of clinical interest in TKI-treated mRCC pts to predict outcome. PMID:21386843

  3. Adenosine Deaminase Activity in Chronic Lymphocytic Leukemia and Healthy Subjects

    PubMed Central

    Ghaderi, Bayazid; Amini, Sabrieh; Maroofi, Farzad; Jalali, Chiya; Javanmardi, Mitra; Roshani, Daem; Abdi, Mohammad

    2016-01-01

    Background B cell chronic lymphocytic leukemia is one of the most frequent hematologic malignancies in the world. Cellular surface CD markers and serum Beta-2-microglobulin may be used as a prognostic tool in CLL patients. Objectives In the present study we introduce serum adenosine deaminase as a diagnostic marker in CLL. Materials and Methods Blood samples were collected from B-CLL and healthy subjects. White blood cell, red blood cell and platelet count and blood Erythrocyte sedimentation rate was recorded and serum Beta-2-microglobulin, Lactate dehydrogenase and total ADA enzyme activity were determined. Results Serum ADA activity was significantly higher in patients group than that of controls. ADA had a significant and direct correlation with B2M, WBC, LDH and ESR. However, there was not any relation between ADA and the stages of disease. Diagnostic cut-off, sensitivity and specificity of the serum ADA test were 27.97 U/L, 91% and 94%, respectively. Conclusions A higher ADA activity in patients group and its correlation with CLL markers were seen in our study. High diagnostic value of serum ADA in our study suggests that it might be considered as a useful screening tool among the other markers in CLL. PMID:27703646

  4. Impact of changes in antigen level on CD38/PD-1 co-expression on HIV-specific CD8 T cells in chronic, untreated HIV-1 infection.

    PubMed

    Vollbrecht, Thomas; Brackmann, Heike; Henrich, Nadja; Roeling, Joerg; Seybold, Ulrich; Bogner, Johannes R; Goebel, Frank D; Draenert, Rika

    2010-03-01

    Excessive immune activation is a hallmark of chronic uncontrolled HIV infection. During the past years, growing evidence suggests that immune inhibitory signals also play an important role in progressive disease. However, the relationship between positive and negative immune signals on HIV-specific CD8 T cells has not been studied in detail so far in chronic HIV-1 infection. In this study, the expression of markers of positive (CD38) and negative (PD-1) immune signals on virus-specific CD8 T cells in chronic, untreated HIV-1 infection was evaluated using intracellular cytokine staining. Viral escape mutations were assessed by autologous virus sequence analysis and subsequent peptide titration assays. Single-epitope CD8 T-cell responses toward Gag, Pol, and Nef were compared in 12 HIV-1 controllers (viral load <5,000 cp/ml) and 12 HIV-1 progressors (viral load >50,000 cp/ml) and a highly significant increase of CD38/PD-1 co-expression on virus-specific CD8 T cells in progressors was found (P < 0.0001). The level of CD38/PD-1 co-expression was independent of epitope specificity. Longitudinal follow-up revealed a clear drop in CD38/PD-1 co-expression on virus-specific CD8 T cells after the suppression of antigen following either viral escape mutation or the initiation of HAART (P = 0.004). Antigen persistence with a fluctuating viral load revealed stable levels of CD38/PD-1 co-expression whereas significant rises in viral load were accompanied or even preceded by substantial increases in CD38/PD-1 co-expression. The CD38/PD-1 phenotype clearly distinguishes HIV-specific CD8 T-cell responses between controllers and progressors. Whether it plays a causative role in disease progression remains debatable. J. Med. Virol. 82:358-370, 2010. (c) 2010 Wiley-Liss, Inc.

  5. Long-Term Expression of Human Adenosine Deaminase in Vascular Smooth Muscle Cells of Rats: A Model for Gene Therapy

    NASA Astrophysics Data System (ADS)

    Lynch, Carmel M.; Clowes, Monika M.; Osborne, William R. A.; Clowes, Alexander W.; Dusty Miller, A.

    1992-02-01

    Gene transfer into vascular smooth muscle cells in animals was examined by using recombinant retroviral vectors containing an Escherichia coli β-galactosidase gene or a human adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) gene. Direct gene transfer by infusion of virus into rat carotid arteries was not observed. However, gene transfer by infection of smooth muscle cells in culture and seeding of the transduced cells onto arteries that had been denuded of endothelial cells was successful. Potentially therapeutic levels of human adenosine deaminase activity were detected over 6 months of observation, indicating the utility of vascular smooth muscle cells for gene therapy in humans.

  6. Reduction of Fermi level pinning and recombination at polycrystalline CdTe surfaces by laser irradiation

    SciTech Connect

    Simonds, Brian J.; Kheraj, Vipul; Palekis, Vasilios; Ferekides, Christos; Scarpulla, Michael A.

    2015-06-14

    Laser processing of polycrystalline CdTe is a promising approach that could potentially increase module manufacturing throughput while reducing capital expenditure costs. For these benefits to be realized, the basic effects of laser irradiation on CdTe must be ascertained. In this study, we utilize surface photovoltage spectroscopy (SPS) to investigate the changes to the electronic properties of the surface of polycrystalline CdTe solar cell stacks induced by continuous-wave laser annealing. The experimental data explained within a model consisting of two space charge regions, one at the CdTe/air interface and one at the CdTe/CdS junction, are used to interpret our SPS results. The frequency dependence and phase spectra of the SPS signal are also discussed. To support the SPS findings, low-temperature spectrally-resolved photoluminescence and time-resolved photoluminescence were also measured. The data show that a modest laser treatment of 250 W/cm{sup 2} with a dwell time of 20 s is sufficient to reduce the effects of Fermi level pinning at the surface due to surface defects.

  7. Evolution of the deaminase fold and multiple origins of eukaryotic editing and mutagenic nucleic acid deaminases from bacterial toxin systems

    PubMed Central

    Iyer, Lakshminarayan M.; Zhang, Dapeng; Rogozin, Igor B.; Aravind, L.

    2011-01-01

    The deaminase-like fold includes, in addition to nucleic acid/nucleotide deaminases, several catalytic domains such as the JAB domain, and others involved in nucleotide and ADP-ribose metabolism. Using sensitive sequence and structural comparison methods, we develop a comprehensive natural classification of the deaminase-like fold and show that its ancestral version was likely to operate on nucleotides or nucleic acids. Consequently, we present evidence that a specific group of JAB domains are likely to possess a DNA repair function, distinct from the previously known deubiquitinating peptidase activity. We also identified numerous previously unknown clades of nucleic acid deaminases. Using inference based on contextual information, we suggest that most of these clades are toxin domains of two distinct classes of bacterial toxin systems, namely polymorphic toxins implicated in bacterial interstrain competition and those that target distantly related cells. Genome context information suggests that these toxins might be delivered via diverse secretory systems, such as Type V, Type VI, PVC and a novel PrsW-like intramembrane peptidase-dependent mechanism. We propose that certain deaminase toxins might be deployed by diverse extracellular and intracellular pathogens as also endosymbionts as effectors targeting nucleic acids of host cells. Our analysis suggests that these toxin deaminases have been acquired by eukaryotes on several independent occasions and recruited as organellar or nucleo-cytoplasmic RNA modifiers, operating on tRNAs, mRNAs and short non-coding RNAs, and also as mutators of hyper-variable genes, viruses and selfish elements. This scenario potentially explains the origin of mutagenic AID/APOBEC-like deaminases, including novel versions from Caenorhabditis, Nematostella and diverse algae and a large class of fast-evolving fungal deaminases. These observations greatly expand the distribution of possible unidentified mutagenic processes catalyzed by

  8. The ADA*2 allele of the adenosine deaminase gene (20q13.11) and recurrent spontaneous abortions: an age-dependent association

    PubMed Central

    Nunes, Daniela Prudente Teixeira; Spegiorin, Lígia Cosentino Junqueira Franco; de Mattos, Cinara Cássia Brandão; Oliani, Antonio Helio; Vaz-Oliani, Denise Cristina Mós; de Mattos, Luiz Carlos

    2011-01-01

    OBJECTIVE: Adenosine deaminase acts on adenosine and deoxyadenosine metabolism and modulates the immune response. The adenosine deaminase G22A polymorphism (20q.11.33) influences the level of adenosine deaminase enzyme expression, which seems to play a key role in maintaining pregnancy. The adenosine deaminase 2 phenotype has been associated with a protective effect against recurrent spontaneous abortions in European Caucasian women. The aim of this study was to investigate whether the G22A polymorphism of the adenosine deaminase gene is associated with recurrent spontaneous abortions in Brazilian women. METHODS: A total of 311 women were recruited to form two groups: G1, with a history of recurrent spontaneous abortions (N = 129), and G2, without a history of abortions (N = 182). Genomic DNA was extracted from peripheral blood with a commercial kit and PCR-RFLP analysis was used to identify the G22A genetic polymorphism. Fisher's exact test and odds ratio values were used to compare the proportions of adenosine deaminase genotypes and alleles between women with and without a history of recurrent spontaneous abortion (p<0.05). The differences between mean values for categorical data were calculated using unpaired t tests. The Hardy-Weinberg equilibrium was assessed with a chi-square test. RESULTS: Statistically significant differences were identified for the frequencies of adenosine deaminase genotypes and alleles between the G1 and G2 groups when adjusted for maternal age. CONCLUSIONS: The results suggest that the adenosine deaminase *2 allele is associated with a low risk for recurrent spontaneous abortions, but this association is dependent on older age. PMID:22086524

  9. Increased levels of the CD40:CD40 ligand dyad in the cerebrospinal fluid of rats with vitamin B12(cobalamin)-deficient central neuropathy.

    PubMed

    Veber, Daniela; Mutti, Elena; Galmozzi, Enrico; Cedrola, Sabrina; Galbiati, Stefania; Morabito, Alberto; Tredici, Giovanni; La Porta, Caterina A; Scalabrino, Giuseppe

    2006-07-01

    The levels of the soluble (s) CD40:sCD40 ligand (L) dyad, which belongs to the tumor necrosis factor (TNF)-alpha:TNF-alpha-receptor superfamily, are significantly increased in the cerebrospinal fluid (CSF), but not the serum of cobalamin (Cbl)-deficient (Cbl-D) rats. They were normalized or significantly reduced after treatment with Cbl, transforming growth factor-beta1 or S-adenosyl-L-methionine, and the normal myelin ultrastructure of the spinal cord was concomitantly restored. The concomitance of the two beneficial effects of these treatments strongly suggests that the increases in CSF sCD40:sCD40L levels may participate in the pathogenesis of purely myelinolytic Cbl-D central neuropathy in the rat. In keeping with this, an anti-CD40 treatment prevented myelin lesions.

  10. The catalase activity of diiron adenine deaminase.

    PubMed

    Kamat, Siddhesh S; Holmes-Hampton, Gregory P; Bagaria, Ashima; Kumaran, Desigan; Tichy, Shane E; Gheyi, Tarun; Zheng, Xiaojing; Bain, Kevin; Groshong, Chris; Emtage, Spencer; Sauder, J Michael; Burley, Stephen K; Swaminathan, Subramanyam; Lindahl, Paul A; Raushel, Frank M

    2011-12-01

    Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn(2+) before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO(4). Inductively coupled plasma mass spectrometry and Mössbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe(II) /Fe(II) ]-ADE catalyzed the conversion of H(2)O(2) to O(2) and H(2)O. The values of k(cat) and k(cat)/K(m) for the catalase activity are 200 s(-1) and 2.4 × 10(4) M(-1) s(-1), respectively. [Fe(II)/Fe(II)]-ADE underwent more than 100 turnovers with H(2)O(2) before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g(ave) = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H(2)O(2) by [Fe(II)/Fe(II)]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS. PMID:21998098

  11. Target sequence accessibility limits activation-induced cytidine deaminase activity in primary mediastinal B-cell lymphoma.

    PubMed

    Popov, Sergey W; Moldenhauer, Gerhard; Wotschke, Beate; Brüderlein, Silke; Barth, Thomas F; Dorsch, Karola; Ritz, Olga; Möller, Peter; Leithäuser, Frank

    2007-07-15

    Activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) in activated B lymphocytes and is potentially implicated in genomic instability of B-cell malignancies. For unknown reasons, B-cell neoplasms often lack SHM and CSR in spite of high AID expression. Here, we show that primary mediastinal B-cell lymphoma (PMBL), an immunoglobulin (Ig)-negative lymphoma that possesses hypermutated, class-switched Ig genes, expresses high levels of AID with an intact primary structure but does not do CSR in 14 of 16 cases analyzed. Absence of CSR coincided with low Ig germ-line transcription, whereas high level germ-line transcription was observed only in those two cases with active CSR. Interleukin-4/CD40L costimulation induced CSR and a marked up-regulation of germ-line transcription in the PMBL-derived cell line MedB-1. In the PMBL cell line Karpas 1106P, CSR was not inducible and germ-line transcription remained low on stimulation. However, Karpas 1106P, but not MedB-1, had ongoing SHM of the Ig gene and BCL6. These genes were transcribed in Karpas 1106P, whereas transcription was undetectable or low in MedB-1 cells. Thus, accessibility of the target sequences seems to be a major limiting factor for AID-dependent somatic gene diversification in PMBL. PMID:17638864

  12. Investigation of deep level defects in CdTe thin films

    SciTech Connect

    Shankar, H.; Castaldini, A.; Dauksta, E.; Medvid, A.; Cavallini, A.

    2014-02-21

    In the past few years, a large body of work has been dedicated to CdTe thin film semiconductors, as the electronic and optical properties of CdTe nanostructures make them desirable for photovoltaic applications. The performance of semiconductor devices is greatly influenced by the deep levels. Knowledge of parameters of deep levels present in as-grown materials and the identification of their origin is the key factor in the development of photovoltaic device performance. Photo Induced Current Transient Spectroscopy technique (PICTS) has proven to be a very powerful method for the study of deep levels enabling us to identify the type of traps, their activation energy and apparent capture cross section. In the present work, we report the effect of growth parameters and LASER irradiation intensity on the photo-electric and transport properties of CdTe thin films prepared by Close-Space Sublimation method using SiC electrical heating element. CdTe thin films were grown at three different source temperatures (630, 650 and 700 °C). The grown films were irradiated with Nd:YAG LASER and characterized by Photo-Induced Current Transient Spectroscopy, Photocurrent measurementand Current Voltage measurements. The defect levels are found to be significantly influenced by the growth temperature.

  13. Progesterone Levels Associate with a Novel Population of CCR5+CD38+ CD4 T Cells Resident in the Genital Mucosa with Lymphoid Trafficking Potential.

    PubMed

    Swaims-Kohlmeier, Alison; Haaland, Richard E; Haddad, Lisa B; Sheth, Anandi N; Evans-Strickfaden, Tammy; Lupo, L Davis; Cordes, Sarah; Aguirre, Alfredo J; Lupoli, Kathryn A; Chen, Cheng-Yen; Ofotukun, Igho; Hart, Clyde E; Kohlmeier, Jacob E

    2016-07-01

    The female genital tract (FGT) provides a means of entry to pathogens, including HIV, yet immune cell populations at this barrier between host and environment are not well defined. We initiated a study of healthy women to characterize resident T cell populations in the lower FGT from lavage and patient-matched peripheral blood to investigate potential mechanisms of HIV sexual transmission. Surprisingly, we observed FGT CD4 T cell populations were primarily CCR7(hi), consistent with a central memory or recirculating memory T cell phenotype. In addition, roughly half of these CCR7(hi) CD4 T cells expressed CD69, consistent with resident memory T cells, whereas the remaining CCR7(hi) CD4 T cells lacked CD69 expression, consistent with recirculating memory CD4 T cells that traffic between peripheral tissues and lymphoid sites. HIV susceptibility markers CCR5 and CD38 were increased on FGT CCR7(hi) CD4 T cells compared with blood, yet migration to the lymphoid homing chemokines CCL19 and CCL21 was maintained. Infection with GFP-HIV showed that FGT CCR7(hi) memory CD4 T cells are susceptible HIV targets, and productive infection of CCR7(hi) memory T cells did not alter chemotaxis to CCL19 and CCL21. Variations of resident CCR7(hi) FGT CD4 T cell populations were detected during the luteal phase of the menstrual cycle, and longitudinal analysis showed the frequency of this population positively correlated to progesterone levels. These data provide evidence women may acquire HIV through local infection of migratory CCR7(hi) CD4 T cells, and progesterone levels predict opportunities for HIV to access these novel target cells.

  14. Progesterone Levels Associate with a Novel Population of CCR5+CD38+ CD4 T Cells Resident in the Genital Mucosa with Lymphoid Trafficking Potential.

    PubMed

    Swaims-Kohlmeier, Alison; Haaland, Richard E; Haddad, Lisa B; Sheth, Anandi N; Evans-Strickfaden, Tammy; Lupo, L Davis; Cordes, Sarah; Aguirre, Alfredo J; Lupoli, Kathryn A; Chen, Cheng-Yen; Ofotukun, Igho; Hart, Clyde E; Kohlmeier, Jacob E

    2016-07-01

    The female genital tract (FGT) provides a means of entry to pathogens, including HIV, yet immune cell populations at this barrier between host and environment are not well defined. We initiated a study of healthy women to characterize resident T cell populations in the lower FGT from lavage and patient-matched peripheral blood to investigate potential mechanisms of HIV sexual transmission. Surprisingly, we observed FGT CD4 T cell populations were primarily CCR7(hi), consistent with a central memory or recirculating memory T cell phenotype. In addition, roughly half of these CCR7(hi) CD4 T cells expressed CD69, consistent with resident memory T cells, whereas the remaining CCR7(hi) CD4 T cells lacked CD69 expression, consistent with recirculating memory CD4 T cells that traffic between peripheral tissues and lymphoid sites. HIV susceptibility markers CCR5 and CD38 were increased on FGT CCR7(hi) CD4 T cells compared with blood, yet migration to the lymphoid homing chemokines CCL19 and CCL21 was maintained. Infection with GFP-HIV showed that FGT CCR7(hi) memory CD4 T cells are susceptible HIV targets, and productive infection of CCR7(hi) memory T cells did not alter chemotaxis to CCL19 and CCL21. Variations of resident CCR7(hi) FGT CD4 T cell populations were detected during the luteal phase of the menstrual cycle, and longitudinal analysis showed the frequency of this population positively correlated to progesterone levels. These data provide evidence women may acquire HIV through local infection of migratory CCR7(hi) CD4 T cells, and progesterone levels predict opportunities for HIV to access these novel target cells. PMID:27233960

  15. Degradation of the cancer genomic DNA deaminase APOBEC3B by SIV Vif

    PubMed Central

    Land, Allison M.; Wang, Jiayi; Law, Emily K.; Aberle, Ryan; Kirmaier, Andrea; Krupp, Annabel; Johnson, Welkin E.; Harris, Reuben S.

    2015-01-01

    APOBEC3B is a newly identified source of mutation in many cancers, including breast, head/neck, lung, bladder, cervical, and ovarian. APOBEC3B is a member of the APOBEC3 family of enzymes that deaminate DNA cytosine to produce the pro-mutagenic lesion, uracil. Several APOBEC3 family members function to restrict virus replication. For instance, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H combine to restrict HIV-1 in human lymphocytes. HIV-1 counteracts these APOBEC3s with the viral protein Vif, which targets the relevant APOBEC3s for proteasomal degradation. While APOBEC3B does not restrict HIV-1 and is not targeted by HIV-1 Vif in CD4-positive T cells, we asked whether related lentiviral Vif proteins could degrade APOBEC3B. Interestingly, several SIV Vif proteins are capable of promoting APOBEC3B degradation, with SIVmac239 Vif proving the most potent. This likely occurs through the canonical polyubiquitination mechanism as APOBEC3B protein levels are restored by MG132 treatment and by altering a conserved E3 ligase-binding motif. We further show that SIVmac239 Vif can prevent APOBEC3B mediated geno/cytotoxicity and degrade endogenous APOBEC3B in several cancer cell lines. Our data indicate that the APOBEC3B degradation potential of SIV Vif is an effective tool for neutralizing the cancer genomic DNA deaminase APOBEC3B. Further optimization of this natural APOBEC3 antagonist may benefit cancer therapy. PMID:26544511

  16. Influence of deep level defects on carrier lifetime in CdZnTe:In

    SciTech Connect

    Guo, Rongrong; Jie, Wanqi Wang, Ning; Zha, Gangqiang; Xu, Yadong; Wang, Tao; Fu, Xu

    2015-03-07

    The defect levels and carrier lifetime in CdZnTe:In crystal were characterized with photoluminescence, thermally stimulated current measurements, as well as contactless microwave photoconductivity decay (MWPCD) technique. An evaluation equation to extract the recombination lifetime and the reemission time from MWPCD signal is developed based on Hornbeck-Haynes trapping model. An excellent agreement between defect level distribution and carrier reemission time in MWPCD signal reveals the tail of the photoconductivity decay is controlled by the defect level reemission effect. Combining {sup 241}Am gamma ray radiation response measurement and laser beam induced transient current measurement, it predicted that defect level with the reemission time shorter than the collection time could lead to better charge collection efficiency of CdZnTe detector.

  17. Marginal Zone B Cells in Neonatal Rats Express Intermediate Levels of CD90 (Thy-1)

    PubMed Central

    Dammers, Peter M.; Lodewijk, Monique E.; Zandvoort, André; Kroese, Frans G. M.

    2002-01-01

    Here we show that marginal zone (MZ)-B cells in rats can already be detected in neonatal spleen from two days after birth. At this time point, morphologically distinct MZs are not present yet and the vast majority of B cells in spleen are located in a concentric area surrounding the T cell zones (PALS). Before MZs are obviously detectable in spleen (14 days after birth), MZ-B cells seem to be enriched at the outer zones of the concentric B cell areas. Similar to adult rats, neonatal MZ-B cells are intermediate-sized cells that express high levels of surface (s)IgM and HIS57 antigen, and low levels of sIgD and CD45R (HIS24). We show here, however, that in contrast to adult MZ-B cells, MZ-B cells (and also recirculating follicular (RF)-B cells) in neonatal rats express higher levels of CD90 (Thy-1). In adult rats, expression of CD90 on the B cell lineage is confined to immature B cells. We speculate that the expression of CD90 on neonatal MZ-B cells may have implications for their responsiveness to polysaccharide (T cell-independent type 2) antigens. PMID:15144015

  18. Development of Novel Anti-Cd20 Monoclonal Antibodies and Modulation in Cd20 Levels on Cell Surface: Looking to Improve Immunotherapy Response

    PubMed Central

    Singh, Vijay; Gupta, Damodar; Almasan, Alexandru

    2016-01-01

    Rituximab has been revolutionized and validated CD20 targeting monoclonal antibody. Although, it is widely used for lymphoma therapy and many patients have been benefited. However significant numbers of patients are refractory or developed resistance to current therapies due to low level of CD20 expression and/or availability on cells surface. Thus development of novel anti-CD20 mAbs with great cell killing ability and enhance CD20 levels on cell surface can potentially exploit lymphoma therapy. In this scenario, we are summarizing the recently developed mAbs against CD20 and compounds that have ability to induce CD20 expression at significant level. We also are providing information regarding combination strategy for use of radiation and anti-CD20 mAbs in vitro. However, it will need to be determined by rigorous at pre-clinical and clinic testing. We hope this review will be beneficial for current research in the area of immunotherapy or radio-immunotherapy. PMID:27413424

  19. Tolerance of transgenic canola expressing 1-aminocyclopropane-1-carboxylic acid deaminase to growth inhibition by nickel.

    PubMed

    Stearns, Jennifer C; Shah, Saleh; Greenberg, Bruce M; Dixon, D George; Glick, Bernard R

    2005-07-01

    Plant growth-promoting bacteria are useful to phytoremediation strategies in that they confer advantages to plants in contaminated soil. When plant growth-promoting bacteria contain the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, the bacterial cell acts as a sink for ACC, the immediate biosynthetic precursor of the plant growth regulator ethylene thereby lowering plant ethylene levels and decreasing the negative effects of various environmental stresses. In an effort to gain the advantages provided by bacterial ACC deaminase in the phytoremediation of metals from the environment two transgenic canola lines with the gene for this enzyme were generated and tested. In these transgenic canola plants, expression of the ACC deaminase gene is driven by either tandem constitutive cauliflower mosaic virus (CaMV) 35S promoters or the root specific rolD promoter from Agrobacterium rhizogenes. Following the growth of transgenic and non-transformed canola in nickel contaminated soil, it was observed that the rolD plants demonstrate significantly increased tolerance to nickel compared to the non-transformed control plants.

  20. Bacteria with ACC deaminase can promote plant growth and help to feed the world.

    PubMed

    Glick, Bernard R

    2014-01-20

    To feed all of the world's people, it is necessary to sustainably increase agricultural productivity. One way to do this is through the increased use of plant growth-promoting bacteria; recently, scientists have developed a more profound understanding of the mechanisms employed by these bacteria to facilitate plant growth. Here, it is argued that the ability of plant growth-promoting bacteria that produce 1-aminocyclopropane-1-carboxylate (ACC) deaminase to lower plant ethylene levels, often a result of various stresses, is a key component in the efficacious functioning of these bacteria. The optimal functioning of these bacteria includes the synergistic interaction between ACC deaminase and both plant and bacterial auxin, indole-3-acetic acid (IAA). These bacteria not only directly promote plant growth, they also protect plants against flooding, drought, salt, flower wilting, metals, organic contaminants, and both bacterial and fungal pathogens. While a considerable amount of both basic and applied work remains to be done before ACC deaminase-producing plant growth-promoting bacteria become a mainstay of plant agriculture, the evidence indicates that with the expected shift from chemicals to soil bacteria, the world is on the verge of a major paradigm shift in plant agriculture. PMID:24095256

  1. Bacteria with ACC deaminase can promote plant growth and help to feed the world.

    PubMed

    Glick, Bernard R

    2014-01-20

    To feed all of the world's people, it is necessary to sustainably increase agricultural productivity. One way to do this is through the increased use of plant growth-promoting bacteria; recently, scientists have developed a more profound understanding of the mechanisms employed by these bacteria to facilitate plant growth. Here, it is argued that the ability of plant growth-promoting bacteria that produce 1-aminocyclopropane-1-carboxylate (ACC) deaminase to lower plant ethylene levels, often a result of various stresses, is a key component in the efficacious functioning of these bacteria. The optimal functioning of these bacteria includes the synergistic interaction between ACC deaminase and both plant and bacterial auxin, indole-3-acetic acid (IAA). These bacteria not only directly promote plant growth, they also protect plants against flooding, drought, salt, flower wilting, metals, organic contaminants, and both bacterial and fungal pathogens. While a considerable amount of both basic and applied work remains to be done before ACC deaminase-producing plant growth-promoting bacteria become a mainstay of plant agriculture, the evidence indicates that with the expected shift from chemicals to soil bacteria, the world is on the verge of a major paradigm shift in plant agriculture.

  2. Combining molecular dynamics and docking simulations of the cytidine deaminase from Mycobacterium tuberculosis H37Rv.

    PubMed

    Timmers, Luís Fernando Saraiva Macedo; Ducati, Rodrigo Gay; Sánchez-Quitian, Zilpa Adriana; Basso, Luiz Augusto; Santos, Diógenes Santiago; de Azevedo, Walter Filgueira

    2012-02-01

    Cytidine Deaminase (CD) is an evolutionarily conserved enzyme that participates in the pyrimidine salvage pathway recycling cytidine and deoxycytidine into uridine and deoxyuridine, respectively. Here, our goal is to apply computational techniques in the pursuit of potential inhibitors of Mycobacterium tuberculosis CD (MtCDA) enzyme activity. Molecular docking simulation was applied to find the possible hit compounds. Molecular dynamics simulations were also carried out to investigate the physically relevant motions involved in the protein-ligand recognition process, aiming at providing estimates for free energy of binding. The proposed approach was capable of identifying a potential inhibitor, which was experimentally confirmed by IC(50) evaluation. Our findings open up the possibility to extend this protocol to different databases in order to find new potential inhibitors for promising targets based on a rational drug design process.

  3. Low CD36 and LOX-1 Levels and CD36 Gene Subexpression Are Associated with Metabolic Dysregulation in Older Individuals with Abdominal Obesity.

    PubMed

    Madrigal-Ruíz, Perla-Monserrat; Navarro-Hernández, Rosa-Elena; Ruíz-Quezada, Sandra-Luz; Corona-Meraz, Fernanda-Isadora; Vázquez-Del Mercado, Mónica; Gómez-Bañuelos, Eduardo; Castro-Albarran, Jorge; Sandoval-García, Flavio; Flores-Alvarado, Luis-Javier; Martín-Marquez, Beatriz-Teresita

    2016-01-01

    Background. Obesity study in the context of scavenger receptors has been linked to atherosclerosis. CD36 and LOX-1 are important, since they have been associated with atherogenic and metabolic disease but not fat redistribution. The aim of our study was to determinate the association between CD36 and LOX-1 in presence of age and abdominal obesity. Methods. This is a cross-sectional study that included 151 healthy individuals, clinically and anthropometrically classified into two groups by age (<30 and ≥30 years old) and abdominal obesity (according to World Health Organization guidelines). We excluded individuals with any chronic and metabolic illness, use of medication, or smoking. Fasting blood samples were taken to perform determination of CD36 mRNA expression by real-time PCR, lipid profile and metabolic and low grade inflammation markers by routine methods, and soluble scavenger receptors (CD36 and LOX-1) by ELISA. Results. Individuals ≥30 years old with abdominal obesity presented high atherogenic index, lower soluble scavenger receptor levels, and subexpression of CD36 mRNA (54% less). On the other hand, individuals <30 years old with abdominal adiposity presented higher levels in the same parameters, except LOX-1 soluble levels. Conclusion. In this study, individuals over 30 years of age presented low soluble scavenger receptors levels pattern and CD36 gene subexpression, which suggest the chronic metabolic dysregulation in abdominal obesity. PMID:27525284

  4. Low CD36 and LOX-1 Levels and CD36 Gene Subexpression Are Associated with Metabolic Dysregulation in Older Individuals with Abdominal Obesity.

    PubMed

    Madrigal-Ruíz, Perla-Monserrat; Navarro-Hernández, Rosa-Elena; Ruíz-Quezada, Sandra-Luz; Corona-Meraz, Fernanda-Isadora; Vázquez-Del Mercado, Mónica; Gómez-Bañuelos, Eduardo; Castro-Albarran, Jorge; Sandoval-García, Flavio; Flores-Alvarado, Luis-Javier; Martín-Marquez, Beatriz-Teresita

    2016-01-01

    Background. Obesity study in the context of scavenger receptors has been linked to atherosclerosis. CD36 and LOX-1 are important, since they have been associated with atherogenic and metabolic disease but not fat redistribution. The aim of our study was to determinate the association between CD36 and LOX-1 in presence of age and abdominal obesity. Methods. This is a cross-sectional study that included 151 healthy individuals, clinically and anthropometrically classified into two groups by age (<30 and ≥30 years old) and abdominal obesity (according to World Health Organization guidelines). We excluded individuals with any chronic and metabolic illness, use of medication, or smoking. Fasting blood samples were taken to perform determination of CD36 mRNA expression by real-time PCR, lipid profile and metabolic and low grade inflammation markers by routine methods, and soluble scavenger receptors (CD36 and LOX-1) by ELISA. Results. Individuals ≥30 years old with abdominal obesity presented high atherogenic index, lower soluble scavenger receptor levels, and subexpression of CD36 mRNA (54% less). On the other hand, individuals <30 years old with abdominal adiposity presented higher levels in the same parameters, except LOX-1 soluble levels. Conclusion. In this study, individuals over 30 years of age presented low soluble scavenger receptors levels pattern and CD36 gene subexpression, which suggest the chronic metabolic dysregulation in abdominal obesity.

  5. Low CD36 and LOX-1 Levels and CD36 Gene Subexpression Are Associated with Metabolic Dysregulation in Older Individuals with Abdominal Obesity

    PubMed Central

    Castro-Albarran, Jorge; Sandoval-García, Flavio; Flores-Alvarado, Luis-Javier

    2016-01-01

    Background. Obesity study in the context of scavenger receptors has been linked to atherosclerosis. CD36 and LOX-1 are important, since they have been associated with atherogenic and metabolic disease but not fat redistribution. The aim of our study was to determinate the association between CD36 and LOX-1 in presence of age and abdominal obesity. Methods. This is a cross-sectional study that included 151 healthy individuals, clinically and anthropometrically classified into two groups by age (<30 and ≥30 years old) and abdominal obesity (according to World Health Organization guidelines). We excluded individuals with any chronic and metabolic illness, use of medication, or smoking. Fasting blood samples were taken to perform determination of CD36 mRNA expression by real-time PCR, lipid profile and metabolic and low grade inflammation markers by routine methods, and soluble scavenger receptors (CD36 and LOX-1) by ELISA. Results. Individuals ≥30 years old with abdominal obesity presented high atherogenic index, lower soluble scavenger receptor levels, and subexpression of CD36 mRNA (54% less). On the other hand, individuals <30 years old with abdominal adiposity presented higher levels in the same parameters, except LOX-1 soluble levels. Conclusion. In this study, individuals over 30 years of age presented low soluble scavenger receptors levels pattern and CD36 gene subexpression, which suggest the chronic metabolic dysregulation in abdominal obesity. PMID:27525284

  6. The catalase activity of diiron adenine deaminase

    SciTech Connect

    Kamat S. S.; Swaminathan S.; Holmes-Hampton, G. P.; Bagaria, A.; Kumaran, D.; Tichy, S. E.; Gheyi, T.; Zheng, X.; Bain, K.; Groshong, C.; Emtage, S.; Sauder, J. M.; Burley, S. K.; Lindahl, P. A.; Raushel, F. M.

    2011-12-01

    Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn{sup 2+} before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO{sub 4}. Inductively coupled plasma mass spectrometry and Moessbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe{sup II}/Fe{sup II}]-ADE catalyzed the conversion of H{sub 2}O{sub 2} to O{sub 2} and H{sub 2}O. The values of k{sub cat} and k{sub cat}/K{sub m} for the catalase activity are 200 s{sup -1} and 2.4 x 10{sup 4} M{sup -1} s{sup -1}, respectively. [Fe{sup II}/Fe{sup II}]-ADE underwent more than 100 turnovers with H{sub 2}O{sub 2} before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g{sub ave} = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H{sub 2}O{sub 2} by [Fe{sup II}/Fe{sup II}]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS.

  7. Endothelial Progenitor Cells Combined with Cytosine Deaminase-Endostatin for Suppression of Liver Carcinoma.

    PubMed

    Chen, Rong; Yu, Hui; An, Yan-Li; Chen, Hua-Jun; Jia, ZhenYu; Teng, Gao-Jun

    2016-06-01

    Transplantation of gene transfected endothelial progenitor cells (EPCs) provides a novel method for treatment of human tumors. To study treatment of hepatocellular carcinoma using cytosine deaminase (CD)- and endostatin (ES)-transfected endothelial progenitor cells (EPCs), mouse bone marrow-derived EPCs were cultured and transfected with Lenti6.3-CD-EGFP and Lenti6.3-ES-Monomer-DsRed labeled with superparamagnetic iron oxide (SPIO) nanoparticles. DiD (lipophilic fluorescent dye)-labeled EPCs were injected into normal mice and mice with liver carcinoma. The EPCs loaded with CD-ES were infused into the mice through caudal veins and tumor volumes were measured. The tumor volumes in the EPC + SPIO + CD/5-Fc + ES group were found to be smaller as a result and grew more slowly than those from the EPC + SPIO + LV (lentivirus, empty vector control) group. Survival times were also measured after infusion of the cells into the mice. The median survival time was found to be longer in the EPC + SPIO + CD/5-Fc + ES group than in the others. In conclusion, the EPCs transfected with CD-ES suppressed the liver carcinoma cells in vitro, migrated primarily to the carcinoma, inhibited tumor growth, and also extended the median survival time for the mice with liver carcinoma. PMID:27319212

  8. Adenoviral-mediated imaging of gene transfer using a somatostatin receptor-cytosine deaminase fusion protein.

    PubMed

    Lears, K A; Parry, J J; Andrews, R; Nguyen, K; Wadas, T J; Rogers, B E

    2015-03-01

    Suicide gene therapy is a process by which cells are administered a gene that encodes a protein capable of converting a nontoxic prodrug into an active toxin. Cytosine deaminase (CD) has been widely investigated as a means of suicide gene therapy owing to the enzyme's ability to convert the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil (5-FU). However, the extent of gene transfer is a limiting factor in predicting therapeutic outcome. The ability to monitor gene transfer, non-invasively, would strengthen the efficiency of therapy. In this regard, we have constructed and evaluated a replication-deficient adenovirus (Ad) containing the human somatostatin receptor subtype 2 (SSTR2) fused with a C-terminal yeast CD gene for the non-invasive monitoring of gene transfer and therapy. The resulting Ad (AdSSTR2-yCD) was evaluated in vitro in breast cancer cells to determine the function of the fusion protein. These studies demonstrated that both the SSTR2 and yCD were functional in binding assays, conversion assays and cytotoxicity assays. In vivo studies similarly demonstrated the functionality using conversion assays, biodistribution studies and small animal positron-emission tomography (PET) imaging studies. In conclusion, the fusion protein has been validated as useful for the non-invasive imaging of yCD expression and will be evaluated in the future for monitoring yCD-based therapy. PMID:25837665

  9. Adenoviral-Mediated Imaging of Gene Transfer Using a Somatostatin Receptor-Cytosine Deaminase Fusion Protein

    PubMed Central

    Lears, Kimberly A.; Parry, Jesse J.; Andrews, Rebecca; Nguyen, Kim; Wadas, Thaddeus J.; Rogers, Buck E.

    2015-01-01

    Suicide gene therapy is a process by which cells are administered a gene that encodes a protein capable of converting a nontoxic prodrug into an active toxin. Cytosine deaminase (CD) has been widely investigated as a means of suicide gene therapy due to the enzyme’s ability to convert the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil (5-FU). However, the extent of gene transfer is a limiting factor in predicting therapeutic outcome. The ability to monitor gene transfer, non-invasively, would strengthen the efficiency of therapy. In this regard, we have constructed and evaluated a replication-deficient adenovirus (Ad) containing the human somatostatin receptor subtype 2 (SSTR2) fused with a C-terminal yeast CD gene for the non-invasive monitoring of gene transfer and therapy. The resulting Ad (AdSSTR2-yCD) was evaluated in vitro in breast cancer cells to determine the function of the fusion protein. These studies demonstrated that the both the SSTR2 and yCD were functional in binding assays, conversion assays, and cytotoxicity assays. In vivo studies similarly demonstrated the functionality using conversion assays, biodistribution studies, and small animal positron-emission tomography (PET) imaging studies. In conclusion, the fusion protein has been validated as useful for the non-invasive imaging of yCD expression and will be evaluated in the future for monitoring yCD-based therapy. PMID:25837665

  10. New high-spin level schemes and excitation modes of 117,118,119,120,122 Cd

    SciTech Connect

    J. D. Cole

    2012-01-01

    High-spin level schemes of {sup 117,118,119,120,122}Cd are expanded by analyzing our high-statistics triple and higher-fold coincidence events of prompt fission {gamma} rays from {sup 252}Cf at Gammasphere. Spin/parity assignments were made based on new {gamma}-{gamma} angular correlation measurements and level systematics in the neighboring isotopes. Stretch-aligned band structures observed in low-lying levels in {sup 117,119,121}Cd are seen to weaken with increasing spins, with a quasi-rotational degree of freedom manifested at higher spins. The{sup 5-}levels in even-N{sup 118,120}Cd were tentatively interpreted as candidates of quadrupole-octupole (QOC) coupling. The model-independent spin versus {h_bar}{omega} curves for even-N and odd-N Cd isotopes imply quasirotational alignment of an h11/2 neutron pair in the even-N Cd isotopes. The relative energies of the lowest 11/{sup 2-}, 9/{sup 2-}, 7/{sup 2-}, and 15/{sup 2-} levels in {sup 117}Cd and {sup 119}Cd suggest triaxial shapes based on Meyer-Ter-Vehn theory for these odd-N Cd nuclei. For the even-N Cd isotopes evidence of triaxiality may also be provided by the Shell Correction version of the Tilted Axis Cranking model (SCTAC).

  11. High serum levels of soluble CD44 variant isoform v5 are associated with favourable clinical outcome in ovarian cancer.

    PubMed Central

    Zeimet, A. G.; Widschwendter, M.; Uhl-Steidl, M.; Müller-Holzner, E.; Daxenbichler, G.; Marth, C.; Dapunt, O.

    1997-01-01

    In 96 ovarian cancer patients, the present study investigates the clinical significance of pretreatment concentrations of soluble CD44 standard (CD44s) and its isoforms v5 and v6 determined in the serum and the ascitic fluid by means of recently developed enzyme-linked immunosorbent assays (ELISAs). Furthermore, CD44 serum concentrations in the ovarian cancer patients were compared with circulating CD44 levels in 50 healthy age-matched female blood donors. Whereas CD44s was found to be higher and CD44v5 to be lower in ovarian cancer patients than healthy control subjects, no statistical difference between the two cohorts was revealed for CD44 isoform v6. In the ascitic fluid samples, variant isoform v5 and v6 were demonstrated at lower concentrations than serum. Multivariate analysis of overall survival demonstrated that a high pretreatment serum level of soluble CD44 isoform v5 is independently associated with favourable clinical outcome in ovarian cancer. When circulating CD44 isoforms were compared with a panel of serum parameters known to be involved in the immunological network, an inverse correlation between serum CD44v5 levels and indicators of cellular immune system activation, such as soluble interleukin 2 receptor, immunostimulatory protein 90K and neopterin, became apparent. PMID:9413956

  12. Capture kinetics at deep-level defects in MBE-grown CdTe layers

    NASA Astrophysics Data System (ADS)

    Olender, Karolina; Wosinski, Tadeusz; Makosa, Andrzej; Kret, Slawomir; Kolkovsky, Valery; Karczewski, Grzegorz

    2011-04-01

    The results of deep-level transient spectroscopy (DLTS) investigations in n-type CdTe layers grown by the molecular-beam epitaxy (MBE) technique on lattice-mismatched GaAs substrates are described. Three electron traps and one hole trap, at rather low concentrations of the order of 1013 cm-3, have been revealed in the DLTS spectra measured under various bias conditions of Schottky diodes prepared on the as-grown CdTe layers. One of the electron traps has been attributed to electron states of dislocations on the ground of the logarithmic capture kinetics for capture of electrons into the trap states. The other three traps, displaying exponential capture kinetics, have been attributed to native point defects produced during the epitaxial growth of CdTe. The microscopic nature of the defects responsible for the traps is discussed taking into account recent results of first-principles calculations of the properties of dominant native defects in CdTe.

  13. Increased 1-aminocyclopropane-1-carboxylate deaminase activity enhances Agrobacterium tumefaciens-mediated gene delivery into plant cells

    PubMed Central

    Someya, Tatsuhiko; Nonaka, Satoko; Nakamura, Kouji; Ezura, Hiroshi

    2013-01-01

    Agrobacterium-mediated transformation is a useful tool for the genetic modification in plants, although its efficiency is low for several plant species. Agrobacterium-mediated transformation has three major steps in laboratory-controlled experiments: the delivery of T-DNA into plant cells, the selection of transformed plant cells, and the regeneration of whole plants from the selected cells. Each of these steps must be optimized to improve the efficiency of Agrobacterium-mediated plant transformation. It has been reported that increasing the number of cells transformed by T-DNA delivery can improve the frequency of stable transformation. Previously, we demonstrated that a reduction in ethylene production by plant cells during cocultivation with A. tumefaciens-expressing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase resulted in increased T-DNA delivery into the plant cells. In this study, to further improve T-DNA delivery by A. tumefaciens, we modified the expression cassette of the ACC deaminase gene using vir gene promoter sequences. The ACC deaminase gene driven by the virD1 promoter was expressed at a higher level, resulting in a higher ACC deaminase activity in this A. tumefaciens strain than in the strain with the lac promoter used in a previous study. The newly developed A. tumefaciens strain improves the delivery of T-DNA into Solanum lycopersicum (tomato) and Erianthus ravennae plants and thus may be a powerful tool for the Agrobacterium-mediated genetic engineering of plants. PMID:24000136

  14. Porcine γδ T lymphocytes can be categorized into two functionally and developmentally distinct subsets according to expression of CD2 and level of TCR.

    PubMed

    Stepanova, Katerina; Sinkora, Marek

    2013-03-01

    Porcine γδ T cells have two levels of TCRγδ expression. Whereas TCRγδ(med) cells are mostly CD2(+)CD8(-) and CD2(+)CD8(+), TCRγδ(hi) cells are highly enriched for CD2(-)CD8(-). This distribution is independent of bacterial colonization and it is already established in the thymus prior to export of γδ cells to the periphery. Sorting and cultivation experiments revealed that CD2(-)CD8(-) γδ cells are unable to acquire CD2 and CD8, whereas CD2(+) subsets can gain or loose CD8. There is also differential susceptibility for proliferation between CD2(+) and CD2(-) γδ cells. Although CD2(-)CD8(-) almost do not proliferate, proliferation of CD2(+)CD8(-) and CD2(+)CD8(+) is substantial. Population of CD2(-) γδ cells is also absent in CD1(+) immature thymocytes. Additionally, subpopulations of CD2(+) and CD2(-) γδ cells in the thymus differ in expression of auxiliary surface molecules such as CD25, CD45RA/RC, and MHC class II. Moreover, TCRγδ(hi) cells can generate TCRγδ(med) cells but never the opposite. The only exception is the thymus, where a few TCRγδ(med) cells can be induced to TCRγδ(hi) but only under IL-2 influence. The repertoire of TCRδ is polyclonal in all subsets, indicating that there is the same extent of diversification and equal capability of immune responses. Results collectively indicate that CD2 expression determines two lineages of γδ cells that differ in many aspects. Because CD2(-) γδ cells are missing in the blood of humans and mice but are obvious in other members of γδ-high species such as ruminants and birds, our findings support the idea that circulating CD2(-) γδ T cells are a specific lineage.

  15. Effect of Co-planted Purslane (Portulaca Oleracea L.) on Cd Accumulation by Sunflower in Different Levels of Cd Contamination and Salinity: A Pot Study.

    PubMed

    Ashrafi, Ali; Zahedi, Morteza; Soleimani, Mohsen

    2015-01-01

    Heavy metal bioaccumulation can be affected by various crop-weed interactions that potentially exist in agroecosystems. A pot experiment was conducted to evaluate the role of rhizosphere interaction of sunflower and purslane (Portulaca oleracea L.) weed on cadmium (Cd) uptake and its allocation to sunflower grains. The experimental treatments consisted of two cropping systems (mono and mixed culture), two adjusted salinity levels (0 and 0.5% NaCl) and three artificial levels of Cd in soil (Control, 3 and 6 mg kg(-1)). The results showed that the growth of sunflower in the presence of purslane in comparison to mono culture of sunflower led to change of total Cd content and Cd allocated to grains only in saline conditions. Promoting effects of salinity on Cd concentration of grain were alleviated where sunflower was co-planted with purslane. Besides, supply of Zn in grains of co-planted sunflower was strongly affected by salinity. Results of this study revealed that although co-planted purslane could alter conditions in the shared rhizosphere, it had no effect on enhancing Cd uptake by neighboring sunflower directly. PMID:26091425

  16. Effect of Co-planted Purslane (Portulaca Oleracea L.) on Cd Accumulation by Sunflower in Different Levels of Cd Contamination and Salinity: A Pot Study.

    PubMed

    Ashrafi, Ali; Zahedi, Morteza; Soleimani, Mohsen

    2015-01-01

    Heavy metal bioaccumulation can be affected by various crop-weed interactions that potentially exist in agroecosystems. A pot experiment was conducted to evaluate the role of rhizosphere interaction of sunflower and purslane (Portulaca oleracea L.) weed on cadmium (Cd) uptake and its allocation to sunflower grains. The experimental treatments consisted of two cropping systems (mono and mixed culture), two adjusted salinity levels (0 and 0.5% NaCl) and three artificial levels of Cd in soil (Control, 3 and 6 mg kg(-1)). The results showed that the growth of sunflower in the presence of purslane in comparison to mono culture of sunflower led to change of total Cd content and Cd allocated to grains only in saline conditions. Promoting effects of salinity on Cd concentration of grain were alleviated where sunflower was co-planted with purslane. Besides, supply of Zn in grains of co-planted sunflower was strongly affected by salinity. Results of this study revealed that although co-planted purslane could alter conditions in the shared rhizosphere, it had no effect on enhancing Cd uptake by neighboring sunflower directly.

  17. Soluble CD14 levels in the serum, synovial fluid, and cerebrospinal fluid of patients with various stages of Lyme disease.

    PubMed

    Lin, B; Noring, R; Steere, A C; Klempner, M S; Hu, L T

    2000-03-01

    Levels of circulating soluble CD14 (sCD14) in patients with various stages of Lyme disease (LD) were examined. Patients with early or untreated late LD had significantly higher levels of sCD14 than did healthy controls (P=.0001 and .0007, respectively); levels returned to normal within 3 months after antibiotic therapy. Patients with persistent posttreatment symptoms of LD had sCD14 levels equivalent to those of healthy controls. Differences in the serum sCD14 levels in patients with various stages of LD are likely to be directly correlated with differences in bacterial burden, suggesting that posttreatment symptoms may not require continued presence of the organism. sCD14 levels in the cerebrospinal fluid (CSF) of patients with any stage of LD were no different from those of control subjects. Levels of synovial fluid sCD14 from patients with Borrelia burgdorferi in their joints were elevated, compared with levels in normal serum, and may play a role in the pathogenesis of arthritis.

  18. ADA (adenosine deaminase) gene therapy enters the competition

    SciTech Connect

    Culliton, B.J.

    1990-08-31

    Around the world, some 70 children are members of a select and deadly club. Born with an immune deficiency so severe that they will die of infection unless their immune systems can be repaired, they have captured the attention of would-be gene therapists who believe that a handful of these kids--the 15 or 20 who lack functioning levels of the enzyme adenosine deaminase (ADA)--could be saved by a healthy ADA gene. A team of gene therapists is ready to put the theory to the test. In April 1987, a team of NIH researchers headed by R. Michael Blaese and W. French Anderson came up with the first formal protocol to introduce a healthy ADA gene into an unhealthy human. After 3 years of line-by-line scrutiny by five review committees, they have permission to go ahead. Two or three children will be treated in the next year, and will be infused with T lymphocytes carrying the gene for ADA. If the experiment works, the ADA gene will begin producing normal amounts of ADA. An interesting feature of ADA deficiency, that makes it ideal for initial gene studies, is that the amount of ADA one needs for a healthy immune system is quite variable. Hence, once inside a patient's T cells, the new ADA gene needs only to express the enzyme in moderate amounts. No precise gene regulation is necessary.

  19. 124In levels populated in the β decay of 124Cd

    NASA Astrophysics Data System (ADS)

    Batchelder, J. C.; Brewer, N. T.; Gross, C. J.; Grzywacz, R.; Hamilton, J. H.; Karny, M.; Fijalkowska, A.; Liu, S. H.; Miernik, K.; Padgett, S. W.; Paulauskas, S. V.; Rykaczewski, K. P.; Ramayya, A. V.; Stracener, D. W.; Wolińska-Cichocka, M.

    2016-08-01

    The β decay of 124Cd into levels in 124In was reinvestigated at the Holifield Radioactive Ion Beam Facility. Fifty-MeV protons were bombarded on uranium targets and the induced fission products were mass separated and deposited on a moving tape in the center of an array of γ detectors. The resulting γ -γ coincidences revealed appreciable disagreement with previous work and has resulted in a revised ordering of the low-energy states in 124In. The resulting partial decay scheme has four energy levels, three of which are new.

  20. Dihydropyrimidine Dehydrogenase Is a Prognostic Marker for Mesenchymal Stem Cell-Mediated Cytosine Deaminase Gene and 5-Fluorocytosine Prodrug Therapy for the Treatment of Recurrent Gliomas

    PubMed Central

    Chung, Taemoon; Na, Juri; Kim, Young-il; Chang, Da-Young; Kim, Young Il; Kim, Hyeonjin; Moon, Ho Eun; Kang, Keon Wook; Lee, Dong Soo; Chung, June-Key; Kim, Sung-Soo; Suh-Kim, Haeyoung; Paek, Sun Ha; Youn, Hyewon

    2016-01-01

    We investigated a therapeutic strategy for recurrent malignant gliomas using mesenchymal stem cells (MSC), expressing cytosine deaminase (CD), and prodrug 5-Fluorocytosine (5-FC) as a more specific and less toxic option. MSCs are emerging as a novel cell therapeutic agent with a cancer-targeting property, and CD is considered a promising enzyme in cancer gene therapy which can convert non-toxic 5-FC to toxic 5-Fluorouracil (5-FU). Therefore, use of prodrug 5-FC can minimize normal cell toxicity. Analyses of microarrays revealed that targeting DNA damage and its repair is a selectable option for gliomas after the standard chemo/radio-therapy. 5-FU is the most frequently used anti-cancer drug, which induces DNA breaks. Because dihydropyrimidine dehydrogenase (DPD) was reported to be involved in 5-FU metabolism to block DNA damage, we compared the survival rate with 5-FU treatment and the level of DPD expression in 15 different glioma cell lines. DPD-deficient cells showed higher sensitivity to 5-FU, and the regulation of DPD level by either siRNA or overexpression was directly related to the 5-FU sensitivity. For MSC/CD with 5-FC therapy, DPD-deficient cells such as U87MG, GBM28, and GBM37 showed higher sensitivity compared to DPD-high U373 cells. Effective inhibition of tumor growth was also observed in an orthotopic mouse model using DPD- deficient U87MG, indicating that DPD gene expression is indeed closely related to the efficacy of MSC/CD-mediated 5-FC therapy. Our results suggested that DPD can be used as a biomarker for selecting glioma patients who may possibly benefit from this therapy. PMID:27446484

  1. Dihydropyrimidine Dehydrogenase Is a Prognostic Marker for Mesenchymal Stem Cell-Mediated Cytosine Deaminase Gene and 5-Fluorocytosine Prodrug Therapy for the Treatment of Recurrent Gliomas.

    PubMed

    Chung, Taemoon; Na, Juri; Kim, Young-Il; Chang, Da-Young; Kim, Young Il; Kim, Hyeonjin; Moon, Ho Eun; Kang, Keon Wook; Lee, Dong Soo; Chung, June-Key; Kim, Sung-Soo; Suh-Kim, Haeyoung; Paek, Sun Ha; Youn, Hyewon

    2016-01-01

    We investigated a therapeutic strategy for recurrent malignant gliomas using mesenchymal stem cells (MSC), expressing cytosine deaminase (CD), and prodrug 5-Fluorocytosine (5-FC) as a more specific and less toxic option. MSCs are emerging as a novel cell therapeutic agent with a cancer-targeting property, and CD is considered a promising enzyme in cancer gene therapy which can convert non-toxic 5-FC to toxic 5-Fluorouracil (5-FU). Therefore, use of prodrug 5-FC can minimize normal cell toxicity. Analyses of microarrays revealed that targeting DNA damage and its repair is a selectable option for gliomas after the standard chemo/radio-therapy. 5-FU is the most frequently used anti-cancer drug, which induces DNA breaks. Because dihydropyrimidine dehydrogenase (DPD) was reported to be involved in 5-FU metabolism to block DNA damage, we compared the survival rate with 5-FU treatment and the level of DPD expression in 15 different glioma cell lines. DPD-deficient cells showed higher sensitivity to 5-FU, and the regulation of DPD level by either siRNA or overexpression was directly related to the 5-FU sensitivity. For MSC/CD with 5-FC therapy, DPD-deficient cells such as U87MG, GBM28, and GBM37 showed higher sensitivity compared to DPD-high U373 cells. Effective inhibition of tumor growth was also observed in an orthotopic mouse model using DPD- deficient U87MG, indicating that DPD gene expression is indeed closely related to the efficacy of MSC/CD-mediated 5-FC therapy. Our results suggested that DPD can be used as a biomarker for selecting glioma patients who may possibly benefit from this therapy. PMID:27446484

  2. Investigation of the origin of deep levels in CdTe doped with Bi

    SciTech Connect

    Saucedo, E.; Franc, J.; Elhadidy, H.; Horodysky, P.; Ruiz, C. M.; Bermudez, V.; Sochinskii, N. V.

    2008-05-01

    Combining optical (low temperature photoluminescence), electrical (thermoelectric effect spectroscopy), and structural (synchrotron X-ray powder diffraction) methods, the defect structure of CdTe doped with Bi was studied in crystals with dopant concentration in the range of 10{sup 17}-10{sup 19} at./cm{sup 3}. The semi-insulating state observed in crystals with low Bi concentration is assigned to the formation of a shallow donor level and a deep donor recombination center. Studying the evolution of lattice parameter with temperature, we postulate that the deep center is formed by a Te-Te dimer and their formation is explained by a tetrahedral to octahedral distortion, due to the introduction of Bi in the CdTe lattice. We also shows that this model agrees with the electrical, optical, and transport charge properties of the samples.

  3. Pyrimidine, purine and nitrogen control of cytosine deaminase synthesis in Escherichia coli K 12. Involvement of the glnLG and purR genes in the regulation of codA expression.

    PubMed

    Andersen, L; Kilstrup, M; Neuhard, J

    1989-01-01

    Cytosine deaminase, encoded by the codA gene in Escherichia coli catalyzes the deamination of cytosine to uracil and ammonia. Regulation of codA expression was studied by determining the level of cytosine deaminase in E. coli K12 grown in various defined media. Addition of either pyrimidine or purine nucleobases to the growth medium caused repressed enzyme levels, whereas growth on a poor nitrogen source such as proline resulted in derepression of cytosine deaminase synthesis. Derepression of codA expression was induced by starvation for either uracil or cytosine nucleotides. Nitrogen control was found to be mediated by the glnLG gene products, and purine repression required a functional purR gene product. Studies with strains harbouring multiple mutations affecting both pyrimidine, purine and nitrogen control revealed that the overall regulation of cytosine deaminase synthesis by the different metabolites is cumulative.

  4. Adenosine deaminase from Streptomyces coelicolor: recombinant expression, purification and characterization.

    PubMed

    Pornbanlualap, Somchai; Chalopagorn, Pornchanok

    2011-08-01

    The sequencing of the genome of Streptomyces coelicolor A3(2) identified seven putative adenine/adenosine deaminases and adenosine deaminase-like proteins, none of which have been biochemically characterized. This report describes recombinant expression, purification and characterization of SCO4901 which had been annotated in data bases as a putative adenosine deaminase. The purified putative adenosine deaminase gives a subunit Mr=48,400 on denaturing gel electrophoresis and an oligomer molecular weight of approximately 182,000 by comparative gel filtration. These values are consistent with the active enzyme being composed of four subunits with identical molecular weights. The turnover rate of adenosine is 11.5 s⁻¹ at 30 °C. Since adenine is deaminated ∼10³ slower by the enzyme when compared to that of adenosine, these data strongly show that the purified enzyme is an adenosine deaminase (ADA) and not an adenine deaminase (ADE). Other adenine nucleosides/nucleotides, including 9-β-D-arabinofuranosyl-adenine (ara-A), 5'-AMP, 5'-ADP and 5'-ATP, are not substrates for the enzyme. Coformycin and 2'-deoxycoformycin are potent competitive inhibitors of the enzyme with inhibition constants of 0.25 and 3.4 nM, respectively. Amino acid sequence alignment of ScADA with ADAs from other organisms reveals that eight of the nine highly conserved catalytic site residues in other ADAs are also conserved in ScADA. The only non-conserved residue is Asn317, which replaces Asp296 in the murine enzyme. Based on these data, it is suggested here that ADA and ADE proteins are divergently related enzymes that have evolved from a common α/β barrel scaffold to catalyze the deamination of different substrates, using a similar catalytic mechanism. PMID:21511036

  5. Determination of 1-aminocycopropane-1-carboxylic acid (ACC) to assess the effects of ACC deaminase-containing bacteria on roots of canola seedlings.

    PubMed

    Penrose, D M; Moffatt, B A; Glick, B R

    2001-01-01

    Previously, it was proposed that plant growth-promoting bacteria that possess the enzyme, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, can reduce the amount of ethylene produced by a plant and thereby promote root elongation. To test this model, canola seeds were imbibed in the presence of the chemical ethylene inhibitor, 2-aminoethoxyvinyl glycine (AVG), various strains of plant growth-promoting bacteria, and a psychrophilic bacterium containing an ACC deaminase gene on a broad host range plasmid. The extent of root elongation and levels of ACC, the immediate precursor of ethylene, were measured in the canola seedling roots. A modification of the Waters AccQ.Tag Amino Acid Analysis Method was used to quantify ACC in the root extracts. It was found that, in the presence of the ethylene inhibitor, AVG, or any one of several ACC deaminase-containing strains of bacteria, the growth of canola seedling roots was enhanced and the ACC levels in these roots were lowered.

  6. B-cell agonists up-regulate AID and APOBEC3G deaminases, which induce IgA and IgG class antibodies and anti-viral function.

    PubMed

    Seidl, Thomas; Whittall, Trevor; Babaahmady, Kaboutar; Lehner, Thomas

    2012-03-01

    B cells express two critical deaminases in the development of adaptive and innate immunity. Activation-induced cytidine deaminase (AID) functions in class switch recombination, somatic hypermutation and may result in affinity maturation of antibodies. Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G; A3G) is an innate anti-retroviral factor that inhibits HIV replication. We have studied a number of B-cell agonists with the aim of identifying the most effective agents that will up-regulate both deaminases and thereby enhance adaptive and innate immunity. CD40 ligand (CD40L) with interleukin-4 or HLA-class II antibodies significantly up-regulated both AID and A3G in isolated human CD19(+) B cells. The functions of these deaminases were demonstrated by enhancement of B-cell surface expression of IgA and IgG and inducing significantly higher IgA and IgG4 antibodies. An enhanced A3G function was then demonstrated by inhibition of HIV-1 replication in co-culture of CD4(+) T cells with autologous B cells, treated with CD40L and CD4 or HLA antibodies, compared with unstimulated human B cells. The dual B-cell-induced deaminase functions may be critical in IgA and IgG antibodies inhibiting pre-entry and A3G that of post-entry HIV-1 transmission and suggests a novel strategy of immunization, especially relevant to mucosal infections.

  7. Cytosine Deaminase/5-Fluorocytosine Exposure Induces Bystander and Radiosensitization Effects in Hypoxic Glioblastoma Cells in vitro

    SciTech Connect

    Chen, Jennifer K.; Hu, Lily J.; Wang Dongfang; Lamborn, Kathleen R.; Deen, Dennis F. . E-mail: dennisdeen@juno.com

    2007-04-01

    Purpose: Treatment of glioblastoma (GBM) is limited by therapeutic ratio; therefore, successful therapy must be specifically cytotoxic to cancer cells. Hypoxic cells are ubiquitous in GBM, and resistant to radiation and chemotherapy, and, thus, are logical targets for gene therapy. In this study, we investigated whether cytosine deaminase (CD)/5-fluorocytosine (5-FC) enzyme/prodrug treatment induced a bystander effect (BE) and/or radiosensitization in hypoxic GBM cells. Methods and Materials: We stably transfected cells with a gene construct consisting of the SV40 minimal promoter, nine copies of a hypoxia-responsive element, and the yeast CD gene. During hypoxia, a hypoxia-responsive element regulates expression of the CD gene and facilitates the conversion of 5-FC to 5-fluorouracil, a highly toxic antimetabolite. We used colony-forming efficiency (CFE) and immunofluorescence assays to assess for BE in co-cultures of CD-expressing clone cells and parent, pNeo- or green fluorescent protein-stably transfected GBM cells. We also investigated the radiosensitivity of CD clone cells treated with 5-FC under hypoxic conditions, and we used flow cytometry to investigate treatment-induced cell cycle changes. Results: Both a large BE and radiosensitization occurred in GBM cells under hypoxic conditions. The magnitude of the BE depended on the number of transfected cells producing CD, the functionality of the CD, the administered concentration of 5-FC, and the sensitivity of cell type to 5-fluorouracil. Conclusion: Hypoxia-inducible CD/5-FC therapy in combination with radiation therapy shows both a pronounced BE and a radiosensitizing effect under hypoxic conditions.

  8. Comparative studies on Pb and Cd levels in parasites of terrestrial and aquatic animals

    SciTech Connect

    Sures, B.; Taraschewski, H.

    1995-12-31

    Several fish parasites (Acanthocephala, Cestoda, Nematoda) and organs of their respective intermediate and final hosts were analyzed for heavy metals by electrothermal atomic absorption spectrometry (ET-AAS). Pb and Cd were also quantified in the liver fluke Fasciola hepatica as well as in different organs of the large intestinal roundworm Ascaris suum. The levels of these heavy metals in the parasites were compared to those of muscle, liver, kidney and intestine of the respective definitive hosts cattle and swine obtained from a slaughter house. Most parasites accumulated significantly higher levels of metals than their final hosts. This was most conspicuous in acanthocephalans which contained up to 3 {times} 10{sup 3} fold more lead than the muscle of their fish hosts and up to 1.1 {times} 10{sup 4} more lead than the water surrounding the fish. In these helminths cadmium was enriched up to 400 fold compared to the muscle of the fish and up to 2.7 {times} 10{sup 4} compared to the water. In contrast to the accumulation capacity of adult acanthocephalans their larvae contained about 30 to 180 times less Pb and Cd. Thus, the predominant accumulation of both metals appears in the adult worms. The cestodes of fish and the liver flukes of cattle accumulated the metals up to 200 fold compared to the muscle of their hosts. The nematodes did not contain higher levels of the metals than their hosts. Thus, parasites, especially acanthocephalans, seem to be sensitive bioindicators of Pb and Cd in their environments.

  9. Higher levels of circulating monocyte-platelet aggregates are correlated with viremia and increased sCD163 levels in HIV-1 infection.

    PubMed

    Liang, Hua; Duan, Zhaojun; Li, Dan; Li, Dongliang; Wang, Zheng; Ren, Li; Shen, Tao; Shao, Yiming

    2015-07-01

    Increased levels of monocyte-platelet aggregates (MPAs) are reported to be highly correlated with cardiovascular events. In this study, the MPA levels in different monocyte subsets and the associations between MPA levels, HIV-1 viremia and monocyte activation were evaluated during HIV-1 infection. The results showed that the percentages of MPAs in all three monocyte subsets were higher in HIV-1-infected subjects than in healthy controls, and were associated with the plasma viral load in the non-classical and intermediate monocyte subsets. The plasma levels of sCD14 and sCD163 were upregulated in HIV-1 infection and were positively associated with viral loads and negatively associated with CD4 counts. P-selectin glycoprotein ligand-1 (PSGL-1) was shown to be expressed at significantly lower levels on all three monocyte subsets and was negatively correlated with the sCD163 level. The MPA level was correlated with the levels of plasma sCD163 but negatively correlated with CD163 and PSGL-1 on all three monocyte subsets. An elevated immune activation status was correlated with increased MPA formation, underlying the potential interaction between monocyte activation and MPA formation. This interaction may be related to a higher thromboembolic risk in patients infected with HIV-1.Cellular & Molecular Immunology advance online publication, 11 August 2014; doi:10.1038/cmi.2014.66.

  10. Elevated plasma-soluble CD16 levels in porcine reproductive and respiratory syndrome virus-infected pigs: correlation with ADAM17-mediated shedding.

    PubMed

    Gu, Weihong; Guo, Longjun; Li, Ren; Niu, Junwei; Luo, Xiaolei; Zhang, Jian; Xu, Yunfei; Tian, Zhijun; Feng, Li; Wang, Yue

    2016-03-01

    Soluble CD16 (sCD16) is closely correlated with chronic diseases in humans. Here, plasma sCD16 levels in pigs were increased by infection with porcine reproductive and respiratory syndrome virus (PRRSV) but not with porcine epidemic diarrhea virus, porcine circovirus type 2 and pseudorabies virus. Of interest, PRRSV attached to blood neutrophils and reduced surface CD16 expression on neutrophils. In vitro data confirmed that PRRSV caused CD16 shedding in neutrophils. Further analyses revealed that ADAM17 was involved in porcine CD16 shedding. Thus, our findings suggest that increase in sCD16 levels may be an indicator of PRRSV infection. PMID:26653711

  11. CD4 count levels and pattern of respiratory complications in HIV seropositive patients in Calabar, Nigeria.

    PubMed

    Peters, E J; Essien, O E; Immananagha, K K; Inah, G A; Philip-Ephraim, E E; Agbulu, R E

    2007-01-01

    A prospective observational study was carried out to describe the pattern of pulmonary complications in hospitalized patients with Human Immune-deficiency Virus (HIV) infection at the University of Calabar Teaching Hospital, Calabar between January 2005 to December 2006. One hundred and twenty-four patients which consists 60 males and 64 females, aged between 20-60 who met the inclusion criteria formed the subjects for the study. The mean age of the subjects was 34.60 +/-1.2 years. A structured questionnaire was used to obtain the demographic data, clinical information and CD4 lymphocyte count. Radiological analysis of chest was done with the chest X-ray of each subject. Chronic productive cough topped the list of respiratory symptoms (89%) followed by chest pain (74%) and dyspnea (62 %). Lung consolidation was the commonest respiratory sign as seen in 44 % of the cases. Hilar lymphadenopathy was seen in (35 %), Pleural effusion (32%), lung fibrosis (21%) and finger clubbing (15%). The clinical and radiological pattern of most patients with chronic cough was highly suggestive of mycobacterial infection such as tuberculosis, although only 40% of cases had positive acid fast bacilli. The mean CD4 lymphocyte count level was 174.8 +/- 5.4 cells/microlitre and this may be responsible for the respiratory findings as opportunistic lung infections are said to be commoner at CD4 count levels below 200 cells/microlitre. However, four patients had mediasternal masses which may suggest neoplasms. Concerted efforts and continuous evaluation of these patients are needed to determine the spectrum of respiratory illnesses among HIV positive patients in Calabar.

  12. Human α(2)β(1)(HI) CD133(+VE) epithelial prostate stem cells express low levels of active androgen receptor.

    PubMed

    Williamson, Stuart C; Hepburn, Anastasia C; Wilson, Laura; Coffey, Kelly; Ryan-Munden, Claudia A; Pal, Deepali; Leung, Hing Y; Robson, Craig N; Heer, Rakesh

    2012-01-01

    Stem cells are thought to be the cell of origin in malignant transformation in many tissues, but their role in human prostate carcinogenesis continues to be debated. One of the conflicts with this model is that cancer stem cells have been described to lack androgen receptor (AR) expression, which is of established importance in prostate cancer initiation and progression. We re-examined the expression patterns of AR within adult prostate epithelial differentiation using an optimised sensitive and specific approach examining transcript, protein and AR regulated gene expression. Highly enriched populations were isolated consisting of stem (α(2)β(1)(HI) CD133(+VE)), transiently amplifying (α(2)β(1)(HI) CD133(-VE)) and terminally differentiated (α(2)β(1)(LOW) CD133(-VE)) cells. AR transcript and protein expression was confirmed in α(2)β(1)(HI) CD133(+VE) and CD133(-VE) progenitor cells. Flow cytometry confirmed that median (±SD) fraction of cells expressing AR were 77% (±6%) in α(2)β(1)(HI) CD133(+VE) stem cells and 68% (±12%) in α(2)β(1)(HI) CD133(-VE) transiently amplifying cells. However, 3-fold lower levels of total AR protein expression (peak and median immunofluorescence) were present in α(2)β(1)(HI) CD133(+VE) stem cells compared with differentiated cells. This finding was confirmed with dual immunostaining of prostate sections for AR and CD133, which again demonstrated low levels of AR within basal CD133(+VE) cells. Activity of the AR was confirmed in prostate progenitor cells by the expression of low levels of the AR regulated genes PSA, KLK2 and TMPRSS2. The confirmation of AR expression in prostate progenitor cells allows integration of the cancer stem cell theory with the established models of prostate cancer initiation based on a functional AR. Further study of specific AR functions in prostate stem and differentiated cells may highlight novel mechanisms of prostate homeostasis and insights into tumourigenesis.

  13. Recent developments in use of 1-aminocyclopropane-1-carboxylate (ACC) deaminase for conferring tolerance to biotic and abiotic stress.

    PubMed

    Gontia-Mishra, Iti; Sasidharan, Shaly; Tiwari, Sharad

    2014-05-01

    Ethylene is an essential plant hormone also known as a stress hormone because its synthesis is accelerated by induction of a variety of biotic and abiotic stress. The plant growth promoting bacteria containing the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase enhances plant growth by decreasing plant ethylene levels under stress conditions. The expression of ACC deaminase (acdS) gene in transgenic plants is an alternative approach to overcome the ethylene-induced stress. Several transgenic plants have been engineered to express both bacterial/plant acdS genes which then lowers the stress-induced ethylene levels, thus efficiently combating the deleterious effects of environmental stresses. This review summarizes the current knowledge of various transgenic plants overexpressing microbial and plant acdS genes and their potential under diverse biotic and abiotic stresses. Transcription regulation mechanism of acdS gene from different bacteria, with special emphasis to nitrogen fixing bacteria is also discussed in this review.

  14. B cells naturally induced during dengue virus infection release soluble CD27, the plasma level of which is associated with severe forms of pediatric dengue.

    PubMed

    Castañeda, Diana M; Salgado, Doris M; Narváez, Carlos F

    2016-10-01

    The CD27 and CD38 antigens are highly expressed on the plasmablast surface, and a massive plasmablast response has been described for dengue virus infection. Soluble CD27 and CD38 forms (sCD27 and sCD38, respectively) increase after immune activation. Here, we show increased sCD27 release in cultures of purified polyclonally stimulated B cells. T and B cells isolated from children with dengue spontaneously produced higher levels of sCD27 but not sCD38, compared with healthy children (P=0.03), and sCD27 levels positively correlated with plasmablast frequency in the cultures (rho=0.58, P=0.01). Children with dengue had higher plasma levels of sCD27 and sCD38 than healthy children, which decreased during convalescence. Plasma sCD27 was higher in severe than with mild dengue, but the opposite was observed for sCD38. These findings support a potential new role for B cells in dengue pathogenesis, and sCD27 and sCD38 are novel biomarkers associated with clinical outcome during dengue virus infection.

  15. ACC (1-aminocyclopropane-1-carboxylate) deaminase activity, a widespread trait in Burkholderia species, and its growth-promoting effect on tomato plants.

    PubMed

    Onofre-Lemus, Janette; Hernández-Lucas, Ismael; Girard, Lourdes; Caballero-Mellado, Jesús

    2009-10-01

    The genus Burkholderia includes pathogens of plants and animals and some human opportunistic pathogens, such as the Burkholderia cepacia complex (Bcc), but most species are nonpathogenic, plant associated, and rhizospheric or endophytic. Since rhizobacteria expressing ACC (1-aminocyclopropane-1-carboxylate) deaminase may enhance plant growth by lowering plant ethylene levels, in this work we investigated the presence of ACC deaminase activity and the acdS gene in 45 strains, most of which are plant associated, representing 20 well-known Burkholderia species. The results demonstrated that ACC deaminase activity is a widespread feature in the genus Burkholderia, since 18 species exhibited ACC deaminase activities in the range from 2 to 15 mumol of alpha-ketobutyrate/h/mg protein, which suggests that these species may be able to modulate ethylene levels and enhance plant growth. In these 18 Burkholderia species the acdS gene sequences were highly conserved (76 to 99% identity). Phylogenetic analysis of acdS gene sequences in Burkholderia showed tight clustering of the Bcc species, which were clearly distinct from diazotrophic plant-associated Burkholderia species. In addition, an acdS knockout mutant of the N(2)-fixing bacterium Burkholderia unamae MTl-641(T) and a transcriptional acdSp-gusA fusion constructed in this strain showed that ACC deaminase could play an important role in promotion of the growth of tomato plants. The widespread ACC deaminase activity in Burkholderia species and the common association of these species with plants suggest that this genus could be a major contributor to plant growth under natural conditions.

  16. The Serum Levels of the Soluble Factors sCD40L and CXCL1 Are Not Indicative of Endometriosis

    PubMed Central

    Pateisky, Petra; Pils, Dietmar; Kuessel, Lorenz; Szabo, Ladislaus; Walch, Katharina; Obwegeser, Reinhard; Wenzl, René; Yotova, Iveta

    2016-01-01

    Endometriosis is a benign but troublesome gynecological condition, characterized by endometrial-like tissue outside the uterine cavity. Lately, the discovery and validation of noninvasive diagnostic biomarkers for endometriosis is one of the main priorities in the field. As the disease elicits a chronic inflammatory reaction, we focused our interest on two factors well known to be involved in inflammation and neoplastic processes, namely, soluble CD40 Ligand and CXCL1, and asked whether differences in the serum levels of sCD40L and CXCL1 in endometriosis patients versus controls can serve as noninvasive disease markers. A total of n = 60 women were included in the study, 31 endometriosis patients and 29 controls, and the serum levels of sCD40L and CXCL1 were measured by enzyme-linked immunosorbent assay. Overall, there were no statistically significant differences in the levels of expression of both sCD40L and CXCL1 between patients and controls. This study adds useful clinical data showing that the serum levels of the soluble factors sCD40L and CXCL1 are not associated with endometriosis and are not suitable as biomarkers for disease diagnosis. However, we found a trend toward lower levels of sCD40L in the deep infiltrating endometriosis subgroup making it a potentially interesting target worth further investigation. PMID:27190986

  17. The Role of Different Supplements in Expression Level of Monoclonal Antibody against Human CD20

    PubMed Central

    Mahboudi, Fereidoun; Abolhassan, Mohammad Reza; Azarpanah, Armita; Aghajani-Lazarjani, Hamideh; Sadeghi-Haskoo, Mohammad Amin; Maleknia, Shaian; Vaziri, Behrouz

    2013-01-01

    Background Recombinant monoclonal antibodies have been marketed in last three decades as the major therapeutic proteins against different cancers. However choosing a proper medium and supplements to reach the high expression is a challenging step. Despite of commercial serum free and chemically defined media, there are still numerous researches seeking the optimum media to gain higher expression titer. Selecting the best basal media followed by proper supplementation to increase the cell density and expression titer needs proper and accurate investigation. Methods In this study, we have determined the expression titer of monoclonal antibody against human CD20 using soy extract, Essential Amino Acid, Non-Essential Amino Acid, Panexin NTS, Peptone, Yeast extract, Insulin-transferrin selenite, Human Serum Albumin, Bovine Serum Albumin, Lipid, and two commercially available supplements, Power and Xtreme feed. In each experiment, the expression level was compared with a well defined media, ProCHO5, RPMI 1640 and DMEM-F12. Results It has been shown that supplementing the ProCHO5 basal medium with 10% power feed or combination of 5% PanexinNTS,1.5 g/L yeast and 1.5g/L peptone results in the best production levels with 450 and 425 mg/L of anti CD20 mAb expression level, respectively. Conclusion Panexin NTS, yeast and peptone cane be proper supplement for fed-batch cell culture instead of commercial Power feed supplement which is a cost effective way to increase expression level. And thereby ProCHO5 may be replaced with common media such as RPMI 1640 and DMEM-F12. PMID:23919117

  18. Biochemistry and genetics of ACC deaminase: a weapon to “stress ethylene” produced in plants

    PubMed Central

    Singh, Rajnish P.; Shelke, Ganesh M.; Kumar, Anil; Jha, Prabhat N.

    2015-01-01

    1-aminocyclopropane-1-carboxylate deaminase (ACCD), a pyridoxal phosphate-dependent enzyme, is widespread in diverse bacterial and fungal species. Owing to ACCD activity, certain plant associated bacteria help plant to grow under biotic and abiotic stresses by decreasing the level of “stress ethylene” which is inhibitory to plant growth. ACCD breaks down ACC, an immediate precursor of ethylene, to ammonia and α-ketobutyrate, which can be further metabolized by bacteria for their growth. ACC deaminase is an inducible enzyme whose synthesis is induced in the presence of its substrate ACC. This enzyme encoded by gene AcdS is under tight regulation and regulated differentially under different environmental conditions. Regulatory elements of gene AcdS are comprised of the regulatory gene encoding LRP protein and other regulatory elements which are activated differentially under aerobic and anaerobic conditions. The role of some additional regulatory genes such as AcdB or LysR may also be required for expression of AcdS. Phylogenetic analysis of AcdS has revealed that distribution of this gene among different bacteria might have resulted from vertical gene transfer with occasional horizontal gene transfer (HGT). Application of bacterial AcdS gene has been extended by developing transgenic plants with ACCD gene which showed increased tolerance to biotic and abiotic stresses in plants. Moreover, distribution of ACCD gene or its homolog's in a wide range of species belonging to all three domains indicate an alternative role of ACCD in the physiology of an organism. Therefore, this review is an attempt to explore current knowledge of bacterial ACC deaminase mediated physiological effects in plants, mode of enzyme action, genetics, distribution among different species, ecological role of ACCD and, future research avenues to develop transgenic plants expressing foreign AcdS gene to cope with biotic and abiotic stressors. Systemic identification of regulatory circuits

  19. Quantification of cells with specific phenotypes II: determination of CD4 expression level on reconstituted lyophilized human PBMC labelled with anti-CD4 FITC antibody.

    PubMed

    Wang, L; Stebbings, R; Gaigalas, A K; Sutherland, J; Kammel, M; John, M; Roemer, B; Kuhne, M; Schneider, R J; Braun, M; Engel, A; Dikshit, D; Abbasi, F; Marti, G E; Sassi, M; Revel, L; Kim, S K; Baradez, M; Lekishvili, T; Marshall, D; Whitby, L; Jing, W; Ost, V; Vonsky, M; Neukammer, J

    2015-03-01

    This report focuses on the characterization of CD4 expression level in terms of equivalent number of reference fluorophores (ERF). Twelve different flow cytometer platforms across sixteen laboratories were utilized in this study. As a first step the participants were asked to calibrate the fluorescein isothiocyanate (FITC) channel of each flow cytometer using commercially available calibration standard consisting of five populations of microspheres. Each population had an assigned value of equivalent fluorescein fluorophores (EFF denotes a special case of the generic term ERF with FITC as the reference fluorophore). The EFF values were assigned at the National Institute of Standards and Technology (NIST). A surface-labelled lyophilized cell preparation was provided by the National Institute of Biological Standards and Control (NIBSC), using human peripheral blood mononuclear cells (PBMC) pre-labeled with a FITC conjugated anti-CD4 monoclonal antibody. Three PBMC sample vials, provided to each participant, were used for the CD4 expression analysis. The PBMC are purported to have a fixed number of surface CD4 receptors. On the basis of the microsphere calibration, the EFF value of the PBMC samples was measured to characterize the population average CD4 expression level of the PBMC preparations. Both the results of data analysis performed by each participant and the results of centralized analysis of all participants' raw data are reported. Centralized analysis gave a mean EFF value of 22,300 and an uncertainty of 750, corresponding to 3.3% (level of confidence 68%) of the mean EFF value. The next step will entail the measurement of the ERF values of the lyophilized PBMC stained with labels for other fluorescence channels. The ultimate goal is to show that lyophilized PBMC is a suitable biological reference cell material for multicolor flow cytometry and that it can be used to present multicolor flow cytometry measurements in terms of ABC (antibodies bound per cell

  20. Pleural effusion adenosine deaminase: a candidate biomarker to discriminate between Gram-negative and Gram-positive bacterial infections of the pleural space

    PubMed Central

    Li, Ruolin; Wang, Junli; Wang, Xinfeng; Wang, Maoshui

    2016-01-01

    OBJECTIVES: Delay in the treatment of pleural infection may contribute to its high mortality. In this retrospective study, we aimed to evaluate the diagnostic accuracy of pleural adenosine deaminase in discrimination between Gram-negative and Gram-positive bacterial infections of the pleural space prior to selecting antibiotics. METHODS: A total of 76 patients were enrolled and grouped into subgroups according to Gram staining: 1) patients with Gram-negative bacterial infections, aged 53.2±18.6 years old, of whom 44.7% had empyemas and 2) patients with Gram-positive bacterial infections, aged 53.5±21.5 years old, of whom 63.1% had empyemas. The pleural effusion was sampled by thoracocentesis and then sent for adenosine deaminase testing, biochemical testing and microbiological culture. The Mann-Whitney U test was used to examine the differences in adenosine deaminase levels between the groups. Correlations between adenosine deaminase and specified variables were also quantified using Spearman’s correlation coefficient. Moreover, receiver operator characteristic analysis was performed to evaluate the diagnostic accuracy of pleural effusion adenosine deaminase. RESULTS: Mean pleural adenosine deaminase levels differed significantly between Gram-negative and Gram-positive bacterial infections of the pleural space (191.8±32.1 U/L vs 81.0±16.9 U/L, p<0.01). The area under the receiver operator characteristic curve was 0.689 (95% confidence interval: 0.570, 0.792, p<0.01) at the cutoff value of 86 U/L. Additionally, pleural adenosine deaminase had a sensitivity of 63.2% (46.0-78.2%); a specificity of 73.7% (56.9-86.6%); positive and negative likelihood ratios of 2.18 and 0.50, respectively; and positive and negative predictive values of 70.6% and 66.7%, respectively. CONCLUSIONS: Pleural effusion adenosine deaminase is a helpful alternative biomarker for early and quick discrimination of Gram-negative from Gram-positive bacterial infections of the pleural space

  1. Adenosine Deaminase Inhibition Prevents Clostridium difficile Toxin A-Induced Enteritis in Mice ▿

    PubMed Central

    de Araújo Junqueira, Ana Flávia Torquato; Dias, Adriana Abalen Martins; Vale, Mariana Lima; Spilborghs, Graziela Machado Gruner Turco; Bossa, Aline Siqueira; Lima, Bruno Bezerra; Carvalho, Alex Fiorini; Guerrant, Richard Littleton; Ribeiro, Ronaldo Albuquerque; Brito, Gerly Anne

    2011-01-01

    Toxin A (TxA) is able to induce most of the classical features of Clostridium difficile-associated disease in animal models. The objective of this study was to determine the effect of an inhibitor of adenosine deaminase, EHNA [erythro-9-(2-hydroxy-3-nonyl)-adenine], on TxA-induced enteritis in C57BL6 mice and on the gene expression of adenosine receptors. EHNA (90 μmol/kg) or phosphate-buffered saline (PBS) was injected intraperitoneally (i.p.) 30 min prior to TxA (50 μg) or PBS injection into the ileal loop. A2A adenosine receptor agonist (ATL313; 5 nM) was injected in the ileal loop immediately before TxA (50 μg) in mice pretreated with EHNA. The animals were euthanized 3 h later. The changes in the tissue were assessed by the evaluation of ileal loop weight/length and secretion volume/length ratios, histological analysis, myeloperoxidase assay (MPO), the local expression of inducible nitric oxide synthase (NOS2), pentraxin 3 (PTX3), NF-κB, tumor necrosis factor alpha (TNF-α), and interleukin-1β (IL-1β) by immunohistochemistry and/or quantitative reverse transcription-PCR (qRT-PCR). The gene expression profiles of A1, A2A, A2B, and A3 adenosine receptors also were evaluated by qRT-PCR. Adenosine deaminase inhibition, by EHNA, reduced tissue injury, neutrophil infiltration, and the levels of proinflammatory cytokines (TNF-α and IL-1β) as well as the expression of NOS2, NF-κB, and PTX3 in the ileum of mice injected with TxA. ATL313 had no additional effect on EHNA action. TxA increased the gene expression of A1 and A2A adenosine receptors. Our findings show that the inhibition of adenosine deaminase by EHNA can prevent Clostridium difficile TxA-induced damage and inflammation possibly through the A2A adenosine receptor, suggesting that the modulation of adenosine/adenosine deaminase represents an important tool in the management of C. difficile-induced disease. PMID:21115723

  2. Adenosine deaminase inhibition prevents Clostridium difficile toxin A-induced enteritis in mice.

    PubMed

    de Araújo Junqueira, Ana Flávia Torquato; Dias, Adriana Abalen Martins; Vale, Mariana Lima; Spilborghs, Graziela Machado Gruner Turco; Bossa, Aline Siqueira; Lima, Bruno Bezerra; Carvalho, Alex Fiorini; Guerrant, Richard Littleton; Ribeiro, Ronaldo Albuquerque; Brito, Gerly Anne

    2011-02-01

    Toxin A (TxA) is able to induce most of the classical features of Clostridium difficile-associated disease in animal models. The objective of this study was to determine the effect of an inhibitor of adenosine deaminase, EHNA [erythro-9-(2-hydroxy-3-nonyl)-adenine], on TxA-induced enteritis in C57BL6 mice and on the gene expression of adenosine receptors. EHNA (90 μmol/kg) or phosphate-buffered saline (PBS) was injected intraperitoneally (i.p.) 30 min prior to TxA (50 μg) or PBS injection into the ileal loop. A(2A) adenosine receptor agonist (ATL313; 5 nM) was injected in the ileal loop immediately before TxA (50 μg) in mice pretreated with EHNA. The animals were euthanized 3 h later. The changes in the tissue were assessed by the evaluation of ileal loop weight/length and secretion volume/length ratios, histological analysis, myeloperoxidase assay (MPO), the local expression of inducible nitric oxide synthase (NOS2), pentraxin 3 (PTX3), NF-κB, tumor necrosis factor alpha (TNF-α), and interleukin-1β (IL-1β) by immunohistochemistry and/or quantitative reverse transcription-PCR (qRT-PCR). The gene expression profiles of A₁, A(2A), A(2B), and A₃ adenosine receptors also were evaluated by qRT-PCR. Adenosine deaminase inhibition, by EHNA, reduced tissue injury, neutrophil infiltration, and the levels of proinflammatory cytokines (TNF-α and IL-1β) as well as the expression of NOS2, NF-κB, and PTX3 in the ileum of mice injected with TxA. ATL313 had no additional effect on EHNA action. TxA increased the gene expression of A₁ and A(2A) adenosine receptors. Our findings show that the inhibition of adenosine deaminase by EHNA can prevent Clostridium difficile TxA-induced damage and inflammation possibly through the A(2A) adenosine receptor, suggesting that the modulation of adenosine/adenosine deaminase represents an important tool in the management of C. difficile-induced disease.

  3. Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase

    SciTech Connect

    Kamat, S.S.; Swaminathan, S.; Bagaria, A.; Kumaran, D.; Holmes-Hampton, G. P.; Fan, H.; Sali, A.; Sauder, J. M.; Burley, S. K.; Lindahl, P. A.; Raushel, F. M.

    2011-03-22

    Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k{sub cat} = 2.0 s{sup -1}; k{sub cat}/K{sub m} = 2.5 x 10{sup 3} M{sup -1} s{sup -1}). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn{sup 2+} prior to induction, the purified enzyme was substantially more active for the deamination of adenine with kcat and kcat/Km values of 200 s{sup -1} and 5 x 10{sup 5} M{sup -1} s{sup -1}, respectively. The apoenzyme was prepared and reconstituted with Fe{sup 2+}, Zn{sup 2+}, or Mn{sup 2+}. In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe{sup II}/Fe{sup II}]-ADE was oxidized to [Fe{sup III}/Fe{sup III}]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe{sup III}/Fe{sup III}]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Moessbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 {angstrom} resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH-rate profiles, and solvent viscosity were utilized to propose a chemical reaction mechanism and the

  4. Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase

    SciTech Connect

    S Kamat; A Bagaria; D Kumaran; G Holmes-Hampton; H Fan; A Sali; J Sauder; S Burley; P Lindahl; et. al.

    2011-12-31

    Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k{sub cat} = 2.0 s{sup -1}; k{sub cat}/K{sub m} = 2.5 x 10{sup 3} M{sup -1} s{sup -1}). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn{sup 2+} prior to induction, the purified enzyme was substantially more active for the deamination of adenine with k{sub cat} and k{sub cat}/K{sub m} values of 200 s{sup -1} and 5 x 10{sup 5} M{sup -1} s{sup -1}, respectively. The apoenzyme was prepared and reconstituted with Fe{sup 2+}, Zn{sup 2+}, or Mn{sup 2+}. In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe{sup II}/Fe{sup II}]-ADE was oxidized to [Fe{sup III}/Fe{sup III}]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe{sup III}/Fe{sup III}]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Moessbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 {angstrom} resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH-rate profiles, and solvent viscosity were utilized to propose a chemical reaction

  5. Levels at Streamflow Gaging Stations--A CD-ROM Based Training Class

    USGS Publications Warehouse

    Nolan, K. Michael; Jacobson, Nathan; Erickson, Robert; Landon, Stanley

    2003-01-01

    Streamgages record the elevation of the water surface above some reference surface, or datum. This datum is assumed to remain unchanged throughout the life of the gage. However, the elevation of gages and their supporting structures often change over time as a result of earthmovement, floods, ice, and debris. The surveying practice of leveling is used to establish datum for new gage structures and to check for vertical movement of those structures over time. Vertical changes in gage structures can affect stage-discharge relations and, thus, could result in incorrect discharge determinations. Datum checks are used to correct stage-discharge relations and allow the USGS to document gage datum throughout the life of a gage. This training presentation describes methods currently used by the U.S. Geological Survey to run levels at gaging stations. The presentation is narrated, but you control the pace of the presentation. If the computer you are using can view 'MPEG' videos you will be able to take advantage of videos found within the presentation. A test, found at the end of the presentation, can be taken to assess how well you understood the training material. The class is registered as class SW1307 with the National Training Center of the U.S. Geologcial Survey. The presentation was developed using Macromedia Director 8.5(1) and is contained in the file 'WRI-4002.exe', which should auto-launch after the CD-ROM is inserted in the PC. The program only runs on a windows-based personal computer (PC). A sound card and speakers are necessary to take advantage of the narration that accompanies the presentation. Text of narrations is provided, if you are unable to listen to the narrations. Instructions for installing and running the presentation are included in the file ' Intro.html'. The file 'Intro.html' is on the CD-ROM containing the presentation and is available from the presentation's help menu.

  6. The ecto-ATPDase CD39 is involved in the acquisition of the immunoregulatory phenotype by M-CSF-macrophages and ovarian cancer tumor-associated macrophages: Regulatory role of IL-27.

    PubMed

    d'Almeida, Sènan M; Kauffenstein, Gilles; Roy, Charlotte; Basset, Laetitia; Papargyris, Loukas; Henrion, Daniel; Catros, Véronique; Ifrah, Norbert; Descamps, Philippe; Croue, Anne; Jeannin, Pascale; Grégoire, Marc; Delneste, Yves; Tabiasco, Julie

    2016-07-01

    Tumor-associated macrophages (TAM) are immunosuppressive cells that can massively accumulate in the tumor microenvironment. In patients with ovarian cancer, their density is correlated with poor prognosis. Targeting mediators that control the generation or the differentiation of immunoregulatory macrophages represents a therapeutic challenge to overcome tumor-associated immunosuppression. The ectonucleotidase CD39 hydrolyzes ATP into extracellular adenosine that exhibits potent immunosuppressive properties when signaling through the A2A adenosine receptor. We report here that CD14(+) CD163(+) TAM isolated from ovarian cancer patients and macrophages generated in vitro with M-CSF, express high levels of the membrane ectonucleotidase CD39 compared to classically activated macrophages. The CD39 inhibitor POM-1 and adenosine deaminase (ADA) diminished some of the immunosuppressive functions of CD14(high) CD163(high) CD39(high) macrophages, such as IL-10 secretion. We identified the cytokine IL-27, secreted by tumor-infiltrating neutrophils, located close to infiltrating CD163(+) macrophages, as a major rheostat of CD39 expression and consequently, on the acquisition of immunoregulatory properties by macrophages. Accordingly, the depletion of IL-27 downregulated CD39 and PD-L1 expression as well as IL-10 secretion by M-CSF-macrophages. Collectively, these data suggest that CD39, drived by IL-27 and CD115 ligands in ovarian cancer, maintains the immunosuppressive phenotype of TAM. This work brings new information on the acquisition of immunosuppressive properties by tumor-infiltrating macrophages. PMID:27622030

  7. Levels of regulatory T cells CD69(+)NKG2D(+)IL-10(+) are increased in patients with autoimmune thyroid disorders.

    PubMed

    Rodríguez-Muñoz, Ana; Vitales-Noyola, Marlen; Ramos-Levi, Ana; Serrano-Somavilla, Ana; González-Amaro, Roberto; Marazuela, Mónica

    2016-03-01

    Regulatory T (Treg) cells play an important role in the pathogenesis of autoimmune thyroid disorders (AITD). New subsets of CD4(+)CD69(+) and CD4(+)NKG2D(+) T lymphocytes that behave as regulatory cells have been recently reported. The role of these immunoregulatory lymphocytes has not been previously explored in AITD. We analyzed by multi-parametric flow cytometry different Treg cell subsets in peripheral blood from 32 patients with AITD and 19 controls, and in thyroid tissue from seven patients. The suppressive activity was measured by an assay of inhibition of lymphocyte activation. We found a significant increased percentage of CD4(+)CD69(+)IL-10(+), CD4(+)CD69(+)NKG2D(+), and CD4(+)CD69(+)IL-10(+)NKG2D(+) cells, in peripheral blood from GD patients compared to controls. The increase in CD4(+)CD69(+)IL-10(+) and CD4(+)CD69(+)IL-10(+)NKG2D(+) T cells was especially remarkable in patients with active Graves' ophthalmopathy (GO), and a significant positive correlation between GO activity and CD4(+)CD69(+)IL-10(+) or CD4(+)CD69(+)IL-10(+)NKG2D(+) cells was also found. In addition, these cells were increased in patients with a more severe and/or prolonged disease. Thyroid from AITD patients showed an increased proportion of CD69(+) regulatory T cells subpopulations compared to autologous peripheral blood. The presence of CD69(+), NKG2D(+), and IL-10(+) cells was confirmed by immunofluorescence microscopy. In vitro functional assays showed that CD69(+) Treg cells exerted an important suppressive effect on the activation of T effector cells in controls, but not in AITD patients. Our findings suggest that the levels of CD69(+) regulatory lymphocytes are increased in AITD patients, but they are apparently unable to down-modulate the autoimmune response and tissue damage.

  8. The effects of deep level traps on the electrical properties of semi-insulating CdZnTe

    SciTech Connect

    Zha, Gangqiang; Yang, Jian; Xu, Lingyan; Feng, Tao; Wang, Ning; Jie, Wanqi

    2014-01-28

    Deep level traps have considerable effects on the electrical properties and radiation detection performance of high resistivity CdZnTe. A deep-trap model for high resistivity CdZnTe was proposed in this paper. The high resistivity mechanism and the electrical properties were analyzed based on this model. High resistivity CdZnTe with high trap ionization energy E{sub t} can withstand high bias voltages. The leakage current is dependent on both the deep traps and the shallow impurities. The performance of a CdZnTe radiation detector will deteriorate at low temperatures, and the way in which sub-bandgap light excitation could improve the low temperature performance can be explained using the deep trap model.

  9. Enzymatic production of 5'-inosinic acid by AMP deaminase from a newly isolated Aspergillus oryzae.

    PubMed

    Li, Shubo; Chen, Leitao; Hu, Yangjun; Fang, Guohui; Zhao, Mouming; Guo, Yuan; Pang, Zongwen

    2017-02-01

    5'-adenylic acid deaminase (AMP deaminase), an important enzyme for the food industry, can catalyze the irreversible hydrolysis of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia. In this study, a new strain was screened that efficiently produces 3191.6U/g of AMP deaminase at 32°C. After purification, the optimal temperature and pH of the AMP deaminase were found to be 40°C and 6.0, respectively, but it was partially inhibited by Fe(3+), Cu(2+), Al(3+), and Zn(2+). With amplification of the AMP deaminase production system, 6mL of crude enzyme could produce 2.00mg/g of IMP from 2.04mg/g of dried yeast with an 84.8% molar yield after 40min. These results provide a new insight into AMP deaminase production and offer a potential platform for producing 5'-IMP. PMID:27596420

  10. Enzymatic production of 5'-inosinic acid by AMP deaminase from a newly isolated Aspergillus oryzae.

    PubMed

    Li, Shubo; Chen, Leitao; Hu, Yangjun; Fang, Guohui; Zhao, Mouming; Guo, Yuan; Pang, Zongwen

    2017-02-01

    5'-adenylic acid deaminase (AMP deaminase), an important enzyme for the food industry, can catalyze the irreversible hydrolysis of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia. In this study, a new strain was screened that efficiently produces 3191.6U/g of AMP deaminase at 32°C. After purification, the optimal temperature and pH of the AMP deaminase were found to be 40°C and 6.0, respectively, but it was partially inhibited by Fe(3+), Cu(2+), Al(3+), and Zn(2+). With amplification of the AMP deaminase production system, 6mL of crude enzyme could produce 2.00mg/g of IMP from 2.04mg/g of dried yeast with an 84.8% molar yield after 40min. These results provide a new insight into AMP deaminase production and offer a potential platform for producing 5'-IMP.

  11. Baseline CD4 T Cell Level Predicts Recovery Rate after Initiation of ART in HIV Infected Nigerians.

    PubMed

    Adewumi, Olubusuyi M; Odaibo, Georgina N; Olaleye, Olufemi D

    2016-01-01

    The most characteristic immunologic disorder in HIV infection is the progressive loss of CD4 T lymphocytes, thus, it remains the most important and commonly used marker for monitoring of immune status of HIV-infected individuals. This study monitored CD4 T lymphocyte cell dynamics among HIV patients on ART, and consequently defined an optimal baseline level required for enhanced ARV treatment. Ninety-eight (M = 33; F = 65) out of 106 consenting HIV-infected ARV-naïve patients enrolled and monitored for 24 months were considered in the analysis. The patients were classified into four groups based on baseline CD4 T lymphocyte cell levels, and specific parameters were evaluated at interval. Median CD4 T lymphocyte increased from 114 (Range: 6-330) at baseline to highest 357 (Range: 15-1036) cells/μL at 18 months of therapy. Fifty (51.0%), 58(59.2%), 75(76.5%), 69(70.4%), 63(64.3%), and 69(70.4%) doubled their preceding CD4 levels during the 3(rd), 6(th), 9(th), 12(th), 18(th), and 24(th) months of ART, respectively. Maximum 337, 302, 360, and 475 cells/μL of blood were attained by groups commenced on ART with baseline CD4 ≤ 50, 51-100, 101-200, and 201-350 cells/μL of blood, respectively. The results show that higher baseline CD4 T lymphocyte cell level correlates with enhanced restoration and plateau after commencement of ART.

  12. Soluble CD23 Levels are Inversely Associated with Atopy and Parasite-Specific IgE Levels but Not with Polyclonal IgE Levels in People Exposed to Helminth Infection

    PubMed Central

    Rujeni, Nadine; Nausch, Norman; Midzi, Nicholas; Gwisai, Reginald; Mduluza, Takafira; Taylor, David W.; Mutapi, Francisca

    2013-01-01

    Background Protective acquired immunity against helminths and allergic sensitisation are both characterised by high IgE antibody levels. Levels of IgE antibodies are naturally tightly regulated by several mechanisms including binding of the CD23 receptor. Following observations that helminth infections and allergic sensitisation may co-present, the current study aims to investigate the relationship between the soluble CD23 (sCD23) receptor, parasite-specific IgE responses and allergic sensitisation in people exposed to the helminth parasite Schistosoma haematobium. Methods A cohort of 434 participants was recruited in two villages with different levels of S. haematobium infection in Zimbabwe. Serum levels of the 25-kDa fragment of sCD23 were related to levels of schistosome infection intensity, allergen (house dust mite, HDM) and schistosome-specific IgE, total IgE and skin sensitisation to HDM. Results sCD23 levels rose significantly with schistosome infection intensity but declined significantly with schistosome-specific IgE levels. Furthermore, sCD23 levels were negatively associated with skin sensitisation and IgE reactivity against HDM, but showed no relationship with total IgE. Conclusion The results are consistent with the suppression of parasite and allergen-specific IgE levels by sCD23. Further mechanistic studies will determine the relevance of this potential regulatory mechanism in the development of helminth-specific immune responses in atopic individuals. PMID:23689700

  13. A 24-Year Enzyme Replacement Therapy in an Adenosine-deaminase-Deficient Patient.

    PubMed

    Tartibi, Hana M; Hershfield, Michael S; Bahna, Sami L

    2016-01-01

    Severe combined immunodeficiency (SCID) is a fatal childhood disease unless immune reconstitution is performed early in life, with either hematopoietic stem cell transplantation or gene therapy. One of its subtypes is caused by adenosine deaminase (ADA) enzyme deficiency, which leads to the accumulation of toxic metabolites that impair lymphocyte development and function. With the development of polyethylene glycol-conjugated adenosine deaminase (PEG-ADA) enzyme replacement therapy, many ADA-deficient children with SCID who could not receive a hematopoietic stem cell transplantation or gene therapy survived and had longer and healthier lives. We report a 24-year course of treatment in a patient who was diagnosed with ADA deficiency at 4 months of age. The patient was treated with PEG-ADA, which was the only therapy available for him. The patient's plasma ADA level was regularly monitored and the PEG-ADA dose adjusted accordingly. This treatment has resulted in near-normalization of lymphocyte counts, and his clinical course has been associated with only minor to moderate infections. Thus far, he has had no manifestations of autoimmune or lymphoproliferative disorders. This patient is among the longest to be maintained on PEG-ADA enzyme replacement therapy. PMID:26684479

  14. Purification and properties of porphobilinogen deaminase from Arabidopsis thaliana.

    PubMed Central

    Jones, R M; Jordan, P M

    1994-01-01

    Porphobilinogen deaminase (EC 4.3.1.8) has been purified to homogeneity (16,000-fold) from the plant Arabidopsis thaliana in yields of 8%. The deaminase is a monomer of M(r) 35,000, as shown by SDS/PAGE, and 31,000, using gel-filtration chromatography. The pure enzyme has a Vmax. of 4.5 mumol/h per mg and a Km of 17 +/- 4 microM. Determination of the pI and pH optimum revealed values of 5.2 and 8.0 respectively. The sequence of the N-terminus was found to be NH2-XVAVEQKTRTAI. The deaminase is heat-stable up to 70 degrees C and is inhibited by NH3 and hydroxylamine. The enzyme is inactivated by arginine-, histidine- and lysine-specific reagents. Incubation with the substrate analogue and suicide inhibitor, 2-bromoporphobilinogen, results in chain termination and in inactivation. Images Figure 1 PMID:8192681

  15. Endogenous tumor-reactive CD8+ T cells are differentiated effector cells expressing high levels of CD11a and PD-1 but are unable to control tumor growth

    PubMed Central

    Liu, Xin; Gibbons, Rachel M; Harrington, Susan M; Krco, Christopher J; Markovic, Svetomir N; Kwon, Eugene D; Dong, Haidong

    2013-01-01

    Immunotherapies aimed at enhancing natural or endogenous antitumor T-cell immunity in patients affected by advanced malignancies are currently being implemented in the clinic with promising results. In order to optimize therapeutic protocols and monitor the effectiveness of such therapies, reliable biomarkers are needed. We used CD11a, an integrin that is upregulated on the surface of effector and memory CD8+ T cells, and PD-1, an immunoregulatory receptor expressed by activated T cells, as biomarkers to identify, quantify and monitor endogenous tumor-reactive cytotoxic T lymphocytes (CTLs) in two mouse tumor models and in the peripheral blood of 12 patients affected by Stage IV melanoma. High expression levels of CD11a and PD-1 were detected among CD8+ T cells residing within primary and metastatic murine tumor sites, as well as in spontaneous murine breast cancer tissues. In the peripheral blood of melanoma patients, tumor antigen-specific CD8+ T cells were associated with a population of CD11ahigh CD8+ T cells that co-expressed high levels of PD-1. Healthy donors exhibited a comparatively much lower frequency of such PD-1+CD11ahighCD8+ T cells. Phenotypic analyses demonstrated that CD11ahighCD8+ T cells are proliferating (Ki67+) and activated (CD62L-CD69+). Increased CD11ahighCD8+ T cells and delayed tumor growth were observed in PD-1 deficient mice, suggesting that the antitumor effector functions of CD8+ T cells is compromised by an elevated expression of PD-1. The CD11ahighCD8+ T-cell population expresses high levels of PD-1 and presumably constitutes the cellular target of PD-1 blockade therapy. The expression level of CD11a and PD-1 by CD8+ T cells may therefore represent a novel biomarker to identify and monitor endogenous tumor-reactive CTLs. This may not only provide an immunological readout for evaluating the efficacy of immunotherapy but also contribute to the selection of cancer patients who are likely to benefit from anti-PD-1 therapy. PMID:23894697

  16. Cell membrane CD44v6 levels in squamous cell carcinoma of the lung: association with high cellular proliferation and high concentrations of EGFR and CD44v5.

    PubMed

    Ruibal, Álvaro; Aguiar, Pablo; Del Río, María Carmen; Nuñez, Matilde Isabel; Pubul, Virginia; Herranz, Michel

    2015-01-01

    Membranous CD44v6 levels in tumors and surrounding samples obtained from 94 patients with squamous cell lung carcinomas were studied and compared to clinical stage, cellular proliferation, membranous CD44v5 levels, epidermal growth factor receptor EGFR and cytoplasmatic concentrations of CYFRA 21.1. CD44v6 positive values were observed in 33/38 non-tumor samples and in 76/94 tumor samples, but there were not statistically significant differences between both subgroups. In CD44v6 positive tumor samples, CD44v6 was not associated with clinical stage, histological grade, ploidy and lymph node involvement, but significant association was found with high cellular proliferation. Likewise, CD44v6 positive tumors had significantly higher levels of EGFR and CD44v5. In patients with squamous cell lung carcinomas and clinical stage I, positive CD44v6 cases were associated with the same parameters. Furthermore, positive CD44v5 squamous tumors were associated significantly with histological grade III and lower levels of CYFRA21.1. Our findings support the value of CD44v6 as a possible indicator of poor outcome in patients with squamous lung carcinomas. PMID:25809603

  17. 1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing rhizobacteria protect Ocimum sanctum plants during waterlogging stress via reduced ethylene generation.

    PubMed

    Barnawal, Deepti; Bharti, Nidhi; Maji, Deepamala; Chanotiya, Chandan Singh; Kalra, Alok

    2012-09-01

    Ocimum sanctum grown as rain-fed crop, is known to be poorly adapted to waterlogged conditions. Many a times the crop suffers extreme damages because of anoxia and excessive ethylene generation due to waterlogging conditions present under heavy rain. The usefulness of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing plant growth promoting rhizobacteria was investigated under waterlogging stress. The comparison of herb yield and stress induced biochemical changes of waterlogged and non-waterlogged plants with and without ACC deaminase-containing microbiological treatments were monitored in this study. Ten plant growth promoting rhizobacteria strains containing ACC-deaminase were isolated and characterized. Four selected isolates Fd2 (Achromobacter xylosoxidans), Bac5 (Serratia ureilytica), Oci9 (Herbaspirillum seropedicae) and Oci13 (Ochrobactrum rhizosphaerae) had the potential to protect Ocimum plants from flood induced damage under waterlogged glass house conditions. Pot experiments were conducted to evaluate the potential of these ACC deaminase-containing selected strains for reducing the yield losses caused by waterlogging conditions. Bacterial treatments protected plants from waterlogging induced detrimental changes like stress ethylene production, reduced chlorophyll concentration, higher lipid peroxidation, proline concentration and reduced foliar nutrient uptake. Fd2 (A. xylosoxidans) induced maximum waterlogging tolerance as treated waterlogged plants recorded maximum growth and herb yield (46.5% higher than uninoculated waterlogged plants) with minimum stress ethylene levels (53% lower ACC concentration as compared to waterlogged plants without bacterial inoculation) whereas under normal non-waterlogged conditions O. rhizosphaerae was most effective in plant growth promotion. PMID:22846334

  18. 1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing rhizobacteria protect Ocimum sanctum plants during waterlogging stress via reduced ethylene generation.

    PubMed

    Barnawal, Deepti; Bharti, Nidhi; Maji, Deepamala; Chanotiya, Chandan Singh; Kalra, Alok

    2012-09-01

    Ocimum sanctum grown as rain-fed crop, is known to be poorly adapted to waterlogged conditions. Many a times the crop suffers extreme damages because of anoxia and excessive ethylene generation due to waterlogging conditions present under heavy rain. The usefulness of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing plant growth promoting rhizobacteria was investigated under waterlogging stress. The comparison of herb yield and stress induced biochemical changes of waterlogged and non-waterlogged plants with and without ACC deaminase-containing microbiological treatments were monitored in this study. Ten plant growth promoting rhizobacteria strains containing ACC-deaminase were isolated and characterized. Four selected isolates Fd2 (Achromobacter xylosoxidans), Bac5 (Serratia ureilytica), Oci9 (Herbaspirillum seropedicae) and Oci13 (Ochrobactrum rhizosphaerae) had the potential to protect Ocimum plants from flood induced damage under waterlogged glass house conditions. Pot experiments were conducted to evaluate the potential of these ACC deaminase-containing selected strains for reducing the yield losses caused by waterlogging conditions. Bacterial treatments protected plants from waterlogging induced detrimental changes like stress ethylene production, reduced chlorophyll concentration, higher lipid peroxidation, proline concentration and reduced foliar nutrient uptake. Fd2 (A. xylosoxidans) induced maximum waterlogging tolerance as treated waterlogged plants recorded maximum growth and herb yield (46.5% higher than uninoculated waterlogged plants) with minimum stress ethylene levels (53% lower ACC concentration as compared to waterlogged plants without bacterial inoculation) whereas under normal non-waterlogged conditions O. rhizosphaerae was most effective in plant growth promotion.

  19. Additions to the spectra and energy levels of the zinc-like ions Y X-Cd XIX

    NASA Astrophysics Data System (ADS)

    Litzén, Ulf; Hansson, Anna

    1989-10-01

    Transitions from 4p4d 1F and 3F have been identified in the spectra Y X-Cd XIX emitted from laserproduced plasmas. Energy levels have been derived, and the term structure has been studied with special emphasis on the 4s4f-4p4d configuration interaction.

  20. Student Interactions with CD-ROM Storybooks: A Look at Potential Relationships between Multiple Intelligence Strengths and Levels of Interaction

    ERIC Educational Resources Information Center

    Huffman, Celia A.

    2012-01-01

    This study looked at the potential relationship that may exist between students' intelligence strengths, in particular their spatial and kinesthetic strengths, and their combined cognitive and metacognitive levels of interaction with a CD-ROM storybook. The multiple intelligence strengths of a sample of students, measured via the MIDAS/My…

  1. Pollution of mycological surfaces in hospital emergency departments correlates positively with blood NKT CD3+ 16+ 56+ and negatively with CD4+ cell levels of their staff

    PubMed Central

    Suska, Milena; Kiepura, Anna; Winnicka, Izabela; Leszczyński, Paweł; Bielawska-Drózd, Agata; Cieślik, Piotr; Kubiak, Leszek; Depczyńska, Daria; Brewczyńska, Aleksandra; Skopińska-Różewska, Ewa; Kocik, Janusz

    2016-01-01

    The aim of the present study was the assessment of the putative influence of yeast and filamentous fungi in healthcare and control (office) workplaces (10 of each kind) on immune system competence measured by NK (natural killer), CD4+, and NKT (natural killer T lymphocyte) cell levels in the blood of the personnel employed at these workplaces. Imprints from floors and walls were collected in winter. The blood was taken in spring the following year, from 40 men, 26 to 53 years old, healthcare workers of hospital emergency departments (HED), who had been working for at least five years in their current positions, and from 36 corresponding controls, working in control offices. Evaluation of blood leukocyte subpopulations was done by flow cytometry. The qualitative analysis of the surface samples revealed a prevalence of strains belonging to Aspergillus spp. and Penicillium spp. genus. There was no statistically significant difference between the level of NKT; however, the percentage of NK cells was lower in the blood of HED workers than in the blood of offices personnel. Spearman analysis revealed the existence of positive correlation (r = 0.4677, p = 0.002) between the total CFU/25 cm2 obtained by imprinting method from walls and floors of HED and the percentage of NKT (CD3+16+56+) lymphocytes collected from the blood of their personnel, and negative correlation (r = –0. 3688, p = 0.019) between this parameter of fungal pollution and the percentage of CD4+ lymphocytes in the blood of HED staff. No other correlations were found. PMID:27095925

  2. Pollution of mycological surfaces in hospital emergency departments correlates positively with blood NKT CD3(+) 16(+) 56(+) and negatively with CD4(+) cell levels of their staff.

    PubMed

    Suska, Milena; Lewicki, Sławomir; Kiepura, Anna; Winnicka, Izabela; Leszczyński, Paweł; Bielawska-Drózd, Agata; Cieślik, Piotr; Kubiak, Leszek; Depczyńska, Daria; Brewczyńska, Aleksandra; Skopińska-Różewska, Ewa; Kocik, Janusz

    2016-01-01

    The aim of the present study was the assessment of the putative influence of yeast and filamentous fungi in healthcare and control (office) workplaces (10 of each kind) on immune system competence measured by NK (natural killer), CD4(+), and NKT (natural killer T lymphocyte) cell levels in the blood of the personnel employed at these workplaces. Imprints from floors and walls were collected in winter. The blood was taken in spring the following year, from 40 men, 26 to 53 years old, healthcare workers of hospital emergency departments (HED), who had been working for at least five years in their current positions, and from 36 corresponding controls, working in control offices. Evaluation of blood leukocyte subpopulations was done by flow cytometry. The qualitative analysis of the surface samples revealed a prevalence of strains belonging to Aspergillus spp. and Penicillium spp. genus. There was no statistically significant difference between the level of NKT; however, the percentage of NK cells was lower in the blood of HED workers than in the blood of offices personnel. Spearman analysis revealed the existence of positive correlation (r = 0.4677, p = 0.002) between the total CFU/25 cm(2) obtained by imprinting method from walls and floors of HED and the percentage of NKT (CD3(+)16(+)56(+)) lymphocytes collected from the blood of their personnel, and negative correlation (r = -0. 3688, p = 0.019) between this parameter of fungal pollution and the percentage of CD4(+) lymphocytes in the blood of HED staff. No other correlations were found.

  3. Increased serum levels of soluble l-selectin (CD62L) in patients with active systemic lupus erythematosus (SLE)

    PubMed Central

    Font, J; Pizcueta, P; Ramos-Casals, M; Cervera, R; García-Carrasco, M; Navarro, M; Ingelmo, M; Engel, P

    2000-01-01

    The adhesion molecule l-selectin (CD62L) mediates lymphocyte recirculation and leucocyte rolling on vascular endothelium at sites of inflammation. Serum levels of soluble l-selectin (sl-selectin) were measured in patients with SLE in order to relate these levels to clinical activity and immunological parameters. An ELISA was used to detect the soluble form of human l-selectin (CD62L) in 42 patients with SLE and in 33 healthy individuals. The mean ± s.e.m. values of sl-selectin were 1285 ± 121 ng/ml for patients with SLE and 986 ± 180 ng/ml for healthy blood donors, but there was no significant difference. When patients with active SLE were analysed, higher levels of circulating sl-selectin were found when compared with patients without activity (1497 ± 167 ng/ml versus 941 ± 150 ng/ml; P = 0.028). We found a significant correlation between the levels of sl-selectin and of dsDNA antibodies (r = 0.36, P = 0.044) and between levels of sl-selectin and SLE Disease Activity Index (SLEDAI) score (r = 0.42, P = 0.003). Patients with active SLE studied cross-sectionally showed significant elevations of sl-selectin (CD62L) compared with controls. Thus, the levels of this soluble adhesion molecule correlated with active disease and levels of anti-dsDNA antibodies. PMID:10606979

  4. Numerical modeling of HgCdTe solidification: effects of phase diagram double-diffusion convection and microgravity level

    NASA Astrophysics Data System (ADS)

    Bune, Andris V.; Gillies, Donald C.; Lehoczky, Sandor L.

    1997-07-01

    A numerical model of HgCdTe solidification was implemented using finite the element code FIDAP. Model verification was done using both experimental data and numerical test problems. The model was used to eluate possible effects of double- diffusion convection in molten material, and microgravity level on concentration distribution in the solidified HgCdTe. Particular attention was paid to incorporation of HgCdTe phase diagram. It was found, that below a critical microgravity amplitude, the maximum convective velocity in the melt appears virtually independent on the microgravity vector orientation. Good agreement between predicted interface shape and an interface obtained experimentally by quenching was achieved. The results of numerical modeling are presented in the form of video film.

  5. Human trophoblast cells induced MDSCs from peripheral blood CD14+ myelomonocytic cells via elevated levels of CCL2

    PubMed Central

    Zhang, Yun; Qu, Daiwei; Sun, Jintang; Zhao, Lei; Wang, Qingjie; Shao, Qianqian; Kong, Beihua; Zhang, Yun; Qu, Xun

    2016-01-01

    Successful human pregnancy requires the maternal immune system to recognize and tolerate the semi-allogeneic fetus. Myeloid-derived suppressor cells (MDSCs), which are capable of inhibiting T-cell responses, are highly increased in the early stages of pregnancy. Although recent reports indicate a role for MDSCs in fetal–maternal tolerance, little is known about the expansion of MDSCs during pregnancy. In the present study, we demonstrated that the trophoblast cell line HTR8/SVneo could instruct peripheral CD14+ myelomonocytic cells toward a novel subpopulation of MDSCs, denoted as CD14+HLA-DR−/low cells, with suppressive activity and increased expression of IDO1, ARG-1, and COX2. After interaction with HTR8/SVneo cells, CD14+ myelomonocytic cells secrete high levels of CCL2, promoting the expression of signal transducer and activator of transcription 3. We utilized a neutralizing monoclonal antibody to reveal the prominent role of CCL2 in the induction of CD14+HLA-DR−/low MDSCs. In combination, the results of the present study support a novel role for the cross-talk between the trophoblast cell line HTR8/SVneo and maternal CD14+ myelomonocytic cells in initiating MDSCs induction, prompting a tolerogenic immune response to ensure a successful pregnancy. PMID:26027727

  6. Circulating CD36 and fractalkine levels are associated with vulnerable plaque progression in patients with unstable angina pectoris.

    PubMed

    Li, Rui Jian; Yang, Ming; Li, Ji Fu; Xue, Li; Chen, Yu Guo; Chen, Wen Qiang

    2014-11-01

    The chemokine, fractalkine, independently enhances the vulnerability of coronary atherosclerotic plaques. The present study investigated the combined effects of CD36 and fractalkine on coronary plaque progression in patients with unstable angina pectoris. In the present study, 120 unstable angina pectoris patients undergoing coronary angiography and intravascular ultrasound were divided into two groups: an intermediate lesion group (lumen diameter stenosis 50-70%, 80 patients) and a severe lesion group (at least one lesion with lumen diameter stenosis > 70%, 40 patients). The control group consisted of 40 healthy age- and sex-matched subjects. Concentrations of CD36 and fractalkine were measured by enzyme-linked immunosorbent assay. Major adverse cardiovascular events were monitored over a 2-year follow up. Intravascular ultrasound showed that patients with severe lesions had more calcified and mixed plaques, and a larger plaque area and plaque burden than patients with intermediate lesions (P < 0.05-0.01). More patients with severe lesions underwent stent deployment (P < 0.05) than those with intermediate lesions. CD36 and fractalkine concentrations were significantly higher in the severe lesion patients (P < 0.05), and both had significant positive correlations (P < 0.05) with the plaque burden of atherosclerotic lesions. Using the matched nested case-control study, we found that CD36 and fractalkine levels were higher in patients with recurrent major adverse cardiovascular events than controls (P < 0.05). In conclusion, CD36 and fractalkine both promote, and might synergistically enhance, the progression of coronary atherosclerotic plaques.

  7. Bacterial Modulation of Plant Ethylene Levels

    PubMed Central

    Gamalero, Elisa; Glick, Bernard R.

    2015-01-01

    A focus on the mechanisms by which ACC deaminase-containing bacteria facilitate plant growth.Bacteria that produce the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, when present either on the surface of plant roots (rhizospheric) or within plant tissues (endophytic), play an active role in modulating ethylene levels in plants. This enzyme activity facilitates plant growth especially in the presence of various environmental stresses. Thus, plant growth-promoting bacteria that express ACC deaminase activity protect plants from growth inhibition by flooding and anoxia, drought, high salt, the presence of fungal and bacterial pathogens, nematodes, and the presence of metals and organic contaminants. Bacteria that express ACC deaminase activity also decrease the rate of flower wilting, promote the rooting of cuttings, and facilitate the nodulation of legumes. Here, the mechanisms behind bacterial ACC deaminase facilitation of plant growth and development are discussed, and numerous examples of the use of bacteria with this activity are summarized. PMID:25897004

  8. Characterization of lead-resistant and ACC deaminase-producing endophytic bacteria and their potential in promoting lead accumulation of rape.

    PubMed

    Zhang, Yan-feng; He, Lin-yan; Chen, Zhao-jin; Zhang, Wen-hui; Wang, Qing-ya; Qian, Meng; Sheng, Xia-fang

    2011-02-28

    Forty-nine lead (Pb)-resistant endophytic bacteria were isolated from metal-tolerant Commelina communis plants grown on lead and zinc mine tailing, of which, seven 1-aminocyclopropane-1-carboxylate (ACC) deaminase-producing endophytic bacteria were initially obtained and characterized with respect to heavy metal resistance and production of ACC deaminase, indole-3-acetic acid (IAA) as well as siderophores. Two isolates (Q2BJ2 and Q2BG1) showing higher ACC deaminase activity were evaluated for promoting plant growth and Pb uptake of rape grown in quartz sand containing 0 and 100 mg kg(-1) of Pb in pot experiments. The seven Pb-resistant and ACC deaminase-producing endophytic bacterial isolates were found to exhibit different multiple heavy metal resistance characteristics and to show different levels of ACC deaminase activity (ranging from 12.8 μM α-KB mg(-1) h(-1) to 121 μM α-KB mg(-1) h(-1)). Among the seven isolates, six isolates produced indole acetic acid, whilst five isolates produced siderophores. In experiments involving rape plants grown in quartz sand containing 100 mg kg(-1) of Pb, inoculation with the isolates resulted in the increased dry weights of above-ground tissues (ranging from 39% to 71%) and roots (ranging from 35% to 123%) compared to the uninoculated control. Increases in above-ground tissue Pb contents of rape cultivated in 100 mg kg(-1) of Pb-contaminated substrates varied from 58% to 62% in inoculated-rape plants compared to the uninoculated control.

  9. Characterization of ACC deaminase-producing endophytic bacteria isolated from copper-tolerant plants and their potential in promoting the growth and copper accumulation of Brassica napus.

    PubMed

    Zhang, Yan-Feng; He, Lin-Yan; Chen, Zhao-Jin; Wang, Qing-Ya; Qian, Meng; Sheng, Xia-Fang

    2011-03-01

    One hundred Cu-resistant-endophytic bacteria were isolated from Cu-tolerant plants grown on Cu mine wasteland, of which, eight Cu-resistant and 1-aminocyclopropane-1-carboxylate (ACC) deaminase-producing endophytic bacteria were obtained based on the ACC deaminase activity of the bacteria and characterized with respect to metal resistance, production of ACC deaminase, indole-3-acetic acid (IAA) as well as siderophores and mineral phosphate solubilization. Ralstonia sp. J1-22-2, Pantoea agglomerans Jp3-3, and Pseudomonas thivervalensis Y1-3-9 with higher ACC deaminase activity (ranging from 213 to 370 μM α-ketobutyrate mg(-1)h(-1)) were evaluated for promoting plant growth and Cu uptake of rape grown in quartz sand containing 0, 2.5, and 5 mg kg(-1) of Cu in pot experiments. The eight bacteria were found to exhibit different multiple heavy metal resistance characteristics, to show different levels of ACC deaminase activity and to produce indole acetic acid. Seven bacteria produced siderophores and solubilized inorganic phosphate. Pot experiments showed that inoculation with the strains (J1-22-2, Jp3-3, and Y1-3-9) was found to increase the biomass of rape. Increases in above-ground tissue Cu contents of rape cultivated in 2.5 and 5 mg kg(-1) of Cu-contaminated substrates varied from 9% to 31% and from 3 to 4-fold respectively in inoculated-rape plants compared to the uninoculated control. The maximum Cu uptake of rape was observed after inoculation with P. agglomerans Jp3-3. The results show that metal-resistant and plant growth promoting endophytic bacteria play an important role in plant growth and Cu uptake which may provide a new endophytic bacterial-assisted phytoremediation of Cu-contaminated environment. PMID:21315404

  10. Characterization of lead-resistant and ACC deaminase-producing endophytic bacteria and their potential in promoting lead accumulation of rape.

    PubMed

    Zhang, Yan-feng; He, Lin-yan; Chen, Zhao-jin; Zhang, Wen-hui; Wang, Qing-ya; Qian, Meng; Sheng, Xia-fang

    2011-02-28

    Forty-nine lead (Pb)-resistant endophytic bacteria were isolated from metal-tolerant Commelina communis plants grown on lead and zinc mine tailing, of which, seven 1-aminocyclopropane-1-carboxylate (ACC) deaminase-producing endophytic bacteria were initially obtained and characterized with respect to heavy metal resistance and production of ACC deaminase, indole-3-acetic acid (IAA) as well as siderophores. Two isolates (Q2BJ2 and Q2BG1) showing higher ACC deaminase activity were evaluated for promoting plant growth and Pb uptake of rape grown in quartz sand containing 0 and 100 mg kg(-1) of Pb in pot experiments. The seven Pb-resistant and ACC deaminase-producing endophytic bacterial isolates were found to exhibit different multiple heavy metal resistance characteristics and to show different levels of ACC deaminase activity (ranging from 12.8 μM α-KB mg(-1) h(-1) to 121 μM α-KB mg(-1) h(-1)). Among the seven isolates, six isolates produced indole acetic acid, whilst five isolates produced siderophores. In experiments involving rape plants grown in quartz sand containing 100 mg kg(-1) of Pb, inoculation with the isolates resulted in the increased dry weights of above-ground tissues (ranging from 39% to 71%) and roots (ranging from 35% to 123%) compared to the uninoculated control. Increases in above-ground tissue Pb contents of rape cultivated in 100 mg kg(-1) of Pb-contaminated substrates varied from 58% to 62% in inoculated-rape plants compared to the uninoculated control. PMID:21227577

  11. Biosynthesis of riboflavin. Characterization of the product of the deaminase.

    PubMed

    Nielsen, P; Bacher, A

    1981-12-15

    The 2'5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate deaminase was partially purified from cell extracts of Candida guilliermondii ATCC 9058. The enzyme requires Mg2+ for activity. Maximal activity was observed at pH 7,3. The enzyme converts its substrate, 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate, to 2,5-diamino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate. This labile compound was treated with diacetyl and the resulting 6,7-dimethyl-8-ribityllumazine 5'-phosphate was identified by comparison with a synthetic sample. PMID:7317443

  12. New Insights into 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase Phylogeny, Evolution and Ecological Significance

    PubMed Central

    Nascimento, Francisco X.; Rossi, Márcio J.; Soares, Cláudio R. F. S.; McConkey, Brendan J.; Glick, Bernard R.

    2014-01-01

    The main objective of this work is the study of the phylogeny, evolution and ecological importance of the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, the activity of which represents one of the most important and studied mechanisms used by plant growth–promoting microorganisms. The ACC deaminase gene and its regulatory elements presence in completely sequenced organisms was verified by multiple searches in diverse databases, and based on the data obtained a comprehensive analysis was conducted. Strain habitat, origin and ACC deaminase activity were taken into account when analyzing the results. In order to unveil ACC deaminase origin, evolution and relationships with other closely related pyridoxal phosphate (PLP) dependent enzymes a phylogenetic analysis was also performed. The data obtained show that ACC deaminase is mostly prevalent in some Bacteria, Fungi and members of Stramenopiles. Contrary to previous reports, we show that ACC deaminase genes are predominantly vertically inherited in various bacterial and fungal classes. Still, results suggest a considerable degree of horizontal gene transfer events, including interkingdom transfer events. A model for ACC deaminase origin and evolution is also proposed. This study also confirms the previous reports suggesting that the Lrp-like regulatory protein AcdR is a common mechanism regulating ACC deaminase expression in Proteobacteria, however, we also show that other regulatory mechanisms may be present in some Proteobacteria and other bacterial phyla. In this study we provide a more complete view of the role for ACC deaminase than was previously available. The results show that ACC deaminase may not only be related to plant growth promotion abilities, but may also play multiple roles in microorganism's developmental processes. Hence, exploring the origin and functioning of this enzyme may be the key in a variety of important agricultural and biotechnological applications. PMID:24905353

  13. HIV-1 Vif Versus the APOBEC3 Cytidine Deaminases: An Intracellular Duel Between Pathogen and Host Restriction Factors

    PubMed Central

    Wissing, Silke; Galloway, Nicole L. K.; Greene, Warner C.

    2010-01-01

    The Vif protein of HIV is essential for the effective propagation of this pathogenic retrovirus in vivo. Vif acts by preventing virion encapsidation of two potent antiviral factors, the APOBEC3G and APOBEC3F cytidine deaminases. Decreased encapsidation in part involves Vif-mediated recruitment of a ubiquitin E3 ligase complex that promotes polyubiquitylation and proteasome-mediated degradation of APOBEC3G/F. The resultant decline in intracellular levels of these enzymes leads to decreased encapsidation of APOBECG/F into budding virions. This review discusses recent advances in our understanding of the dynamic interplay of Vif with the antiviral APOBEC3 enzymes. PMID:20538015

  14. HIV-1 Vif versus the APOBEC3 cytidine deaminases: an intracellular duel between pathogen and host restriction factors.

    PubMed

    Wissing, Silke; Galloway, Nicole L K; Greene, Warner C

    2010-10-01

    The Vif protein of HIV is essential for the effective propagation of this pathogenic retrovirus in vivo. Vif acts by preventing virion encapsidation of two potent antiviral factors, the APOBEC3G and APOBEC3F cytidine deaminases. Decreased encapsidation in part involves Vif-mediated recruitment of a ubiquitin E3 ligase complex that promotes polyubiquitylation and proteasome-mediated degradation of APOBEC3G/F. The resultant decline in intracellular levels of these enzymes leads to decreased encapsidation of APOBECG/F into budding virions. This review discusses recent advances in our understanding of the dynamic interplay of Vif with the antiviral APOBEC3 enzymes. PMID:20538015

  15. The Severity of Visceral Leishmaniasis Correlates with Elevated Levels of Serum IL-6, IL-27 and sCD14

    PubMed Central

    dos Santos, Priscila L.; de Oliveira, Fabrícia A.; Santos, Micheli Luize B.; Cunha, Luana Celina S.; Lino, Michelle T. B.; de Oliveira, Michelle F. S.; Bomfim, Manuela O. M.; Silva, Angela Maria; de Moura, Tatiana R.; de Jesus, Amélia R.; Duthie, Malcolm S.; Reed, Steven G.; de Almeida, Roque P.

    2016-01-01

    Background Visceral leishmaniasis (VL) is a severe disease caused by infection with protozoa of the genus Leishmania. Classic VL is characterized by a systemic infection of phagocytic cells and an intense activation of the inflammatory response. It is unclear why 90% of infected individuals do not develop the disease while a minority develop the classical form. Furthermore, among those that develop disease, a small group progresses to more severe form that is unresponsive to treatment. The presence of inflammatory mediators in serum could theoretically help to control the infection. However, there is also a release of anti-inflammatory mediators that could interfere with the control of parasite multiplication. In this study, we took advantage of the spectrum of outcomes to test the hypothesis that the immune profile of individuals infected with Leishmania (L.) infantum is associated with the development and severity of disease. Methodology/Principal Findings Sera from patients with confirmed diagnosis of VL were evaluated for the presence of numerous molecules, and levels compared with healthy control and asymptomatic infected individuals. Conclusions/Principal Findings Although differences were not observed in LPS levels, higher levels of sCD14 were detected in VL patients. Our data suggest that L. infantum may activate the inflammatory response via CD14, stimulating a generalized inflammatory response with production of several cytokines and soluble molecules, including IFN-γ, IL-27, IL-10, IL-6 and sCD14. These molecules were strongly associated with hepatosplenomegaly, neutropenia and thrombocytopenia. We also observed that IL-6 levels greater than 200 pg/ml were strongly associated with death. Together our data reinforce the close relationship of IFN-γ, IL-10, IL-6, TNF-α and IL-27 in the immune dynamics of VL and suggest the direct participation of sCD14 in the activation of the immune response against L. infantum. PMID:26814478

  16. Intracellular localization of differentially regulated RNA-specific adenosine deaminase isoforms in inflammation.

    PubMed

    Yang, Jing-Hua; Nie, Yongzhan; Zhao, Qingchuan; Su, Yingjun; Pypaert, Marc; Su, Haili; Rabinovici, Reuven

    2003-11-14

    Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional process that amplifies the repertoire of protein production. Recently, the induction of this process through up-regulation of the editing enzyme RNA-specific adenosine deaminase 1 (ADAR1) was documented during acute inflammation. Here we report that the inflammation-induced up-regulation of ADAR1 involves differential production and intracellular localization of several isoforms with distinct RNA-binding domains and localization signals. These include the full-length ADAR1 (p150) and two functionally active short isoforms (p80 and p110). ADAR1 p80 starts at a methionine 519 (M519) due to alternative splicing in exon 2, which deletes the putative nuclear localization signal, the Z-DNA binding domain, and the entire RNA binding domain I. ADAR1 p110 is the mouse homologue of the human ADAR1 110-kDa variant (M246), which retains the second half of the Z-DNA binding domain, all RNA binding domains, and the deaminase domain. Additional variations are found in the third RNA binding domain of ADAR1; they are differentially regulated during inflammation, generating isoforms with different levels of activities. Studies in several cell types transfected with ADAR1-EGFP chimeras demonstrated that the p150 and p80 variants are localized in the cytoplasm and nucleolus, respectively. In agreement with this observation, endogenous ADAR1 was identified in the cytoplasm and nucleolus of mouse splenocytes and HeLa cells. Since the ADAR1 variants are differentially regulated during acute inflammation, it suggests that the localization of these variants and of A-to-I RNA editing in the cytoplasm, nucleus, and nucleolus is intracellularly reorganized in response to inflammatory stimulation. PMID:12954622

  17. Critical role of activation induced cytidine deaminase in experimental autoimmune encephalomyelitis.

    PubMed

    Sun, Yonglian; Peng, Ivan; Senger, Kate; Hamidzadeh, Kajal; Reichelt, Mike; Baca, Miriam; Yeh, Ronald; Lorenzo, Maria N; Sebrell, Andrew; Dela Cruz, Christopher; Tam, Lucinda; Corpuz, Racquel; Wu, Jiansheng; Sai, Tao; Roose-Girma, Merone; Warming, Søren; Balazs, Mercedesz; Gonzalez, Lino C; Caplazi, Patrick; Martin, Flavius; Devoss, Jason; Zarrin, Ali A

    2013-03-01

    Multiple Sclerosis (MS) is a neurodegenerative autoimmune disorder caused by chronic inflammation and demyelination within the central nervous system (CNS). Clinical studies in MS patients have demonstrated efficacy with B cell targeted therapies such as anti-CD20. However, the exact role that B cells play in the disease process is unclear. Activation Induced cytidine deaminase (AID) is an essential enzyme for the processes of antibody affinity maturation and isotype switching. To evaluate the impact of affinity maturation and isotype switching, we have interrogated the effect of AID-deficiency in an animal model of MS. Here, we show that the severity of experimental autoimmune encephalomyelitis (EAE) induced by the extracellular domain of human myelin oligodendrocyte glycoprotein (MOG1-125) is significantly reduced in Aicda deficient mice, which, unlike wild-type mice, lack serum IgG to myelin associated antigens. MOG specific T cell responses are comparable between wild-type and Aicda knockout mice suggesting an active role for antigen experienced B cells. Thus affinity maturation and/or class switching are critical processes in the pathogenesis of EAE. PMID:23167594

  18. Critical role of activation induced cytidine deaminase in Experimental Autoimmune Encephalomyelitis

    PubMed Central

    2013-01-01

    Multiple Sclerosis (MS) is a neurodegenerative autoimmune disorder caused by chronic inflammation and demyelination within the central nervous system (CNS). Clinical studies in MS patients have demonstrated efficacy with B cell targeted therapies such as anti-CD20. However, the exact role that B cells play in the disease process is unclear. Activation Induced cytidine deaminase (AID) is an essential enzyme for the processes of antibody affinity maturation and isotype switching. To evaluate the impact of affinity maturation and isotype switching, we have interrogated the effect of AID-deficiency in an animal model of MS. Here, we show that the severity of experimental autoimmune encephalomyelitis (EAE) induced by the extracellular domain of human myelin oligodendrocyte glycoprotein (MOG1-125) is significantly reduced in Aicda deficient mice, which, unlike wild-type mice, lack serum IgG to myelin associated antigens. MOG specific T cell responses are comparable between wild-type and Aicda knockout mice suggesting an active role for antigen experienced B cells. Thus affinity maturation and/or class switching are critical processes in the pathogenesis of EAE. PMID:23167594

  19. Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast

    PubMed Central

    Mallano, Alessandra; Zamboni, Silvia; Carpinelli, Giulia; Santoro, Filippo; Flego, Michela; Ascione, Alessandro; Gellini, Mara; Tombesi, Marina; Podo, Franca; Cianfriglia, Maurizio

    2008-01-01

    Background The ability of cytosine deaminase (CD) to convert the antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT/ADEPT) aiming to improve the therapeutic ratio (benefit versus toxic side-effects) of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv) format represents a powerful reagent to allow in in vitro and in vivo detection of CD expression in GDEPT/ADEPT studies. Results An enzymatic active recombinant CD from yeast (yCD) was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU. Conclusion The construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function. PMID:18783590

  20. The effect of therapeutic drugs and other pharmacologic agents on activity of porphobilinogen deaminase, the enzyme that is deficient in intermittent acute porphyria.

    PubMed

    Tishler, P V

    1999-01-01

    Drugs and toxins precipitate life-threatening acute attacks in patients with intermittent acute porphyria. These materials may act by directly inhibiting enzyme activity, thus further reducing porphobilinogen (PBG) deaminase activity below the ca. 50% level that results from the gene defect. To test this, we studied the effects of drugs that precipitate acute attacks (lead, phenobarbital, griseofulvin, phenytoin, sulfanilamide, sulfisoxazole, 17alpha-ethinyl estradiol, 5beta-pregnan-3alpha-ol-20-one), drugs that are safe (lithium, magnesium, chlorpromazine, promethazine), and those with uncertain effects (ethyl alcohol, imipramine, diazepam, haloperidol) on activity of PBG deaminase in vitro and in vivo. In the in vitro studies, of PBG deaminase from human erythrocytes from normals and individuals with IAP, only lead (> or = .01 mM) inhibited enzyme activity. Chlorpromazine (> or = .01 mM), promethazine (> or = .01 mM) and imipramine (1 mM) seemed to increase enzyme activity. In most in vivo experiments, male rats were injected intraperitoneally with test material twice daily for 3 days and once on day four; and erythrocyte and hepatic PBG deaminase activity was assayed thereafter. Effects on enzyme activity were observed only with 17alpha-ethinyl estradiol (0.05 microg/kg/day; reduction of 11% in erythrocyte enzyme [NS], and of 20% in liver enzyme [P=.02]), and imipramine (12.5 mg/kg/day; reduction in erythrocyte enzyme activity of 13% [P<.001]). Rats given lead acetate in their drinking water (10 mg/ml) for the first 60 days of life, resulting in high blood and liver lead levels, had increased erythrocyte PBG deaminase (167% of control; P=.004). Thus, enzyme inhibition by lead in vitro was not reflected in a similar in vivo inhibition. The only inhibitory effects in vivo, with ethinyl estradiol and imipramine, appear to be mild and biologically inconsequential. We conclude that inhibition of PBG deaminase activity by materials that precipitate acute attacks is an

  1. Hot electron extraction from CdTe quantum dots via beta carotene molecular energy levels

    NASA Astrophysics Data System (ADS)

    Pazhanivel, T.; Nataraj, D.; Devarajan, V. P.; Senthil, K.; Seol, M.; Yong, K.

    2012-06-01

    We report our findings related to hot electron extraction from CdTe quantum dots, and we were able to do this by using beta carotene as an electron acceptor. Transient absorption spectra with two slow recovering negative bleaches at the absorption maximum of the molecule and quantum dot have indicated the slowing down of cooling process and the existence of hot carriers in this hybrid system.

  2. Self inhibition of phagocytosis: the affinity of ‘Marker of Self’ CD47 for SIRPα dictates potency of inhibition but only at low expression levels

    PubMed Central

    Tsai, Richard K.; Rodriguez, Pia L.; Discher, Dennis E.

    2010-01-01

    Phagocytes engulf foreign cells but not ‘self’ in part because self cells express CD47 as a ligand for signal regulatory protein SIRPα, which inhibits phagocytosis. Motivated by reports of upregulation of CD47 on both normal and cancerous stem cells [1] and also by polymorphisms in SIRPα [2], we show here that inhibition of engulfment correlates with affinity of CD47 for SIRPα – but only at low levels of CD47. One common human polymorph of SIRPα is studied and binds more strongly to human-CD47 than to mouse-CD47 (Kd ≈ 0.12 μM and 6.9 μM, respectively) and does not bind sheep red blood cells (RBC) – which are well-established targets of human macrophages; in comparison, a common mouse polymorph of SIRPα binds with similar affinity to human and mouse CD47 (Kd ≈ 0.22 μM). Using immunoglobulin (IgG)-opsonized particles with varying levels of either human- or mouse-CD47, the effective inhibition constants Ki for blocking phagocytosis are then determined with both human- and mouse-derived macrophages. Only human phagocytes show significant differences in man versus mouse Ki's and only at CD47 levels below normal densities for RBCs. While phospo-signaling through human-SIRPα shows similar trends, consistent again with the affinity differences, saturating levels of CD47 (> Ki) can signal and inhibit phagocytosis regardless of man versus mouse. Quantitative analyses here prompt more complete characterizations of both CD47 levels and SIRPα polymorphisms when attempting to study in vivo effects of these key proteins in innate immunity. PMID:20299253

  3. Southern blight disease of tomato control by 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing Paenibacillus lentimorbus B-30488

    PubMed Central

    Dixit, Ritu; Agrawal, Lalit; Gupta, Swati; Kumar, Manoj; Yadav, Sumit; Chauhan, Puneet Singh; Nautiyal, Chandra Shekhar

    2016-01-01

    abstract Tomato cultivation is highly susceptible for soil born diseases and among them southern blight disease caused by Scelerotium rolfsii is very common. For its management use of chemical fungicides is not very successful as their spores are able to survive for many years in the soil. As an alternative eco-friendly approach to control the disease antagonistic microbes are being characterized.Among them plant growth promoting rhizobacteria Paenibacillus lentimorbus B-30488 (B-30488) with antagonistic properties, multiple PGP attributes stress tolerance and ACC deaminase enzyme activity is characterized to decipher its mode of action against S. rolfsii under in vitro and in vivo conditions. In vitro results obtained from this study clearly demonstrate that B-30488 has ability to show antagonistic properties under different abiotic stresses against S. rolfsii. Similar results were also obtained from in vivo experiments where B-30488 inoculation has efficiently controlled the disease caused by S. rolfsii and improve the plant growth. Deleterious enhanced ethylene level in S. rolfsii infected plants was also ameliorated by inoculation of ACC deaminase producing B-30488. The ACC accumulation, ACO and ACS activities were also modulated in S. rolfsii infected plants. Results from defense enzymes and other biochemical attributes were also support the role of B-30488 inoculation in ameliorating the biotic stress caused by S. rolfsii in tomato plants. These results were further validated by pathogen related gene expression analysis by real time PCR. Overall results from the present study may be concluded that ACC deaminase producing B-30488 has ability to control the southern blight disease caused by S. rolfsii and commercial bioinoculant package may be developed. PMID:26825539

  4. Processive DNA Demethylation via DNA Deaminase-Induced Lesion Resolution

    PubMed Central

    Morgan, Hugh; Incorvaia, Elisabetta; Rangam, Gopinath; Dean, Wendy; Santos, Fatima; Reik, Wolf; Petersen-Mahrt, Svend K.

    2014-01-01

    Base modifications of cytosine are an important aspect of chromatin biology, as they can directly regulate gene expression, while DNA repair ensures that those modifications retain genome integrity. Here we characterize how cytosine DNA deaminase AID can initiate DNA demethylation. In vitro, AID initiated targeted DNA demethylation of methyl CpGs when in combination with DNA repair competent extracts. Mechanistically, this is achieved by inducing base alterations at or near methyl-cytosine, with the lesion being resolved either via single base substitution or a more efficient processive polymerase dependent repair. The biochemical findings are recapitulated in an in vivo transgenic targeting assay, and provide the genetic support of the molecular insight into DNA demethylation. This targeting approach supports the hypothesis that mCpG DNA demethylation can proceed via various pathways and mCpGs do not have to be targeted to be demethylated. PMID:25025377

  5. AMP-deaminase from thymus of patients with myasthenia gravis.

    PubMed

    Rybakowska, I; Szydłowska, M; Szrok, S; Bakuła, S; Kaletha, K

    2015-01-01

    Myasthenia gravis (MG) is characterized clinically by skeletal muscle fatigue following the excessive exercise. Interestingly most of MG patients manifest parallely also some abnormalities of the thymus.AMP-deaminase (AMPD) from human thymus was not a subject of studies up to now. In this paper, mRNA expression and some physico-chemical and immunological properties of AMPD purified from the thymus of MG patients were described. Experiments performed identified the liver isozyme (AMPD2) as the main isoform of AMPD expressed in this organ. The activity of AMPD found in this organ was higher than in other human non-(skeletal) muscle tissues indicating on role the enzyme may play in supplying of guanylates required for the intensive multiplication of thymocytes.

  6. Photodynamic therapy-driven induction of suicide cytosine deaminase gene.

    PubMed

    Bil, Jacek; Wlodarski, Pawel; Winiarska, Magdalena; Kurzaj, Zuzanna; Issat, Tadeusz; Jozkowicz, Alicja; Wegiel, Barbara; Dulak, Jozef; Golab, Jakub

    2010-04-28

    Photodynamic therapy (PDT) of tumors is associated with induction of hypoxia that results in activation of hypoxia-inducible factors (HIFs). Several observations indicate that increased HIFs transcriptional activity in tumor cells is associated with cytoprotective responses that limit cytotoxic effectiveness of PDT. Therefore, we decided to examine whether this cytoprotective mechanism could be intentionally used for designing more efficient tumor cell cytotoxicity. To this end we transfected tumor cells with a plasmid vector carrying a suicide cytosine deaminase gene driven by a promoter containing hypoxia response elements (HRE). The presence of such a genetic molecular beacon rendered tumor cells sensitive to cytotoxic effects of a non-toxic prodrug 5-fluorocytosine (5-FC). The results of this study provides a proof of concept that inducible cytoprotective mechanisms can be exploited to render tumor cells more susceptible to cytotoxic effects of prodrugs activated by products of suicide genes.

  7. β decay of semi-magic 130Cd: Revision and extension of the level scheme of 130In

    NASA Astrophysics Data System (ADS)

    Jungclaus, A.; Grawe, H.; Nishimura, S.; Doornenbal, P.; Lorusso, G.; Simpson, G. S.; Söderström, P.-A.; Sumikama, T.; Taprogge, J.; Xu, Z. Y.; Baba, H.; Browne, F.; Fukuda, N.; Gernhäuser, R.; Gey, G.; Inabe, N.; Isobe, T.; Jung, H. S.; Kameda, D.; Kim, G. D.; Kim, Y.-K.; Kojouharov, I.; Kubo, T.; Kurz, N.; Kwon, Y. K.; Li, Z.; Sakurai, H.; Schaffner, H.; Shimizu, Y.; Steiger, K.; Suzuki, H.; Takeda, H.; Vajta, Zs.; Watanabe, H.; Wu, J.; Yagi, A.; Yoshinaga, K.; Benzoni, G.; Bönig, S.; Chae, K. Y.; Coraggio, L.; Daugas, J.-M.; Drouet, F.; Gadea, A.; Gargano, A.; Ilieva, S.; Itaco, N.; Kondev, F. G.; Kröll, T.; Lane, G. J.; Montaner-Pizá, A.; Moschner, K.; Mücher, D.; Naqvi, F.; Niikura, M.; Nishibata, H.; Odahara, A.; Orlandi, R.; Patel, Z.; Podolyák, Zs.; Wendt, A.

    2016-08-01

    The β decay of the semi-magic nucleus 130Cd has been studied at the RIBF facility at the RIKEN Nishina Center. The high statistics of the present experiment allowed for a revision of the established level scheme of 130In and the observation of additional β feeding to high-lying core-excited states in 130In. The experimental results are compared to shell-model calculations employing a model space consisting of the full major N =50 - 82 neutron and Z =28 - 50 proton shells and the NA-14 interaction, and good agreement is found.

  8. Elimination of cadmium from Cd-contaminated Tilapia zilli in media containing EDTA and freshwater: Changes in protein levels

    SciTech Connect

    Kargin, F.

    1996-10-01

    Cadmium is not an essential metal for aquatic organisms. It enters the aquatic environment through anthropogenic sources such as industry and agriculture. Kay et al. indicated that in the USA and Belgium 40-50% of Salmo gairdneri contained 1-20 {mu}g Cd/L. Various agents are known to reduce metal accumulation in tissues of aquantic animals. This study investigates cadmium elimination from the tissues of cadmium contaminated Tilapia zilli and changes in protein levels in the tissues after exposures to cadmium, EDTA and freshwater. 18 refs., 2 tabs.

  9. Serine/threonine-protein phosphatase 1 α levels are paralleling olfactory memory formation in the CD1 mouse.

    PubMed

    Winding, Christiana; Sun, Yanwei; Höger, Harald; Bubna-Littitz, Hermann; Pollak, Arnold; Schmidt, Peter; Lubec, Gert

    2011-06-01

    Although olfactory discrimination has already been studied in several mouse strains, data on protein levels linked to olfactory memory are limited. Wild mouse strains Mus musculus musculus, Mus musculus domesticus and CD1 laboratory outbred mice were tested in a conditioned odor preference task and trained to discriminate between two odors, Rose and Lemon, by pairing one odor with a sugar reward. Six hours following the final test, mice were sacrificed and olfactory bulbs (OB) were taken for gel-based proteomics analyses and immunoblotting. OB proteins were extracted, separated by 2-DE and quantified using specific software (Proteomweaver). Odor-trained mice showed a preference for the previously rewarded odor suggesting that conditioned odor preference occurred. In CD1 mice levels, one out of 482 protein spots was significantly increased in odor-trained mice as compared with the control group; it was in-gel digested by trypsin and chymotrypsin and analyzed by tandem mass spectrometry (nano-ESI-LC-MS/MS). The spot was unambiguously identified as serine/threonine-protein phosphatase PP1-α catalytic subunit (PP-1A) and differential levels observed in gel-based proteomic studies were verified by immunoblotting. PP-1A is a key signalling element in synaptic plasticity and memory processes and is herein shown to be paralleling olfactory discrimination representing olfactory memory.

  10. Trace level determination of u, zn, cd, pb and cu in drinking water samples.

    PubMed

    Kumar, Mukesh; Singh, Surinder; Mahajan, Rakesh Kumar

    2006-01-01

    The concentration of uranium has been assessed in drinking water samples collected from different locations in Bathinda district, Punjab, India. The water samples are taken from hand pumps and tube wells. Uranium is determined using fission track technique. Uranium concentration in the water samples varies from 2.23+/- 0.05 to 87.05+/- 0.29 microg/L. These values are compared with safe limit values recommended for drinking water. The uranium concentration in almost all drinking water samples is found to be more than the safe limit. Analysis of some heavy metals viz. Zn, Cd, Pb and Cu in water is made. The concentration of sodium, potassium, calcium, magnesium, chlorine and total hardness along with the pH value and conductivity of the water samples are measured. Some of the samples show stunningly high values of these parameters.

  11. Cytidine deaminase activity in synovial fluid of patients with rheumatoid arthritis: relation to lactoferrin, acidosis, and cartilage proteoglycan release.

    PubMed Central

    Månsson, B; Geborek, P; Saxne, T; Björnsson, S

    1990-01-01

    It is claimed that cytidine deaminase activity reflects local granulocyte turnover or activity in the synovial fluid of patients with rheumatoid arthritis, but cytidine deaminase is not a granulocyte specific enzyme. Lactoferrin is a granulocyte specific protein that is released from the secondary granulae during activation. We measured cytidine deaminase activity and lactoferrin concentrations in 33 rheumatic synovial fluid samples. Cytidine deaminase activity and lactoferrin concentrations correlated closely, indicating that both analyses reflect similar events in the joint-that is, result in their release from granulocytes. Cytidine deaminase activity and granulocyte concentrations correlated less closely, suggesting that there are additional factors besides the cell number which contribute to this release. Joint acidosis may be one such factor, as pH and cytidine deaminase activity correlated inversely. There was no association with synovial fluid proteoglycan concentrations, a marker of cartilage degradation. PMID:2396864

  12. Characterization of plant growth promoting rhizobacteria isolated from polluted soils and containing 1-aminocyclopropane-1-carboxylate deaminase.

    PubMed

    Belimov, A A; Safronova, V I; Sergeyeva, T A; Egorova, T N; Matveyeva, V A; Tsyganov, V E; Borisov, A Y; Tikhonovich, I A; Kluge, C; Preisfeld, A; Dietz, K J; Stepanok, V V

    2001-07-01

    Fifteen bacterial strains containing 1-aminocyclopropane-1-carboxylate (ACC) deaminase were isolated from the rhizoplane of pea (Pisum sativum L.) and Indian mustard (Brassica juncea L.) grown in different soils and a long-standing sewage sludge contaminated with heavy metals. The isolated strains were characterized and assigned to various genera and species, such as Pseudomonas brassicacearum, Pseudomonas marginalis, Pseudomonas oryzihabitans, Pseudomonas putida, Pseudomonas sp., Alcaligenes xylosoxidans, Alcaligenes sp., Variovorax paradoxus, Bacillus pumilus, and Rhodococcus sp. by determination of 16S rRNA gene sequences. The root elongation of Indian mustard and rape (Brassica napus var. oleifera L.) germinating seedlings was stimulated by inoculation with 8 and 13 isolated strains, respectively. The bacteria were tolerant to cadmium toxicity and stimulated root elongation of rape seedlings in the presence of 300 microM CdCl2 in the nutrient solution. The effect of ACC-utilising bacteria on root elongation correlated with the impact of aminoethoxyvinylglycine and silver ions, chemical inhibitors of ethylene biosynthesis. A significant improvement in the growth of rape caused by inoculation with certain selected strains was also observed in pot experiments, when the plants were cultivated in cadmium-supplemented soil. The biomass of pea cv. Sparkle and its ethylene sensitive mutant E2 (sym5), in particular, was increased through inoculation with certain strains of ACC-utilising bacteria in pot experiments in quartz sand culture. The beneficial effect of the bacteria on plant growth varied significantly depending on individual bacterial strains, plant genotype, and growth conditions. The results suggest that plant growth promoting rhizobacteria containing ACC deaminase are present in various soils and offer promise as a bacterial inoculum for improvement of plant growth, particularly under unfavourable environmental conditions.

  13. Characterization of plant growth promoting rhizobacteria isolated from polluted soils and containing 1-aminocyclopropane-1-carboxylate deaminase.

    PubMed

    Belimov, A A; Safronova, V I; Sergeyeva, T A; Egorova, T N; Matveyeva, V A; Tsyganov, V E; Borisov, A Y; Tikhonovich, I A; Kluge, C; Preisfeld, A; Dietz, K J; Stepanok, V V

    2001-07-01

    Fifteen bacterial strains containing 1-aminocyclopropane-1-carboxylate (ACC) deaminase were isolated from the rhizoplane of pea (Pisum sativum L.) and Indian mustard (Brassica juncea L.) grown in different soils and a long-standing sewage sludge contaminated with heavy metals. The isolated strains were characterized and assigned to various genera and species, such as Pseudomonas brassicacearum, Pseudomonas marginalis, Pseudomonas oryzihabitans, Pseudomonas putida, Pseudomonas sp., Alcaligenes xylosoxidans, Alcaligenes sp., Variovorax paradoxus, Bacillus pumilus, and Rhodococcus sp. by determination of 16S rRNA gene sequences. The root elongation of Indian mustard and rape (Brassica napus var. oleifera L.) germinating seedlings was stimulated by inoculation with 8 and 13 isolated strains, respectively. The bacteria were tolerant to cadmium toxicity and stimulated root elongation of rape seedlings in the presence of 300 microM CdCl2 in the nutrient solution. The effect of ACC-utilising bacteria on root elongation correlated with the impact of aminoethoxyvinylglycine and silver ions, chemical inhibitors of ethylene biosynthesis. A significant improvement in the growth of rape caused by inoculation with certain selected strains was also observed in pot experiments, when the plants were cultivated in cadmium-supplemented soil. The biomass of pea cv. Sparkle and its ethylene sensitive mutant E2 (sym5), in particular, was increased through inoculation with certain strains of ACC-utilising bacteria in pot experiments in quartz sand culture. The beneficial effect of the bacteria on plant growth varied significantly depending on individual bacterial strains, plant genotype, and growth conditions. The results suggest that plant growth promoting rhizobacteria containing ACC deaminase are present in various soils and offer promise as a bacterial inoculum for improvement of plant growth, particularly under unfavourable environmental conditions. PMID:11547884

  14. Intracellular calcium levels are differentially regulated in T lymphocytes triggered by anti-CD2 and anti-CD3 monoclonal antibodies.

    PubMed

    Spinozzi, F; Agea, E; Bistoni, O; Belia, S; Travetti, A; Gerli, R; Muscat, C; Bertotto, A

    1995-03-01

    Antigen and/or mitogen-driven T-cell activation is mediated by a rise in intracellular free Ca2+, as second messenger. A regulatory key role for this process is represented by membrane-associated [Ca2+/Mg2+] ATP-ase that is mainly devoted to extrusion of intracellular ion excess. In the present study we have investigated the kinetics of CA2+ fluxes in both resting and already activated (Jurkat T-cell line) T lymphocytes after CD3 and CD2 (T11(2) and T11(3)) triggering and focused our attention on plasma membrane [Ca2+/Mg2+] ATP-ase activity. In both resting T cells and Jurkat cell line, the CD2 stimulation was able to determine a rise in intracellular free Ca2+ higher than that observed after CD3 triggering. In addition, this calcium signal was independent of negative feedback control exerted by [Ca2+/Mg2+] ATP-ase, as well as of IP3 generation. Thus the CD2 molecular system may, together with cell-adhesion properties, act as an amplifier of Ca2+ signals that, if delivered in the context of other molecular systems, such as CD3 or MHC class II antigens, are essentially devoted to the polyclonal co-stimulatory recruitment of a larger cellular repertoire. PMID:7662514

  15. Diminished CD4+/CD25+ T cell and increased IFN-gamma levels occur in dogs vaccinated with Leishmune in an endemic area for visceral leishmaniasis.

    PubMed

    de Lima, Valéria Marçal Felix; Ikeda, Fabiana Augusta; Rossi, Cláudio N; Feitosa, Mary Marcondes; Vasconcelos, Rosemeride Oliveira; Nunes, Caris Maroni; Goto, Hiro

    2010-06-15

    The Leishmune vaccine has been used in endemic areas to prevent canine visceral leishmaniasis in Brazil, but cytokine production induced by vaccination has rarely been investigated in dogs. This study aimed to evaluate the immune response of dogs vaccinated with Leishmune FML vaccine (Fort Dodge) against total antigen of Leishmania (Leishmania) chagasi (TAg) and FML. Twenty healthy dogs from Araçatuba, São Paulo, Brazil, an endemic leishmaniasis area, received three consecutive subcutaneous injection of Leishmune vaccine at 21-day intervals. PBMC were isolated before and 10 days after completing vaccination and lymphoproliferative response and antibody production against FML or total promastigote antigen were tested. Cytokines IFN-gamma, IL-4 and TNF-alpha were measured in culture supernatant and CD4+/CD25+ and CD8+/CD25+ T cell presence was determined. Analysis of the data indicated that the vaccine conferred humoral responses (100%) against both antigens and cellular immunity to FML (85%) and total antigen (80%), the supernatant of cultured cells stimulated with TAg and FML showed an increase in IFN-gamma (P<0.05), and the vaccine reduced CD4+/CD25+ T cell presence compared to that observed before vaccination. These responses may constitute part of the immune mechanism induced by Leishmune.

  16. Correlation between viral load, plasma levels of CD4 - CD8 T lymphocytes and AIDS-related oral diseases: a multicentre study on 30 HIV+ children in the HAART era.

    PubMed

    Nesti, M; Carli, E; Giaquinto, C; Rampon, O; Nastasio, S; Giuca, M R

    2012-01-01

    This experimental retrospective multicenter study carried out on 30 seropositive children treated with Highly Active Antiretroviral Therapy (HAART), between the ages of 18 months and 14 years, in the clinical categories Centers for Disease Control (CDC) classification 1993 A (mildly symptomatic), B (moderately symptomatic) and C (severely symptomatic) aims to: 1) clinically and immunologically demonstrate the therapeutic benefits of HAART; 2) monitor the frequency of AIDS-related oral diseases in seropositive children with HAART therapy; 3) monitor the plasma levels of total CD4, CD4 percent, CD8 percent, CD4-CD8 lymphocytes and viral load from 1997 to 30 April, 2011. The statistic methods used are the analysis of covariance and the Bonferroni Test. More than 100 AIDS-related oral diseases were found in the study samples, the most frequent being: oral candidiasis, oropharyngeal candidiasis, HSV-1 herpetic esophagyitis, herpetic gingivolstomatitis (RHOG), recurrent aphthous stomatitis (RAS), parotid swelling, oral hairy leukoplakia (OHL), Herpes simplex 1 (HSV-1), linear gingival erythema (LGE), necrotizing gingivitis (NUG), facial lipodistrophy, facial-cervical lymphadenopathy (FCL), xerostomia, dysgeusia, hyposmia, oral mucosa hyperpigmentation (OMP). The Bonferroni test showed a significant difference between the mean plasma values (mpVTL) of total CD4, CD4 percentage, CD4-CD8 T lymphocytes and Viral Load (VL) of the various oral diseases found in the study samples. The therapeutic benefits of HAART are: immune reconstitution; reduction of the HIV/AIDS-related stomatology diseases; prevention and cure of the AIDS correlated neoplasias; reduction in maternal-fetal transmission of the HIV virus. The negative effects of HAART in relation to odontostomatolgy are: increase in oral lesions from HPV; xerostomia; dysgeusia/ageusia, hyposmia, perioral paresthesia; hyperpigmentation of oral mucosa; facial lipodystrophy, recurrent aphthous stomatitis (RAS). No case of

  17. Ovarian hormone level alterations during rat post-reproductive life-span influence CD8 + T-cell homeostasis

    PubMed Central

    Arsenović-Ranin, Nevena; Kosec, Duško; Nacka-Aleksić, Mirjana; Pilipović, Ivan; Stojić-Vukanić, Zorica; Djikić, Jasmina; Bufan, Biljana

    2015-01-01

    The study examined the putative role of ovarian hormones in shaping of rat peripheral T-cell compartment during post-reproductive period. In 20-month-old rats ovariectomized (Ox) at the very end of reproductive period, thymic output, cellularity and composition of major TCRαβ + peripheral blood lymphocyte and splenocyte subsets were analyzed. Ovariectomy led to the enlargement of CD8 + peripheral blood lymphocyte and splenocyte subpopulations. This reflected: (i) a more efficient thymic generation of CD8 + cells as indicated by increased number of CD4+CD8 + double positive and the most mature CD4-CD8+TCRαβhigh thymocytes and CD8 + recent thymic emigrants (RTEs) in peripheral blood, but not in the spleen of Ox rats, and (ii) the expansion of CD8 + memory/activated peripheral blood lymphocytes and splenocytes. The latter was consistent with a greater frequency of proliferating cells among freshly isolated memory/activated CD8 + peripheral blood lymphocytes and splenocytes and increased proliferative response of CD8 + splenocytes to stimulation with plate-bound anti-CD3 antibody. The former could be related to the rise in splenic IL-7 and IL-15 mRNA expression. Although ovariectomy affected the overall number of CD4 + T cells in none of the examined compartments, it increased CD4+FoxP3 + peripheral blood lymphocyte and splenocyte counts by enhancing their generation in periphery. Collectively, the results suggest that ovariectomy-induced long-lasting disturbances in ovarian hormone levels (mirrored in diminished progesterone serum level in 20-month-old rats) affects both thymic CD8 + cell generation and peripheral homeostasis and leads to the expansion of CD4+FoxP3 + cells in the periphery, thereby enhancing autoreactive cell control on account of immune system efficacy to combat infections and tumors. PMID:25716018

  18. Determination of contamination levels of Pb, Cd, Cu, Ni, and Mn caused by former lead mining gallery.

    PubMed

    Bakırdere, Sezgin; Bölücek, Cemal; Yaman, Mehmet

    2016-03-01

    In the present study, levels of metal contamination caused by former lead mining area were figured out. For this purpose, Pb, Cd, Cu, Ni, and Mn were determined not only in sediment samples taken from different places of the mining area but also in some plants taken around the mining place. In the digestion of plant samples, dry ashing procedure was applied. Flame atomic absorption spectrophotometer (FAAS) was used in the determination of analytes of interest. All the parameters in digestion and detection procedures were optimized to obtain efficient digestion and high sensitivities for analytes. Standard addition and direct calibration methods were applied to find whether there was any matrix interference to affect the determination of analytes. Mn concentration was found to be the highest for each sample analyzed. Lead concentration was found to be between 41 and 249 mg/kg in soil/sediment samples and between 2.2 and 1003 mg/kg in plant samples. The highest contamination levels for all of the analytes with the exception of Cd were found in current sediment sample.

  19. Determination of contamination levels of Pb, Cd, Cu, Ni, and Mn caused by former lead mining gallery.

    PubMed

    Bakırdere, Sezgin; Bölücek, Cemal; Yaman, Mehmet

    2016-03-01

    In the present study, levels of metal contamination caused by former lead mining area were figured out. For this purpose, Pb, Cd, Cu, Ni, and Mn were determined not only in sediment samples taken from different places of the mining area but also in some plants taken around the mining place. In the digestion of plant samples, dry ashing procedure was applied. Flame atomic absorption spectrophotometer (FAAS) was used in the determination of analytes of interest. All the parameters in digestion and detection procedures were optimized to obtain efficient digestion and high sensitivities for analytes. Standard addition and direct calibration methods were applied to find whether there was any matrix interference to affect the determination of analytes. Mn concentration was found to be the highest for each sample analyzed. Lead concentration was found to be between 41 and 249 mg/kg in soil/sediment samples and between 2.2 and 1003 mg/kg in plant samples. The highest contamination levels for all of the analytes with the exception of Cd were found in current sediment sample. PMID:26837380

  20. Discovery and Structure Determination of the Orphan Enzyme Isoxanthopterin Deaminase

    SciTech Connect

    Hall, R.S.; Swaminathan, S.; Agarwal, R.; Hitchcock, D.; Sauder, J. M.; Burley, S. K.; Raushel, F. M.

    2010-05-25

    Two previously uncharacterized proteins have been identified that efficiently catalyze the deamination of isoxanthopterin and pterin 6-carboxylate. The genes encoding these two enzymes, NYSGXRC-9339a (gi|44585104) and NYSGXRC-9236b (gi|44611670), were first identified from DNA isolated from the Sargasso Sea as part of the Global Ocean Sampling Project. The genes were synthesized, and the proteins were subsequently expressed and purified. The X-ray structure of Sgx9339a was determined at 2.7 {angstrom} resolution (Protein Data Bank entry 2PAJ). This protein folds as a distorted ({beta}/{alpha}){sub 8} barrel and contains a single zinc ion in the active site. These enzymes are members of the amidohydrolase superfamily and belong to cog0402 within the clusters of orthologous groups (COG). Enzymes in cog0402 have previously been shown to catalyze the deamination of guanine, cytosine, S-adenosylhomocysteine, and 8-oxoguanine. A small compound library of pteridines, purines, and pyrimidines was used to probe catalytic activity. The only substrates identified in this search were isoxanthopterin and pterin 6-carboxylate. The kinetic constants for the deamination of isoxanthopterin with Sgx9339a were determined to be 1.0 s{sup -1}, 8.0 {micro}M, and 1.3 x 10{sup 5} M{sup -1} s{sup -1} (k{sub cat}, K{sub m}, and k{sub cat}/K{sub m}, respectively). The active site of Sgx9339a most closely resembles the active site for 8-oxoguanine deaminase (Protein Data Bank entry 2UZ9). A model for substrate recognition of isoxanthopterin by Sgx9339a was proposed on the basis of the binding of guanine and xanthine in the active site of guanine deaminase. Residues critical for substrate binding appear to be conserved glutamine and tyrosine residues that form hydrogen bonds with the carbonyl oxygen at C4, a conserved threonine residue that forms hydrogen bonds with N5, and another conserved threonine residue that forms hydrogen bonds with the carbonyl group at C7. These conserved active site

  1. Discovery and structure determination of the orphan enzyme isoxanthopterin deaminase .

    PubMed

    Hall, Richard S; Agarwal, Rakhi; Hitchcock, Daniel; Sauder, J Michael; Burley, Stephen K; Swaminathan, Subramanyam; Raushel, Frank M

    2010-05-25

    Two previously uncharacterized proteins have been identified that efficiently catalyze the deamination of isoxanthopterin and pterin 6-carboxylate. The genes encoding these two enzymes, NYSGXRC-9339a ( gi|44585104 ) and NYSGXRC-9236b ( gi|44611670 ), were first identified from DNA isolated from the Sargasso Sea as part of the Global Ocean Sampling Project. The genes were synthesized, and the proteins were subsequently expressed and purified. The X-ray structure of Sgx9339a was determined at 2.7 A resolution (Protein Data Bank entry 2PAJ ). This protein folds as a distorted (beta/alpha)(8) barrel and contains a single zinc ion in the active site. These enzymes are members of the amidohydrolase superfamily and belong to cog0402 within the clusters of orthologous groups (COG). Enzymes in cog0402 have previously been shown to catalyze the deamination of guanine, cytosine, S-adenosylhomocysteine, and 8-oxoguanine. A small compound library of pteridines, purines, and pyrimidines was used to probe catalytic activity. The only substrates identified in this search were isoxanthopterin and pterin 6-carboxylate. The kinetic constants for the deamination of isoxanthopterin with Sgx9339a were determined to be 1.0 s(-1), 8.0 muM, and 1.3 x 10(5) M(-1) s(-1) (k(cat), K(m), and k(cat)/K(m), respectively). The active site of Sgx9339a most closely resembles the active site for 8-oxoguanine deaminase (Protein Data Bank entry 2UZ9 ). A model for substrate recognition of isoxanthopterin by Sgx9339a was proposed on the basis of the binding of guanine and xanthine in the active site of guanine deaminase. Residues critical for substrate binding appear to be conserved glutamine and tyrosine residues that form hydrogen bonds with the carbonyl oxygen at C4, a conserved threonine residue that forms hydrogen bonds with N5, and another conserved threonine residue that forms hydrogen bonds with the carbonyl group at C7. These conserved active site residues were used to identify 24 other genes

  2. ADAR1 Facilitates HIV-1 Replication in Primary CD4+ T Cells

    PubMed Central

    van Hamme, John L.; Jansen, Machiel H.; van Dort, Karel A.; Vanderver, Adeline; Rice, Gillian I.; Crow, Yanick J.; Kootstra, Neeltje A.; Kuijpers, Taco W.

    2015-01-01

    Unlike resting CD4+ T cells, activated CD4+T cells are highly susceptible to infection of human immunodeficiency virus 1 (HIV-1). HIV-1 infects T cells and macrophages without activating the nucleic acid sensors and the anti-viral type I interferon response. Adenosine deaminase acting on RNA 1 (ADAR1) is an RNA editing enzyme that displays antiviral activity against several RNA viruses. Mutations in ADAR1 cause the autoimmune disorder Aicardi-Goutieères syndrome (AGS). This disease is characterized by an inappropriate activation of the interferon-stimulated gene response. Here we show that HIV-1 replication, in ADAR1-deficient CD4+T lymphocytes from AGS patients, is blocked at the level of protein translation. Furthermore, viral protein synthesis block is accompanied by an activation of interferon-stimulated genes. RNA silencing of ADAR1 in Jurkat cells also inhibited HIV-1 protein synthesis. Our data support that HIV-1 requires ADAR1 for efficient replication in human CD4+T cells. PMID:26629815

  3. Glucose metabolism during fasting is altered in experimental porphobilinogen deaminase deficiency.

    PubMed

    Collantes, María; Serrano-Mendioroz, Irantzu; Benito, Marina; Molinet-Dronda, Francisco; Delgado, Mercedes; Vinaixa, María; Sampedro, Ana; Enríquez de Salamanca, Rafael; Prieto, Elena; Pozo, Miguel A; Peñuelas, Iván; Corrales, Fernando J; Barajas, Miguel; Fontanellas, Antonio

    2016-04-01

    Porphobilinogen deaminase (PBGD) haploinsufficiency (acute intermittent porphyria, AIP) is characterized by neurovisceral attacks when hepatic heme synthesis is activated by endogenous or environmental factors including fasting. While the molecular mechanisms underlying the nutritional regulation of hepatic heme synthesis have been described, glucose homeostasis during fasting is poorly understood in porphyria. Our study aimed to analyse glucose homeostasis and hepatic carbohydrate metabolism during fasting in PBGD-deficient mice. To determine the contribution of hepatic PBGD deficiency to carbohydrate metabolism, AIP mice injected with a PBGD-liver gene delivery vector were included. After a 14 h fasting period, serum and liver metabolomics analyses showed that wild-type mice stimulated hepatic glycogen degradation to maintain glucose homeostasis while AIP livers activated gluconeogenesis and ketogenesis due to their inability to use stored glycogen. The serum of fasted AIP mice showed increased concentrations of insulin and reduced glucagon levels. Specific over-expression of the PBGD protein in the liver tended to normalize circulating insulin and glucagon levels, stimulated hepatic glycogen catabolism and blocked ketone body production. Reduced glucose uptake was observed in the primary somatosensorial brain cortex of fasted AIP mice, which could be reversed by PBGD-liver gene delivery. In conclusion, AIP mice showed a different response to fasting as measured by altered carbohydrate metabolism in the liver and modified glucose consumption in the brain cortex. Glucose homeostasis in fasted AIP mice was efficiently normalized after restoration of PBGD gene expression in the liver. PMID:26908609

  4. Plasma Prostaglandin E2 Levels Correlated with the Prevention of Intravenous Immunoglobulin Resistance and Coronary Artery Lesions Formation via CD40L in Kawasaki Disease

    PubMed Central

    Kuo, Ho-Chang; Wang, Chih-Lu; Yang, Kuender D.; Lo, Mao-Hung; Hsieh, Kai-Sheng; Li, Sung-Chou

    2016-01-01

    Background A form of systemic vasculitis, Kawasaki disease (KD) occurs most frequently in children under the age of five years old. Previous studies have found that Prostaglandin E2 (PGE2) correlates with KD, although the related mechanisms are still unknown. CD40L may also be a marker of vasculitis in KD, so this study focuses on PGE2 and CD40L expression in KD. Materials and Methods This study consisted of a total of 144 KD patients, whose intravenous immunoglobulin (IVIG)/coronary arterial lesion (CAL) formation resistance was evaluated. PGE2 levels were evaluated in vitro to study the effect of CD40L on CD4+ T lymphocytes. Results PGE2 levels significantly increased after IVIG treatment (p<0.05), especially in patients who responded to initial IVIG treatment (p = 0.004) and for patients without CAL formation (p = 0.016). Furthermore, an in vitro study revealed that IVIG acted as a trigger for PGE2 expression in the acute-stage mononuclear cells of KD patients. According to our findings, both IVIG and PGE2 can impede surface CD40L expressions on CD4+ T lymphocytes (p<0.05). Conclusions The results of this study are among the first to find that plasma PGE2 is correlated with the prevention of IVIG resistance and CAL formation through CD40L in KD. PMID:27525421

  5. Crystal Structure of DNA Cytidine Deaminase ABOBEC3G Catalytic Deamination Domain Suggests a Binding Mode of Full-length Enzyme to Single-stranded DNA*

    PubMed Central

    Lu, Xiuxiu; Zhang, Tianlong; Xu, Zeng; Liu, Shanshan; Zhao, Bin; Lan, Wenxian; Wang, Chunxi; Ding, Jianping; Cao, Chunyang

    2015-01-01

    APOBEC3G (A3G) is a DNA cytidine deaminase (CD) that demonstrates antiviral activity against human immunodeficiency virus 1 (HIV-1) and other pathogenic virus. It has an inactive N-terminal CD1 virus infectivity factor (Vif) protein binding domain (A3G-CD1) and an actively catalytic C-terminal CD2 deamination domain (A3G-CD2). Although many studies on the structure of A3G-CD2 and enzymatic properties of full-length A3G have been reported, the mechanism of how A3G interacts with HIV-1 single-stranded DNA (ssDNA) is still not well characterized. Here, we reported a crystal structure of a novel A3G-CD2 head-to-tail dimer (in which the N terminus of the monomer H (head) interacts with the C terminus of monomer T (tail)), where a continuous DNA binding groove was observed. By constructing the A3G-CD1 structural model, we found that its overall fold was almost identical to that of A3G-CD2. We mutated the residues located in or along the groove in monomer H and the residues in A3G-CD1 that correspond to those seated in or along the groove in monomer T. Then, by performing enzymatic assays, we confirmed the reported key elements and the residues in A3G necessary to the catalytic deamination. Moreover, we identified more than 10 residues in A3G essential to DNA binding and deamination reaction. Therefore, this dimer structure may represent a structural model of full-length A3G, which indicates a possible binding mode of A3G to HIV-1 ssDNA. PMID:25542899

  6. Crystal structure of DNA cytidine deaminase ABOBEC3G catalytic deamination domain suggests a binding mode of full-length enzyme to single-stranded DNA.

    PubMed

    Lu, Xiuxiu; Zhang, Tianlong; Xu, Zeng; Liu, Shanshan; Zhao, Bin; Lan, Wenxian; Wang, Chunxi; Ding, Jianping; Cao, Chunyang

    2015-02-13

    APOBEC3G (A3G) is a DNA cytidine deaminase (CD) that demonstrates antiviral activity against human immunodeficiency virus 1 (HIV-1) and other pathogenic virus. It has an inactive N-terminal CD1 virus infectivity factor (Vif) protein binding domain (A3G-CD1) and an actively catalytic C-terminal CD2 deamination domain (A3G-CD2). Although many studies on the structure of A3G-CD2 and enzymatic properties of full-length A3G have been reported, the mechanism of how A3G interacts with HIV-1 single-stranded DNA (ssDNA) is still not well characterized. Here, we reported a crystal structure of a novel A3G-CD2 head-to-tail dimer (in which the N terminus of the monomer H (head) interacts with the C terminus of monomer T (tail)), where a continuous DNA binding groove was observed. By constructing the A3G-CD1 structural model, we found that its overall fold was almost identical to that of A3G-CD2. We mutated the residues located in or along the groove in monomer H and the residues in A3G-CD1 that correspond to those seated in or along the groove in monomer T. Then, by performing enzymatic assays, we confirmed the reported key elements and the residues in A3G necessary to the catalytic deamination. Moreover, we identified more than 10 residues in A3G essential to DNA binding and deamination reaction. Therefore, this dimer structure may represent a structural model of full-length A3G, which indicates a possible binding mode of A3G to HIV-1 ssDNA.

  7. Adenosine Deaminase Deficiency – More Than Just an Immunodeficiency

    PubMed Central

    Whitmore, Kathryn V.; Gaspar, Hubert B.

    2016-01-01

    Adenosine deaminase (ADA) deficiency is best known as a form of severe combined immunodeficiency (SCID) that results from mutations in the gene encoding ADA. Affected patients present with clinical and immunological manifestations typical of a SCID. Therapies are currently available that can target these immunological disturbances and treated patients show varying degrees of clinical improvement. However, there is now a growing body of evidence that deficiency of ADA has significant impact on non-immunological organ systems. This review will outline the impact of ADA deficiency on various organ systems, starting with the well-understood immunological abnormalities. We will discuss possible pathogenic mechanisms and also highlight ways in which current treatments could be improved. In doing so, we aim to present ADA deficiency as more than an immunodeficiency and suggest that it should be recognized as a systemic metabolic disorder that affects multiple organ systems. Only by fully understanding ADA deficiency and its manifestations in all organ systems can we aim to deliver therapies that will correct all the clinical consequences. PMID:27579027

  8. The ONIOM molecular dynamics method for biochemical applications: cytidine deaminase

    SciTech Connect

    Matsubara, Toshiaki; Dupuis, Michel; Aida, Misako

    2007-03-22

    Abstract We derived and implemented the ONIOM-molecular dynamics (MD) method for biochemical applications. The implementation allows the characterization of the functions of the real enzymes taking account of their thermal motion. In this method, the direct MD is performed by calculating the ONIOM energy and gradients of the system on the fly. We describe the first application of this ONOM-MD method to cytidine deaminase. The environmental effects on the substrate in the active site are examined. The ONIOM-MD simulations show that the product uridine is strongly perturbed by the thermal motion of the environment and dissociates easily from the active site. TM and MA were supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan. MD was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, and by the Office of Biological and Environmental Research of the U.S. Department of Energy DOE. Battelle operates Pacific Northwest National Laboratory for DOE.

  9. Adenosine Deaminase Deficiency - More Than Just an Immunodeficiency.

    PubMed

    Whitmore, Kathryn V; Gaspar, Hubert B

    2016-01-01

    Adenosine deaminase (ADA) deficiency is best known as a form of severe combined immunodeficiency (SCID) that results from mutations in the gene encoding ADA. Affected patients present with clinical and immunological manifestations typical of a SCID. Therapies are currently available that can target these immunological disturbances and treated patients show varying degrees of clinical improvement. However, there is now a growing body of evidence that deficiency of ADA has significant impact on non-immunological organ systems. This review will outline the impact of ADA deficiency on various organ systems, starting with the well-understood immunological abnormalities. We will discuss possible pathogenic mechanisms and also highlight ways in which current treatments could be improved. In doing so, we aim to present ADA deficiency as more than an immunodeficiency and suggest that it should be recognized as a systemic metabolic disorder that affects multiple organ systems. Only by fully understanding ADA deficiency and its manifestations in all organ systems can we aim to deliver therapies that will correct all the clinical consequences. PMID:27579027

  10. Three-Dimensional Structure and Catalytic Mechanism of Cytosine Deaminase

    SciTech Connect

    R Hall; A Fedorov; C Xu; E Fedorov; S Almo; F Raushel

    2011-12-31

    Cytosine deaminase (CDA) from E. coli is a member of the amidohydrolase superfamily. The structure of the zinc-activated enzyme was determined in the presence of phosphonocytosine, a mimic of the tetrahedral reaction intermediate. This compound inhibits the deamination of cytosine with a K{sub i} of 52 nM. The zinc- and iron-containing enzymes were characterized to determine the effect of the divalent cations on activation of the hydrolytic water. Fe-CDA loses activity at low pH with a kinetic pKa of 6.0, and Zn-CDA has a kinetic pKa of 7.3. Mutation of Gln-156 decreased the catalytic activity by more than 5 orders of magnitude, supporting its role in substrate binding. Mutation of Glu-217, Asp-313, and His-246 significantly decreased catalytic activity supporting the role of these three residues in activation of the hydrolytic water molecule and facilitation of proton transfer reactions. A library of potential substrates was used to probe the structural determinants responsible for catalytic activity. CDA was able to catalyze the deamination of isocytosine and the hydrolysis of 3-oxauracil. Large inverse solvent isotope effects were obtained on k{sub cat} and k{sub cat}/K{sub m}, consistent with the formation of a low-barrier hydrogen bond during the conversion of cytosine to uracil. A chemical mechanism for substrate deamination by CDA was proposed.

  11. Creative deaminases, self-inflicted damage, and genome evolution.

    PubMed

    Conticello, Silvestro G

    2012-09-01

    Organisms minimize genetic damage through complex pathways of DNA repair. Yet a gene family--the AID/APOBECs--has evolved in vertebrates with the sole purpose of producing targeted damage in DNA/RNA molecules through cytosine deamination. They likely originated from deaminases involved in A>I editing in tRNAs. AID, the archetypal AID/APOBEC, is the trigger of the somatic diversification processes of the antibody genes. Its homologs may have been associated with the immune system even before the evolution of the antibody genes. The APOBEC3s, arising from duplication of AID, are involved in the restriction of exogenous/endogenous threats such as retroviruses and mobile elements. Another family member, APOBEC1, has (re)acquired the ability to target RNA while maintaining its ability to act on DNA. The AID/APOBECs have shaped the evolution of vertebrate genomes, but their ability to mutate nucleic acids is a double-edged sword: AID is a key player in lymphoproliferative diseases by triggering mutations and chromosomal translocations in B cells, and there is increasing evidence suggesting that other AID/APOBECs could be involved in cancer development as well.

  12. In utero exposure to benzene increases embryonic c-Myb and Pim-1 protein levels in CD-1 mice

    SciTech Connect

    Wan, Joanne; Winn, Louise M.

    2008-05-01

    Benzene is a known human leukemogen, but its role as an in utero leukemogen remains controversial. Epidemiological studies have correlated parental exposure to benzene with an increased incidence of childhood leukemias. We hypothesize that in utero exposure to benzene may cause leukemogenesis by affecting the embryonic c-Myb/Pim-1 signaling pathway and that this is mediated by oxidative stress. To investigate this hypothesis, pregnant CD-1 mice were treated with either 800 mg/kg of benzene or corn oil (i.p.) on days 10 and 11 of gestation and in some cases pretreated with 25 kU/kg of PEG-catalase. Phosphorylated and total embryonic c-Myb and Pim-1 protein levels were assessed using Western blotting and maternal and embryonic oxidative stress were assessed by measuring reduced to oxidized glutathione ratios. Our results show increased oxidative stress at 4 and 24 h after exposure, increased phosphorylated Pim-1 protein levels 4 h after benzene exposure, and increased Pim-1 levels at 24 and 48 h after benzene exposure. Embryonic c-Myb levels were elevated at 24 h after exposure. PEG-catalase pretreatment prevented benzene-mediated increases in embryonic c-Myb and Pim-1 protein levels, and benzene-induced oxidative stress. These results support a role for ROS in c-Myb and Pim-1 alterations after in utero benzene exposure.

  13. B cell Rab7 mediates induction of activation-induced cytidine deaminase expression and class-switching in T-dependent and T-independent antibody responses.

    PubMed

    Pone, Egest J; Lam, Tonika; Lou, Zheng; Wang, Rui; Chen, Yuhui; Liu, Dongfang; Edinger, Aimee L; Xu, Zhenming; Casali, Paolo

    2015-04-01

    Class switch DNA recombination (CSR) is central to the maturation of the Ab response because it diversifies Ab effector functions. Like somatic hypermutation, CSR requires activation-induced cytidine deaminase (AID), whose expression is restricted to B cells, as induced by CD40 engagement or dual TLR-BCR engagement (primary CSR-inducing stimuli). By constructing conditional knockout Igh(+/C)γ(1-cre)Rab7(fl/fl) mice, we identified a B cell-intrinsic role for Rab7, a small GTPase involved in intracellular membrane functions, in mediating AID induction and CSR. Igh(+/C)γ(1-cre)Rab7(fl/fl) mice displayed normal B and T cell development and were deficient in Rab7 only in B cells undergoing Igh(C)γ(1-cre) Iγ1-Sγ1-Cγ1-cre transcription, as induced--like Igh germline Iγ1-Sγ1-Cγ1 and Iε-Sε-Cε transcription--by IL-4 in conjunction with a primary CSR-inducing stimulus. These mice could not mount T-independent or T-dependent class-switched IgG1 or IgE responses while maintaining normal IgM levels. Igh(+/C)γ(1-cre)Rab7(fl/fl) B cells showed, in vivo and in vitro, normal proliferation and survival, normal Blimp-1 expression and plasma cell differentiation, as well as intact activation of the noncanonical NF-κB, p38 kinase, and ERK1/2 kinase pathways. They, however, were defective in AID expression and CSR in vivo and in vitro, as induced by CD40 engagement or dual TLR1/2-, TLR4-, TLR7-, or TLR9-BCR engagement. In Igh(+/C)γ(1-cre)Rab7(fl/fl) B cells, CSR was rescued by enforced AID expression. These findings, together with our demonstration that Rab7-mediated canonical NF-κB activation, as critical to AID induction, outline a novel role of Rab7 in signaling pathways that lead to AID expression and CSR, likely by promoting assembly of signaling complexes along intracellular membranes.

  14. Greek Mythology: Literature Curriculum, Levels C-D [Grades Three and Four]; Teacher's Guide.

    ERIC Educational Resources Information Center

    Oregon Univ., Eugene. Oregon Elementary English Project.

    This curriculum guide is intended to introduce elementary school students to Greek mythology. The authors suggest that the selections be presented by the teacher as lively and imaginative stories; the more abstract aspects of the myths should be largely ignored until students reach the junior high school level. In addition to the myths themselves,…

  15. Macrophage entry mediated by HIV Envs from brain and lymphoid tissues is determined by the capacity to use low CD4 levels and overall efficiency of fusion

    SciTech Connect

    Thomas, Elaine R.; Dunfee, Rebecca L.; Stanton, Jennifer; Bogdan, Derek; Taylor, Joann; Kunstman, Kevin; Bell, Jeanne E.; Wolinsky, Steven M.; Gabuzda, Dana . E-mail: dana_gabuzda@dfci.harvard.edu

    2007-03-30

    HIV infects macrophages and microglia in the central nervous system (CNS), which express lower levels of CD4 than CD4+ T cells in peripheral blood. To investigate mechanisms of HIV neurotropism, full-length env genes were cloned from autopsy brain and lymphoid tissues from 4 AIDS patients with HIV-associated dementia (HAD). Characterization of 55 functional Env clones demonstrated that Envs with reduced dependence on CD4 for fusion and viral entry are more frequent in brain compared to lymphoid tissue. Envs that mediated efficient entry into macrophages were frequent in brain but were also present in lymphoid tissue. For most Envs, entry into macrophages correlated with overall fusion activity at all levels of CD4 and CCR5. gp160 nucleotide sequences were compartmentalized in brain versus lymphoid tissue within each patient. Proline at position 308 in the V3 loop of gp120 was associated with brain compartmentalization in 3 patients, but mutagenesis studies suggested that P308 alone does not contribute to reduced CD4 dependence or macrophage-tropism. These results suggest that HIV adaptation to replicate in the CNS selects for Envs with reduced CD4 dependence and increased fusion activity. Macrophage-tropic Envs are frequent in brain but are also present in lymphoid tissues of AIDS patients with HAD, and entry into macrophages in the CNS and other tissues is dependent on the ability to use low receptor levels and overall efficiency of fusion.

  16. Improvement of radiopurity level of enriched 116CdWO4 and ZnWO4 crystal scintillators by recrystallization

    NASA Astrophysics Data System (ADS)

    Barabash, A. S.; Belli, P.; Bernabei, R.; Borovlev, Yu. A.; Cappella, F.; Caracciolo, V.; Cerulli, R.; Danevich, F. A.; Incicchitti, A.; Kobychev, V. V.; Konovalov, S. I.; Laubenstein, M.; Mokina, V. M.; Polischuk, O. G.; Safonova, O. E.; Shlegel, V. N.; Tretyak, V. I.; Tupitsyna, I. A.; Umatov, V. I.; Zhdankov, V. N.

    2016-10-01

    As low as possible radioactive contamination of a detector plays a crucial role to improve sensitivity of a double beta decay experiment. The radioactive contamination of a sample of 116CdWO4 crystal scintillator by thorium was reduced by a factor ≈10, down to the level 0.01 mBq/kg (228Th), by exploiting the recrystallization procedure. The total alpha activity of uranium and thorium daughters was reduced by a factor ≈3, down to 1.6 mBq/kg. No change in the specific activity (the total α activity and 228Th) was observed in a sample of ZnWO4 crystal produced by recrystallization after removing ≈0.4 mm surface layer of the crystal.

  17. Structural and biological function of NYD-SP15 as a new member of cytidine deaminases.

    PubMed

    Xu, Yidan; Li, Lei; Li, Jianmin; Liu, Qinghuai

    2016-05-25

    Recent studies were mainly focus on the cytidine deaminase family genes, which contained a lot of members that varied on the function of catalytic deamination in RNA or DNA and were involved in the process of growth maintenance, host immunity, retroviral infection, tumorigenesis, and drug resistance with a feature of C-U deamination. In this study, we identified a new member of cytidine deaminase family, NYD-SP15. Previous work showed that the deduced structure of the protein contained two dCMP_cyt_deam domains, which were involved in zinc ion binding. NYD-SP15 was expressed variably in a wide range of tissues, indicating its worthy biological function and creative significances. Sequence analysis, RT-PCR, western blot, flow cytometry, direct-site mutation and GST pull-down assay were performed to analyze the construction and function of NYD-SP15. The results in our studies showed that NYD-SP15 was closely related to deoxycytidylate deaminase and cytidine deaminase, with authentic cytidine deaminase activity in vivo and vitro as well as homo dimerization effects. NYD-SP15 contained nuclear localization sequence (NLS) and nuclear export-signal (NES) and could dynamically shuttle between the nucleus and cytoplasm. Furthermore, NYD-SP15 gene over-expression reduced the cells growth and blocked G1 to S phase, which implied a potential inhibition effect on cell growth. PMID:26945630

  18. AMP deaminase histochemical activity and immunofluorescent isozyme localization in rat skeletal muscle

    NASA Technical Reports Server (NTRS)

    Thompson, J. L.; Sabina, R. L.; Ogasawara, N.; Riley, D. A.

    1992-01-01

    The cellular distribution of AMP deaminase (AMPda) isozymes was documented for rat soleus and plantaris muscles, utilizing immunofluorescence microscopy and immunoprecipitation methods. AMPda is a ubiquitous enzyme existing as three distinct isozymes, A, B and C, which were initially purified from skeletal muscle, liver (and kidney), and heart, respectively. AMPda-A is primarily concentrated subsarcolemmally and intermyofibrillarly within muscle cells, while isozymes B and C are concentrated within non-myofiber elements of muscle tissue. AMPda-B is principally associated with connective tissues surrounding neural elements and the muscle spindle capsule, and AMPda-C is predominantly associated with circulatory elements, such as arterial and venous walls, capillary endothelium, and red blood cells. These specific localizations, combined with documented differences in kinetic properties, suggest multiple functional roles for the AMPda isozymes or temporal segregation of similar AMPda functions. Linkage of the AMPda substrate with adenosine production pathways at the AMP level and the localization of isozyme-C in vascular tissue suggest a regulatory role in the microcirculation.

  19. Purine nucleoside metabolism in the erythrocytes of patients with adenosine deaminase deficiency and severe combined immunodeficiency.

    PubMed Central

    Agarwal, R P; Crabtree, G W; Parks, R E; Nelson, J A; Keightley, R; Parkman, R; Rosen, F S; Stern, R C; Polmar, S H

    1976-01-01

    Deficiency of erythrocytic and lymphocytic adenosine deaminase (ADA) occurs in some patients with severe combined immunodeficiency disease (SCID). SCID with ADA deficiency is inherited as an autosomal recessive trait. ADA is markedly reduced or undetectable in affected patients (homozygotes), and approximately one-half normal levels are found in individuals heterozygous for ADA deficiency. The metabolism of purine nucleosides was studied in erythrocytes from normal individuals, four ADA-deficiency patients, and two heterozygous individuals. ADA deficiency in intake erythrocytes was confirmed by a very sensitive ammonia-liberation technique. Erythrocytic ADA activity in three heterozygous individuals (0.07,0.08, and 0.14 mumolar units/ml of packed cells) was between that of the four normal controls (0.20-0.37 mumol/ml) and the ADA-deficient patients (no activity). In vitro, adenosine was incorporated principally into IMP in the heterozygous and normal individuals but into the adenosine nucleotides in the ADa-deficient patients. Coformycin (3-beta-D-ribofuranosyl-6,7,8-trihydroimidazo[4,5-4] [1,3] diazepin-8 (R)-ol), a potent inhibitor of ADA, made possible incorporation of adenosine nucleotides in the ADA-deficient patients... PMID:947948

  20. ADENOSINE DEAMINASE ACTIVITY AND SERUM C-REACTIVE PROTEIN AS PROGNOSTIC MARKERS OF CHAGAS DISEASE SEVERITY.

    PubMed

    Bravo-Tobar, Iván Darío; Nello-Pérez, Carlota; Fernández, Alí; Mogollón, Nora; Pérez, Mary Carmen; Verde, Juan; Concepción, Juan Luis; Rodriguez-Bonfante, Claudina; Bonfante-Cabarcas, Rafael

    2015-01-01

    Chagas disease is a public health problem worldwide. The availability of diagnostic tools to predict the development of chronic Chagas cardiomyopathy is crucial to reduce morbidity and mortality. Here we analyze the prognostic value of adenosine deaminase serum activity (ADA) and C-reactive protein serum levels (CRP) in chagasic individuals. One hundred and ten individuals, 28 healthy and 82 chagasic patients were divided according to disease severity in phase I (n = 35), II (n = 29), and III (n = 18). A complete medical history, 12-lead electrocardiogram, chest X-ray, and M-mode echocardiogram were performed on each individual. Diagnosis of Chagas disease was confirmed by ELISA and MABA using recombinant antigens; ADA was determined spectrophotometrically and CRP by ELISA. The results have shown that CRP and ADA increased linearly in relation to disease phase, CRP being significantly higher in phase III and ADA at all phases. Also, CRP and ADA were positively correlated with echocardiographic parameters of cardiac remodeling and with electrocardiographic abnormalities, and negatively with ejection fraction. CRP and ADA were higher in patients with cardiothoracic index ≥ 50%, while ADA was higher in patients with ventricular repolarization disturbances. Finally, CRP was positively correlated with ADA. In conclusion, ADA and CRP are prognostic markers of cardiac dysfunction and remodeling in Chagas disease.

  1. Activation-induced cytidine deaminase (AID) is localized to subnuclear domains enriched in splicing factors

    SciTech Connect

    Hu, Yi Ericsson, Ida Doseth, Berit Liabakk, Nina B. Krokan, Hans E. Kavli, Bodil

    2014-03-10

    Activation-induced cytidine deaminase (AID) is the mutator enzyme in adaptive immunity. AID initiates the antibody diversification processes in activated B cells by deaminating cytosine to uracil in immunoglobulin genes. To some extent other genes are also targeted, which may lead to genome instability and B cell malignancy. Thus, it is crucial to understand its targeting and regulation mechanisms. AID is regulated at several levels including subcellular compartmentalization. However, the complex nuclear distribution and trafficking of AID has not been studied in detail previously. In this work, we examined the subnuclear localization of AID and its interaction partner CTNNBL1 and found that they associate with spliceosome-associated structures including Cajal bodies and nuclear speckles. Moreover, protein kinase A (PKA), which activates AID by phosphorylation at Ser38, is present together with AID in nuclear speckles. Importantly, we demonstrate that AID physically associates with the major spliceosome subunits (small nuclear ribonucleoproteins, snRNPs), as well as other essential splicing components, in addition to the transcription machinery. Based on our findings and the literature, we suggest a transcription-coupled splicing-associated model for AID targeting and activation. - Highlights: • AID and its interaction partner CTNNBL1 localize to Cajal bodies and nuclear speckles. • AID associates with its activating kinase PKA in nuclear speckles. • AID is linked to the splicing machinery in switching B-cells. • Our findings suggest a transcription-coupled splicing associated mechanism for AID targeting and activation.

  2. ADENOSINE DEAMINASE ACTIVITY AND SERUM C-REACTIVE PROTEIN AS PROGNOSTIC MARKERS OF CHAGAS DISEASE SEVERITY

    PubMed Central

    BRAVO-TOBAR, Iván Darío; NELLO-PÉREZ, Carlota; FERNÁNDEZ, Alí; MOGOLLÓN, Nora; PÉREZ, Mary Carmen; VERDE, Juan; CONCEPCIÓN, Juan Luis; RODRIGUEZ-BONFANTE, Claudina; BONFANTE-CABARCAS, Rafael

    2015-01-01

    SUMMARY Chagas disease is a public health problem worldwide. The availability of diagnostic tools to predict the development of chronic Chagas cardiomyopathy is crucial to reduce morbidity and mortality. Here we analyze the prognostic value of adenosine deaminase serum activity (ADA) and C-reactive protein serum levels (CRP) in chagasic individuals. One hundred and ten individuals, 28 healthy and 82 chagasic patients were divided according to disease severity in phase I (n = 35), II (n = 29), and III (n = 18). A complete medical history, 12-lead electrocardiogram, chest X-ray, and M-mode echocardiogram were performed on each individual. Diagnosis of Chagas disease was confirmed by ELISA and MABA using recombinant antigens; ADA was determined spectrophotometrically and CRP by ELISA. The results have shown that CRP and ADA increased linearly in relation to disease phase, CRP being significantly higher in phase III and ADA at all phases. Also, CRP and ADA were positively correlated with echocardiographic parameters of cardiac remodeling and with electrocardiographic abnormalities, and negatively with ejection fraction. CRP and ADA were higher in patients with cardiothoracic index ≥ 50%, while ADA was higher in patients with ventricular repolarization disturbances. Finally, CRP was positively correlated with ADA. In conclusion, ADA and CRP are prognostic markers of cardiac dysfunction and remodeling in Chagas disease. PMID:26603224

  3. LINE-1 retroelements complexed and inhibited by activation induced cytidine deaminase.

    PubMed

    Metzner, Mirjam; Jäck, Hans-Martin; Wabl, Matthias

    2012-01-01

    LINE-1 (abbreviated L1) is a major class of retroelements in humans and mice. If unrestricted, retroelements accumulate in the cytoplasm and insert their DNA into the host genome, with the potential to cause autoimmune disease and cancer. Retroviruses and other retroelements are inhibited by proteins of the APOBEC family, of which activation-induced cytidine deaminase (AID) is a member. Although AID is mainly known for being a DNA mutator shaping the antibody repertoire in B lymphocytes, we found that AID also restricts de novo L1 integrations in B- and non-B-cell lines. It does so by decreasing the protein level of open reading frame 1 (ORF1) of both exogenous and endogenous L1. In activated B lymphocytes, AID deficiency increased L1 mRNA 1.6-fold and murine leukemia virus (MLV) mRNA 2.7-fold. In cell lines and activated B lymphocytes, AID forms cytoplasmic high-molecular-mass complexes with L1 mRNA, which may contribute to L1 restriction. Because AID-deficient activated B lymphocytes do not express ORF1 protein, we suggest that ORF1 protein expression is inhibited by additional restriction factors in these cells. The greater increase in MLV compared to L1 mRNA in AID-deficient activated B lymphocytes may indicate less strict surveillance of retrovirus. PMID:23133680

  4. A low level of dietary selenium has both beneficial and toxic effects and is protective against Cd-toxicity in the least killifish Heterandria formosa.

    PubMed

    Xie, Lingtian; Wu, Xing; Chen, Hongxing; Dong, Wu; Cazan, Alfy Morales; Klerks, Paul L

    2016-10-01

    As an essential element, selenium (Se) is beneficial at low levels yet toxic at high levels. The present study assessed the effects of dietary exposure to Se in the least killifish Heterandria formosa, and investigated how this exposure influences the effects of a subsequent exposure to cadmium (Cd). The fish were pre-exposed to an environmentally relevant concentration (2 μg g(-1) dry wt) of dietary selenite (Se(4+)) or seleno-l-methionine (Se-Met) for 10 d. The same fish were then exposed to 0.5 mg L(-1) of Cd for 5 d. Both Se(IV) and Se-Met rapidly accumulated in H. formosa. Results for the two Se species were generally similar in this study. Fish exposed to Se had lower levels of lipid peroxidation (measured as levels of thiobarbituric acid reactive substances or TBARS) and a higher catalase (CAT) activity. In contrast, their Na(+)/K(+)-ATPase activity was reduced. The Cd exposure resulted in an increase in lipid peroxidation and decreases in the activities of catalase and Na(+)/K(+)-ATPase. The Cd-exposed H. formosa that were pre-exposed to Se had lower Cd body burdens, less lipid peroxidation, and higher catalase activity, than did fish not pre-exposed to Se. The Se exposure did not have a protective effect on the Cd-induced reduction in Na(+)/K(+)-ATPase activity. These results clearly demonstrate that a Se-enriched diet reduces some (but not all) forms of Cd-toxicity and that Se can simultaneously have beneficial and detrimental effects, making it difficult to predict the net outcome of changes in dietary Se levels for fish.

  5. Association of CD14-260 Polymorphisms, Red-complex Periodontopathogens and Gingival Crevicular Fluid Cytokine Levels with Cyclosporine A-induced Gingival Overgrowth in Renal Transplant Patients

    PubMed Central

    Gong, Yiming; Bi, Wei; Cao, Lingfeng; Yang, Yi; Chen, Jake; Yu, Youcheng

    2012-01-01

    Backgroud and Objective Genetic factors may influence the colonization of pathogenic bacteria, therefore increasing the risk for the initiation and development of periodontal disease. The present study was carried out to investigate the association of CD14-260 polymorphisms, subgingival microbiota, and gingival crevicular fluid (GCF) cytokine levels with cyclosporine A (CsA)-induced gingival overgrowth (GO) in renal transplant patients. Material and Methods 204 patients were dichotomized into two groups: 124 with GO and 80 without GO. The CD14-260 polymorphisms were measured using an allele-specific PCR method. The levels of periodontal pathogens were determined by real-time PCR of subgingival samples. GCF levels of IL-1β and sCD14 were detected by ELISA. Results The frequency of CD14-260 genotype CT + TT was found to be similar in both groups. Patients with GO presented increased prevalence of Pg, Td, and Tf (red complex) and significantly higher levels of IL-1β than those without GO. GO patients carrying CT+TT genotypes were found to have higher frequencies of Pg, Td, and Tf than those carrying CC genotype. Furthermore, in the presence of red complex, CT+TT genotypes were associated with higher IL-1β levels and severe GO. Multiple logistic regression analysis demonstrated that the severity of GO is not dependent on age, gender and pharmacological variables, being only associated with CD14-260 genotype and red complex periodontopathogens. Conclusions No association between CD14-260 polymorphisms and the prevalence of GO was revealed in renal transplant patients administered CsA. However, CD14-260 CT+TT genotypes are associated with the prevalence of red complex periodontopathogens in GO patients, and may thus play some role in the development of severe CsA-induced GO. PMID:22934794

  6. A low level of dietary selenium has both beneficial and toxic effects and is protective against Cd-toxicity in the least killifish Heterandria formosa.

    PubMed

    Xie, Lingtian; Wu, Xing; Chen, Hongxing; Dong, Wu; Cazan, Alfy Morales; Klerks, Paul L

    2016-10-01

    As an essential element, selenium (Se) is beneficial at low levels yet toxic at high levels. The present study assessed the effects of dietary exposure to Se in the least killifish Heterandria formosa, and investigated how this exposure influences the effects of a subsequent exposure to cadmium (Cd). The fish were pre-exposed to an environmentally relevant concentration (2 μg g(-1) dry wt) of dietary selenite (Se(4+)) or seleno-l-methionine (Se-Met) for 10 d. The same fish were then exposed to 0.5 mg L(-1) of Cd for 5 d. Both Se(IV) and Se-Met rapidly accumulated in H. formosa. Results for the two Se species were generally similar in this study. Fish exposed to Se had lower levels of lipid peroxidation (measured as levels of thiobarbituric acid reactive substances or TBARS) and a higher catalase (CAT) activity. In contrast, their Na(+)/K(+)-ATPase activity was reduced. The Cd exposure resulted in an increase in lipid peroxidation and decreases in the activities of catalase and Na(+)/K(+)-ATPase. The Cd-exposed H. formosa that were pre-exposed to Se had lower Cd body burdens, less lipid peroxidation, and higher catalase activity, than did fish not pre-exposed to Se. The Se exposure did not have a protective effect on the Cd-induced reduction in Na(+)/K(+)-ATPase activity. These results clearly demonstrate that a Se-enriched diet reduces some (but not all) forms of Cd-toxicity and that Se can simultaneously have beneficial and detrimental effects, making it difficult to predict the net outcome of changes in dietary Se levels for fish. PMID:27448316

  7. Mapping Zn, Cu and Cd contents at the small catchment level after dispersion of contaminants by agricultural practices

    NASA Astrophysics Data System (ADS)

    Vidal Vázquez, E.; Mirás-Avalos, J. M.; Paz-Ferreiro, J.

    2009-04-01

    Dispersion of trace metals into the rural environment through the use of sewage sludge, fertilizers and manure has been worldwide reported. In El Abelar (Coruña province, Spain), pig slurry was discharged during years intensively into an agricultural field by means of a device which constituted a point source of contamination. The application point was located near the head of an elementary basin, so that slurry was dispersed by runoff into neighboring grassland and maize fields. In addition, diffuse pollution was also present in the study area as a consequence of cattle grazing. Water quality was monitored during and after slurry application at the outlet of a small catchment (about 10.7 ha in surface) draining the study fields. High levels of nutrients, including heavy metals, were found in drainage water. The main objectives of this paper are to determine the spatial variability of Cu, Zn and Cd as extracted by NO3H, EDTA and Ca2Cl and to evaluate the risk of accumulation of these heavy metals at the small catchment level. A set of 55 soil samples were taken from the top soil layer (0-20 cm) of the studied catchment, following a random sampling scheme. Fe, Mn, Cu, Zn and Cd contents were determined i) after digestion by nitric acid in a microwave (USEPA-SW-846 3051) ii) after extraction with EDTA and iii) after extraction with Cl2Ca. Element contents in the extracts were determined by ICP-MS. Summary statistics indicate that variability in Cu, Zn and Cd contents over the study area was very high. For example, after NO3H digestion Zn contents ranged from 29.66 to 141.77 3 mg kg-1 and Cu contents varied from 10.45 to 72.7 3 mg kg-1. High Cu and Zn contents result from accumulation as a consequence of slurry discharge. Also, some hot spots with high levels of Cd (> 3 mg kg-1 after NO3H) with respect to background values were recorded. Geostatistics provides all necessary tools to analyze the spatial variability of soil properties over a landscape. The spatial

  8. Adenosine Deaminase Enzyme Therapy Prevents and Reverses the Heightened Cavernosal Relaxation in Priapism

    PubMed Central

    Wen, Jiaming; Jiang, Xianzhen; Dai, Yingbo; Zhang, Yujin; Tang, Yuxin; Sun, Hong; Mi, Tiejuan; Kellems, Rodney E.; Blackburn, Michael R.; Xia, Yang

    2010-01-01

    Introduction Priapism featured with painful prolonged penile erection is dangerous and commonly seen in sickle cell disease (SCD). The preventive approaches or effective treatment options for the disorder are limited because of poor understanding of its pathogenesis. Recent studies have revealed a novel role of excess adenosine in priapism caused by heightened cavernosal relaxation, and therefore present an intriguing mechanism-based therapeutic possibility. Aim The aim of this study was to determine the therapeutic effects of adenosine deaminase (ADA) enzyme therapy to lower adenosine in priapism. Methods Both ADA-deficient mice and SCD transgenic (Tg) mice display priapism caused by excessive adenosine. Thus, we used these two distinct lines of mouse models of priapism as our investigative tools. Specifically, we treated both of these mice with different dosages of polyethylene glycol–modified ADA (PEG–ADA) to reduce adenosine levels in vivo. At the end points of the experiments, we evaluated the therapeutic effects of PEG–ADA treatment by measuring adenosine levels and monitoring the cavernosal relaxation. Main Outcome Measures Adenosine levels in penile tissues were measured by high-performance liquid chromatography, and cavernosal relaxation was quantified by electrical field stimulation (EFS)-induced corporal cavernosal strip (CCS) assays. Results We found that lowering adenosine levels in penile tissues by PEG–ADA treatment from birth in ADA-deficient mice prevented the increased EFS-induced CCS relaxation associated with priapism. Intriguingly, in both ADA-deficient mice and SCD Tg mice with established priapism, we found that normalization of adenosine levels in penile tissues by PEG–ADA treatment relieved the heightened EFS-induced cavernosal relaxation in priapism. Conclusions Our studies have identified that PEG–ADA is a novel, safe, and mechanism-based drug to prevent and correct excess adenosine-mediated increased cavernosal relaxation

  9. Structural and Metabolic Specificity of Methylthiocoformycin for Malarial Adenosine Deaminases

    SciTech Connect

    Ho, M.; Cassera, M; Madrid, D; Ting, L; Tyler, P; Kim, K; Almo, S; Schramm, V

    2009-01-01

    Plasmodium falciparum is a purine auxotroph requiring hypoxanthine as a key metabolic precursor. Erythrocyte adenine nucleotides are the source of the purine precursors, making adenosine deaminase (ADA) a key enzyme in the pathway of hypoxanthine formation. Methylthioadenosine (MTA) is a substrate for most malarial ADAs, but not for human ADA. The catalytic site specificity of malarial ADAs permits methylthiocoformycin (MT-coformycin) to act as a Plasmodium-specific transition state analogue with low affinity for human ADA. The structural basis for MTA and MT-coformycin specificity in malarial ADAs is the subject of speculation. Here, the crystal structure of ADA from Plasmodium vivax (PvADA) in a complex with MT-coformycin reveals an unprecedented binding geometry for 5?-methylthioribosyl groups in the malarial ADAs. Compared to malarial ADA complexes with adenosine or deoxycoformycin, 5?-methylthioribosyl groups are rotated 130 degrees. A hydrogen bonding network between Asp172 and the 3?-hydroxyl of MT-coformycin is essential for recognition of the 5?-methylthioribosyl group. Water occupies the 5?-hydroxyl binding site when MT-coformycin is bound. Mutagenesis of Asp172 destroys the substrate specificity for MTA and MT-coformycin. Kinetic, mutagenic, and structural analyses of PvADA and kinetic analysis of five other Plasmodium ADAs establish the unique structural basis for its specificity for MTA and MT-coformycin. Plasmodium gallinaceum ADA does not use MTA as a substrate, is not inhibited by MT-coformycin, and is missing Asp172. Treatment of P. falciparum cultures with coformycin or MT-coformycin in the presence of MTA is effective in inhibiting parasite growth.

  10. Biological function of activation-induced cytidine deaminase (AID).

    PubMed

    Kumar, Ritu; DiMenna, Lauren J; Chaudhuri, Jayanta; Evans, Todd

    2014-01-01

    Activation-induced Cytidine Deaminase (AID) is an essential regulator of B cell diversification, but its full range of action has until recently been an enigma. Based on homology, it was originally proposed to be an RNA-editing enzyme, but so far, no RNA substrates are known. Rather, it functions by deaminating cytidine, and in this manner, coupled with base-excision repair or mismatch repair machinery, it is a natural mutator. This allows it to play a central role in adaptive immunity, whereby it initiates the processes of class switch recombination and somatic hypermutation to help generate a diverse and high-affinity repertoire of immunoglobulin isotypes. More recently, it has been appreciated that methylated cytidine, already known as a key epigenetic mark on DNA controlling gene expression, can also be a target for AID modification. Coupled with repair machinery, this can facilitate the active removal of methylated DNA. This activity can impact the process of cellular reprogramming, including transition of a somatic cell to pluripotency, which requires major reshuffling of epigenetic memory. Thus, seemingly disparate roles for AID in controlling immune diversity and epigenetic memory have a common mechanistic basis. However, the very activity that is so useful for B cell diversity and cellular reprogramming is dangerous for the integrity of the genome. Thus, AID expression and activity is tightly regulated, and deregulation is associated with diseases including cancer. Here, we review the range of AID functions with a focus on its mechanisms of action and regulation. Major questions remain to be answered concerning how and when AID is targeted to specific loci and how this impacts development and disease.

  11. Genetic heterogeneity in Cornelia de Lange syndrome (CdLS) and CdLS-like phenotypes with observed and predicted levels of mosaicism

    PubMed Central

    Ansari, Morad; Poke, Gemma; Ferry, Quentin; Williamson, Kathleen; Aldridge, Roland; Meynert, Alison M; Bengani, Hemant; Chan, Cheng Yee; Kayserili, Hülya; Avci, Şahin; Hennekam, Raoul C M; Lampe, Anne K; Redeker, Egbert; Homfray, Tessa; Ross, Alison; Falkenberg Smeland, Marie; Mansour, Sahar; Parker, Michael J; Cook, Jacqueline A; Splitt, Miranda; Fisher, Richard B; Fryer, Alan; Magee, Alex C; Wilkie, Andrew; Barnicoat, Angela; Brady, Angela F; Cooper, Nicola S; Mercer, Catherine; Deshpande, Charu; Bennett, Christopher P; Pilz, Daniela T; Ruddy, Deborah; Cilliers, Deirdre; Johnson, Diana S; Josifova, Dragana; Rosser, Elisabeth; Thompson, Elizabeth M; Wakeling, Emma; Kinning, Esther; Stewart, Fiona; Flinter, Frances; Girisha, Katta M; Cox, Helen; Firth, Helen V; Kingston, Helen; Wee, Jamie S; Hurst, Jane A; Clayton-Smith, Jill; Tolmie, John; Vogt, Julie; Tatton–Brown, Katrina; Chandler, Kate; Prescott, Katrina; Wilson, Louise; Behnam, Mahdiyeh; McEntagart, Meriel; Davidson, Rosemarie; Lynch, Sally-Ann; Sisodiya, Sanjay; Mehta, Sarju G; McKee, Shane A; Mohammed, Shehla; Holden, Simon; Park, Soo-Mi; Holder, Susan E; Harrison, Victoria; McConnell, Vivienne; Lam, Wayne K; Green, Andrew J; Donnai, Dian; Bitner-Glindzicz, Maria; Donnelly, Deirdre E; Nellåker, Christoffer; Taylor, Martin S; FitzPatrick, David R

    2014-01-01

    Background Cornelia de Lange syndrome (CdLS) is a multisystem disorder with distinctive facial appearance, intellectual disability and growth failure as prominent features. Most individuals with typical CdLS have de novo heterozygous loss-of-function mutations in NIPBL with mosaic individuals representing a significant proportion. Mutations in other cohesin components, SMC1A, SMC3, HDAC8 and RAD21 cause less typical CdLS. Methods We screened 163 affected individuals for coding region mutations in the known genes, 90 for genomic rearrangements, 19 for deep intronic variants in NIPBL and 5 had whole-exome sequencing. Results Pathogenic mutations [including mosaic changes] were identified in: NIPBL 46 [3] (28.2%); SMC1A 5 [1] (3.1%); SMC3 5 [1] (3.1%); HDAC8 6 [0] (3.6%) and RAD21 1 [0] (0.6%). One individual had a de novo 1.3 Mb deletion of 1p36.3. Another had a 520 kb duplication of 12q13.13 encompassing ESPL1, encoding separase, an enzyme that cleaves the cohesin ring. Three de novo mutations were identified in ANKRD11 demonstrating a phenotypic overlap with KBG syndrome. To estimate the number of undetected mosaic cases we used recursive partitioning to identify discriminating features in the NIPBL-positive subgroup. Filtering of the mutation-negative group on these features classified at least 18% as ‘NIPBL-like’. A computer composition of the average face of this NIPBL-like subgroup was also more typical in appearance than that of all others in the mutation-negative group supporting the existence of undetected mosaic cases. Conclusions Future diagnostic testing in ‘mutation-negative’ CdLS thus merits deeper sequencing of multiple DNA samples derived from different tissues. PMID:25125236

  12. Enthalpy and enzyme activity of modified histidine residues of adenosine deaminase and diethyl pyrocarbonate complexes.

    PubMed

    Ataie, G; Moosavi-Movahedi, A A; Saboury, A A; Hakimelahi, G H; Hwu, J R; Tsay, S C

    2000-03-16

    Kinetic and thermodynamic studies have been made on the effect of diethyl pyrocarbonate as a histidine modifier on the active site of adenosine deaminase in 50 mM sodium phosphate buffer pH 6.8, at 27 degrees C using UV spectrophotometry and isothermal titration calorimetry (ITC). Inactivation of adenosine deaminase by diethyl pyrocarbonate is correlated with modification of histidyl residues. The number of modified histidine residues complexed to active site of adenosine deaminase are equivalent to 4. The number and energy of histidine binding sets are determined by enthalpy curve, which represents triple stages. These stages are composed of 3,1 and 1 sites of histidyl modified residues at diethyl pyrocarbonate concentrations, 0.63, 1.8, 3.3 mM. The heat contents corresponding to the first, second and third sets are found to be 18000, 22000 and 21900 kJ mol(-1) respectively.

  13. Cortisol increases CXCR4 expression but does not affect CD62L and CCR7 levels on specific T cell subsets in humans.

    PubMed

    Besedovsky, Luciana; Linz, Barbara; Dimitrov, Stoyan; Groch, Sabine; Born, Jan; Lange, Tanja

    2014-06-01

    Glucocorticoids are well known to affect T cell migration, leading to a redistribution of the cells from blood to the bone marrow, accompanied by a concurrent suppression of lymph node homing. Despite numerous studies in this context, with most of them employing synthetic glucocorticoids in nonphysiological doses, the mechanisms of this redistribution are not well understood. Here, we investigated in healthy men the impact of cortisol at physiological concentrations on the expression of different migration molecules on eight T cell subpopulations in vivo and in vitro. Hydrocortisone (cortisol, 22 mg) infused during nocturnal rest when endogenous cortisol levels are low, compared with placebo, differentially reduced numbers of T cell subsets, with naive CD4(+) and CD8(+) subsets exhibiting the strongest reduction. Hydrocortisone in vivo and in vitro increased CXCR4 expression, which presumably mediates the recruitment of T cells to the bone marrow. Expression of the lymph node homing receptor CD62L on total CD3(+) and CD8(+) T cells appeared reduced following hydrocortisone infusion. However, this was due to a selective extravasation of CD62L(+) T cell subsets, as hydrocortisone affected neither CD62L expression on a subpopulation level nor CD62L expression in vitro. Corresponding results in the opposite direction were observed after blocking of endogenous cortisol synthesis by metyrapone. CCR7, another lymph node homing receptor, was also unaffected by hydrocortisone in vitro. Thus, cortisol seems to redirect T cells to the bone marrow by upregulating their CXCR4 expression, whereas its inhibiting effect on T cell homing to lymph nodes is apparently regulated independently of the expression of classical homing receptors.

  14. Oxidative Stress Biomarkers and Adenosine Deaminase over the Alopecic Area of the Patients with Alopecia Areata

    PubMed Central

    Öztürk, Perihan; Arıcan, Özer; Kurutaş, Ergül Belge; Mülayim, Kamil

    2016-01-01

    Background: Alopecia areata (AA) is an autoimmune, T-cell mediated, and chronic inflammatory disorder. The pathological mechanisms of disease are unclear, but oxidative stress may be involved. To our knowledge, no studies have examined the oxidative stress levels or biomarkers within the lesional area and skin surface in patients with AA. Similarly, adenosine deaminase (ADA) has not been characterized in AA. Aims: Therefore, we aimed to define ADA levels and the factors involved in oxidative stress from scalp-scrapes of patients with AA. Study Design: Case-control study. Method: A total of 60 patients (30 diagnosed AA patients and 30 healthy controls) were included in the study. ADA as well as oxidative stress factors, including malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) were analyzed from scalp-scrapes in both groups and quantified by spectrophotometry. Results: Activities of SOD (p=0.000), CAT (p=0.033), and ADA (p=0.004) as well as levels of GSH (p=0.000) and MDA (p=0.032) in patients with AA were higher than the controls statistically significant. Conclusion: Based on these results, factors associated with oxidative stress were elevated in AA patient scalp-scrapes compared to controls and may have a defined role the disease pathogenesis. Alterations in the activities of antioxidant enzymes from AA patient scraping samples may be a local effect of elevated oxidative stress levels. In this disease, oxidative stress may affect not only hair follicle but also any layers of the skin. PMID:27403388

  15. CXCL8, IL-1β and sCD200 are pro-inflammatory cytokines and their levels increase in the circulation of breast carcinoma patients

    PubMed Central

    Celik, Betul; Yalcin, Arzu Didem; Genc, Gizem Esra; Bulut, Tangul; Kuloglu Genc, Sibel; Gumuslu, Saadet

    2016-01-01

    The influence of biomarkers on carcinogenesis has been investigated extensively. Whether they promote carcinogenesis or work against cancer development remains to be elucidated. To the best of our knowledge, the novel molecule cluster of differentiation 200 (CD200) has not been studied on human breast cancer subjects. The present study aimed to evaluate interleukin-1β (IL-1β), C-X-C motif chemokine ligand 8 (CXCL8), cancer antigen 15.3 (CA 15.3) and the soluble CD200 (sCD200) levels in the serum samples of breast carcinoma patients in order to predict their role in breast carcinoma. The subjects included individuals with early and advanced stage breast cancers, as well as healthy controls. Commercially available ELISA kits were used to measure the serum concentrations of sCD200, IL-1β, CXCL8, CA 15.3, C-reactive protein (CRP) and leukocyte count. A total of 130 subjects were recruited; 50 early stage cancer, 50 advanced stage and 30 control subjects. Serum sCD200, CXCL8, IL-1β and CRP levels were significantly higher in the early as well as the advanced stage breast cancer patients compared to the control group. The level of CA 15.3 was statistically different between early and advanced stage. There were significant positive correlations between IL-1β and CXCL8, and IL-1β and serum sCD200 levels in the control group. These correlations did not persist in the early or the advanced stage cancer groups except CRP and CA 15.3, but new correlations appeared between serum sCD200 level and leukocyte count for advanced stage breast cancer group. Multivariate regression correlation analysis revealed positive correlation between IL-1β and sCD200; and IL-1β and CXCL8. In conclusion, sCD200, CXCL8, CA 15.3 and IL-1β are proinflammatory molecules and their levels are influenced in breast cancer patients. PMID:27446554

  16. Cyclosporin A inhibits CD11a/CD18 adhesion molecules due to inhibition of TNFα and IL-1β levels in the mouse model of pleurisy induced by carrageenan

    PubMed Central

    Dalmarco, Eduardo Monguilhott; Medeiros, Yara Santos

    2008-01-01

    The mouse model of pleurisy induced by carrageenan is characterized by a significant enhancement of cell migration due to neutrophils 4 h after pleurisy induction. Forty-eight hours after pleurisy induction, a significant increase in cell migration due to mononuclear cells occurs. Recently, studies in our laboratory have demonstrated that cyclosporine A (CsA) inhibits leukocyte migration in the pleural cavity and lungs in the mouse model of pleurisy induced by carrageenan. In the present work we evaluated whether CsA was able to downregulate CD11a/CD18 adhesion molecule in the lungs, as well as TNFα and IL-1β levels in the fluid leakage of the pleural cavity in this model. Our results showed that CsA significantly decreased CD11a/CD18 in the lungs, as well as TNFα and IL-1β levels in the fluid leakage of the pleural cavity 4 h and 48 h after pleurisy induction. It is our hypothesis that the inhibitory effect elicited by CsA upon these adhesion molecules may be also be attributed to the downregulation of TNFα and IL-1β cytokines. PMID:19262158

  17. Down-regulation of promoter methylation level of CD4 gene after MDV infection in MD-susceptible chicken line

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Marek’s disease virus (MDV) is an oncovirus that induces lymphoid tumors in susceptible chickens, and may affect the epigenetic stability of the CD4 gene. The purpose of this study was to find how the effect of MDV infection on DNA methylation status of the CD4 gene differed between MD-resistant (L6...

  18. Increased CD4+ T cell levels during IL-7 administration of antiretroviral therapy-treated simian immunodeficiency virus-positive macaques are not dependent on strong proliferative responses.

    PubMed

    Leone, Amanda; Rohankhedkar, Mukta; Okoye, Afam; Legasse, Alfred; Axthelm, Michael K; Villinger, Francois; Piatak, Michael; Lifson, Jeffrey D; Assouline, Brigitte; Morre, Michel; Picker, Louis J; Sodora, Donald L

    2010-08-01

    CD4(+) T cell depletion is a fundamental component of HIV infection and AIDS pathogenesis and is not always reversed following antiretroviral therapy (ART). In this study, the SIV-infected rhesus macaque model was used to assess recombinant simian IL-7 in its glycosylated form (rsIL-7gly) to enhance regeneration of CD4(+) T cells, particularly the crucial central memory compartment, after ART. We assessed the impact of rsIL-7gly administration as single injections and as a cluster of three doses. Irrespective of the dosing strategy used, the rsIL-7gly administration transiently increased proliferation of both central memory and naive cells, in both CD4(+) and CD8(+) subsets, without increasing SIV levels in the blood. Administration of rsIL-7gly at intervals of 4-6 wk maximized the proliferative response to therapy but resulted in only transient increases in peripheral blood T cell counts. Although more frequent rsIL-7gly "clustered" dosing (three times weekly with 2 wk of rest and then repeat) induced only an initial proliferative burst by CD4(+) T cells, this dosing strategy resulted in sustained increases in peripheral blood CD4(+) T cell counts. The clustered rsIL-7gly treatment regimen was shown to increase the half-life of a BrdU label among memory T cells in the blood when compared with that of macaques treated with ART alone, which is consistent with enhanced cell survival. These results indicate that dosing intervals have a major impact on the response to rsIL-7gly in SIV-positive ART-treated rhesus macaques and that optimum dosing strategies may be ones that induce CD4(+) T cell proliferation initially and provide increased CD4(+) T cell survival.

  19. Carrier Density and Compensation in Semiconductors with Multi Dopants and Multi Transition Energy Levels: The Case of Cu Impurity in CdTe: Preprint

    SciTech Connect

    Wei, S. H.; Ma, J.; Gessert, T. A.; Chin, K. K.

    2011-07-01

    Doping is one of the most important issues in semiconductor physics. The charge carrier generated by doping can profoundly change the properties of semiconductors and their performance in optoelectronic device applications, such as solar cells. Using detailed balance theory and first-principles calculated defect formation energies and transition energy levels, we derive general formulae to calculate carrier density for semiconductors with multi dopants and multi transition energy levels. As an example, we studied CdTe doped with Cu, in which VCd, CuCd, and Cui are the dominant defects/impurities. We show that in this system, when Cu concentration increases, the doping properties of the system can change from a poor p-type, to a poorer p-type, to a better p-type, and then to a poor p-type again, in good agreement with experimental observation of CdTe-based solar cells.

  20. High aminopeptidase N/CD13 levels characterize human amniotic mesenchymal stem cells and drive their increased adipogenic potential in obese women.

    PubMed

    Iaffaldano, Laura; Nardelli, Carmela; Raia, Maddalena; Mariotti, Elisabetta; Ferrigno, Maddalena; Quaglia, Filomena; Labruna, Giuseppe; Capobianco, Valentina; Capone, Angela; Maruotti, Giuseppe Maria; Pastore, Lucio; Di Noto, Rosa; Martinelli, Pasquale; Sacchetti, Lucia; Del Vecchio, Luigi

    2013-08-15

    Maternal obesity is associated to increased fetal risk of obesity and other metabolic diseases. Human amniotic mesenchymal stem cells (hA-MSCs) have not been characterized in obese women. The aim of this study was to isolate and compare hA-MSC immunophenotypes from obese (Ob-) and normal weight control (Co-) women, to identify alterations possibly predisposing the fetus to obesity. We enrolled 16 Ob- and 7 Co-women at delivery (mean/SEM prepregnancy body mass index: 40.3/1.8 and 22.4/1.0 kg/m2, respectively), and 32 not pregnant women. hA-MSCs were phenotyped by flow cytometry; several maternal and newborn clinical and biochemical parameters were also measured. The expression of membrane antigen CD13 was higher on Ob-hA-MSCs than on Co-hA-MSCs (P = 0.005). Also, serum levels of CD13 at delivery were higher in Ob- versus Co-pregnant women and correlated with CD13 antigen expression on Ob-hA-MSCs (r2 = 0.84, P < 0.0001). Adipogenesis induction experiments revealed that Ob-hA-MSCs had a higher adipogenic potential than Co-hA-MSCs as witnessed by higher peroxisome proliferator-activated receptor gamma and aP2 mRNA levels (P = 0.05 and P = 0.05, respectively), at postinduction day 14 associated with increased CD13 mRNA levels from baseline to day 4 postinduction (P < 0.05). Adipogenesis was similar in the two sets of hA-MSCs after CD13 silencing, whereas it was increased in Co-hA-MSCs after CD13 overexpression. CD13 expression was high also in Ob-h-MSCs from umbilical cords or visceral adipose tissue of not pregnant women. In conclusion, antigen CD13, by influencing the adipogenic potential of hA-MSCs, could be an in utero risk factor for obesity. Our data strengthen the hypothesis that high levels of serum and MSC CD13 are obesity markers.

  1. High Aminopeptidase N/CD13 Levels Characterize Human Amniotic Mesenchymal Stem Cells and Drive Their Increased Adipogenic Potential in Obese Women

    PubMed Central

    Iaffaldano, Laura; Nardelli, Carmela; Raia, Maddalena; Mariotti, Elisabetta; Ferrigno, Maddalena; Quaglia, Filomena; Labruna, Giuseppe; Capobianco, Valentina; Capone, Angela; Maruotti, Giuseppe Maria; Pastore, Lucio; Di Noto, Rosa; Martinelli, Pasquale; Del Vecchio, Luigi

    2013-01-01

    Maternal obesity is associated to increased fetal risk of obesity and other metabolic diseases. Human amniotic mesenchymal stem cells (hA-MSCs) have not been characterized in obese women. The aim of this study was to isolate and compare hA-MSC immunophenotypes from obese (Ob-) and normal weight control (Co-) women, to identify alterations possibly predisposing the fetus to obesity. We enrolled 16 Ob- and 7 Co-women at delivery (mean/SEM prepregnancy body mass index: 40.3/1.8 and 22.4/1.0 kg/m2, respectively), and 32 not pregnant women. hA-MSCs were phenotyped by flow cytometry; several maternal and newborn clinical and biochemical parameters were also measured. The expression of membrane antigen CD13 was higher on Ob-hA-MSCs than on Co-hA-MSCs (P=0.005). Also, serum levels of CD13 at delivery were higher in Ob- versus Co-pregnant women and correlated with CD13 antigen expression on Ob-hA-MSCs (r2=0.84, P<0.0001). Adipogenesis induction experiments revealed that Ob-hA-MSCs had a higher adipogenic potential than Co-hA-MSCs as witnessed by higher peroxisome proliferator-activated receptor gamma and aP2 mRNA levels (P=0.05 and P=0.05, respectively), at postinduction day 14 associated with increased CD13 mRNA levels from baseline to day 4 postinduction (P<0.05). Adipogenesis was similar in the two sets of hA-MSCs after CD13 silencing, whereas it was increased in Co-hA-MSCs after CD13 overexpression. CD13 expression was high also in Ob-h-MSCs from umbilical cords or visceral adipose tissue of not pregnant women. In conclusion, antigen CD13, by influencing the adipogenic potential of hA-MSCs, could be an in utero risk factor for obesity. Our data strengthen the hypothesis that high levels of serum and MSC CD13 are obesity markers. PMID:23488598

  2. Interleukin-1 and tumor necrosis factor-α trigger restriction of hepatitis B virus infection via a cytidine deaminase activation-induced cytidine deaminase (AID).

    PubMed

    Watashi, Koichi; Liang, Guoxin; Iwamoto, Masashi; Marusawa, Hiroyuki; Uchida, Nanako; Daito, Takuji; Kitamura, Kouichi; Muramatsu, Masamichi; Ohashi, Hirofumi; Kiyohara, Tomoko; Suzuki, Ryosuke; Li, Jisu; Tong, Shuping; Tanaka, Yasuhito; Murata, Kazumoto; Aizaki, Hideki; Wakita, Takaji

    2013-11-01

    Virus infection is restricted by intracellular immune responses in host cells, and this is typically modulated by stimulation of cytokines. The cytokines and host factors that determine the host cell restriction against hepatitis B virus (HBV) infection are not well understood. We screened 36 cytokines and chemokines to determine which were able to reduce the susceptibility of HepaRG cells to HBV infection. Here, we found that pretreatment with IL-1β and TNFα remarkably reduced the host cell susceptibility to HBV infection. This effect was mediated by activation of the NF-κB signaling pathway. A cytidine deaminase, activation-induced cytidine deaminase (AID), was up-regulated by both IL-1β and TNFα in a variety of hepatocyte cell lines and primary human hepatocytes. Another deaminase APOBEC3G was not induced by these proinflammatory cytokines. Knockdown of AID expression impaired the anti-HBV effect of IL-1β, and overexpression of AID antagonized HBV infection, suggesting that AID was one of the responsible factors for the anti-HBV activity of IL-1/TNFα. Although AID induced hypermutation of HBV DNA, this activity was dispensable for the anti-HBV activity. The antiviral effect of IL-1/TNFα was also observed on different HBV genotypes but not on hepatitis C virus. These results demonstrate that proinflammatory cytokines IL-1/TNFα trigger a novel antiviral mechanism involving AID to regulate host cell permissiveness to HBV infection.

  3. Interleukin-1 and Tumor Necrosis Factor-α Trigger Restriction of Hepatitis B Virus Infection via a Cytidine Deaminase Activation-induced Cytidine Deaminase (AID)*

    PubMed Central

    Watashi, Koichi; Liang, Guoxin; Iwamoto, Masashi; Marusawa, Hiroyuki; Uchida, Nanako; Daito, Takuji; Kitamura, Kouichi; Muramatsu, Masamichi; Ohashi, Hirofumi; Kiyohara, Tomoko; Suzuki, Ryosuke; Li, Jisu; Tong, Shuping; Tanaka, Yasuhito; Murata, Kazumoto; Aizaki, Hideki; Wakita, Takaji

    2013-01-01

    Virus infection is restricted by intracellular immune responses in host cells, and this is typically modulated by stimulation of cytokines. The cytokines and host factors that determine the host cell restriction against hepatitis B virus (HBV) infection are not well understood. We screened 36 cytokines and chemokines to determine which were able to reduce the susceptibility of HepaRG cells to HBV infection. Here, we found that pretreatment with IL-1β and TNFα remarkably reduced the host cell susceptibility to HBV infection. This effect was mediated by activation of the NF-κB signaling pathway. A cytidine deaminase, activation-induced cytidine deaminase (AID), was up-regulated by both IL-1β and TNFα in a variety of hepatocyte cell lines and primary human hepatocytes. Another deaminase APOBEC3G was not induced by these proinflammatory cytokines. Knockdown of AID expression impaired the anti-HBV effect of IL-1β, and overexpression of AID antagonized HBV infection, suggesting that AID was one of the responsible factors for the anti-HBV activity of IL-1/TNFα. Although AID induced hypermutation of HBV DNA, this activity was dispensable for the anti-HBV activity. The antiviral effect of IL-1/TNFα was also observed on different HBV genotypes but not on hepatitis C virus. These results demonstrate that proinflammatory cytokines IL-1/TNFα trigger a novel antiviral mechanism involving AID to regulate host cell permissiveness to HBV infection. PMID:24025329

  4. Preexisting Levels of CD4 T Cells Expressing PD-1 Are Related to Overall Survival in Prostate Cancer Patients Treated with Ipilimumab.

    PubMed

    Kwek, Serena S; Lewis, Jera; Zhang, Li; Weinberg, Vivian; Greaney, Samantha K; Harzstark, Andrea L; Lin, Amy M; Ryan, Charles J; Small, Eric J; Fong, Lawrence

    2015-09-01

    Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) blockade can induce tumor regression and improved survival in cancer patients. This treatment can enhance adaptive immune responses without an exogenous vaccine, but the immunologic biomarkers associated with improved clinical outcome in cancer patients are not fully established. A phase Ib trial in patients with metastatic, castration-resistant prostate cancer was performed combining ipilimumab with sargramostim (GM-CSF). In addition to evaluating ipilimumab dose, patients were followed clinically for response and overall survival, and for immunomodulation of circulating T cells. PSA declines of ≥50% and radiographic responses were observed at doses of ≥3 mg/kg/dose. Timing of clinical responses could be either immediate or delayed. Durable responses were also observed off treatment. A subset of patients experienced long-term survival with or without objective clinical responses. The relationship between T-cell phenotype in peripheral blood and overall survival was examined retrospectively. We found that the treatment induced an increase in the levels of CD4(+) effector T (Teff) cells, regulatory T cells, PD-1(+) CD4 Teff cells, and PD-1(+) CD8 T cells. However, these increased levels were not associated with overall survival. Instead, low pretreatment baseline levels of PD-1(+) CD4 Teff cells were found to correlate with longer overall survival. Furthermore, baseline levels of PD-1(+) CD4 Teff cells from patients with shorter overall survival were higher than from cancer-free male control subjects. These results suggest that preexisting expression of immunologic checkpoint marker PD-1 on CD4 Teff cells may help identify patients that may benefit from ipilimumab treatment. PMID:25968455

  5. Effects of acupotomylysis on basic fibroblast growth factor and CD34 levels in rabbits with third lumbar vertebral transverse foramen syndrome.

    PubMed

    Li, X-H; Liu, N-G; Guo, C-Q; Sun, H-M; Wu, H-X; Xu, H; Zhang, Y

    2015-08-19

    This study observed the local tissue homogenates in rabbits with third lumbar vertebral transverse foramen syndrome and explored the mechanism of acupotomylysis in local tissue revascularization. Thirty Japanese white rabbits were randomly divided into the following 5 groups of 6 rabbits each: normal, model, acupotomy, electroacupuncture (EA), and acupotomy-EA groups. All except the normal group were comprised of animal models of third lumbar vertebral transverse foramen syndrome prepared by embedding sponge in the left third lumbar transverse process. The rabbits in the acupotomy and EA groups underwent bilateral acupotomylysis intervention; those in the acupotomy-EA group underwent acupotomylysis and EA interventions. On the 28th day after modeling, the double-antibody ELISA was used to detect b-FGF and CD34 levels in the serum and homogenates of a muscle tissue sample from the left side of the third lumbar transverse process. The b-FGF levels in local muscle homogenates were significantly higher in the modeled rabbits than in the normal rabbits (P < 0.01), and the CD34 levels in the modeled group were significantly lower than in the normal group (P < 0.01). The b-FGF and CD34 levels in the EA, acutopomy, and acutopomy-EA groups were significantly lower than those in the modeled group (P < 0.01); the CD34 levels were significantly higher in the acupotomy-EA group than in the model group (P < 0.05); and the differences among the EA, acupotomy, and acupotomy-EA groups were not significant (P > 0.05). In conclusion, acupotomylysis regulates the levels of b-FGF and CD34 levels in serum and muscle tissue as well as local tissue revascularization.

  6. Evaluation of Serum Adenosine Deaminase in Cystic Fibrosis Patients in an Iranian Referral Hospital

    PubMed Central

    Farahmand, Fatemeh; Tajdini, Parisa; Falahi, Gholamhossein; Shams, Sedigheh; Mahmoudi, Shima

    2016-01-01

    Background: Adenosine, a signaling nucleoside, is controlled in part by the enzyme adenosine deaminase (ADA). There are rare reports on the role of adenosine levels and ADA in cystic fibrosis (CF) patients. Objectives: The aim of this study was to assess serum ADA in CF patients in order to find whether the severity of lung disease in CF is related to significant changes of ADA or not. Patients and Methods: Venous blood serum ADA was measured in CF patients (3-15 years) and 49 healthy children (3-15 years) referred to Children’s Medical Center. Classification of respiratory and gastrointestinal disease severity in CF patients as well as Body Mass Index (BMI) was performed. The results were compared with values obtained from healthy children matched for age and gender. Results: This study included 49 children of both genders (20 females and 29 males) with CF (mean age: 6.36 ± 2.22 years). Mean serum ADA in CF patients group and control group was 9.38 ± 2.72 and 16.04 ± 1.27, respectively (P value = 0.001). Mean serum ADA in CF patients with normal BMI was higher than in patients with low BMI (P value = 0.002). Conclusions: In this study the lower serum level of ADA was seen in CF patients compared to control group. The clinical symptoms, especially respiratory symptoms, in CF patients might be associated with reduction of serum ADA and rising serum adenosine; therefore, further studies on the use of ADA enzyme therapy in CF patients are highly recommended. PMID:27617063

  7. Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection

    PubMed Central

    Doria, Margherita; Neri, Francesca; Gallo, Angela; Farace, Maria Giulia; Michienzi, Alessandro

    2009-01-01

    Adenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent and editing-independent mechanisms. We show that over-expression of ADAR1 in HIV-1 producer cells increases viral protein accumulation in an editing-independent manner. Moreover, HIV-1 virions generated in the presence of over-expressed ADAR1 but not an editing-inactive ADAR1 mutant are released more efficiently and display enhanced infectivity, as demonstrated by challenge assays performed with T cell lines and primary CD4+ T lymphocytes. Finally, we report that ADAR1 associates with HIV-1 RNAs and edits adenosines in the 5′ untranslated region (UTR) and the Rev and Tat coding sequence. Overall these results suggest that HIV-1 has evolved mechanisms to take advantage of specific RNA editing activity of the host cell and disclose a stimulatory function of ADAR1 in the spread of HIV-1. PMID:19651874

  8. Serum Levels of Soluble CD26/Dipeptidyl Peptidase-IV in Type 2 Diabetes Mellitus and Its Association with Metabolic Syndrome and Therapy with Antidiabetic Agents in Malaysian Subjects

    PubMed Central

    Ahmed, Radwan H.; Huri, Hasniza Zaman; Al-Hamodi, Zaid; Salem, Sameer D.; Muniandy, Sekaran

    2015-01-01

    Background A soluble form of CD26/dipeptidyl peptidase-IV (sCD26/DPP-IV) induces DPP-IV enzymatic activity that degrades incretin. We investigated fasting serum levels of sCD26/DPP-IV and active glucagon-like peptide-1 (GLP-1) in Malaysian patients with type 2 diabetes mellitus (T2DM) with and without metabolic syndrome (MetS), as well as the associations between sCD26/DPP-IV levels, MetS, and antidiabetic therapy. Methods We assessed sCD26/DPP-IV levels, active GLP-1 levels, body mass index (BMI), glucose, insulin, A1c, glucose homeostasis indices, and lipid profiles in 549 Malaysian subjects (including 257 T2DM patients with MetS, 57 T2DM patients without MetS, 71 non-diabetics with MetS, and 164 control subjects without diabetes or metabolic syndrome). Results Fasting serum levels of sCD26/DPP-IV were significantly higher in T2DM patients with and without MetS than in normal subjects. Likewise, sCD26/DPP-IV levels were significantly higher in patients with T2DM and MetS than in non-diabetic patients with MetS. However, active GLP-1 levels were significantly lower in T2DM patients both with and without MetS than in normal subjects. In T2DM subjects, sCD26/DPP-IV levels were associated with significantly higher A1c levels, but were significantly lower in patients using monotherapy with metformin. In addition, no significant differences in sCD26/DPP-IV levels were found between diabetic subjects with and without MetS. Furthermore, sCD26/DPP-IV levels were negatively correlated with active GLP-1 levels in T2DM patients both with and without MetS. In normal subjects, sCD26/DPP-IV levels were associated with increased BMI, cholesterol, and LDL-cholesterol (LDL-c) levels. Conclusion Serum sCD26/DPP-IV levels increased in T2DM subjects with and without MetS. Active GLP-1 levels decreased in T2DM patients both with and without MetS. In addition, sCD26/DPP-IV levels were associated with Alc levels and negatively correlated with active GLP-1 levels. Moreover, metformin

  9. Retinoic acid regulates CD1d gene expression at the transcriptional level in human and rodent monocytic cells.

    PubMed

    Chen, Qiuyan; Ross, A Catharine

    2007-04-01

    CD1d belongs to a group of nonclassical antigen-presenting molecules that present glycolipid antigens and thereby activate natural killer T (NKT) cells, a subset of bifunctional T cells. Little is known so far regarding the expression and physiologic regulation of CD1d. Here we show that all-trans-retinoic acid (RA), the active metabolite of vitamin A, rapidly (1 hr after treatment) increases CD1d mRNA in human and rodent monocytic cells at a physiologic dose (10 nM). The induction is RA specific and RA receptor (RAR) dependent-RA and an RARalpha agonist, Am580, both had a pronounced positive effect, whereas the addition of RARalpha antagonist partially blocked the increase in CD1d mRNA induced by RA and Am580. The induction was also completely blocked by the presence of actinomycin D. A putative RA-response element was identified in the distal 5' flanking region of the CD1d gene, which binds nuclear retinoid receptors and was responsive to RA in both gel mobility shift assay and transient transfection assay in THP-1 cells. These results further confirmed the transcriptional regulation of RA in CD1d gene expression. Moreover, RA significantly increased alpha-galactosylceramide-induced spleen cell proliferation. These studies together provide evidence for a previously unknown mechanism of CD1d gene expression regulation by RA and suggest that RA is a significant modulator of NKT cell activation.

  10. Hyperglycemia alters E-NTPDases, ecto-5'-nucleotidase, and ectosolic and cytosolic adenosine deaminase activities and expression from encephala of adult zebrafish (Danio rerio).

    PubMed

    Capiotti, Katiucia Marques; Siebel, Anna Maria; Kist, Luiza Wilges; Bogo, Maurício Reis; Bonan, Carla Denise; Da Silva, Rosane Souza

    2016-06-01

    Hyperglycemia is the main feature for the diagnosis of diabetes mellitus (DM). Some studies have demonstrated the relationship between DM and dysfunction on neurotransmission systems, such as the purinergic system. In this study, we evaluated the extracellular nucleotide hydrolysis and adenosine deamination activities from encephalic membranes of hyperglycemic zebrafish. A significant decrease in ATP, ADP, and AMP hydrolyses was observed at 111-mM glucose-treated group, which returned to normal levels after 7 days of glucose withdrawal. A significant increase in ecto-adenosine deaminase activity was observed in 111-mM glucose group, which remain elevated after 7 days of glucose withdrawal. The soluble-adenosine deaminase activity was significantly increased just after 7 days of glucose withdrawal. We also evaluated the gene expressions of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-5'-nucleotidase, ADA, and adenosine receptors from encephala of adult zebrafish. The entpd 2a.1, entpd 2a.2, entpd 3, and entpd 8 mRNA levels from encephala of adult zebrafish were decreased in 111-mM glucose-treated and glucose withdrawal groups. The gene expressions of adenosine receptors (adora 1 , adora 2aa , adora 2ab , and adora 2b ) were decreased in 111-mM glucose-treated and glucose withdrawal groups. The gene expression of ADA (ada 2a.1) was decreased in glucose withdrawal group. Maltodextrin, used as a control, did not affect the expression of adenosine receptors, ADA and E-NTPDases 2, 3, and 8, while the expression of ecto-5'-nucleotidase was slightly increased and the E-NTPDases 1 decreased. These findings demonstrated that hyperglycemia might affect the ecto-nucleotidase and adenosine deaminase activities and gene expression in zebrafish, probably through a mechanism involving the osmotic effect, suggesting that the modifications caused on purinergic system may also contribute to the diabetes-induced progressive cognitive impairment.

  11. Hyperglycemia alters E-NTPDases, ecto-5'-nucleotidase, and ectosolic and cytosolic adenosine deaminase activities and expression from encephala of adult zebrafish (Danio rerio).

    PubMed

    Capiotti, Katiucia Marques; Siebel, Anna Maria; Kist, Luiza Wilges; Bogo, Maurício Reis; Bonan, Carla Denise; Da Silva, Rosane Souza

    2016-06-01

    Hyperglycemia is the main feature for the diagnosis of diabetes mellitus (DM). Some studies have demonstrated the relationship between DM and dysfunction on neurotransmission systems, such as the purinergic system. In this study, we evaluated the extracellular nucleotide hydrolysis and adenosine deamination activities from encephalic membranes of hyperglycemic zebrafish. A significant decrease in ATP, ADP, and AMP hydrolyses was observed at 111-mM glucose-treated group, which returned to normal levels after 7 days of glucose withdrawal. A significant increase in ecto-adenosine deaminase activity was observed in 111-mM glucose group, which remain elevated after 7 days of glucose withdrawal. The soluble-adenosine deaminase activity was significantly increased just after 7 days of glucose withdrawal. We also evaluated the gene expressions of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-5'-nucleotidase, ADA, and adenosine receptors from encephala of adult zebrafish. The entpd 2a.1, entpd 2a.2, entpd 3, and entpd 8 mRNA levels from encephala of adult zebrafish were decreased in 111-mM glucose-treated and glucose withdrawal groups. The gene expressions of adenosine receptors (adora 1 , adora 2aa , adora 2ab , and adora 2b ) were decreased in 111-mM glucose-treated and glucose withdrawal groups. The gene expression of ADA (ada 2a.1) was decreased in glucose withdrawal group. Maltodextrin, used as a control, did not affect the expression of adenosine receptors, ADA and E-NTPDases 2, 3, and 8, while the expression of ecto-5'-nucleotidase was slightly increased and the E-NTPDases 1 decreased. These findings demonstrated that hyperglycemia might affect the ecto-nucleotidase and adenosine deaminase activities and gene expression in zebrafish, probably through a mechanism involving the osmotic effect, suggesting that the modifications caused on purinergic system may also contribute to the diabetes-induced progressive cognitive impairment. PMID:26769247

  12. Expression and characterization of 1-aminocyclopropane-1-carboxylate deaminase from the rhizobacterium Pseudomonas putida UW4: a key enzyme in bacterial plant growth promotion.

    PubMed

    Hontzeas, Nikos; Zoidakis, Jérôme; Glick, Bernard R; Abu-Omar, Mahdi M

    2004-12-01

    The enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACCD) converts ACC, the precursor of the plant hormone ethylene, to alpha-ketobutyrate and ammonium. This enzyme has been identified in soil bacteria and has been proposed to play a key role in microbe-plant association. A soluble recombinant ACCD from Pseudomonas putida UW4 of molecular weight 41 kDa has been cloned, expressed, and purified. It showed selectivity and high activity towards the substrate ACC: K(M)=3.4+/-0.2 mM and k(cat)=146+/-5 min(-1) at pH 8.0 and 22 degrees C. The enzyme displayed optimal activity at pH 8.0 with a sharp decline to essentially no activity below pH 6.5 and a slightly less severe tapering in activity at higher pH resulting in loss of activity at pH>10. The major component of the enzyme's secondary structure was determined to be alpha-helical by circular dichroism (CD). P. putida UW4 ACCD unfolded at 60 degrees C as determined by its CD temperature profile as well as by differential scanning microcalorimetry (DSC). Enzyme activity was knocked out in the point mutant Gly44Asp. Modeling this mutation into the known yeast ACCD structure shed light on the role this highly conserved residue plays in allowing substrate accessibility to the active site. This enzyme's biochemical and biophysical properties will serve as an important reference point to which newly isolated ACC deaminases from other organisms can be compared. PMID:15588698

  13. Infusing Interactive, Multimedia CD-ROM Technology into the First-Year College-Level Geology Curriculum: Recent Examples from Radford University, United States.

    ERIC Educational Resources Information Center

    Sethi, Parvinder S.; Newbill, Phyllis Leary

    In recent years, several CD-ROM-based instructional technology applications have been developed for use in both high school and college level classrooms. As multimedia authoring techniques evolve as important tools for teaching, it is imperative that teachers and multimedia authors understand the importance of focusing on specifically how the…

  14. High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase

    PubMed Central

    Gajula, Kiran S.; Huwe, Peter J.; Mo, Charlie Y.; Crawford, Daniel J.; Stivers, James T.; Radhakrishnan, Ravi; Kohli, Rahul M.

    2014-01-01

    Antibody maturation is a critical immune process governed by the enzyme activation-induced deaminase (AID), a member of the AID/APOBEC DNA deaminase family. AID/APOBEC deaminases preferentially target cytosine within distinct preferred sequence motifs in DNA, with specificity largely conferred by a small 9–11 residue protein loop that differs among family members. Here, we aimed to determine the key functional characteristics of this protein loop in AID and to thereby inform our understanding of the mode of DNA engagement. To this end, we developed a methodology (Sat-Sel-Seq) that couples saturation mutagenesis at each position across the targeting loop, with iterative functional selection and next-generation sequencing. This high-throughput mutational analysis revealed dominant characteristics for residues within the loop and additionally yielded enzymatic variants that enhance deaminase activity. To rationalize these functional requirements, we performed molecular dynamics simulations that suggest that AID and its hyperactive variants can engage DNA in multiple specific modes. These findings align with AID's competing requirements for specificity and flexibility to efficiently drive antibody maturation. Beyond insights into the AID-DNA interface, our Sat-Sel-Seq approach also serves to further expand the repertoire of techniques for deep positional scanning and may find general utility for high-throughput analysis of protein function. PMID:25064858

  15. Improved cytotoxic effects of Salmonella-producing cytosine deaminase in tumour cells

    PubMed Central

    Mesa-Pereira, Beatriz; Medina, Carlos; Camacho, Eva María; Flores, Amando; Santero, Eduardo

    2015-01-01

    In order to increase the cytotoxic activity of a Salmonella strain carrying a salicylate-inducible expression system that controls cytosine deaminase production, we have modified both, the vector and the producer bacterium. First, the translation rates of the expression module containing the Escherichia coli codA gene cloned under the control of the Pm promoter have been improved by using the T7 phage gene 10 ribosome binding site sequence and replacing the original GUG start codon by AUG. Second, to increase the time span in which cytosine deaminase may be produced by the bacteria in the presence of 5-fluorocytosine, a 5-fluorouracyl resistant Salmonella strain has been constructed by deleting its upp gene sequence. This new Salmonella strain shows increased cytosine deaminase activity and, after infecting tumour cell cultures, increased cytotoxic and bystander effects under standard induction conditions. In addition, we have generated a purD mutation in the producer strain to control its intracellular proliferation by the presence of adenine and avoid the intrinsic Salmonella cell death induction. This strategy allows the analysis and comparison of the cytotoxic effects of cytosine deaminase produced by different Salmonella strains in tumour cell cultures. PMID:25227763

  16. Role for Free Isoleucine or Glycyl-Leucine in the Repression of Threonine Deaminase in Escherichia coli

    PubMed Central

    Wasmuth, John J.; Umbarger, H. E.

    1974-01-01

    In Escherichia coli, the three branched-chain amino acid activating enzymes appear to be essential for multivalent repression of the isoleucine- and valine-forming enzymes. The results of experiments with a mutant, strain CU18, having an altered threonine deaminase, indicate that free isoleucine and some form of threonine deaminase (the product of the ilvA gene) are also involved in multivalent repression. This strain exhibits abnormally high derepressibility but normal repressibility of its ilv gene products, and its threonine deaminase is inhibited only by high concentrations of isoleucine. In strain CU18, the isoleucine analogue, thiaisoleucine, is incapable of replacing isoleucine in the multivalent repression of the ilv genes, whereas the analogue can fully replace the natural amino acid in repression in other strains examined. The dipeptide, glycyl-leucine, which, like isoleucine, is a heterotropic negative effector of threonine deaminase but is not a substrate for isoleucyl-transfer ribonucleic acid synthetase, can completely prevent the accumulation of threonine deaminase-forming potential during isoleucine starvation in strains with normal threonine deaminases. It can not, however, prevent such accumulation in strain CU18 or in other strains in which threonine deaminase is insensitive to any concentration of isoleucine. PMID:4587610

  17. Prolonged ethanol ingestion enhances benzene myelotoxicity and lowers urinary concentrations of benzene metabolite levels in CD-1 male mice.

    PubMed

    Marrubini, Giorgio; Castoldi, Anna F; Coccini, Teresa; Manzo, Luigi

    2003-09-01

    Benzene toxicity is attributed to its metabolism, which is primarily mediated by the ethanol-inducible cytochrome P450 2E1 isoform (CYP2E1). The present study investigated the myelotoxicity and urinary concentrations of major benzene metabolites in adult CD-1 male mice treated with low levels of benzene vapors, ethanol, or a combination of the two. Groups of ethanol-treated (5% in a Lieber-DeCarli liquid diet, 3 weeks) or pair-fed control mice were exposed to 10 ppm benzene, 6 h per day, 5 days per week for 2 weeks, starting from the second week of ethanol administration. On the last day of treatment, the number of early and late erythroid progenitors (BFU-E and CFU-E) was reduced by 55%, while the number of granulocyte/macrophage progenitors (CFU-GM) was reduced by 36% in benzene-treated mice. Ethanol lowered the CFU-E, BFU-E, and CFU-GM colony formation by 33, 28, and 12%, respectively. In animals coexposed to benzene and ethanol, the CFU-E colony counts were decreased by 70%, the BFU-E by 80%, and the CFU-GM by 45%. Phenol (Ph), hydroquinone (HQ), catechol (Cat), and trans,trans-muconic acid (MA) were measured by HPLC-UV in urine samples collected weekly during the last 6-h benzene/air exposure session. In benzene-exposed mice urinary metabolite levels peaked at the end of the first week of treatment (microg/kg body weight (bw): Ph: 4931 +/- 1055; Cat: 109 +/- 17; HQ: 784 +/- 137; MA: 534 +/- 92) and significantly decreased at the end of the second week (microg/kg bw: Ph: 3909 +/- 984; Cat: 82 +/- 24; HQ: 337 +/- 72; MA: 235 +/- 55). In mice given benzene and ethanol, the urinary levels of Ph, Cat, HQ, and MA were significantly lower than those measured in the group given benzene alone. The urinary levels of Ph and Cat showed a decreasing trend, again, from the first to the second week of benzene exposure. These data indicate that chronic ethanol ingestion exacerbates benzene myelotoxicity and, in addition, reduces the urinary excretion of benzene metabolites in

  18. Allosteric kinetics of the isoform 1 of human glucosamine-6-phosphate deaminase.

    PubMed

    Alvarez-Añorve, Laura I; Alonzo, Diego A; Mora-Lugo, Rodrigo; Lara-González, Samuel; Bustos-Jaimes, Ismael; Plumbridge, Jacqueline; Calcagno, Mario L

    2011-12-01

    The human genome contains two genes encoding for two isoforms of the enzyme glucosamine-6-phosphate deaminase (GNPDA, EC 3.5.99.6). Isoform 1 has been purified from several animal sources and the crystallographic structure of the human recombinant enzyme was solved at 1.75Å resolution (PDB ID: 1NE7). In spite of their great structural similarity, human and Escherichia coli GNPDAs show marked differences in their allosteric kinetics. The allosteric site ligand, N-acetylglucosamine 6-phosphate (GlcNAc6P), which is an activator of the K-type of E. coli GNPDA has an unusual mixed allosteric effect on hGNPDA1, behaving as a V activator and a K inhibitor (antiergistic or crossed mixed K(-)V(+) effect). In the absence of GlcNAc6P, the apparent k(cat) of the enzyme is so low, that GlcNAc6P behaves as an essential activator. Additionally, substrate inhibition, dependent on GlcNAc6P concentration, is observed. All these kinetic properties can be well described within the framework of the Monod allosteric model with some additional postulates. These unusual kinetic properties suggest that hGNPDA1 could be important for the maintenance of an adequate level of the pool of the UDP-GlcNAc6P, the N-acetylglucosylaminyl donor for many reactions in the cell. In this research we have also explored the possible functional significance of the C-terminal extension of hGNPDA1 enzyme, which is not present in isoform 2, by constructing and studying two mutants truncated at positions 268 and 275.

  19. RNA editing of hepatitis B virus transcripts by activation-induced cytidine deaminase.

    PubMed

    Liang, Guoxin; Kitamura, Kouichi; Wang, Zhe; Liu, Guangyan; Chowdhury, Sajeda; Fu, Weixin; Koura, Miki; Wakae, Kousho; Honjo, Tasuku; Muramatsu, Masamichi

    2013-02-01

    Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. The mechanism by which AID triggers SHM and CSR has been explained by two distinct models. In the DNA deamination model, AID converts cytidine bases in DNA into uridine. The uridine is recognized by the DNA repair system, which produces DNA strand breakages and point mutations. In the alternative model, RNA edited by AID is responsible for triggering CSR and SHM. However, RNA deamination by AID has not been demonstrated. Here we found that C-to-T and G-to-A mutations accumulated in hepatitis B virus (HBV) nucleocapsid DNA when AID was expressed in HBV-replicating hepatic cell lines. AID expression caused C-to-T mutations in the nucleocapsid DNA of RNase H-defective HBV, which does not produce plus-strand viral DNA. Furthermore, the RT-PCR products of nucleocapsid viral RNA from AID-expressing cells exhibited significant C-to-T mutations, whereas viral RNAs outside the nucleocapsid did not accumulate C-to-U mutations. Moreover, AID was packaged within the nucleocapsid by forming a ribonucleoprotein complex with HBV RNA and the HBV polymerase protein. The encapsidation of the AID protein with viral RNA and DNA provides an efficient environment for evaluating AID's RNA and DNA deamination activities. A bona fide RNA-editing enzyme, apolipoprotein B mRNA editing catalytic polypeptide 1, induced a similar level of C-to-U mutations in nucleocapsid RNA as AID. Taken together, the results indicate that AID can deaminate the nucleocapsid RNA of HBV.

  20. Diagnostic Algorithm for Glycogenoses and Myoadenylate Deaminase Deficiency Based on Exercise Testing Parameters: A Prospective Study

    PubMed Central

    Rannou, Fabrice; Uguen, Arnaud; Scotet, Virginie; Le Maréchal, Cédric; Rigal, Odile; Marcorelles, Pascale; Gobin, Eric; Carré, Jean-Luc; Zagnoli, Fabien; Giroux-Metges, Marie-Agnès

    2015-01-01

    Aim Our aim was to evaluate the accuracy of aerobic exercise testing to diagnose metabolic myopathies. Methods From December 2008 to September 2012, all the consecutive patients that underwent both metabolic exercise testing and a muscle biopsy were prospectively enrolled. Subjects performed an incremental and maximal exercise testing on a cycle ergometer. Lactate, pyruvate, and ammonia concentrations were determined from venous blood samples drawn at rest, during exercise (50% predicted maximal power, peak exercise), and recovery (2, 5, 10, and 15 min). Biopsies from vastus lateralis or deltoid muscles were analysed using standard techniques (reference test). Myoadenylate deaminase (MAD) activity was determined using p-nitro blue tetrazolium staining in muscle cryostat sections. Glycogen storage was assessed using periodic acid-Schiff staining. The diagnostic accuracy of plasma metabolite levels to identify absent and decreased MAD activity was assessed using Receiver Operating Characteristic (ROC) curve analysis. Results The study involved 51 patients. Omitting patients with glycogenoses (n = 3), MAD staining was absent in 5, decreased in 6, and normal in 37 subjects. Lactate/pyruvate at the 10th minute of recovery provided the greatest area under the ROC curves (AUC, 0.893 ± 0.067) to differentiate Abnormal from Normal MAD activity. The lactate/rest ratio at the 10th minute of recovery from exercise displayed the best AUC (1.0) for discriminating between Decreased and Absent MAD activities. The resulting decision tree achieved a diagnostic accuracy of 86.3%. Conclusion The present algorithm provides a non-invasive test to accurately predict absent and decreased MAD activity, facilitating the selection of patients for muscle biopsy and target appropriate histochemical analysis. PMID:26207760

  1. Fermi level stabilization and band edge energies in Cd{sub x}Zn{sub 1−x}O alloys

    SciTech Connect

    Detert, Douglas M.; Tom, Kyle B.; Dubon, Oscar D.; Battaglia, Corsin; Javey, Ali; Denlinger, Jonathan D.; Lim, Sunnie H. N.; Anders, André; Yu, Kin M.; Walukiewicz, Wladek

    2014-06-21

    We have measured the band edge energies of Cd{sub x}Zn{sub 1−x}O thin films as a function of composition by three independent techniques: we determine the Fermi level stabilization energy by pinning the Fermi level with ion irradiation, measure the binding energy of valence band states and core levels by X-ray photoelectron spectroscopy, and probe shifts in the conduction band and valence band density of states using soft X-ray absorption and emission spectroscopy, respectively. The three techniques find consensus in explaining the origin of compositional trends in the optical-bandgap narrowing upon Cd incorporation in wurtzite ZnO and widening upon Zn incorporation in rocksalt CdO. The conduction band minimum is found to be stationary for both wurtzite and rocksalt alloys, and a significant upward rise of the valence band maximum accounts for the majority of these observed bandgap changes. Given these band alignments, alloy disorder scattering is found to play a negligible role in decreasing the electron mobility for all alloys. These band alignment details, combined with the unique optical and electrical properties of the two phase regimes, make CdZnO alloys attractive candidates for photoelectrochemical water splitting applications.

  2. Inducing salt tolerance in mung bean through coinoculation with rhizobia and plant-growth-promoting rhizobacteria containing 1-aminocyclopropane-1-carboxylate deaminase.

    PubMed

    Ahmad, Maqshoof; Zahir, Zahir A; Asghar, H Naeem; Asghar, M

    2011-07-01

    Twenty-five strains of plant-growth-promoting rhizobacteria (PGPR) containing 1-aminocyclopropane-1-carboxylate (ACC) deaminase and 10 strains of rhizobia were isolated from rhizosphere soil samples and nodules of mung bean. They were screened in separate trials under salt-stressed axenic conditions. The three most effective strains of PGPR (Mk1, Pseudomonas syringae ; Mk20, Pseudomonas fluorescens ; and Mk25, Pseudomonas fluorescens biotype G) and Rhizobium phaseoli strains M1, M6, and M9 were evaluated in coinoculation for their growth-promoting activity at three salinity levels (original, 4 dS·m(-1), and 6 dS·m(-1)) under axenic conditions. The results showed that salinity stress significantly reduced plant growth but inoculation with PGPR containing ACC deaminase and rhizobia enhanced plant growth, thus reducing the inhibitory effect of salinity. However, their combined application was more effective under saline conditions, and the combination Mk20 × M6 was the most efficient for improving seedling growth and nodulation. The effect of high ethylene concentrations on plant growth and the performance of these strains for reducing the negative impact of saline stress was also evaluated by conducting a classical triple-response bioassay. The intensity of the classical triple response decreased owing to inoculation with these strains, with the root and shoot lengths of inoculated mung bean seedlings increasing and stem diameter decreasing, which is a typical response to the dilution in a classical triple response bioassay. Thus, coinoculation with PGPR containing ACC deaminase and Rhizobium spp. could be a useful approach for inducing salt tolerance and thus improving growth and nodulation in mung bean under salt-affected conditions.

  3. Integrated Pharmacokinetic/Pharmacodynamic Analysis for Determining the Minimal Anticipated Biological Effect Level of a Novel Anti-CD28 Receptor Antagonist BMS-931699.

    PubMed

    Yang, Zheng; Wang, Haiqing; Salcedo, Theodora W; Suchard, Suzanne J; Xie, Jenny H; Schneeweis, Lumelle A; Fleener, Catherine A; Calore, James D; Shi, Rong; Zhang, Sean X Y; Rodrigues, A David; Car, Bruce D; Marathe, Punit H; Nadler, Steven G

    2015-12-01

    BMS-931699 (lulizumab pegol), a domain antibody (dAb) conjugated with 40-kDa branched polyethylene glycol, is a human anti-CD28 receptor antagonist under development for the treatment of inflammatory and autoimmune diseases. In the present work, the minimal anticipated biologic effect level (MABEL) was determined for BMS-931699 by integrating all the available preclinical data. The relevance of the in vitro mixed lymphocyte reaction (MLR) assay to a whole blood CD28 receptor occupancy (RO) assessment, as well as the relationship between the CD28 RO and the inhibition of T-cell-dependent antibody response to keyhole limpet hemocyanin in vivo, was demonstrated through an integrated pharmacokinetic/pharmacodynamic analysis using anti-hCD28 dAb-001 (differing from BMS-931699 by two additional amino acids at the N-terminus) and a mouse surrogate. Based on this analysis, the EC10 value (0.32 nM) from the human MLR assay and the human plasma volume (0.04 l/kg) were employed to calculate the MABEL (0.01 mg) of BMS-931699 in humans, with a CD28 RO predicted to be ≤10%. The estimated MABEL dose was threefold higher than the value derived from the binding constant and twofold less than the MABEL converted from animal efficacy studies based on the body surface area. Furthermore, it was 2900-fold lower than the human equivalent dose derived from the no observed adverse effect level in monkeys (15 mg/kg/week for 5 doses, intravenous dosing) with a 10-fold safety factor applied. Therefore, the MABEL dose represented a sound approach to mitigate any potential risk in targeting CD28 and was successfully used as the first-in-human starting dose for BMS-931699. PMID:26442523

  4. Interplay between T Cell Receptor Binding Kinetics and the Level of Cognate Peptide Presented by Major Histocompatibility Complexes Governs CD8+ T Cell Responsiveness*

    PubMed Central

    Irving, Melita; Zoete, Vincent; Hebeisen, Michael; Schmid, Daphné; Baumgartner, Petra; Guillaume, Philippe; Romero, Pedro; Speiser, Daniel; Luescher, Immanuel; Rufer, Nathalie; Michielin, Olivier

    2012-01-01

    Through a rational design approach, we generated a panel of HLA-A*0201/NY-ESO-1157–165-specific T cell receptors (TCR) with increasing affinities of up to 150-fold from the wild-type TCR. Using these TCR variants which extend just beyond the natural affinity range, along with an extreme supraphysiologic one having 1400-fold enhanced affinity, and a low-binding one, we sought to determine the effect of TCR binding properties along with cognate peptide concentration on CD8+ T cell responsiveness. Major histocompatibility complexes (MHC) expressed on the surface of various antigen presenting cells were peptide-pulsed and used to stimulate human CD8+ T cells expressing the different TCR via lentiviral transduction. At intermediate peptide concentration we measured maximum cytokine/chemokine secretion, cytotoxicity, and Ca2+ flux for CD8+ T cells expressing TCR within a dissociation constant (KD) range of ∼1–5 μm. Under these same conditions there was a gradual attenuation in activity for supraphysiologic affinity TCR with KD < ∼1 μm, irrespective of CD8 co-engagement and of half-life (t1/2 = ln 2/koff) values. With increased peptide concentration, however, the activity levels of CD8+ T cells expressing supraphysiologic affinity TCR were gradually restored. Together our data support the productive hit rate model of T cell activation arguing that it is not the absolute number of TCR/pMHC complexes formed at equilibrium, but rather their productive turnover, that controls levels of biological activity. Our findings have important implications for various immunotherapies under development such as adoptive cell transfer of TCR-engineered CD8+ T cells, as well as for peptide vaccination strategies. PMID:22549784

  5. Inhibition of G-Protein βγ Signaling Decreases Levels of Messenger RNAs Encoding Proinflammatory Cytokines in T Cell Receptor-Stimulated CD4+ T Helper Cells

    PubMed Central

    Hynes, Thomas R.; Yost, Evan A.; Hartle, Cassandra M.; Ott, Braden J.

    2015-01-01

    Background: Inhibition of G-protein βγ (Gβγ) signaling was found previously to enhance T cell receptor (TCR)-stimulated increases in interleukin 2 (IL-2) mRNA in CD4+ T helper cells, suggesting that Gβγ might be a useful drug target for treating autoimmune diseases, as low dose IL-2 therapy can suppress autoimmune responses. Because IL-2 may counteract autoimmunity in part by shifting CD4+ T helper cells away from the Type 1 T helper cell (TH1) and TH17 subtypes towards the TH2 subtype, the purpose of this study was to determine if blocking Gβγ signaling affected the balance of TH1, TH17, and TH2 cytokine mRNAs produced by CD4+ T helper cells. Methods: Gallein, a small molecule inhibitor of Gβγ, and siRNA-mediated silencing of the G-protein β1 subunit (Gβ1) were used to test the effect of blocking Gβγ on mRNA levels of cytokines in primary human TCR-stimulated CD4+ T helper cells. Results: Gallein and Gβ1 siRNA decreased interferon-γ (IFN-γ) and IL-17A mRNA levels in TCR-stimulated CD4+ T cells grown under TH1-promoting conditions. Inhibiting Gβγ also decreased mRNA levels of STAT4, which plays a positive role in TH1 differentiation and IL-17A production. Moreover, mRNA levels of the STAT4-regulated TH1-associated proteins, IL-18 receptor β chain (IL-18Rβ), mitogen-activated protein kinase kinase kinase 8 (MAP3K8), lymphocyte activation gene 3 (LAG-3), natural killer cell group 7 sequence (NKG7), and oncostatin M (OSM) were also decreased upon Gβγ inhibition. Gallein also increased IL-4, IL-5, IL-9, and IL-13 mRNA levels in TCR-stimulated memory CD4+ T cells grown in TH2-promoting conditions. Conclusions: Inhibiting Gβγ to produce these shifts in cytokine mRNA production might be beneficial for patients with autoimmune diseases such as rheumatoid arthritis (RA), Crohn’s disease (CD), psoriasis, multiple sclerosis (MS), and Hashimoto’s thyroiditis (HT), in which both IFN-γ and IL-17A are elevated. PMID:27095999

  6. Neopterin and Soluble CD14 Levels as Indicators of Immune Activation in Cases with Indeterminate Pattern and True Positive HIV-1 Infection

    PubMed Central

    Uysal, Hayriye Kırkoyun; Sohrabi, Pari; Habip, Zafer; Saribas, Suat; Kocazeybek, Emre; Seyhan, Fatih; Calışkan, Reyhan; Bonabi, Esad; Yuksel, Pelin; Birinci, Ilhan; Uysal, Omer; Kocazeybek, Bekir

    2016-01-01

    Background We aimed to evaluate the roles of the plasma immune activation biomarkers neopterin and soluble CD14 (sCD14) in the indirect assessment of the immune activation status of patients with the indeterminate HIV-1 (IHIV-1) pattern and a true HIV-1-positive infection (PCG). Methods This cross-sectional and descriptive study included eighty-eight patients with the IHIV-1 pattern, 100 patients in the PCG, and 100 people in a healthy control group (HCG). Neopterin and sCD14 levels were determined by competitive and sandwich ELISA methods, respectively. Results Mean neopterin and sCD14 levels among those with the IHIV-1 pattern were significantly lower than among the PCG (p < 0.001 and p = 0.001, respectively), but they were similiar to those in the HCG (p = 0.57 and p = 0.66, respectively. Mean neopterin and sCD14 levels among the PCG were found to be significantly higher than among those with the IHIV-1 pattern (p < 0.001 and p = 0.001, respectively) and among those in the HCG (p = 0.001, p < 0.001, respectively). Neopterin did not have adequate predictive value for identifying those in the PCG (area under the curve [AUC] = 0.534; 95% CI, 0.463–0.605; p = 0.4256); sCD14 also had poor predictive value but high specificity (100%) for identifying those in the PCG (AUC = 0.627; 95% CI, 0.556–0.694; p = 0.0036). Conclusions While low levels of these two biomarkers were detected among those with the IHIV-1 pattern, they were found in high levels among those in the PCG. These two markers obviously cannot be used as a sceening test because they have low sensitivies. Taken together, we suggest that neopterin and sCD14 may be helpful because they both have high specificity (92%-100%) as indirect non-specific markers for predicting the immune activation status of individuals, whether or not they have true positive HIV-1. PMID:27031691

  7. Alirocumab, a Therapeutic Human Antibody to PCSK9, Does Not Affect CD81 Levels or Hepatitis C Virus Entry and Replication into Hepatocytes

    PubMed Central

    Ramanathan, Aarti; Gusarova, Viktoria; Stahl, Neil; Gurnett-Bander, Anne; Kyratsous, Christos A.

    2016-01-01

    Background Proprotein convertase subtilisin/kexin type 9 (PSCK9) is secreted mainly from the liver and binds to the low-density lipoprotein receptor (LDLR), reducing LDLR availability and thus resulting in an increase in LDL-cholesterol. While the LDLR has been implicated in the cell entry process of the hepatitis C virus (HCV), overexpression of an artificial non-secreted, cell membrane-bound form of PCSK9 has also been shown to reduce surface expression of CD81, a major component of the HCV entry complex, leading to concerns that pharmacological inhibition of PCSK9 may increase susceptibility to HCV infection by increasing either CD81 or LDLR availability. Here, we evaluated effects of PCSK9 and PCSK9 blockade on CD81 levels and HCV entry with a physiologically relevant model using native secreted PCSK9 and a monoclonal antibody to PCSK9, alirocumab. Methods and Results Flow cytometry and Western blotting of human hepatocyte Huh-7 cells showed that, although LDLR levels were reduced when cells were exposed to increasing PCSK9 concentrations, there was no correlation between total or surface CD81 levels and the presence and amount of soluble PCSK9. Moreover, inhibiting PCSK9 with the monoclonal antibody alirocumab did not affect expression levels of CD81. In an in vitro model of HCV entry, addition of soluble PCSK9 or treatment with alirocumab had no effect on the ability of either lentiviral particles bearing the HCV glycoproteins or JFH-1 based cell culture virus to enter hepatocytes. Consistent with these in vitro findings, no differences were observed in hepatic CD81 levels using in vivo mouse models, including Pcsk9-/- mice compared with wild-type controls and hyperlipidemic mice homozygous for human Pcsk9 and heterozygous for Ldlr deletion, treated with either alirocumab or isotype control antibody. Conclusion These results suggest that inhibition of PCSK9 with alirocumab has no effect on CD81 and does not result in increased susceptibility to HCV entry

  8. Self-Reported Sleep Disturbance is associated with Lower CD4 Count and 24-Hour Urinary Dopamine Levels in Ethnic Minority Women Living with HIV

    PubMed Central

    Seay, Julia S.; McIntosh, Roger; Fekete, Erin M.; Fletcher, Mary Ann; Kumar, Mahendra; Schneiderman, Neil; Antoni, Michael H.

    2013-01-01

    Background Sleep disturbance is associated with dopamine dysregulation, which can negatively impact immune status. Individuals living with HIV experience more sleep difficulties, and poor sleep may compound immune decrements associated with HIV infection. Little research has examined associations between sleep, dopamine, and immune status (CD4 count) in individuals with HIV. As ethnic minority women living with HIV (WLWH) are at heightened risk for HIV disease progression, we related sleep reports to both CD4 count and dopamine levels in a cohort of ethnic minority WLWH. Methods Participants were 139 low-income WLWH (ages 20–62; 78.3% African-American or Caribbean) who reported both overall sleep quality and sleep disturbance on the Pittsburgh Sleep Quality Index (PSQI). CD4 count and HIV viral load were measured via morning peripheral venous blood samples, and concentrations of dopamine were measured via 24-hour urine collection. Covariates included HIV viral load, length of time since HIV diagnosis, HAART adherence, perceived stress and depression. Results After controlling for all covariates, greater sleep disturbance was associated with significantly lower CD4 count (β = −.20, p = .03) and lower levels of dopamine (β = −.25, p = .04). Poorer overall sleep quality was marginally associated with lower CD4 count (β = −.16, p = .08), and was not associated with dopamine. Conclusion Our analyses suggest that sleep disturbance is independently related with immune status and dopamine levels in WLWH. Lower levels of dopamine may indicate neuroendocrine dysregulation and may impact immune and health status. Results highlight sleep disturbance rather than overall sleep quality as potentially salient to neuroendocrine and immune status in ethnic minority WLWH. PMID:23850225

  9. Plasma Levels of Presepsin (Soluble CD14-subtype) as a Novel Prognostic Marker for Hemophagocytic Syndrome in Hematological Malignancies.

    PubMed

    Nanno, Satoru; Koh, Hideo; Katayama, Takako; Hashiba, Masamichi; Sato, Ayumi; Makuuchi, Yosuke; Nagasaki, Joji; Kuno, Masatomo; Yoshimura, Takuro; Okamura, Hiroshi; Nishimoto, Mitsutaka; Hirose, Asao; Nakamae, Mika; Nakane, Takahiko; Hino, Masayuki; Nakamae, Hirohisa

    2016-01-01

    Objective Recent studies suggest that presepsin (soluble CD14-subtype) is a useful diagnostic and prognostic marker for sepsis, with secretion by activated macrophages potentially dependent on phagocytosis of microorganisms. As "hemophagocytosis" is one of the major characteristics in patients with hemophagocytic syndrome (HPS), we hypothesized that presepsin may reflect the phagocytic activity and be a useful prognostic marker for HPS. Therefore, we aimed to assess the prognostic potential of presepsin in secondary HPS in adult patients with hematological malignancies. Methods Between April 2006 and August 2014, we retrospectively examined consecutive patients with HPS whose blood samples were available at our institution and compared the prognostic value of the following in HPS, singly and in combination: plasma presepsin, serum soluble interleukin (IL)-2 receptor (sIL-2R), ferritin, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), IL-6 and IL-10. Results A total of 14 patients were enrolled. The median age of the patients was 46.5 years (range, 22-65). In univariable Cox models, there were no significant variables associated with the prognosis. However, in 12 evaluable patients, only the combination of higher median values of presepsin (>1,935 pg/mL) and sIL-2R (>4,585 U/mL) at the onset of HPS was significantly associated with the 90-day mortality (hazard ratio 14.5; 95% CI, 1.47-143.36; p=0.02). Conclusion These results suggest that a composite model of plasma presepsin and serum sIL-2R levels at the onset of HPS might be a novel predictor of the prognosis of patients with hematological malignancies and secondary HPS. PMID:27522992

  10. High conservation level of CD8(+) T cell immunogenic regions within an unusual H1N2 human influenza variant.

    PubMed

    Komadina, Naomi; Quiñones-Parra, Sergio M; Kedzierska, Katherine; McCaw, James M; Kelso, Anne; Leder, Karin; McVernon, Jodie

    2016-10-01

    Current seasonal influenza vaccines require regular updates due to antigenic drift causing loss of effectiveness and therefore providing little or no protection against novel influenza A subtypes. Next generation vaccines capable of eliciting CD8(+) T cell (CTL) mediated cross-protective immunity may offer a long-term alternative strategy. However, measuring pre- and existing levels of CTL cross-protection in humans is confounded by differences in infection histories across individuals. During 2000-2003, H1N2 viruses circulated persistently in the human population for the first time and we hypothesized that the viral nucleoprotein (NP) contained novel CTL epitopes that may have contributed to the survival of the viruses. This study describes the immunogenic NP peptides of H1N1, H2N2, and H3N2 influenza viruses isolated from humans over the past century, 1918-2003, by comparing this historical dataset to reference NP peptides from H1N2 that circulated in humans during 2000-2003. Observed peptides sequences ranged from highly conserved (15%) to highly variable (12%), with variation unrelated to reported immunodominance. No unique NP peptides which were exclusive to the H1N2 viruses were noted. However, the virus had inherited the NP from a recently emerged H3N2 variant containing novel peptides, which may have assisted its persistence. Any advantage due to this novelty was subsequently lost with emergence of a newer H3N2 variant in 2003. Our approach has potential to provide insight into the population context in which influenza viruses emerge, and may help to inform immunogenic peptide selection for CTL-inducing influenza vaccines. J. Med. Virol. 88:1725-1732, 2016. © 2016 Wiley Periodicals, Inc.

  11. Toxic metal (Pb, Cd, Cr, and Hg) levels in Rapana venosa (Valenciennes, 1846), Eriphia verrucosa (Forskal, 1775), and sediment samples from the Black Sea littoral (Thrace, Turkey).

    PubMed

    Mülayim, A; Balkıs, H

    2015-06-15

    Rapana venosa (Valenciennes, 1846) and Eriphia verrucosa (Forskal, 1775) are the dominant benthic invertebrate species along the Thrace Coast of the western Black Sea. The aim of this study was to determine toxic metal (Hg, Cr, Cd, and Pb) accumulation levels in these species, as well as within littoral sediments from this area. Our results showed that all of the metals, except for Cd, were below that in average shale. The measured accumulation levels were mostly within the range of what is naturally found within the earth's crust. However, some study stations did have increased concentrations, indicating anthropogenic pollution in these areas. The Cd contents of E. verrucosa collected from all our study stations were well above the limits set by the Turkish Food Codex, especially in Kıyıköy, whereas Pb content was close to the limit at all stations and exceeded the limit in Kıyıköy, but Hg content was below the limit at all stations. Cd content of R. venosa exceeded the limit only in Kumköy. Pb content was below the limit, and Hg was at or slightly above the limit at all stations. PMID:25913797

  12. Toxic metal (Pb, Cd, Cr, and Hg) levels in Rapana venosa (Valenciennes, 1846), Eriphia verrucosa (Forskal, 1775), and sediment samples from the Black Sea littoral (Thrace, Turkey).

    PubMed

    Mülayim, A; Balkıs, H

    2015-06-15

    Rapana venosa (Valenciennes, 1846) and Eriphia verrucosa (Forskal, 1775) are the dominant benthic invertebrate species along the Thrace Coast of the western Black Sea. The aim of this study was to determine toxic metal (Hg, Cr, Cd, and Pb) accumulation levels in these species, as well as within littoral sediments from this area. Our results showed that all of the metals, except for Cd, were below that in average shale. The measured accumulation levels were mostly within the range of what is naturally found within the earth's crust. However, some study stations did have increased concentrations, indicating anthropogenic pollution in these areas. The Cd contents of E. verrucosa collected from all our study stations were well above the limits set by the Turkish Food Codex, especially in Kıyıköy, whereas Pb content was close to the limit at all stations and exceeded the limit in Kıyıköy, but Hg content was below the limit at all stations. Cd content of R. venosa exceeded the limit only in Kumköy. Pb content was below the limit, and Hg was at or slightly above the limit at all stations.

  13. Theoretical Analysis of Effects of Deep Level, Back Contact, and Absorber Thickness on Capacitance-Voltage Profiling of CdTe Thin-Film Solar Cells

    SciTech Connect

    Li, J. V.; Halverson, A. F.; Sulima, O. V.; Bansal, S.; Burst, J. M.; Barnes, T. M.; Gessert, T. A.; Levi, D. H.

    2012-05-01

    The apparent carrier density profile measured by the capacitance-voltage technique in CdTe thin-film solar cells frequently displays a distinctive U-shape. We show that, even assuming a uniform carrier density, such a U-shape may arise from deep levels, a non-ohmic back-contact, and a thin absorber, which are commonly present in practical CdTe thin-film solar cells. A thin CdTe absorber contributes to the right branch of the U-shape due to a punch-through effect at reverse or zero biases, when the CdTe absorber is nearly fully depleted. A rectifying back-contact contributes to both branches of the U-shape due to voltage sharing with the front junction under a forward bias and early punch-through under a reverse bias. Deep levels contribute to the right branch, but also raise the bottom of the U-shape, leading to an overestimate of carrier density.

  14. Anti-CMV-IgG positivity of donors is beneficial for alloHSCT recipients with respect to the better short-term immunological recovery and high level of CD4+CD25high lymphocytes.

    PubMed

    Jaskula, Emilia; Dlubek, Dorota; Tarnowska, Agnieszka; Lange, Janusz; Mordak-Domagala, Monika; Suchnicki, Krzysztof; Sedzimirska, Mariola; Borowik, Agata; Mizia, Sylwia; Lange, Andrzej

    2015-03-01

    Hematopoietic stem cell transplantation from anti-cytomegalovirus immunoglobulin G (anti-CMV-IgG) positive donors facilitated immunological recovery post-transplant, which may indicate that chronic CMV infection has an effect on the immune system. This can be seen in the recipients after reconstitution with donor lymphocytes. We evaluated the composition of lymphocytes at hematologic recovery in 99 patients with hematologic malignancies post hematopoietic stem cell transplantation (HSCT). Anti-CMV-IgG seropositivity of the donor was associated with higher proportions of CD4+ (227.963 ± 304.858 × 106 vs. 102.050 ± 17.247 × 106 cells/L, p = 0.009) and CD4+CD25high (3.456 ± 0.436 × 106 vs. 1.589 ± 0.218 × 106 cells/L, p = 0.003) lymphocytes in the blood at hematologic recovery. The latter parameter exerted a diverse influence on the risk of acute graft-versus-host disease (GvHD) if low (1.483 ± 0.360 × 106 vs. 3.778 ± 0.484 × 106 cells/L, p < 0.001) and de novo chronic GvHD (cGvHD) if high (3.778 ± 0.780 × 106 vs. 2.042 ± 0.261 × 106 cells/L, p = 0.041). Higher values of CD4+ lymphocytes in patients who received transplants from anti-CMV-IgG-positive donors translated into a reduced demand for IgG support (23/63 vs. 19/33, p = 0.048), and these patients also exhibited reduced susceptibility to cytomegalovirus (CMV), Epstein-Barr virus (EBV) and/or human herpes 6 virus (HHV6) infection/reactivation (12/50 vs. 21/47, p = 0.032). Finally, high levels (³0.4%) of CD4+CD25high lymphocytes were significantly associated with better post-transplant survival (56% vs. 38%, four-year survival, p = 0.040). Donors who experience CMV infection/reactivation provide the recipients with lymphocytes, which readily reinforce the recovery of the transplanted patients' immune system. PMID:25807050

  15. Plant Purine Nucleoside Catabolism Employs a Guanosine Deaminase Required for the Generation of Xanthosine in Arabidopsis[W

    PubMed Central

    Dahncke, Kathleen; Witte, Claus-Peter

    2013-01-01

    Purine nucleotide catabolism is common to most organisms and involves a guanine deaminase to convert guanine to xanthine in animals, invertebrates, and microorganisms. Using metabolomic analysis of mutants, we demonstrate that Arabidopsis thaliana uses an alternative catabolic route employing a highly specific guanosine deaminase (GSDA) not reported from any organism so far. The enzyme is ubiquitously expressed and deaminates exclusively guanosine and 2’-deoxyguanosine but no other aminated purines, pyrimidines, or pterines. GSDA belongs to the cytidine/deoxycytidylate deaminase family of proteins together with a deaminase involved in riboflavin biosynthesis, the chloroplastic tRNA adenosine deaminase Arg and a predicted tRNA-specific adenosine deaminase 2 in A. thaliana. GSDA is conserved in plants, including the moss Physcomitrella patens, but is absent in the algae and outside the plant kingdom. Our data show that xanthosine is exclusively generated through the deamination of guanosine by GSDA in A. thaliana, excluding other possible sources like the dephosphorylation of xanthosine monophosphate. Like the nucleoside hydrolases NUCLEOSIDE HYDROLASE1 (NSH1) and NSH2, GSDA is located in the cytosol, indicating that GMP catabolism to xanthine proceeds in a mostly cytosolic pathway via guanosine and xanthosine. Possible implications for the biosynthetic route of purine alkaloids (caffeine and theobromine) and ureides in other plants are discussed. PMID:24130159

  16. The mutagenic effects of low level sub-acute inhalation exposure to benzene in CD-1 mice.

    PubMed

    Ward, J B; Ammenheuser, M M; Ramanujam, V M; Morris, D L; Whorton, E B; Legator, M S

    1992-07-01

    Benzene is a widely used chemical and common environmental contaminant. It is carcinogenic in man and animals and is genotoxic in mice, rats, and occupationally exposed humans at doses above one part per million. In order to evaluate the genotoxic effects of prolonged exposures to very low concentrations of benzene, we exposed CD-1 mice to benzene by inhalation for 22 h per day, seven days per week for six weeks at 40, 100 and 1000 parts per billion (ppb). Additional groups were exposed to purified air or were housed in standard plastic cages. The effects of in vivo exposure to benzene were evaluated by using an autoradiographic assay to determine the frequency of mutants which represent mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in spleen lymphocytes. At the end of the six weeks exposure period lymphocytes were recovered from the spleens of the mice and cryopreserved prior to assay. Mutant cells were selected on the basis of their ability to incorporate tritiated thymidine in the presence of 6-thioguanine. The weighted mean variant (mutant) frequencies (Vf) of female mice (three per group) were 7.2 x 10(-6) at 0 ppb; 29.2 x 10(-6) at 40 ppb; 62.5 x 10(-6) at 100 ppb and 25.0 x 10(-6) at 1000 ppb. The Vf of unexposed mice housed in standard cages was 13.2 x 10(-6). In male mice the same pattern of response was observed, but the increases in Vf in response to benzene were not as great. In both sexes of mice, the increases at 40 and 100 ppb were significantly greater than at 0 ppb (P less than 0.05). The increase in Vf with exposure to 100 ppb and the decline at 1000 ppb parallel the results observed for chromosome damage in spleen lymphocytes from the same animals (Au et al., Mutation Res., 260 (1991) 219-224). These results indicate that sub-chronic exposure to benzene at levels below the current Occupational Safety and Health Administration Permitted Exposure Limit may induce gene mutations in lymphocytes in mice.

  17. Syzygium cumini extract decrease adenosine deaminase, 5'nucleotidase activities and oxidative damage in platelets of diabetic patients.

    PubMed

    De Bona, Karine S; Bellé, Luziane P; Sari, Marcel H; Thomé, Gustavo; Schetinger, Maria R C; Morsch, Vera M; Boligon, Aline; Athayde, Margareth L; Pigatto, Aline S; Moretto, Maria B

    2010-01-01

    Diabetes mellitus, a chronic metabolic disorder, has assumed epidemic proportions and its long-term complications can have devastating consequences. The oxidative stress in diabetes was greatly increased due to prolonged exposure to hyperglycemia and impairment of oxidant/antioxidant equilibrium. Syzygium cumini is being widely used to treat diabetes by the traditional practitioners over many centuries. Adenosine deaminase (ADA) and 5'-Nucleotidase (5'NT) are enzymes of purine nucleoside metabolism that play an important role in the regulation of adenosine (Ado) levels. In this study, we investigated the effect of Syzygium cumini aqueous leaves extract (ASc) on ADA and 5'NT activities and on parameters of oxidative stress under in vitro conditions, using platelets of patients with Type 2 diabetes mellitus. Platelet-Rich Plasma (PRP) was assayed by ADA, 5'NT, Catalase (CAT), Superoxide Dismutase (SOD) activities and Thiobarbituric acid reactive substances (TBARS) levels. We observed that ADA, 5'NT activities and TBARS levels were significantly higher when compared to the control group, and ASc (100 and 200 μg/mL) prevented these effects. Our study demonstrates that ASc was able to remove oxidant species generated in diabetic conditions and modulates in the Ado levels. Then, ASc may promote a compensatory response in platelet function, improving the susceptibility-induced by the diabetes mellitus. PMID:21063110

  18. A Study on the Serum Adenosine Deaminase Activity in Patients with Typhoid Fever and Other Febrile Illnesses

    PubMed Central

    Ketavarapu, Sameera; Ramani G., Uma; Modi, Prabhavathi

    2013-01-01

    Background: Adenosine Deaminase (ADA) has been suggested to be an important enzyme which is associated with the cell mediated immunity, but its clinical significance in typhoid fever has not yet been characterized. The present study was taken up to evaluate the serum ADA activity in patients of typhoid fever. The levels of ADA were also measured in the patients who were suffering from other febrile illnesses. Material and Method: This was a case control study. The subjects who were included in this study were divided into 3 groups. Group A consisted of 50 normal healthy individuals who served as the controls. Group B consisted of 50 patients, both males and females of all age groups, who were suffering from culture positive typhoid fever. Group C consisted of 50 patients who were suffering from febrile illnesses other than typhoid fever like viral fever, gastro enteritis, malaria, tonsillitis, upper respiratory tract infections, etc. The serum levels of ADA were estimated in all the subjects who were under study. Results: The serum ADA level was found to be increased in the patients of typhoid fever as compared to that in those with other febrile illnesses and in the controls. Conclusion: From the present study, it can be concluded that there was a statistically significant increase in the serum ADA levels in the patients with typhoid. PMID:23730630

  19. Strong mucosal immune responses in SIV infected macaques contribute to viral control and preserved CD4+ T-cell levels in blood and mucosal tissues

    PubMed Central

    2011-01-01

    Background Since there is still no protective HIV vaccine available, better insights into immune mechanism of persons effectively controlling HIV replication in the absence of any therapy should contribute to improve further vaccine designs. However, little is known about the mucosal immune response of this small unique group of patients. Using the SIV-macaque-model for AIDS, we had the rare opportunity to analyze 14 SIV-infected rhesus macaques durably controlling viral replication (controllers). We investigated the virological and immunological profile of blood and three different mucosal tissues and compared their data to those of uninfected and animals progressing to AIDS-like disease (progressors). Results Lymphocytes from blood, bronchoalveolar lavage (BAL), and duodenal and colonic biopsies were phenotypically characterized by polychromatic flow cytometry. In controllers, we observed higher levels of CD4+, CD4+CCR5+ and Gag-specific CD8+ T-cells as well as lower immune activation in blood and all mucosal sites compared to progressors. However, we could also demonstrate that immunological changes are distinct between these three mucosal sites. Intracellular cytokine staining demonstrated a significantly higher systemic and mucosal CD8+ Gag-specific cellular immune response in controllers than in progressors. Most remarkable was the polyfunctional cytokine profile of CD8+ lymphocytes in BAL of controllers, which significantly dominated over their blood response. The overall suppression of viral replication in the controllers was confirmed by almost no detectable viral RNA in blood and all mucosal tissues investigated. Conclusion A strong and complex virus-specific CD8+ T-cell response in blood and especially in mucosal tissue of SIV-infected macaques was associated with low immune activation and an efficient suppression of viral replication. This likely afforded a repopulation of CD4+ T-cells in different mucosal compartments to almost normal levels. We

  20. Decreased plasma glutamine level and CD4+ T cell number in response to 8 wk of anaerobic training.

    PubMed

    Hack, V; Weiss, C; Friedmann, B; Suttner, S; Schykowski, M; Erbe, N; Benner, A; Bärtsch, P; Dröge, W

    1997-05-01

    The purpose of the present study was to investigate the role of plasma amino acids and glutathione (GSH) on the absolute number of leukocyte and lymphocyte subpopulations in response to different training programs. Healthy untrained subjects were randomly assigned to an 8-wk aerobic (AET) or anaerobic (ANT) exercise training program. Absolute number of cell counts did not significantly change in AET, whereas a decrease of CD4+ T cell counts (P < 0.05), a fall in cells expressing CD45RA+ antigen (P < 0.05), and a marked increase in CD8+ T cell numbers (P < 0.01) were noted in ANT at the end of the training period compared with baseline values. Furthermore, ANT demonstrated a marked rise (P < 0.001) in plasma glutamate from 27.6 +/- 2.8 to 49.8 +/- 5.2 microM and a considerable reduction (P < 0.001) of the plasma glutamine pool from 713 +/- 22 to 601 +/- 30 microM after 8 wk of training. The decrease in glutamine showed a strong positive correlation to the individual loss of CD4+ T cells (r = 0.67, P < 0.001). AET demonstrated a rise (P < 0.05) in GSH from 20.7 +/- 2.5 to 28.1 +/- 1.5 nmol/mg protein at terminal examination. In conclusion, our data indicate impairment of the number and activity of CD4+ T cells in response to 8 wk of ANT, which might be linked to metabolic factors such as glutamine.

  1. Low dietary levels of Al, Pb and Cd may affect the non-enzymatic antioxidant capacity in caged honey bees (Apis mellifera).

    PubMed

    Gauthier, Maxime; Aras, Philippe; Jumarie, Catherine; Boily, Monique

    2016-02-01

    Several hypotheses have been proposed to explain the abnormally high mortality rate observed in bee populations in Europe and North America. While studies based on the effects of pesticides are paramount, the metals present in agroecosystems are often overlooked. Sources of metals are linked to the nature of soils and to agricultural practices, namely the use of natural or chemical nutrients as well as residual materials from waste-water treatment sludge. The aim of this study was to investigate the effects of metals on honey bees exposed for 10 days to environmentally realistic concentrations of Al, Pb and Cd (dissolved in syrup). The monitoring of syrup consumption combined with the quantification of metals in bees revealed the following order for metal bioconcentration ratios: Cd > Pb > Al. Alpha-tocopherol, metallothionein-like proteins (MTLPs) and lipid peroxidation were quantified. When bees were exposed to increasing amounts of Cd, a marked augmentation of MTLPs levels was found. Lead (Pb) and Cd caused an increase in α-tocopherol content, while alteration of lipid peroxidation was observed only with Al exposure. These findings raise concerns about the bioavailability and the additional threat posed by metals for pollinators in agricultural areas while providing new insights for potential use of the honey bee as a sentinel species for metal exposure. PMID:26421624

  2. Low dietary levels of Al, Pb and Cd may affect the non-enzymatic antioxidant capacity in caged honey bees (Apis mellifera).

    PubMed

    Gauthier, Maxime; Aras, Philippe; Jumarie, Catherine; Boily, Monique

    2016-02-01

    Several hypotheses have been proposed to explain the abnormally high mortality rate observed in bee populations in Europe and North America. While studies based on the effects of pesticides are paramount, the metals present in agroecosystems are often overlooked. Sources of metals are linked to the nature of soils and to agricultural practices, namely the use of natural or chemical nutrients as well as residual materials from waste-water treatment sludge. The aim of this study was to investigate the effects of metals on honey bees exposed for 10 days to environmentally realistic concentrations of Al, Pb and Cd (dissolved in syrup). The monitoring of syrup consumption combined with the quantification of metals in bees revealed the following order for metal bioconcentration ratios: Cd > Pb > Al. Alpha-tocopherol, metallothionein-like proteins (MTLPs) and lipid peroxidation were quantified. When bees were exposed to increasing amounts of Cd, a marked augmentation of MTLPs levels was found. Lead (Pb) and Cd caused an increase in α-tocopherol content, while alteration of lipid peroxidation was observed only with Al exposure. These findings raise concerns about the bioavailability and the additional threat posed by metals for pollinators in agricultural areas while providing new insights for potential use of the honey bee as a sentinel species for metal exposure.

  3. Energy Band Gap, Intrinsic Carrier Concentration and Fermi Level of CdTe Bulk Crystal between 304 K and 1067 K

    NASA Technical Reports Server (NTRS)

    Su, Ching-Hua

    2007-01-01

    Optical transmission measurements were performed on CdTe bulk single crystal. It was found that when a sliced and polished CdTe wafer was used, a white film started to develop when the sample was heated above 530 K and the sample became opaque. Therefore, a bulk crystal of CdTe was first grown in the window area by physical vapor transport; the optical transmission was then measured and from which the energy band gap was derived between 304 and 1067 K. The band gaps of CdTe can be fit well as a function of temperature using the Varshini expression: Eg (e V) = 1.5860 - 5.9117xl0(exp -4) T(sup 2)/(T + 160). Using the band gap data, the high temperature electron-hole equilibrium was calculated numerically by assuming the Kane's conduction band structure and a heavy-hole parabolic valance band. The calculated intrinsic carrier concentrations agree well with the experimental data reported previously. The calculated intrinsic Fermi levels between 270 and 1200 K were also presented.

  4. Metal uptake of tomato and alfalfa plants as affected by water source, salinity, and Cd and Zn levels under greenhouse conditions.

    PubMed

    Gharaibeh, Mamoun A; Marschner, Bernd; Heinze, Stefanie

    2015-12-01

    Irrigation with wastewater is a promising option to improve crop yields and to reduce pressure on freshwater sources. However, heavy metal concentrations in wastewater may cause health concerns. A greenhouse pot experiment was conducted in order to determine cadmium (Cd) and zinc (Zn) concentrations in sandy soil and plant tissues of tomato (Lycopersicon esculentum L.) and alfalfa (Medicago sativa L.). A 2 × 2 × 4 × 2 factorial treatment arrangement was utilized. Two water sources, fresh (FW) or treated wastewater (TWW), at two salinity levels (1 and 3 dS m(-1)) containing different levels of Cd and Zn were used. Samples were collected after a 90-day growth period. It was observed that the growth of both plants was depressed at the highest metal level (L3). Metal accumulation in plant parts increased with the increase of metal concentration and salinity in irrigation water. At low salinity, water source was the main factor which controlled metal accumulation, whereas, at high salinity, chloride appeared to be the principal factor controlling metal uptake regardless of water source. Metal translocation from roots to shoots increased in TWW-irrigated plants, even in the controls. Tomatoes accumulated Cd up to and above critical levels safe for human consumption, even though Cd concentration in irrigation water did not exceed the current recommended values. Therefore, food production in sandy soils may well pose a health hazard when irrigated with TWW containing heavy metals. Complexation with dissolved organic compounds (DOC) in TWW may be to be the principal factor responsible for increased metal uptake and transfer at low salinity, thereby increasing the risk of heavy metal contamination of food and forage crops.

  5. A Cytidine Deaminase Edits C to U in Transfer RNAs in Archaea

    PubMed Central

    Randau, Lennart; Stanley, Bradford J.; Kohlway, Andrew; Mechta, Sarah; Xiong, Yong; Söll, Dieter

    2010-01-01

    All canonical transfer RNAs (tRNAs) have a uridine at position 8, involved in maintaining tRNA tertiary structure. However, the hyperthermophilic archaeon Methanopyrus kandleri harbors 30 (out of 34) tRNA genes with cytidine at position 8. Here, we demonstrate C-to-U editing at this location in the tRNA’s tertiary core, and present the crystal structure of a tRNA-specific cytidine deaminase, CDAT8, which has the cytidine deaminase domain linked to a tRNA-binding THUMP domain. CDAT8 is specific for C deamination at position 8, requires only the acceptor stem hairpin for activity, and belongs to a unique family within the “cytidine deaminase–like” superfamily. The presence of this C-to-U editing enzyme guarantees the proper folding and functionality of all M. kandleri tRNAs. PMID:19407206

  6. AID and APOBEC deaminases: balancing DNA damage in epigenetics and immunity.

    PubMed

    Franchini, Don-Marc; Petersen-Mahrt, Svend K

    2014-01-01

    DNA mutations and genomic recombinations are the origin of oncogenesis, yet parts of developmental programs as well as immunity are intimately linked to, or even depend on, such DNA damages. Therefore, the balance between deleterious DNA damages and organismal survival utilizing DNA editing (modification and repair) is in continuous flux. The cytosine deaminases AID/APOBEC are a DNA editing family and actively participate in various biological processes. In conjunction with altered DNA repair, the mutagenic potential of the family allows for APOBEC3 proteins to restrict viral infection and transposons propagation, while AID can induce somatic hypermutation and class switch recombination in antibody genes. On the other hand, the synergy between effective DNA repair and the nonmutagenic potential of the DNA deaminases can induce local DNA demethylation to support epigenetic cellular identity. Here, we review the current state of knowledge on the mechanisms of action of the AID/APOBEC family in immunity and epigenetics.

  7. Metal-induced stress in bivalves living along a gradient of Cd contamination: relating sub-cellular metal distribution to population-level responses.

    PubMed

    Perceval, Olivier; Couillard, Yves; Pinel-Alloul, Bernadette; Giguère, Anik; Campbell, Peter G C

    2004-09-20

    The use of biomarkers to assess the impacts of contaminants on aquatic ecosystems has noticeably increased over the past few years. Few of these studies, however, have contributed to the prediction of ecologically significant effects (i.e., at the population or community levels). The present field study was designed to evaluate the potential of metallothionein (MT) and sub-cellular metal partitioning measurements for predicting toxic effects at higher levels of the biological organization in freshwater bivalves (Pyganodon grandis) chronically exposed to Cd. For that purpose, we quantitatively sampled P. grandis populations in the littoral zone of nine lakes on the Precambrian Canadian Shield during two consecutive summers (1998 and 1999); lakes were characterized by contrasting Cd levels but similar trophic status. We tested relationships between the population status of P. grandis (i.e., growth parameters, density, biomass, secondary production, turnover ratio and cumulative fecundity) and (i) ambient Cd concentrations, (ii) sub-organismal responses (MT concentrations in the gill cytosol of individuals and Cd concentrations in three metal-ligand pools identified as M-HMW, the high molecular weight pool, M-MT, the metallothionein-like pool and M-LMW, the low molecular weight pool) and (iii) ecological confounding factors (food resources, presence of host fishes for the obligatory parasitic larval stage of P. grandis). Our results show that littoral density, live weight, dry viscera biomass, production and cumulative fecundity decreased with increasing concentrations of the free-cadmium ion in the environment (Pearson's r ranging from -0.63 to -0.78). On the other hand, theoretical maximum shell lengths (L( infinity )) in our populations were related to both the dissolved Ca concentration and food quality (sestonic C and N concentrations). Overall, Cd concentrations in the gill cytosolic HMW pool of the individual molluscs were the biomarker response that was most

  8. Metal-induced stress in bivalves living along a gradient of Cd contamination: relating sub-cellular metal distribution to population-level responses.

    PubMed

    Perceval, Olivier; Couillard, Yves; Pinel-Alloul, Bernadette; Giguère, Anik; Campbell, Peter G C

    2004-09-20

    The use of biomarkers to assess the impacts of contaminants on aquatic ecosystems has noticeably increased over the past few years. Few of these studies, however, have contributed to the prediction of ecologically significant effects (i.e., at the population or community levels). The present field study was designed to evaluate the potential of metallothionein (MT) and sub-cellular metal partitioning measurements for predicting toxic effects at higher levels of the biological organization in freshwater bivalves (Pyganodon grandis) chronically exposed to Cd. For that purpose, we quantitatively sampled P. grandis populations in the littoral zone of nine lakes on the Precambrian Canadian Shield during two consecutive summers (1998 and 1999); lakes were characterized by contrasting Cd levels but similar trophic status. We tested relationships between the population status of P. grandis (i.e., growth parameters, density, biomass, secondary production, turnover ratio and cumulative fecundity) and (i) ambient Cd concentrations, (ii) sub-organismal responses (MT concentrations in the gill cytosol of individuals and Cd concentrations in three metal-ligand pools identified as M-HMW, the high molecular weight pool, M-MT, the metallothionein-like pool and M-LMW, the low molecular weight pool) and (iii) ecological confounding factors (food resources, presence of host fishes for the obligatory parasitic larval stage of P. grandis). Our results show that littoral density, live weight, dry viscera biomass, production and cumulative fecundity decreased with increasing concentrations of the free-cadmium ion in the environment (Pearson's r ranging from -0.63 to -0.78). On the other hand, theoretical maximum shell lengths (L( infinity )) in our populations were related to both the dissolved Ca concentration and food quality (sestonic C and N concentrations). Overall, Cd concentrations in the gill cytosolic HMW pool of the individual molluscs were the biomarker response that was most

  9. Plasma Adenosine Deaminase Enzyme Reduces with Treatment of Pulmonary Tuberculosis in Nigerian Patients: Indication for Diagnosis and Treatment Monitoring.

    PubMed

    Ige, O; Edem, V F; Arinola, O G

    2016-01-01

    Tuberculosis(TB)-specific host biomarkers for diagnosis and monitoring of treatment response have been identified as priorities for TB research. Macrophage and T cell lymphocytes play vital roles in Mycobacterium tuberculosis immune response and their associated biomarkers could form good candidates for diagnosis and treatment monitoring. The enzyme adenosine deaminase (ADA) is produced mainly by monocytes and macrophages and increase in biological fluids in the course of infection with microorganisms infecting macrophages. This study comprised sixty-eight (68) participants; twenty-four (24) multi-drug-resistant TB(MDR-TB) patients, twenty-four (24) drug-sensitive TB patients(DS-TB) and twenty (20) non-TB apparently healthy individuals. Five (5) milliliters of blood was drawn before commencement of chemotherapy and 6 anti-TB therapy. In DSTB and MDR-TB patients before commencement of chemotherapy and 6 months of anti-TB treatment, the mean plasma levels of ADA were significantly increased compared with control. At 6 months of anti-TB chemotherapy of DSTB or MDR TB patients, ADA level was significantly decreased compared with before chemotherapy. Plasma ADA in DSTB patients before and 6 months of chemotherapy were not significantly different compared MDR TB patients. Plasma ADA level is a promising biomarker for the screening and treatment monitoring of pulmonary tuberculosis but not to differentiate MDR TB from DSTB patients. PMID:27574764

  10. Various effects of fluorescent bacteria of the genus Pseudomonas containing ACC deaminase on wheat seedling growth.

    PubMed

    Magnucka, Elżbieta G; Pietr, Stanisław J

    2015-12-01

    The study evaluates the effect of rhizobacteria having 1-aminocyclopropane-1-carboxylate deaminase (ACCd) on the development of wheat seedlings. This enzyme has been proposed to play a key role in microbe-plant association. Three fluorescent pseudomonads containing this deaminase were selected from 70 strains of pseudomonads isolated from rhizosphere of wheat (Triticum aestivum L.) and rape (Brassica napus L.). These bacteria, varied significantly in the ability to both biosynthesize auxins and hydrolyze ACC. Among them, Pseudomonas brassicacearum subsp. brassicacearum strain RZ310 presented the highest activities of ACC deaminase during 96h of growth in liquid Dworkin and Foster (DF) salt medium. Additionally, this rape rhizosphere strain did not produce indoles. Two other isolates, Pseudomonas sp. PO283 and Pseudomonas sp. PO366, secreted auxins only in the presence of their precursor. Phylogenetic analysis of the 16S rRNA gene and four other protein-encoding genes indicated that these wheat rhizosphere isolates belonged to the fluorescent Pseudomonas group. Moreover, the effects of these strains on wheat seedling growth under in vitro conditions were markedly dependent on both their cell suspensions used to grain inoculation and nutrient conditions. Strains tested had beneficial influence on wheat seedlings mainly at low cell densities. In addition, access to nutrients markedly changed bacteria action on cereal growth. Their presence generally favored the positive effects of pseudomonads on length and the estimated biomasses of wheat coleoptiles. Despite these general rules, impacts of each isolate on the growth parameters of cereal seedlings were unique. PMID:25983132

  11. Structure of the DNA deaminase domain of the HIV-1 restriction factor APOBEC3G.

    PubMed

    Chen, Kuan-Ming; Harjes, Elena; Gross, Phillip J; Fahmy, Amr; Lu, Yongjian; Shindo, Keisuke; Harris, Reuben S; Matsuo, Hiroshi

    2008-03-01

    The human APOBEC3G (apolipoprotein B messenger-RNA-editing enzyme, catalytic polypeptide-like 3G) protein is a single-strand DNA deaminase that inhibits the replication of human immunodeficiency virus-1 (HIV-1), other retroviruses and retrotransposons. APOBEC3G anti-viral activity is circumvented by most retroelements, such as through degradation by HIV-1 Vif. APOBEC3G is a member of a family of polynucleotide cytosine deaminases, several of which also target distinct physiological substrates. For instance, APOBEC1 edits APOB mRNA and AID deaminates antibody gene DNA. Although structures of other family members exist, none of these proteins has elicited polynucleotide cytosine deaminase or anti-viral activity. Here we report a solution structure of the human APOBEC3G catalytic domain. Five alpha-helices, including two that form the zinc-coordinating active site, are arranged over a hydrophobic platform consisting of five beta-strands. NMR DNA titration experiments, computational modelling, phylogenetic conservation and Escherichia coli-based activity assays combine to suggest a DNA-binding model in which a brim of positively charged residues positions the target cytosine for catalysis. The structure of the APOBEC3G catalytic domain will help us to understand functions of other family members and interactions that occur with pathogenic proteins such as HIV-1 Vif.

  12. Beyond SHM and CSR: AID and related cytidine deaminases in the host response to viral infection.

    PubMed

    Rosenberg, Brad R; Papavasiliou, F Nina

    2007-01-01

    As the primary effector of immunoglobulin somatic hypermutation (SHM) and class switch recombination (CSR), activation-induced cytidine deaminase (AID) serves an important function in the adaptive immune response. Recent advances have demonstrated that AID and a group of closely related cytidine deaminases, the APOBEC3 proteins, also act in the innate host response to viral infection. Antiviral activity was first attributed to APOBEC3G as a potent inhibitor of HIV. It is now apparent that the targets of the APOBEC3 proteins extend beyond HIV, with family members acting against a wide variety of viruses as well as host-encoded retrotransposable genetic elements. Although it appears to function through a different mechanism, AID also possesses antiviral properties. Independent of its antibody diversification functions, AID protects against transformation by Abelson murine leukemia virus (Ab-MLV), an oncogenic retrovirus. Additionally, AID has been implicated in the host response to other pathogenic viruses. These emerging roles for the AID/APOBEC cytidine deaminases in viral infection suggest an intriguing evolutionary connection of innate and adaptive immune mechanisms.

  13. Alternative Induction of Meiotic Recombination From Single-Base Lesions of DNA Deaminases

    PubMed Central

    Pauklin, Siim; Burkert, Julia S.; Martin, Julie; Osman, Fekret; Weller, Sandra; Boulton, Simon J.; Whitby, Matthew C.; Petersen-Mahrt, Svend K.

    2009-01-01

    Meiotic recombination enhances genetic diversity as well as ensures proper segregation of homologous chromosomes, requiring Spo11-initiated double-strand breaks (DSBs). DNA deaminases act on regions of single-stranded DNA and deaminate cytosine to uracil (dU). In the immunoglobulin locus, this lesion will initiate point mutations, gene conversion, and DNA recombination. To begin to delineate the effect of induced base lesions on meiosis, we analyzed the effect of expressing DNA deaminases (activation-induced deaminase, AID, and APOBEC3C) in germ cells. We show that meiotic dU:dG lesions can partially rescue a spo11Δ phenotype in yeast and worm. In rec12 Schizosaccharomyces pombe, AID expression increased proper chromosome segregation, thereby enhancing spore viability, and induced low-frequency meiotic crossovers. Expression of AID in the germ cells of Caenorhabditis elegans spo-11 induced meiotic RAD-51 foci formation and chromosomal bivalency and segregation, as well as an increase in viability. RNAi experiments showed that this rescue was dependent on uracil DNA-glycosylase (Ung). Furthermore, unlike ionizing radiation-induced spo-11 rescue, AID expression did not induce large numbers of DSBs during the rescue. This suggests that the products of DNA deamination and base excision repair, such as uracil, an abasic site, or a single-stranded nick, are sufficient to initiate and alter meiotic recombination in uni- and multicellular organisms. PMID:19237686

  14. Various effects of fluorescent bacteria of the genus Pseudomonas containing ACC deaminase on wheat seedling growth.

    PubMed

    Magnucka, Elżbieta G; Pietr, Stanisław J

    2015-12-01

    The study evaluates the effect of rhizobacteria having 1-aminocyclopropane-1-carboxylate deaminase (ACCd) on the development of wheat seedlings. This enzyme has been proposed to play a key role in microbe-plant association. Three fluorescent pseudomonads containing this deaminase were selected from 70 strains of pseudomonads isolated from rhizosphere of wheat (Triticum aestivum L.) and rape (Brassica napus L.). These bacteria, varied significantly in the ability to both biosynthesize auxins and hydrolyze ACC. Among them, Pseudomonas brassicacearum subsp. brassicacearum strain RZ310 presented the highest activities of ACC deaminase during 96h of growth in liquid Dworkin and Foster (DF) salt medium. Additionally, this rape rhizosphere strain did not produce indoles. Two other isolates, Pseudomonas sp. PO283 and Pseudomonas sp. PO366, secreted auxins only in the presence of their precursor. Phylogenetic analysis of the 16S rRNA gene and four other protein-encoding genes indicated that these wheat rhizosphere isolates belonged to the fluorescent Pseudomonas group. Moreover, the effects of these strains on wheat seedling growth under in vitro conditions were markedly dependent on both their cell suspensions used to grain inoculation and nutrient conditions. Strains tested had beneficial influence on wheat seedlings mainly at low cell densities. In addition, access to nutrients markedly changed bacteria action on cereal growth. Their presence generally favored the positive effects of pseudomonads on length and the estimated biomasses of wheat coleoptiles. Despite these general rules, impacts of each isolate on the growth parameters of cereal seedlings were unique.

  15. Two-Year Follow-up Study of Mycobacterium tuberculosis Antigen-Driven IFN-γ Responses and Macrophage sCD14 Levels After Tuberculosis Contact.

    PubMed

    Druszczynska, Magdalena; Wlodarczyk, Marcin; Kielnierowski, Grzegorz; Kawka, Malwina; Rudnicka, Wieslawa

    2016-06-01

    Clinical data regarding the prediction of active tuberculosis (TB) development in close TB contacts are scarce. To address this problem, we performed a 2-year follow-up study of Mycobacterium tuberculosis (M.tb) antigen-driven IFN-gamma responses and serum levels of soluble macrophage CD14 receptor in individuals with recent or prolonged M.tb exposure. Between June 2011 and June 2013, we studied 60 healthy Polish adults with recent household or long-term work TB contact and individuals without known M.tb exposure. All of them underwent baseline and repeated testing with IGRA (IFN-gamma release assay) and serum sCD14 ELISA quantification. Frequencies of IGRA results differed at the baseline and follow-up testing. IGRA reversions were noticed in almost one-third of Work TB Contacts and no participants from the Household TB Contact group. IGRA conversions were found in 40 % of Household TB Contacts. No correlation between the IGRA result and the sCD14 level was observed. IFN-γ variability has important implications for clinical practice and requires caution in interpreting the results to distinguish new infections from nonspecific inter-individual variations in cytokine responses. The impairment of IFN-γ response in some individuals with prolonged M.tb exposure representing a resistant immune status does not allow considering IGRA results as reliable and credible. Monitoring the serum sCD14 level can reduce the likelihood of a false prediction of active TB development in close TB contacts showing an M.tb-specific increase in the IFN-gamma production in repeated IGRA testing. PMID:27570313

  16. Fractionated neem leaf extract is safe and increases CD4+ cell levels in HIV/AIDS patients.

    PubMed

    Mbah, A U; Udeinya, I J; Shu, E N; Chijioke, C P; Nubila, T; Udeinya, F; Muobuike, Angela; Mmuobieri, Ancila; Obioma, Maria S

    2007-01-01

    The safety and effect of an acetone-water neem leaf extract (IRAB) on CD4 cells was investigated in 60 HIV/AIDS patients as part of an ongoing study to determine the influence of neem on immunity and viral load in HIV/AIDS. Patients were confirmed as HIV I or II positive, as having CD4 cell count, less than 300 cells/microL, and as antiretrovirally naïve. They were given oral IRAB (1.0 g daily for 12 weeks). Clinical and laboratory tests were carried out at baseline and at 4 weekly intervals. Thus, the patients served as their own controls. Sixty patients completed treatment. Fifty (83.33%) were completely compliant with respect to laboratory tests. Increase in mean CD4 cells, 266 cells/microL (159%), for the 50 patients was significant (P < 0.001) between baseline and week 12. Erythrocyte sedimentation rate (64 mm/hr at baseline) was 16 mm/hr at week 12, whereas total number of incidences of HIV/AIDS-related pathologies decreased from 120 at baseline to 5. Mean bodyweight, hemoglobin concentration, and lymphocyte differential count increased significantly by 12% (P < 0.05), 24% (P < 0.0001), and 20% (P < 0.0001), respectively. There were no adverse effects and no abnormalities in kidney and liver function parameters. The results support the safety of IRAB in HIV/AIDS, and its significant influence on CD4 cells may be useful in the formulation of multidrug combination therapies for HIV/AIDS. However, its antiretroviral activity is being evaluated in our laboratory.

  17. Fractionated neem leaf extract is safe and increases CD4+ cell levels in HIV/AIDS patients.

    PubMed

    Mbah, A U; Udeinya, I J; Shu, E N; Chijioke, C P; Nubila, T; Udeinya, F; Muobuike, Angela; Mmuobieri, Ancila; Obioma, Maria S

    2007-01-01

    The safety and effect of an acetone-water neem leaf extract (IRAB) on CD4 cells was investigated in 60 HIV/AIDS patients as part of an ongoing study to determine the influence of neem on immunity and viral load in HIV/AIDS. Patients were confirmed as HIV I or II positive, as having CD4 cell count, less than 300 cells/microL, and as antiretrovirally naïve. They were given oral IRAB (1.0 g daily for 12 weeks). Clinical and laboratory tests were carried out at baseline and at 4 weekly intervals. Thus, the patients served as their own controls. Sixty patients completed treatment. Fifty (83.33%) were completely compliant with respect to laboratory tests. Increase in mean CD4 cells, 266 cells/microL (159%), for the 50 patients was significant (P < 0.001) between baseline and week 12. Erythrocyte sedimentation rate (64 mm/hr at baseline) was 16 mm/hr at week 12, whereas total number of incidences of HIV/AIDS-related pathologies decreased from 120 at baseline to 5. Mean bodyweight, hemoglobin concentration, and lymphocyte differential count increased significantly by 12% (P < 0.05), 24% (P < 0.0001), and 20% (P < 0.0001), respectively. There were no adverse effects and no abnormalities in kidney and liver function parameters. The results support the safety of IRAB in HIV/AIDS, and its significant influence on CD4 cells may be useful in the formulation of multidrug combination therapies for HIV/AIDS. However, its antiretroviral activity is being evaluated in our laboratory. PMID:17667213

  18. Characterization of ZnO/CdS/CuInSe 2 Thin-Film Solar Cells by Deep-Level Transient Spectroscopy

    NASA Astrophysics Data System (ADS)

    Kuranouchi, Shin'ichi; Konagai, Makoto

    1995-05-01

    Trap energy levels in CuInSe2 (CIS) films have been measured by means of deep-level transient spectroscopy (DLTS). ZnO/CdS/CIS solar cells with conversion efficiencies of 5 11% were used as samples. Hole traps were detected for all samples measured in this study. Activation energy of traps in each sample was estimated to be in the range from 78 to 226 meV, which is shallower than any other reported values. Characteristics of the trap were thought to depend on the fabrication process of CIS films.

  19. Rapid generation of stable cell lines expressing high levels of erythropoietin, factor VIII, and an antihuman CD20 antibody using lentiviral vectors.

    PubMed

    Baranyi, Lajos; Doering, Christopher B; Denning, Gabriella; Gautney, Richard E; Harris, Kyle T; Spencer, H Trent; Roy, Andre; Zayed, Hatem; Dropulic, Boro

    2013-08-01

    Lentiviral vectors (LVs) are widely recognized as the most efficient method for the stable delivery of nucleic acid sequences into mammalian cells. Using erythropoietin (EPO), recombinant factor VIII (fVIII), and an anti-CD20 antibody as model proteins, we demonstrate advantages of LV-based gene delivery to achieve high production levels by transduced cells. Highly productive cell clones were able to incorporate up to 100 vector copies per cellular genome, without selection or gene amplification, and were isolated without extensive screening of a large number of clones. The LV transgenes were shown to be distributed throughout the genome, as visualized by fluorescent in situ hybridization. High-expressing clones producing 100-200 pg/cell/day of EPO were isolated and characterized. EPO production was demonstrated for at least 5½ months of continuous culture without selection, during which all the clones displayed high levels of glycosylation despite production levels at 10-20 g/liter. To demonstrate the utility of LV technology for multiple classes of proteins, cell lines producing fVIII and an anti-CD20 antibody were also developed. Cell clones demonstrating high levels of fVIII (100 clot units/ml and anti-CD20 antibody as high as 40-100 pg/cell/day) were isolated and characterized. LV-transduced cells and plasmid-transfected cells were compared for protein production per transgene copy. LV-transduced cells produced significantly higher levels of protein per copy of transgene than plasmid-transfected cells did. This study demonstrates the utility of LV technology for rapid generation of highly productive and stable cell lines over conventional plasmid transfection methods, significantly decreasing the time, cost, and risk of the manufacture of proteins and other complex biological molecules.

  20. The DNA cytosine deaminase APOBEC3H haplotype I likely contributes to breast and lung cancer mutagenesis.

    PubMed

    Starrett, Gabriel J; Luengas, Elizabeth M; McCann, Jennifer L; Ebrahimi, Diako; Temiz, Nuri A; Love, Robin P; Feng, Yuqing; Adolph, Madison B; Chelico, Linda; Law, Emily K; Carpenter, Michael A; Harris, Reuben S

    2016-01-01

    Cytosine mutations within TCA/T motifs are common in cancer. A likely cause is the DNA cytosine deaminase APOBEC3B (A3B). However, A3B-null breast tumours still have this mutational bias. Here we show that APOBEC3H haplotype I (A3H-I) provides a likely solution to this paradox. A3B-null tumours with this mutational bias have at least one copy of A3H-I despite little genetic linkage between these genes. Although deemed inactive previously, A3H-I has robust activity in biochemical and cellular assays, similar to A3H-II after compensation for lower protein expression levels. Gly105 in A3H-I (versus Arg105 in A3H-II) results in lower protein expression levels and increased nuclear localization, providing a mechanism for accessing genomic DNA. A3H-I also associates with clonal TCA/T-biased mutations in lung adenocarcinoma suggesting this enzyme makes broader contributions to cancer mutagenesis. These studies combine to suggest that A3B and A3H-I, together, explain the bulk of 'APOBEC signature' mutations in cancer. PMID:27650891

  1. The DNA cytosine deaminase APOBEC3H haplotype I likely contributes to breast and lung cancer mutagenesis

    PubMed Central

    Starrett, Gabriel J.; Luengas, Elizabeth M.; McCann, Jennifer L.; Ebrahimi, Diako; Temiz, Nuri A.; Love, Robin P.; Feng, Yuqing; Adolph, Madison B.; Chelico, Linda; Law, Emily K.; Carpenter, Michael A.; Harris, Reuben S

    2016-01-01

    Cytosine mutations within TCA/T motifs are common in cancer. A likely cause is the DNA cytosine deaminase APOBEC3B (A3B). However, A3B-null breast tumours still have this mutational bias. Here we show that APOBEC3H haplotype I (A3H-I) provides a likely solution to this paradox. A3B-null tumours with this mutational bias have at least one copy of A3H-I despite little genetic linkage between these genes. Although deemed inactive previously, A3H-I has robust activity in biochemical and cellular assays, similar to A3H-II after compensation for lower protein expression levels. Gly105 in A3H-I (versus Arg105 in A3H-II) results in lower protein expression levels and increased nuclear localization, providing a mechanism for accessing genomic DNA. A3H-I also associates with clonal TCA/T-biased mutations in lung adenocarcinoma suggesting this enzyme makes broader contributions to cancer mutagenesis. These studies combine to suggest that A3B and A3H-I, together, explain the bulk of ‘APOBEC signature' mutations in cancer. PMID:27650891

  2. Safety and Efficacy of Suicide Gene Therapy with Adenosine Deaminase 5-Fluorocytosine Silmutaneously in in Vitro Cultures of Melanoma and Retinal Cell Lines

    PubMed Central

    Sakkas, Antonios; Zarogoulidis, Paul; Domvri, Kalliopi; Hohenforst-Schmidt, Wolfgang; Bougiouklis, Dimitris; Kakolyris, Stylianos; Zarampoukas, Thomas; Kioumis, Ioannis; Pitsiou, Georgia; Huang, Haidong; Li, Qiang; Meditskou, Soultana; Tsiouda, Theodora; Pezirkianidis, Nikolaos; Zarogoulidis, Konstantinos

    2014-01-01

    Local treatment as a treatment modality is gaining increased general acceptance over time. Novel drugs and methodologies of local administration are being investigated in an effort to achieve disease local control. Suicide gene therapy is a method that has been investigated as a local treatment with simultaneously distant disease control. In our current experiment we purchased HTB-70 (melanoma cell line, derived from metastatic axillary node) and CRL-2302 (human retinal epithelium) were from ATCC LGC Standards and Ancotil®, 2.5 g/250 ml (1 g/00ml) (5-Flucytosine) MEDA; Pharmaceuticals Ltd. UK. Adenosine Cytosine Deaminase (Ad.CD) was also used in order to convert the pro-drug 5-Flucytosine to the active 5-Fluoracil. Three different concentrations of 5-Flucytosine (5-FC) were administered (0.2ml, 0.8ml and 1.2ml). At indicated time-points (4h, 8h and 24h) cell viability and apoptosis were measured. Our concept was to investigate whether suicide gene therapy with Ad. CD-5-FC could be used with safety and efficiency as a future local treatment for melanoma located in the eye cavity. Indeed, our results indicated that in every 5-FC administration had mild cytotoxicity for the retinal cells, while increased apoptosis was observed for the melanoma cell line. PMID:24799955

  3. Synergistic effect of therapeutic stem cells expressing cytosine deaminase and interferon-beta via apoptotic pathway in the metastatic mouse model of breast cancer.

    PubMed

    Yi, Bo-Rim; Kim, Seung U; Choi, Kyung-Chul

    2016-02-01

    As an approach to improve treatment of breast cancer metastasis to the brain, we employed genetically engineered stem cells (GESTECs, HB1.F3 cells) consisting of neural stem cells (NSCs) expressing cytosine deaminase and the interferon-beta genes, HB1.F3.CD and HB1.F3.CD.IFN-β. In this model, MDA-MB-231/Luc breast cancer cells were implanted in the right hemisphere of the mouse brain, while pre-stained GESTECs with redfluorescence were implanted in the contralateral brain. Two days after stem cells injection, 5-fluorocytosine (5-FC) was administrated via intraperitoneal injection. Histological analysis of extracted brain confirmed the therapeutic efficacy of GESTECs in the presence of 5-FC based on reductions in density and aggressive tendency of breast cancer cells, as well as pyknosis, karyorrhexis, and karyolysis relative to a negative control. Additionally, expression of PCNA decreased in the stem cells treated group. Treatment of breast cancer cells with 5-fluorouracil (5-FU) increased the expression of pro-apoptotic and anti-proliferative factor, BAX and p21 protein through phosphorylation of p53 and p38. Moreover, analysis of stem cell migratory ability revealed that MDA-MB-231 cells endogenously secreted VEGF, and stem cells expressed their receptor (VEGFR2). To confirm the role of VEGF/VEGFR2 signaling in tumor tropism of stem cells, samples were treated with the VEGFR2 inhibitor, KRN633. The number of migrated stem cells decreased significantly in response to KRN633 due to Erk1/2 activation and PI3K/Akt inhibition. Taken together, these results indicate that treatment with GESTECs, particularly HB1.F3.CD.IFN-β co-expressing CD.IFN-β, may be a useful strategy for treating breast cancer metastasis to the brain in the presence of a prodrug.

  4. Vitamin D, d-dimer, Interferon γ, and sCD14 Levels are Independently Associated with Immune Reconstitution Inflammatory Syndrome: A Prospective, International Study☆

    PubMed Central

    Musselwhite, Laura W.; Andrade, Bruno B.; Ellenberg, Susan S.; Tierney, Ann; Belaunzaran-Zamudio, Pablo F.; Rupert, Adam; Lederman, Michael M.; Sanne, Ian; Sierra Madero, Juan G.; Sereti, Irini

    2016-01-01

    To determine the immunological profile most important for IRIS prediction, we evaluated 20 baseline plasma biomarkers in Acquired Immunodeficiency Syndrome (AIDS) patients initiating antiretroviral therapy (ART). Patients were enrolled in a randomized, placebo-controlled ART initiation trial in South Africa and Mexico to test whether maraviroc could prevent IRIS. Participants were classified prospectively as having IRIS within 6 months of ART initiation. Twenty plasma biomarkers were measured at study enrollment for 267 participants. Biomarkers were tested for predicting IRIS with adjustment for covariates chosen through forward stepwise selection. Sixty-two participants developed IRIS and of these 19 were tuberculosis (TB)-IRIS. Baseline levels of vitamin D and higher d-dimer, interferon gamma (IFNγ), and sCD14 were independently associated with risk of IRIS in multivariate analyses. TB-IRIS cases exhibited a distinct biosignature from IRIS related to other pathogens, with increased levels of C-reactive protein (CRP), sCD14, IFNγ, and lower levels of Hb that could be captured by a composite risk score. Elevated markers of Type 1 T helper (Th1) response, monocyte activation, coagulation and low vitamin D were independently associated with IRIS risk. Interventions that decrease immune activation and increase vitamin D levels warrant further study. PMID:26981576

  5. Influence of a Diester Glucocorticoid Spray on the Cortisol Level and the CCR4+ CD4+ Lymphocytes in Dogs with Atopic Dermatitis: Open Study

    PubMed Central

    Fujimura, Masato; Ishimaru, Hironobu

    2014-01-01

    This study investigated the influence of 0.00584% hydrocortisone aceponate spray (HCA; Cortavance Virbac SA, Carros, France) on blood serum cortisol levels and peripheral blood CCR4+ CD4+ T-lymphocyte levels in dogs with atopic dermatitis. Patients were randomly divided into group I (N = 8) and group II (N = 8). The dogs in group I were sprayed with HCA on the affected skin once a day for three weeks. The dogs in group II were treated once a day for 3 days followed by no treatment for 4 days for a total of three weeks. For the dogs in group I and group II the CADESI-03 scores before and after use of HCA showed significant reduction (P < 0.01). The postcortisol level after the use of HCA in group I showed 36.0% decrease and showed significant suppression (P < 0.01). By comparison, the use of HCA on group II did not show decrease in postcortisol levels. There was a tendency of suppression for hypothalamus—pituitary gland—adrenal gland system, but it was not serious influence. In addition, there was no influence on peripheral blood CCR4+ CD4+ lymphocytes percentage in dogs in group I after treatment with HCA. PMID:26464935

  6. Isolation and characterization of ACC deaminase-producing fluorescent pseudomonads, to alleviate salinity stress on canola (Brassica napus L.) growth.

    PubMed

    Jalili, Farzad; Khavazi, Kazem; Pazira, Ebrahim; Nejati, Alireza; Rahmani, Hadi Asadi; Sadaghiani, Hasan Rasuli; Miransari, Mohammad

    2009-04-01

    Salinity stress is of great importance in arid and semi-arid areas of the world due to its impact in reducing crop yield. Under salinity stress, the amount of 1-aminocyclopropane-1-carboxylate (ACC), a precursor for ethylene production in plants, increases. Here, we conducted research under the hypothesis that isolated ACC deaminase-producing Pseudomonas fluorescens and Pseudomonas putida can alleviate the stressful effects of salinity on canola (Brassica napus L.) growth. The experiments were conducted in the Soil and Water Research Institute, Tehran, Iran. Seven experimental stages were conducted to isolate and characterize ACC deaminase-producing Pseudomonas fluorescens strains and to determine factors enhancing their growth and, consequently, their effects on the germination of canola seeds. Under salinity stress, in 14% of the isolates, ACC deaminase activity was observed, indicating that they were able to utilize ACC as the sole N-source. Bacterial strains differed in their ability to synthesize auxin and hydrogen cyanide compounds, as well as in their ACC deaminase activity. Under salinity stress, the rate of germinating seeds inoculated with the strains of ACC deaminase-producing Pseudomonas fluorescens and Pseudomonas putida, and seedling growth was significantly higher. These results indicate the significance of soil biological activities, including the activities of plant growth-promoting bacteria, in the alleviation of soil stresses such as salinity on plant growth.

  7. Manifestation of the Se, Cd and Mo levels in different components of the peripheral blood of Sprague-Dawley rats poisoned via the respiratory tract.

    PubMed

    Wang, Dong-Fang; Sun, Xuan; Cao, Bing; Wen, Hua; Zhang, Yu; Liu, Duo-Jian; Yan, Lai-Lai; Liu, Ya-Qiong; Lu, Qing-Bin; Wang, Jing-Yu

    2015-01-01

    This study aimed to explore the effects of exogenous element exposure via the respiratory tract on the Se, Cd and Mo concentrations in different components of the peripheral blood in rats as well as to determine the correlations of the three trace elements concentrations among the components. The Sprague-Dawley rats were randomly divided into a control group and several experimental groups treated with different doses. The rats were exposed to a mixed trace element solution through 10 days of intratracheal instillation. The whole blood of all rats was collected and separated into three parts with Percoll density gradient centrifugation. The Se, Cd and Mo levels in whole blood, plasma, red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) were determined by inductively coupled plasma mass spectrometry. The concentrations of the three trace elements increased together with the increase of the given doses (P<0.05), except Cd and Mo in the PBMCs. The three trace elements lacked linearity with the exposure doses in the PBMCs (r, 0.249-0.508), while the opposite was the case for the other components of the peripheral blood (r, 0.806-0.934). The correlation coefficients were higher (0.842-0.962) among the whole blood, plasma and RBCs than between PBMCs and other components, such as Se (0.376-0.529), Cd (0.495-0.604) and, especially, Mo (0.160-0.257). In conclusion, PBMCs might provide information about endogenous factors, and whole blood could more accurately reflect the effects of exogenous factors compared to other blood components. PMID:26770359

  8. High levels of TSP1+/CD142+ platelet-derived microparticles characterise young patients with high cardiovascular risk and subclinical atherosclerosis.

    PubMed

    Suades, Rosa; Padró, Teresa; Alonso, Rodrigo; Mata, Pedro; Badimon, Lina

    2015-11-25

    Circulating microparticles (cMPs) play important roles in cellular crosstalk and are messengers of cell activation. We have previously reported that platelet-released microparticles (pMPs) stimulate thrombosis and that lipid-lowering treatment as per guidelines in patients with familial hypercholesterolaemia (FH) is not sufficiently effective in reducing pro-inflammatory cell activation and, consequently, CD45+/CD3+-lymphocyte-derived cMP shedding. FH patients, due to life-long vascular exposure to high LDL-cholesterol levels, are at high cardiovascular risk (HCVR) and develop premature coronary artery disease. Our objectives were to investigate a) whether patients with HCVR have cMPs with a prothrombotic phenotype, and b) whether patients with magnetic resonance imaging (MRI) evidence of lipid-rich atherosclerotic lesions have a specific cMP profile regarding prothrombotic protein cargos. cMPs were isolated from HCVR-patients and from age/gender/treatment-matched control patients. cMP phenotype was characterised by triple-labelling flow cytometry. HCVR--patients have higher numbers of pMPs derived from activated platelets as well as of tissue factor-rich microparticles (TF+-cMPs) than controls (P< 0.0001). TF+-cMPs showed procoagulant activity, which associate with atherosclerotic plaque burden, indicating that TF in the cMPs is functional. In HCVR-patients, overall TF+-cMPs (monocyte-derived [CD142+/CD14+] and platelet-derived [CD142+/TSP1+]) and activated pMPs directly correlate with MRI-detected lipid-rich atherosclerotic plaques while inversely correlate with MRI-detected calcified plaques. C-statistics analysis showed that prothrombotic cMPs add significant prognostic value to a risk factor model for the prediction of lipid-rich plaques. In conclusion, the activation status of blood cells in HCVR-patients differed markedly from controls as shown by higher circulating levels of prothrombotic and TF+-cMPs. Prothrombotic cMP numbers identify subclinical

  9. A combined nuclear and nucleolar localization motif in activation-induced cytidine deaminase (AID) controls immunoglobulin class switching.

    PubMed

    Hu, Yi; Ericsson, Ida; Torseth, Kathrin; Methot, Stephen P; Sundheim, Ottar; Liabakk, Nina B; Slupphaug, Geir; Di Noia, Javier M; Krokan, Hans E; Kavli, Bodil

    2013-01-23

    Activation-induced cytidine deaminase (AID) is a DNA mutator enzyme essential for adaptive immunity. AID initiates somatic hypermutation and class switch recombination (CSR) by deaminating cytosine to uracil in specific immunoglobulin (Ig) gene regions. However, other loci, including cancer-related genes, are also targeted. Thus, tight regulation of AID is crucial to balance immunity versus disease such as cancer. AID is regulated by several mechanisms including nucleocytoplasmic shuttling. Here we have studied nuclear import kinetics and subnuclear trafficking of AID in live cells and characterized in detail its nuclear localization signal. Importantly, we find that the nuclear localization signal motif also directs AID to nucleoli where it colocalizes with its interaction partner, catenin-β-like 1 (CTNNBL1), and physically associates with nucleolin and nucleophosmin. Moreover, we demonstrate that release of AID from nucleoli is dependent on its C-terminal motif. Finally, we find that CSR efficiency correlates strongly with the arithmetic product of AID nuclear import rate and DNA deamination activity. Our findings suggest that directional nucleolar transit is important for the physiological function of AID and demonstrate that nuclear/nucleolar import and DNA cytosine deamination together define the biological activity of AID. This is the first study on subnuclear trafficking of AID and demonstrates a new level in its complex regulation. In addition, our results resolve the problem related to dissociation of deamination activity and CSR activity of AID mutants.

  10. High-yield production of apoplast-directed human adenosine deaminase in transgenic tobacco BY-2 cell suspensions.

    PubMed

    Singhabahu, Sanjeewa; George, John; Bringloe, David

    2015-01-01

    Adenosine deaminase (ADA) deficiency, where a deleterious mutation in the ADA gene of patients results in a dysfunctional immune system, is ultimately caused by an absence of ADA. Over the last 25 years the disease has been treated with PEG-ADA, made from purified bovine ADA coupled with polyethylene glycol (PEG). However, it is thought that an enzyme replacement therapy protocol based on recombinant human ADA would probably be a more effective treatment. With this end in mind, a human ADA cDNA was inserted into plant expression vectors used to transform tobacco plant cell suspensions. Transgenic calli expressing constructs containing apoplast-directing signals showed significantly higher levels of recombinant ADA expression than calli transformed with cytosolic constructs. The most significant ADA activities, however, were measured in the media of transgenic cell suspensions prepared from high expressing transformed calli: where incorporation of a signal for arabinogalactan addition to ADA led to a recombinant protein yield of approximately 16 mg L(-1) , a 336-fold increase over ADA produced by cell suspensions transformed with a cytosolic construct.

  11. Aberrant activation-induced cytidine deaminase expression in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia.

    PubMed

    Shi, Yang; Zhao, Xiaoxian; Durkin, Lisa; Rogers, Heesun Joyce; Hsi, Eric D

    2016-06-01

    Activation-induced cytidine deaminase (AID) is expressed in germinal center B cells and plays a critical role in somatic hypermutation and class-switch recombination of immunoglobulin genes. Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) carries a poor prognosis and is specifically treated with tyrosine kinase inhibitors. Interestingly, AID has been shown to be aberrantly expressed and functional in Ph+ ALL and is thought to contribute to genetic instability. We hypothesized that AID might be detectable in routinely processed bone marrow biopsies by immunohistochemistry (IHC) and assist in identifying Ph+ ALL. We found that AID was expressed in 26 (70%) of 37 cases of Ph+ ALL but only 1 (2.9%) of 38 cases of Ph- ALL cases. There was a significant difference in AID expression between these 2 ALL groups (P < .001, Fisher exact test). The expression of AID was confirmed by RT-PCR (reverse-transcriptase polymerase chain reaction) and correlated with IHC scoring. AID protein is expressed in a large proportion of Ph+ ALL cases at levels detectable by IHC in clinical samples and might be useful to rapidly identify cases likely to have a BCR/ABL1 fusion. PMID:26980048

  12. High-yield production of apoplast-directed human adenosine deaminase in transgenic tobacco BY-2 cell suspensions.

    PubMed

    Singhabahu, Sanjeewa; George, John; Bringloe, David

    2015-01-01

    Adenosine deaminase (ADA) deficiency, where a deleterious mutation in the ADA gene of patients results in a dysfunctional immune system, is ultimately caused by an absence of ADA. Over the last 25 years the disease has been treated with PEG-ADA, made from purified bovine ADA coupled with polyethylene glycol (PEG). However, it is thought that an enzyme replacement therapy protocol based on recombinant human ADA would probably be a more effective treatment. With this end in mind, a human ADA cDNA was inserted into plant expression vectors used to transform tobacco plant cell suspensions. Transgenic calli expressing constructs containing apoplast-directing signals showed significantly higher levels of recombinant ADA expression than calli transformed with cytosolic constructs. The most significant ADA activities, however, were measured in the media of transgenic cell suspensions prepared from high expressing transformed calli: where incorporation of a signal for arabinogalactan addition to ADA led to a recombinant protein yield of approximately 16 mg L(-1) , a 336-fold increase over ADA produced by cell suspensions transformed with a cytosolic construct. PMID:24825606

  13. Estradiol induces gene proximity and MLL-MLLT3 fusion in an activation-induced cytidine deaminase-mediated pathway.

    PubMed

    Wright, Rebecca L; Slemmons, Katherine K; Vaughan, Andrew T M

    2015-05-01

    Epidemiological data have linked birth control formulations to an increased risk of infant acute leukemia involving MLL rearrangements. Reverse transcription polymerase chain reaction (RT-PCR) studies showed that 10 nM estradiol enhanced MLL transcription in addition to its common translocation partners, MLLT2 (AF4) and MLLT3 (AF9). The same concentration of estradiol triggered MLL and MLLT3 co-localization without affecting the interaction of genes located on the same chromosomes. Estradiol also stimulated the generation of MLL-MLLT3 fusion transcripts as seen by RT-PCR. RNAi knockdown of activation-induced cytidine deaminase (AICDA) suppressed the induction of MLL-MLLT3 fusion transcript formation observed with estradiol. Additionally, chromatin immunoprecipitation (ChIP) analysis showed estradiol dependent localization of AICDA in MLL intron 11, upstream of a hotspot for both DNA cleavage and rearrangement, but not downstream within intron 12. Combined, these studies show that levels of estradiol consistent with that observed during pregnancy have the potential to initiate MLL fusions through an AICDA-mediated mechanism.

  14. TLR9 Ligand (CpG Oligodeoxynucleotide) Induces CLL B-Cells to Differentiate into CD20(+) Antibody-Secreting Cells.

    PubMed

    Ghamlouch, Hussein; Ouled-Haddou, Hakim; Guyart, Aude; Regnier, Aline; Trudel, Stéphanie; Claisse, Jean-François; Fuentes, Vincent; Royer, Bruno; Marolleau, Jean-Pierre; Gubler, Brigitte

    2014-01-01

    B-cell chronic lymphocytic leukemia (CLL) is the most frequent adult leukemia in the Western world. It is a heterogeneous disease characterized by clonal proliferation and the accumulation of CD5(+) mature B lymphocytes. However, the normal counterpart from which the latter cells arise has not yet been identified. CD27 expression and gene expression profiling data suggest that CLL cells are related to memory B-cells. In vitro, memory B-cells differentiate into plasma cells when stimulated with CpG oligodeoxynucleotide (CpG). The objective of the present study was therefore to investigate the ability of CpG, in the context of CD40 ligation, to induce the differentiation of CLL B-cells into antibody-secreting cells (ASCs). CD20(+)CD38(-) CLL B-cells were stimulated with a combination of CpG, CD40 ligand and cytokines (CpG/CD40L/c) in a two-step, 7-day culture system. We found that the CpG/CD40L/c culture system prompted CLL B-cells to differentiate into CD19(+)CD20(+)CD27(+)CD38(-)ASCs. These cells secreted large amounts of IgM and had the same shape as plasma cells. However, only IgMs secreted by ASCs that had differentiated from unmutated CLL B-cells were poly/autoreactive. Class-switch recombination (CSR) to IgG and IgA was detected in cells expressing the activation-induced cytidine deaminase gene (AICDA). Although these ASCs expressed high levels of the transcription factors PRDM1 (BLIMP1), IRF4, and XBP1s, they did not downregulate expression of PAX5. Our results suggest that CLL B-cells can differentiate into ASCs, undergo CSR and produce poly/autoreactive antibodies. Furthermore, our findings may be relevant for (i) identifying the normal counterpart of CLL B-cells and (ii) developing novel treatment strategies in CLL. PMID:24982661

  15. Yin and yang of cytidine deaminase roles in clinical response to azacitidine in the elderly: a pharmacogenetics tale.

    PubMed

    Fanciullino, Raphaelle; Mercier, Cédric; Serdjebi, Cindy; Venton, Geoffroy; Colle, Julien; Fina, Frédéric; Ouafik, L'Houcine; Lacarelle, Bruno; Ciccolini, Joseph; Costello, Régis

    2015-11-01

    Azacitidine is a mainstay for treating hematological disorders. Azacitidine is metabolized by cytidine deaminase, coded by a highly polymorphic gene. Here, we present two elderly patients with opposite clinical outcomes after azacitidine treatment. First, an acute myeloid leukemia patient showed life-threatening toxicities, but outstanding complete remission, after a single round of azacitidine. Further investigations showed that this patient was cytidine deaminase 79A>C (rs2072671) homozygous with a marked deficient phenotype. Next, a chronic myelomonocytic leukemia patient displayed complete lack of response despite several cycles of azacitidine. This patient had a rapid-deaminator phenotype linked to the -31delC deletion (rs3215400). These polymorphisms lead to opposite clinical outcomes in patients with myelodysplastic syndromes treated with azacitidine, thus suggesting that determining cytidine deaminase status could help to forecast clinical outcome.

  16. PRRSV-infected monocyte-derived dendritic cells express high levels of SLA-DR and CD80/86 but do not stimulate PRRSV-naïve regulatory T cells to proliferate.

    PubMed

    Rodríguez-Gómez, Irene M; Käser, Tobias; Gómez-Laguna, Jaime; Lamp, Benjamin; Sinn, Leonie; Rümenapf, Till; Carrasco, Librado; Saalmüller, Armin; Gerner, Wilhelm

    2015-01-01

    In vitro generated monocyte-derived dendritic cells (moDCs) have frequently been used to study the influence of porcine reproductive and respiratory syndrome virus (PRRSV) infection on antigen presenting cells. However, obtained results have often been conflicting in regard to expression of co-stimulatory molecules and interaction with T cells. In this study we performed a detailed phenotypic characterisation of PRRSV-infected moDCs and non-infected moDCs. For CD163 and CD169, which are involved in PRRSV-entry into host cells, our results show that prior to infection porcine moDCs express high levels of CD163 but only very low levels for CD169. Following infection with either PRRSV-1 or PRRSV-2 strains after 24 h, PRRSV-nucleoprotein (N-protein)(+) and N-protein(-) moDCs derived from the same microculture were analyzed for expression of swine leukocyte antigen-DR (SLA-DR) and CD80/86. N-protein(+) moDCs consistently expressed higher levels of SLA-DR and CD80/86 compared to N-protein(-) moDCs. We also investigated the influence of PRRSV-infected moDCs on proliferation and frequency of Foxp3(+) regulatory T cells present within CD4(+) T cells in in vitro co-cultures. Neither CD3-stimulated nor unstimulated CD4(+) T cells showed differences in regard to proliferation and frequency of Foxp3(+) T cells following co-cultivation with either PRRSV-1 or PRRSV-2 infected moDCs. Our results suggest that a more detailed characterisation of PRRSV-infected moDCs will lead to more consistent results across different laboratories and PRRSV strains as indicated by the major differences in SLA-DR and CD80/86 expression between PRRSV-infected and non-infected moDCs present in the same microculture. PMID:25990845

  17. PRRSV-infected monocyte-derived dendritic cells express high levels of SLA-DR and CD80/86 but do not stimulate PRRSV-naïve regulatory T cells to proliferate.

    PubMed

    Rodríguez-Gómez, Irene M; Käser, Tobias; Gómez-Laguna, Jaime; Lamp, Benjamin; Sinn, Leonie; Rümenapf, Till; Carrasco, Librado; Saalmüller, Armin; Gerner, Wilhelm

    2015-05-20

    In vitro generated monocyte-derived dendritic cells (moDCs) have frequently been used to study the influence of porcine reproductive and respiratory syndrome virus (PRRSV) infection on antigen presenting cells. However, obtained results have often been conflicting in regard to expression of co-stimulatory molecules and interaction with T cells. In this study we performed a detailed phenotypic characterisation of PRRSV-infected moDCs and non-infected moDCs. For CD163 and CD169, which are involved in PRRSV-entry into host cells, our results show that prior to infection porcine moDCs express high levels of CD163 but only very low levels for CD169. Following infection with either PRRSV-1 or PRRSV-2 strains after 24 h, PRRSV-nucleoprotein (N-protein)(+) and N-protein(-) moDCs derived from the same microculture were analyzed for expression of swine leukocyte antigen-DR (SLA-DR) and CD80/86. N-protein(+) moDCs consistently expressed higher levels of SLA-DR and CD80/86 compared to N-protein(-) moDCs. We also investigated the influence of PRRSV-infected moDCs on proliferation and frequency of Foxp3(+) regulatory T cells present within CD4(+) T cells in in vitro co-cultures. Neither CD3-stimulated nor unstimulated CD4(+) T cells showed differences in regard to proliferation and frequency of Foxp3(+) T cells following co-cultivation with either PRRSV-1 or PRRSV-2 infected moDCs. Our results suggest that a more detailed characterisation of PRRSV-infected moDCs will lead to more consistent results across different laboratories and PRRSV strains as indicated by the major differences in SLA-DR and CD80/86 expression between PRRSV-infected and non-infected moDCs present in the same microculture.

  18. Direct deamination of AMP, ADP, ATP and NADH by non-specific adenylate deaminase in the foot muscle of the snail Helix pomatia.

    PubMed Central

    Stankiewicz, A J

    1983-01-01

    Homogeneous adenylate deaminase from snail foot muscle deaminated 5'-AMP, 5'-ADP, 5'-ATP and NADH with similar velocity and affinity to all substrates. At millimolar concentration NAD+ was also deaminated to a comparable extent, but NADP+, NADPH and FAD were not substrates for the snail enzyme. The amount of deaminase activity per g of fresh tissue is 5-10 times greater than in the muscle of any other species studied. The activity of the snail deaminase is regulated by pH, KCl and buffer concentrations, and Pi; however, regulation seems to be very poor in comparison with that of muscle deaminases from other species, specific to 5'-AMP. Snail enzyme appears as the first animal deaminase so far described that has such characteristics. It offers also some opportunities as an analytical tool as a consequence of its very high affinity toward adenylates. PMID:6626180

  19. Modulation of AMP deaminase in rat hearts subjected to ischemia and reperfusion by purine riboside.

    PubMed

    Borkowski, T; Lipinski, M; Kaminski, R; Krzyminska-Stasiuk, E; Langowska, M; Raczak, G; Slominska, E M; Smolenski, R T

    2008-06-01

    Changes in AMP deaminase (AMPD) activity influence heart function and progression of heart disease, but the underlying mechanism is unknown. We evaluated the effect of purine riboside (Purr) on the activity of AMPD in perfused rat hearts and in isolated rat cardiomyocytes. Brief perfusion of the pre-ischemic heart with 200 micro M Purr resulted in activation of AMPD, more pronounced degradation of the adenine nucleotides, and reduced recovery of the adenine nucleotide pool during reperfusion. Brief incubation of rat cardiomyocytes with 200 micro M Purr also activated AMPD, while prolonged exposure resulted in enzyme inhibition. We conclude that Purr activates AMPD, whereas metabolites of this compound may inhibit the enzyme.

  20. Threonine deaminase from extremely halophilic bacteria - Cooperative substrate kinetics and salt dependence.

    NASA Technical Reports Server (NTRS)

    Lieberman, M. M.; Lanyi, J. K.

    1972-01-01

    The effect of salt on the activity, stability, and allosteric properties of catabolic threonine deaminase from Halobacterium cutirubrum was studied. The enzyme exhibits sigmoidal kinetics with the substrate, threonine. The Hill slope is 1.55 at pH 10. The enzyme is activated by ADP at low substrate concentrations. In the presence of this effector, sigmoidal kinetics are no longer observed. At pH 10, in the absence of ADP, enzyme activity increases with increasing NaCl concentration from 0 to 4 M.

  1. Disruption of the adenosine deaminase gene causes hepatocellular impairment and perinatal lethality in mice.

    PubMed Central

    Wakamiya, M; Blackburn, M R; Jurecic, R; McArthur, M J; Geske, R S; Cartwright, J; Mitani, K; Vaishnav, S; Belmont, J W; Kellems, R E

    1995-01-01

    We have generated mice with a null mutation at the Ada locus, which encodes the purine catabolic enzyme adenosine deaminase (ADA, EC 3.5.4.4). ADA-deficient fetuses exhibited hepatocellular impairment and died perinatally. Their lymphoid tissues were not largely affected. Accumulation of ADA substrates was detectable in ADA-deficient conceptuses as early as 12.5 days postcoitum, dramatically increasing during late in utero development, and is the likely cause of liver damage and fetal death. The results presented here demonstrate that ADA is important for the homeostatic maintenance of purines in mice. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7731963

  2. Deoxycytidine monophosphate deaminase in Acetabularia: properties and regulation in the early generative phase.

    PubMed

    Bannwarth, H; Ikehara, N; Schweiger, H G

    1982-06-01

    The occurrence of a dCMP deaminase in Acetabularia mediterranea has been demonstrated. The enzyme which is found in a particulate fraction is substantially stimulated by the addition of dCTP. The activity of the enzyme is increased at the beginning of the generative phase in nucleate as well as in anucleate cells. This regulation is due to de novo synthesis of the enzyme. By means of inhibitor studies, it has been shown that the enzyme is translated on 70S ribosomes of and coced for in cell organelles.

  3. L-nucleoside analogues as potential antimalarials that selectively target Plasmodium falciparum adenosine deaminase.

    PubMed

    Brown, D M; Netting, A G; Chun, B K; Choi, Y; Chu, C K; Gero, A M

    1999-01-01

    The L-stereoisomer analogues of D-coformycin selectively inhibited P. falciparum adenosine deaminase (ADA) in the picomolar range (L-isocoformycin, Ki 7 pM; L-coformycin, Ki 250 pM). While the L-nucleoside analogues, L-adenosine, 2,6-diamino-9-(L-ribofuranosyl)purine and 4-amino-1-(L-ribofuranosyl)pyrazolo[3,4-d]-pyrimidine were selectively deaminated by P. falciparum ADA, L-thioinosine and L-thioguanosine were not. This is the first example of 'non-physiological' L-nucleosides that serve as either substrates or inhibitors of malarial ADA and are not utilised by mammalian ADA.

  4. Cytomegalovirus Replication in Semen Is Associated with Higher Levels of Proviral HIV DNA and CD4+ T Cell Activation during Antiretroviral Treatment

    PubMed Central

    Massanella, Marta; Richman, Douglas D.; Little, Susan J.; Spina, Celsa A.; Vargas, Milenka V.; Lada, Steven M.; Daar, Eric S.; Dube, Michael P.; Haubrich, Richard H.; Morris, Sheldon R.; Smith, Davey M.

    2014-01-01

    ABSTRACT Asymptomatic cytomegalovirus (CMV) replication occurs frequently in the genital tract in untreated HIV-infected men and is associated with increased immune activation and HIV disease progression. To determine the connections between CMV-associated immune activation and the size of the viral reservoir, we evaluated the interactions between (i) asymptomatic seminal CMV replication, (ii) levels of T cell activation and proliferation in blood, and (iii) the size and transcriptional activity of the HIV DNA reservoir in blood from 53 HIV-infected men on long-term antiretroviral therapy (ART) with suppressed HIV RNA in blood plasma. We found that asymptomatic CMV shedding in semen was associated with significantly higher levels of proliferating and activated CD4+ T cells in blood (P < 0.01). Subjects with detectable CMV in semen had approximately five times higher average levels of HIV DNA in blood CD4+ T cells than subjects with no CMV. There was also a trend for CMV shedders to have increased cellular (multiply spliced) HIV RNA transcription (P = 0.068) compared to participants without CMV, but it is unclear if this transcription pattern is associated with residual HIV replication. In multivariate analysis, the presence of seminal plasma CMV (P = 0.04), detectable 2-long terminal repeat (2-LTR), and lower nadir CD4+ (P < 0.01) were independent predictors of higher levels of proviral HIV DNA in blood. Interventions aimed at reducing seminal CMV and associated immune activation may be important for HIV curative strategies. Future studies of anti-CMV therapeutics will help to establish causality and determine the mechanisms underlying these described associations. IMPORTANCE Almost all individuals infected with HIV are also infected with cytomegalovirus (CMV), and the replication dynamics of the two viruses likely influence each other. This study investigated interactions between asymptomatic CMV replication within the male genital tract, levels of inflammation in

  5. Significance of the D-serine-deaminase and D-serine metabolism of Staphylococcus saprophyticus for virulence.

    PubMed

    Korte-Berwanger, Miriam; Sakinc, Türkan; Kline, Kimberly; Nielsen, Hailyn V; Hultgren, Scott; Gatermann, Sören G

    2013-12-01

    Staphylococcus saprophyticus is the only species of Staphylococcus that is typically uropathogenic and possesses a gene coding for a D-serine-deaminase (DsdA). As D-serine is prevalent in urine and toxic or bacteriostatic to many bacteria, it is not surprising that the D-serine-deaminase gene is found in the genome of uropathogens. It has been suggested that D-serine-deaminase or the ability to respond to or to metabolize D-serine is important for virulence. For uropathogenic Escherichia coli (UPEC), a high intracellular D-serine concentration affects expression of virulence factors. S. saprophyticus is able to grow in the presence of high D-serine concentrations; however, its D-serine metabolism has not been described. The activity of the D-serine-deaminase was verified by analyzing the formation of pyruvate from D-serine in different strains with and without D-serine-deaminase. Cocultivation experiments were performed to show that D-serine-deaminase confers a growth advantage to S. saprophyticus in the presence of D-serine. Furthermore, in vivo coinfection experiments showed a disadvantage for the ΔdsdA mutant during urinary tract infection. Expression analysis of known virulence factors by reverse transcription-quantitative PCR (RT-qPCR) showed that the surface-associated lipase Ssp is upregulated in the presence of D-serine. In addition, we show that S. saprophyticus is able to use D-serine as the sole carbon source, but interestingly, D-serine had a negative effect on growth when glucose was also present. Taken together, D-serine metabolism is associated with virulence in S. saprophyticus, as at least one known virulence factor is upregulated in the presence of D-serine and a ΔdsdA mutant was attenuated in virulence murine model of urinary tract infection.

  6. Significance of the d-Serine-Deaminase and d-Serine Metabolism of Staphylococcus saprophyticus for Virulence

    PubMed Central

    Sakinc, Türkan; Kline, Kimberly; Nielsen, Hailyn V.; Hultgren, Scott; Gatermann, Sören G.

    2013-01-01

    Staphylococcus saprophyticus is the only species of Staphylococcus that is typically uropathogenic and possesses a gene coding for a d-serine-deaminase (DsdA). As d-serine is prevalent in urine and toxic or bacteriostatic to many bacteria, it is not surprising that the d-serine-deaminase gene is found in the genome of uropathogens. It has been suggested that d-serine-deaminase or the ability to respond to or to metabolize d-serine is important for virulence. For uropathogenic Escherichia coli (UPEC), a high intracellular d-serine concentration affects expression of virulence factors. S. saprophyticus is able to grow in the presence of high d-serine concentrations; however, its d-serine metabolism has not been described. The activity of the d-serine-deaminase was verified by analyzing the formation of pyruvate from d-serine in different strains with and without d-serine-deaminase. Cocultivation experiments were performed to show that d-serine-deaminase confers a growth advantage to S. saprophyticus in the presence of d-serine. Furthermore, in vivo coinfection experiments showed a disadvantage for the ΔdsdA mutant during urinary tract infection. Expression analysis of known virulence factors by reverse transcription-quantitative PCR (RT-qPCR) showed that the surface-associated lipase Ssp is upregulated in the presence of d-serine. In addition, we show that S. saprophyticus is able to use d-serine as the sole carbon source, but interestingly, d-serine had a negative effect on growth when glucose was also present. Taken together, d-serine metabolism is associated with virulence in S. saprophyticus, as at least one known virulence factor is upregulated in the presence of d-serine and a ΔdsdA mutant was attenuated in virulence murine model of urinary tract infection. PMID:24082071

  7. Developmental forms of human skeletal-muscle AMP deaminase. The kinetic and regulatory properties of the enzyme.

    PubMed Central

    Kaletha, K; Nowak, G

    1988-01-01

    AMP deaminase isoforms from human skeletal muscle can be separated chromatographically [Kaletha, Spychała & Nowak (1987) Experientia 43, 440-443]. In adult tissue nearly all the AMP deaminase activity was eluted from phosphocellulose with 0.75 M-KCl ('adult' isoform), and the remaining activity could be eluted with 2.0 M-KCl. Conversely, most of the AMP deaminase activity from 11-week-old fetal tissue was eluted from phosphocellulose with 2.0 M-KCl ('fetal' isoform). In the present paper the kinetic and regulatory properties of AMP deaminase extracted from 11- and 16-week-old fetal skeletal muscle are reported. The two isoforms from 11-week-old human fetus differed distinctly in these properties. The 'fetal' isoform had about 5-fold higher half-saturation constant (S0.5) value than the 'adult' form. It was also more sensitive to the influence of some important regulatory ligands (ADP, ATP and Pi), and exhibited a different pH/activity profile. The 'adult' isoform of AMP deaminase from fetal muscle and the enzyme from mature muscle possessed similar kinetic and regulatory properties. This isoform seems not to be subject to any major modifications during further ontogenesis. This is not true, however, for the 'fetal' isoform. In the muscle of 16-week-old human fetus, the 'fetal' isoform showed a peculiar, biphasic, type of substrate-saturation kinetics. This phenomenon may reflect appearance of the next, developmentally programmed, isoform of human skeletal-muscle AMP deaminase. PMID:3342010

  8. Potentially therapeutic levels of anti-sickling globin gene expression following lentivirus-mediated gene transfer in sickle cell disease bone marrow CD34+ cells.

    PubMed

    Urbinati, Fabrizia; Hargrove, Phillip W; Geiger, Sabine; Romero, Zulema; Wherley, Jennifer; Kaufman, Michael L; Hollis, Roger P; Chambers, Christopher B; Persons, Derek A; Kohn, Donald B; Wilber, Andrew

    2015-05-01

    Sickle cell disease (SCD) can be cured by allogeneic hematopoietic stem cell transplant. However, this is only possible when a matched donor is available, making the development of gene therapy using autologous hematopoietic stem cells a highly desirable alternative. We used a culture model of human erythropoiesis to directly compare two insulated, self-inactivating, and erythroid-specific lentiviral vectors, encoding for γ-globin (V5m3-400) or a modified β-globin (βAS3-FB) for production of antisickling hemoglobin (Hb) and correction of red cell deformability after deoxygenation. Bone marrow CD34+ cells from three SCD patients were transduced using V5m3-400 or βAS3-FB and compared with mock-transduced SCD or healthy donor CD34+ cells. Lentiviral transduction did not impair cell growth or differentiation, as gauged by proliferation and acquisition of erythroid markers. Vector copy number averaged approximately one copy per cell, and corrective globin mRNA levels were increased more than sevenfold over mock-transduced controls. Erythroblasts derived from healthy donor and mock-transduced SCD cells produced a low level of fetal Hb that was increased to 23.6 ± 4.1% per vector copy for cells transduced with V5m3-400. Equivalent levels of modified normal adult Hb of 17.6 ± 3.8% per vector copy were detected for SCD cells transduced with βAS3-FB. These levels of antisickling Hb production were sufficient to reduce sickling of terminal-stage red blood cells upon deoxygenation. We concluded that the achieved levels of fetal Hb and modified normal adult Hb would likely prove therapeutic to SCD patients who lack matched donors.

  9. Lymphatic endothelial cells induce tolerance via PD-L1 and lack of costimulation leading to high-level PD-1 expression on CD8 T cells

    PubMed Central

    Tewalt, Eric F.; Cohen, Jarish N.; Rouhani, Sherin J.; Guidi, Cynthia J.; Qiao, Hui; Fahl, Shawn P.; Conaway, Mark R.; Bender, Timothy P.; Tung, Kenneth S.; Vella, Anthony T.; Adler, Adam J.; Chen, Lieping

    2012-01-01

    Lymphatic endothelial cells (LECs) induce peripheral tolerance by direct presentation to CD8 T cells (TCD8). We demonstrate that LECs mediate deletion only via programmed cell death-1 (PD-1) ligand 1, despite expressing ligands for the CD160, B- and T-lymphocyte attenuator, and lymphocyte activation gene-3 inhibitory pathways. LECs induce activation and proliferation of TCD8, but lack of costimulation through 4-1BB leads to rapid high-level expression of PD-1, which in turn inhibits up-regulation of the high-affinity IL-2 receptor that is necessary for TCD8 survival. Rescue of tyrosinase-specific TCD8 by interference with PD-1 or provision of costimulation results in autoimmune vitiligo, demonstrating that LECs are significant, albeit suboptimal, antigen-presenting cells. Because LECs express numerous peripheral tissue antigens, lack of costimulation coupled to rapid high-level up-regulation of inhibitory receptors may be generally important in systemic peripheral tolerance. PMID:22993390

  10. Reduced activity of CD13/aminopeptidase N (APN) in aggressive meningiomas is associated with increased levels of SPARC.

    PubMed

    Mawrin, Christian; Wolke, Carmen; Haase, Daniela; Krüger, Sabine; Firsching, Raimund; Keilhoff, Gerburg; Paulus, Werner; Gutmann, David H; Lal, Anita; Lendeckel, Uwe

    2010-01-01

    Meningiomas are the second most common brain tumors in adults, and meningiomas exhibit a tendency to invade adjacent structures. Compared with high-grade gliomas, little is known about the molecular changes that potentially underlie the invasive behavior of meningiomas. In this study, we examined the expression and function of the membrane alanyl-aminopeptidase [mAAP, aminopeptidase N (APN), CD13, EC3.4.11.2] zinc-dependent ectopeptidase in meningiomas and meningioma cell lines, based on its prior association with tumor invasion in colorectal and renal carcinomas. We found a significant reduction of APNmRNA and protein expression, as well as enzymatic activity, in high-grade meningiomas. While meningioma tumor cell proliferation was not affected by either pharmacologic APN inhibition or siRNA-mediated APN silencing, APN pharmacologic and siRNA knockdown significantly reduced meningioma cell invasion in vitro. Next, we employed pathway-specific cDNA microarray analyses to identify extracellular matrix and adhesion molecules regulated by APN, and found that APN-siRNA knockdown substantially increased the expression of secreted protein, acidic and rich in cysteine (SPARC)/osteonectin. Finally, we demonstrated that SPARC, which has been previously associated with meningioma invasiveness, was increased in aggressive meningiomas. Collectively, these results suggest that APN expression and enzymatic function is reduced in aggressive meningiomas, and that alterations in the balance between APN and SPARC might favor meningioma invasion.

  11. Relatively Low Level of Antigen-specific Monocytes Detected in Blood from Untreated Tuberculosis Patients Using CD4+ T-cell Receptor Tetramers

    PubMed Central

    Fang, Yimin; Wang, Juan; Li, Yan; Wang, Nan; Zhang, Jianbo; Gao, Ming; Huang, Lirong; Yang, Fangfang; Wang, Cong; Lin, Shuxian; Yao, Yanan; Ren, Liangliang; Chen, Yi; Du, Xuanjing; Xie, Dan; Wu, Rongshun; Zhang, Kouxing; Jiang, Lifang; Yu, Xinbing; Lai, Xiaomin

    2012-01-01

    The in vivo kinetics of antigen-presenting cells (APCs) in patients with advanced and convalescent tuberculosis (TB) is not well characterized. In order to target Mycobacterium tuberculosis (MTB) peptides- and HLA-DR-holding monocytes and macrophages, 2 MTB peptide-specific CD4+ T-cell receptor (TCR) tetramers eu and hu were successfully constructed. Peripheral blood (PBL) samples from inpatients with advanced pulmonary TB (PTB) were analyzed using flow cytometry, and the percentages of tetramer-bound CD14+ monocytes ranged from 0.26–1.44% and 0.21–0.95%, respectively; significantly higher than those measured in PBL samples obtained from non-TB patients, healthy donors, and umbilical cords. These tetramers were also able to specifically detect macrophages in situ via immunofluorescent staining. The results of the continuous time-point tracking of the tetramer-positive rates in PBL samples from active PTB outpatients undergoing treatment show that the median percentages were at first low before treatment, increased to their highest levels during the first month, and then began to decrease during the second month until finally reaching and maintaining a relatively low level after 3–6 months. These results suggest that there is a relatively low level of MTB-specific monocytes in advanced and untreated patients. Further experiments show that MTB induces apoptosis in CD14+ cells, and the percentage of apoptotic monocytes dramatically decreases after treatment. Therefore, the relatively low level of MTB-specific monocytes is probably related to the apoptosis or necrosis of APCs due to live bacteria and their growth. The bactericidal effects of anti-TB drugs, as well as other unknown factors, would induce a peak value during the first month of treatment, and a relatively low level would be subsequently reached and maintained until all of the involved factors reached equilibrium. These tetramers have diagnostic potential and can provide valuable insights into the

  12. Cloning of the cDNA encoding adenosine 5'-monophosphate deaminase 1 and its mRNA expression in Japanese flounder Paralichthys olivaceus

    NASA Astrophysics Data System (ADS)

    Jiang, Keyong; Sun, Shujuan; Liu, Mei; Wang, Baojie; Meng, Xiaolin; Wang, Lei

    2013-01-01

    AMP deaminase catalyzes the conversion of AMP into IMP and ammonia. In the present study, a full-length cDNA of AMPD1 from skeletal muscle of Japanese flounder Paralichthys olivaceus was cloned and characterized. The 2 526 bp cDNA contains a 5'-UTR of 78 bp, a 3'-UTR of 237 bp and an open reading frame (ORF) of 2 211 bp, which encodes a protein of 736 amino acids. The predicted protein contains a highly conserved AMP deaminase motif (SLSTDDP) and an ATP-binding site sequence (EPLMEEYAIAAQVFK). Phylogenetic analysis showed that the AMPD1 and AMPD3 genes originate from the same branch, but are evolutionarily distant from the AMPD2 gene. RT-PCR showed that the flounder AMPD1 gene was expressed only in skeletal muscle. QRT-PCR analysis revealed a statistically significant 2.54 fold higher level of AMPD1 mRNA in adult muscle (750±40 g) compared with juvenile muscle (7.5±2 g) ( P<0.05). HPLC analysis showed that the IMP content in adult muscle (3.35±0.21 mg/g) was also statistically significantly higher than in juvenile muscle (1.08±0.04 mg/g) ( P<0.05). There is a direct relationship between the AMPD1 gene expression level and IMP content in the skeletal muscle of juvenile and adult flounders. These results may provide useful information for quality improvement and molecular breeding of aquatic animals.

  13. Comparative effectiveness of ACC-deaminase and/or nitrogen-fixing rhizobacteria in promotion of maize (Zea mays L.) growth under lead pollution.

    PubMed

    Hassan, Waseem; Bano, Rizwana; Bashir, Farhat; David, Julie

    2014-09-01

    Lead (Pb) pollution is appearing as an alarming threat nowadays. Excessive Pb concentrations in agricultural soils result in minimizing the soil fertility and health which affects the plant growth and leads to decrease in crop production. Plant growth promoting rhizobacteria (PGPR) are beneficial bacteria which can protect the plants against many abiotic stresses, and enhance the growth. The study aimed to identify important rhizobacterial strains by using the 1-aminocyclopropane-1-carboxylate (ACC) enrichment technique and examine their inoculation effects in the growth promotion of maize, under Pb pollution. A pot experiment was conducted and six rhizobacterial isolates were used. Pb was added to 2 kg soil in each pot (with 4 seeds/pot) using Pb(NO3)2 at the rate of 0, 100, 200, 300, and 400 mg kg(-1) Pb with three replications in completely randomized design. Rhizobacterial isolates performed significantly better under all Pb levels, i.e., 100 to 400 Pb mg kg(-1) soil, compared to control. Comparing the efficacy of the rhizobacterial isolates under different Pb levels, rhizobacterial isolates having both ACC-deaminase and nitrogen-fixing activities (AN8 and AN12) showed highest increase in terms of the physical, chemical and enzymatic growth parameters of maize, followed by the rhizobacterial isolates having ACC-deaminase activity only (ACC5 and ACC8), and then the nitrogen-fixing rhizobia (Azotobacter and RN5). However, the AN8 isolate showed maximum efficiency, and highest shoot and root length (14.2 and 6.1 cm), seedling fresh and dry weights (1.91 and 0.14 g), chlorophyll a, b, and carotenoids (24.1, 30.2 and 77.7 μg/l), protein (0.82 mg/g), proline (3.42 μmol/g), glutathione S-transferase, peroxidase and catalase (12.3, 4.2 and 7.2 units/mg protein), while the lowest Pb uptake in the shoot and root (0.83 and 0.48 mg/kg) were observed under this rhizobial isolate at the highest Pb level (i.e., 400 Pb mg kg(-1) soil). The results revealed that PGPR

  14. Comparative effectiveness of ACC-deaminase and/or nitrogen-fixing rhizobacteria in promotion of maize (Zea mays L.) growth under lead pollution.

    PubMed

    Hassan, Waseem; Bano, Rizwana; Bashir, Farhat; David, Julie

    2014-09-01

    Lead (Pb) pollution is appearing as an alarming threat nowadays. Excessive Pb concentrations in agricultural soils result in minimizing the soil fertility and health which affects the plant growth and leads to decrease in crop production. Plant growth promoting rhizobacteria (PGPR) are beneficial bacteria which can protect the plants against many abiotic stresses, and enhance the growth. The study aimed to identify important rhizobacterial strains by using the 1-aminocyclopropane-1-carboxylate (ACC) enrichment technique and examine their inoculation effects in the growth promotion of maize, under Pb pollution. A pot experiment was conducted and six rhizobacterial isolates were used. Pb was added to 2 kg soil in each pot (with 4 seeds/pot) using Pb(NO3)2 at the rate of 0, 100, 200, 300, and 400 mg kg(-1) Pb with three replications in completely randomized design. Rhizobacterial isolates performed significantly better under all Pb levels, i.e., 100 to 400 Pb mg kg(-1) soil, compared to control. Comparing the efficacy of the rhizobacterial isolates under different Pb levels, rhizobacterial isolates having both ACC-deaminase and nitrogen-fixing activities (AN8 and AN12) showed highest increase in terms of the physical, chemical and enzymatic growth parameters of maize, followed by the rhizobacterial isolates having ACC-deaminase activity only (ACC5 and ACC8), and then the nitrogen-fixing rhizobia (Azotobacter and RN5). However, the AN8 isolate showed maximum efficiency, and highest shoot and root length (14.2 and 6.1 cm), seedling fresh and dry weights (1.91 and 0.14 g), chlorophyll a, b, and carotenoids (24.1, 30.2 and 77.7 μg/l), protein (0.82 mg/g), proline (3.42 μmol/g), glutathione S-transferase, peroxidase and catalase (12.3, 4.2 and 7.2 units/mg protein), while the lowest Pb uptake in the shoot and root (0.83 and 0.48 mg/kg) were observed under this rhizobial isolate at the highest Pb level (i.e., 400 Pb mg kg(-1) soil). The results revealed that PGPR

  15. HIV-1 RNA levels in an African population-based cohort and their relation to CD4 lymphocyte counts and World Health Organization clinical staging.

    PubMed

    Morgan, D; Rutebemberwa, A; Malamba, S; Ross, A; Whitworth, J; Kaleebu, P; Gotch, F

    1999-10-01

    Apart from a small number of reports from people who are based in hospitals, data on viral load in HIV-infected people in sub-Saharan Africa, where most infections occur, are lacking. We report serum HIV-1 RNA levels in a population-based cohort in rural Uganda using the nucleic acid sequence-based amplification procedure (NASBA) test kit and describe their relation to CD4 counts and World Health Organization (WHO) clinical staging. The median (interquartile range [IQR]) viral loads were 87,000 copies/ml (37,500-295,000 copies/ml) in 40 prevalent cases infected for >6 years, and 31,000 copies/ml (7800-174,000 copies/ml) in 65 incident cases with seroconversion dates within the previous 6 years. Although we found a correlation between viral load and absolute CD4 count (p < .0001), there was no evidence for an association with CD4 decline (p = .1). Overall, there was a significant trend of increasing viral load with worsening clinical stage from a median viral load of 15,000 for those in WHO stage 1 (asymptomatic) to 150,600 copies/ml for those in stage 4 (AIDS; p < .001). However, the association was seen only in incident cases. Thus, we found that the NASBA test on serum was a useful indicator of disease stage especially in persons known to be infected for <6 years. Such baseline data are important for vaccine research, and if antiretroviral drugs become available to more than a few people in Africa, it will be important that accurate viral load estimations are available at least in a proportion of people to monitor the effectiveness of treatment, and measure the compliance and emerging resistance to these drugs. PMID:10843531

  16. Pleural effusion: Role of pleural fluid cytology, adenosine deaminase level, and pleural biopsy in diagnosis

    PubMed Central

    Biswas, Biswajit; Sharma, Sudershan Kumar; Negi, Rameshwar Singh; Gupta, Neelam; Jaswal, Virender Mohan Singh; Niranjan, Narsimhalu

    2016-01-01

    Objective: The present study is designed to evaluate the role of pleural fluid analysis in diagnosing pleural diseases and to study the advantages and disadvantages of thoracocentasis and pleural biopsy. Materials and Methods: We prospectively included 66 consecutive indoor patients over a duration of 1 year. Pleural fluid was collected and cytological smears were made from the fluid. Plural biopsy was done in the same patient by Cope needle. Adequate pleural biopsy tissue yielding specific diagnosis was obtained in 47 (71.2%) cases. Results: Tuberculosis was the commonest nonneoplastic lesion followed by chronic nonspecific pleuritis comprising 60% and 33.3% of the nonneoplastic cases respectively and tuberculosis was predominantly diagnosed in the younger age group. Majority (70.8%) of malignancy cases were in the age group of >50-70. Adenocarcinoma was found to be the commonest (66.7%) malignant neoplasm in the pleurae followed by small-cell carcinoma (20.8%). Conclusion: Pleural biopsy is a useful and minimally invasive procedure. It is more sensitive and specific than pleural fluid smears.

  17. Ab Initio ONIOM-Molecular Dynamics (MD) Study on the Deamination Reaction by Cytidine Deaminase

    SciTech Connect

    Matsubara, Toshiaki; Dupuis, Michel; Aida, Misako

    2007-08-23

    We applied the ONIOM-molecular dynamics (MD) method to the hydrolytic deamination of cytidine by cytidine deaminase, which is an essential step of the activation process of the anticancer drug inside the human body. The direct MD simulations were performed for the realistic model of cytidine deaminase calculating the energy and its gradient by the ab initio ONIOM method on the fly. The ONIOM-MD calculations including the thermal motion show that the neighboring amino acid residue is an important factor of the environmental effects and significantly affects not only the geometry and energy of the substrate trapped in the pocket of the active site but also the elementary step of the catalytic reaction. We successfully simulate the second half of the catalytic cycle, which has been considered to involve the rate-determining step, and reveal that the rate-determing step is the release of the NH3 molecule. TM and MA were supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan. MD was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, and by the Office of Biological and Environmental Research of the U.S. Department of Energy DOE. Battelle operates Pacific Northwest National Laboratory for DOE.

  18. Heat shock protein 70 (Hsp70)-stimulated deoxycytidine deaminases from a human lymphoma cell but not the activation-induced cytidine deaminase (AID) from Ramos 6.4 human Burkitt's lymphoma cells.

    PubMed

    Bases, Robert

    2011-01-01

    Deoxycytidine deaminase enzyme activity was reduced in lysates of human leukemic THP1 cells 24 h after transfection with siRNA designed to inhibit cell synthesis of heat shock protein 70 (Hsp70)1a and Hsp701b. The cytidine deaminase enzyme activity from the cell lysates was purified from an affinity column which contained bound single-stranded oligodeoxycytidylic acid. Deficient enzyme activity in certain elution fractions from the siRNA-transfected cells was restored by including recombinant HSP 70 in the assays. Enzyme activity in some other fractions was increased after siRNA transfection. Activation-induced cytidine deaminase (AID) is a central factor in the immune response. A more specific assay for AID was used to study the influence of Hsp70 on AID activity. Unlike Hsp70's ability to stimulate certain enzymes of DNA base excision repair and other cytidine deaminases, it had little effect on AID activity in vitro, or was weakly inhibitory.

  19. T. vaginalis Infection Is Associated with Increased IL-8 and TNFr1 Levels but with the Absence of CD38 and HLADR Activation in the Cervix of ESN

    PubMed Central

    Brady, Kirsten E.; Plants, Jill; Landay, Alan L.; Ghassemi, Mahmood; Golub, Elizabeth T.; Spear, Greg T.

    2015-01-01

    Introduction Trichomonas vaginalis infection is associated with an increased risk of HIV infection in exposed-seronegative women (ESN) despite their unique immune quiescent profile. It is important to understand possible mechanisms, such as recruitment of activated T cells, by which T. vaginalis could facilitate HIV infection in this population. Methods We conducted a cross-sectional study exploring the relationships between T. vaginalis infection, inflammatory markers and T cell activation in the cervix of ESN. During scheduled study visits, participants completed a behavioral questionnaire and physical exam, including sexually transmitted infection (STI) screening and collection of endocervical sponge and cytobrush specimens. T cell and monocyte phenotypes were measured in cervical cytobrush specimens using multi-parameter flow cytometry. Cervical sponge specimens were used to measure cytokines (IL-6, IL-8,IL-10, IP-10, RANTES) using Luminex immunoassays and the immune activation marker soluble TNF receptor 1 using ELISA. Results Specimens of 65 women were tested. Twenty-one of these women were infected with T. vaginalis. T. vaginalis infection was associated with significantly increased concentrations of IL-8 (1275pg/ml vs. 566pg/ml, p=.02) and sTNFr1 (430 pg/ml vs. 264 pg/ml, p=.005). However, T. vaginalis infection was not associated with increased percent expression of CCR5+ T cells nor increased CD38 and HLADR activation compared to uninfected women. It was also not associated with increased expression of CCR5+ monocytes. Conclusions Among ESN T. vaginalis infection is associated with increased levels of genital pro-inflammatory/immune activation markers IL-8 and TNFr1, but was not associated with an increased percentage of activated endocervical T cells along the CD38 and HLADR pathways. Thus, while T.vaginalis infection may result in some reversal of the immune quiescent profile of ESN, enhanced recruitment of activated CD38 and HLADR expressing CD4+ cells

  20. Infection with Marek’s disease virus induces high levels of CD8a/a cells in chickens resistant to Marek’s disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The CD8a/a cells have non-traditional roles. CD8a/a cells (T'' cells) usually recognize monomorphic antigen like bacterial lipids presented by non-traditional class I glycoproteins such as BF1 (minor) or Rfp-Y. CD8a/a cells have also been implicated in natural killing with approximately 80% of NK ...

  1. Pyrimidine Pool Disequilibrium Induced by a Cytidine Deaminase Deficiency Inhibits PARP-1 Activity, Leading to the Under Replication of DNA

    PubMed Central

    Gemble, Simon; Ahuja, Akshay; Buhagiar-Labarchède, Géraldine; Onclercq-Delic, Rosine; Dairou, Julien; Biard, Denis S. F.; Lambert, Sarah; Lopes, Massimo; Amor-Guéret, Mounira

    2015-01-01

    Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA) deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation: the intracellular accumulation of dCTP inhibits PARP-1 activity. CDA deficiency results in incomplete DNA replication when cells enter mitosis, leading to the formation of ultrafine anaphase bridges between sister-chromatids at “difficult-to-replicate” sites such as centromeres and fragile sites. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. The stimulation of PARP-1 activity in CDA-deficient cells restores replication and, thus, chromosome segregation. Moreover, increasing intracellular dCTP levels generates under-replication-induced sister-chromatid bridges as efficiently as PARP-1 knockdown. These results have direct implications for Bloom syndrome (BS), a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3’-5’ DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of ultrafine anaphase bridges due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our study describes previously unknown pathological consequences of the distortion of dNTP pools and reveals an unexpected role for PARP-1 in preventing DNA under-replication and chromosome segregation defects. PMID:26181065

  2. Effects of co-infection with hepatitis C virus and GB virus C on CD4 cell count and HIV-RNA level among HIV-infected patients treated with highly active antiretroviral therapy.

    PubMed

    Voirin, Nicolas; Trépo, Christian; Estève, Jacques; Chevallier, Philippe; Ritter, Jacques; Fabry, Jacques; Vanhems, Philippe

    2002-07-26

    The effects of co-infection with hepatitis C virus (HCV) and GB virus C (GBV-C) on CD4 cell counts and plasma HIV-RNA levels has been investigated in HIV-infected patients treated with highly active antiretroviral therapy (HAART). Patients co-infected with HCV and GBV-C experienced a CD4 cell increase during 4 years of HAART, whereas the increase stopped after 2 years in the other groups.

  3. Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase

    SciTech Connect

    Scott, A.I.; Roessner, C.A.; Stolowich, N.J.; Karuso, P.; Williams, H.J.; Grant, S.K.; Gonzalez, M.D.; Hoshino, T. )

    1988-10-18

    The active site of porphobilinogen (PBG){sup 1} deaminase from Escherichia coli has been found to contain an unusual dipyrromethane derived from four molecules of 5-aminolevulinic acid (ALA) covalently linked to Cys-242, one of the two cysteine residues conserved in E. coli and human deaminase. By use of a hemA{sup {minus}} strain of E. coli the enzyme was enriched from (5-{sup 13}C)ALA and examined by {sup 1}H-detected multiple quantum coherence spectroscopy, which revealed all of the salient features of a dipyrromethane composed of two PBG units linked heat to tail and terminating in a CH{sub 2}-S bond to a cysteine residue. Site-specific mutagenesis of Cys-99 and Cys-242, respectively, has shown that substitution of Ser for Cys-99 does not affect the enzymatic activity, whereas substitution of Ser for Cys-242 removes essentially all of the catalytic activity as measured by the conversion of the substrate PBG to uro'gen I. The NMR spectrum of the covalent complex of deaminase with the suicide inhibitor 2-bromo-(2,11-{sup 13}C{sub 2})PBG reveals that the aminomethyl terminus of the inhibitor reacts with the enzyme's cofactor at the {alpha}-free pyrrole. NMR spectroscopy of the ES{sub 2} complex confirmed a PBG-derived head-to-tail dipyrromethane attached to the {alpha}-free pyrrole position of the enzyme. A mechanistic rationale for deaminase is presented.

  4. 1-Aminocyclopropane-1-Carboxylate Deaminase from Pseudomonas stutzeri A1501 Facilitates the Growth of Rice in the Presence of Salt or Heavy Metals.

    PubMed

    Han, Yunlei; Wang, Rui; Yang, Zhirong; Zhan, Yuhua; Ma, Yao; Ping, Shuzhen; Zhang, Liwen; Lin, Min; Yan, Yongliang

    2015-07-01

    1-Aminocyclopropane-1-carboxylate (ACC) deaminase, which is encoded by some bacteria, can reduce the amount of ethylene, a root elongation inhibitor, and stimulate the growth of plants under various environmental stresses. The presence of ACC deaminase activity and the regulation of ACC in several rhizospheric bacteria have been reported. The nitrogen-fixing Pseudomonas stutzeri A1501 is capable of endophytic association with rice plants and promotes the growth of rice. However, the functional identification of ACC deaminase has not been performed. In this study, the proposed effect of ACC deaminase in P. stutzeri A1501 was investigated. Genome mining showed that P. stutzeri A1501 carries a single gene encoding ACC deaminase, designated acdS. The acdS mutant was devoid of ACC deaminase activity and was less resistant to NaCl and NiCl2 compared with the wild-type. Furthermore, inactivation of acdS greatly impaired its nitrogenase activity under salt stress conditions. It was also observed that mutation of the acdS gene led to loss of the ability to promote the growth of rice under salt or heavy metal stress. Taken together, this study illustrates the essential role of ACC deaminase, not only in enhancing the salt or heavy metal tolerance of bacteria but also in improving the growth of plants, and provides a theoretical basis for studying the interaction between plant growth-promoting rhizobacteria and plants. PMID:25674802

  5. 1-Aminocyclopropane-1-Carboxylate Deaminase from Pseudomonas stutzeri A1501 Facilitates the Growth of Rice in the Presence of Salt or Heavy Metals.

    PubMed

    Han, Yunlei; Wang, Rui; Yang, Zhirong; Zhan, Yuhua; Ma, Yao; Ping, Shuzhen; Zhang, Liwen; Lin, Min; Yan, Yongliang

    2015-07-01

    1-Aminocyclopropane-1-carboxylate (ACC) deaminase, which is encoded by some bacteria, can reduce the amount of ethylene, a root elongation inhibitor, and stimulate the growth of plants under various environmental stresses. The presence of ACC deaminase activity and the regulation of ACC in several rhizospheric bacteria have been reported. The nitrogen-fixing Pseudomonas stutzeri A1501 is capable of endophytic association with rice plants and promotes the growth of rice. However, the functional identification of ACC deaminase has not been performed. In this study, the proposed effect of ACC deaminase in P. stutzeri A1501 was investigated. Genome mining showed that P. stutzeri A1501 carries a single gene encoding ACC deaminase, designated acdS. The acdS mutant was devoid of ACC deaminase activity and was less resistant to NaCl and NiCl2 compared with the wild-type. Furthermore, inactivation of acdS greatly impaired its nitrogenase activity under salt stress conditions. It was also observed that mutation of the acdS gene led to loss of the ability to promote the growth of rice under salt or heavy metal stress. Taken together, this study illustrates the essential role of ACC deaminase, not only in enhancing the salt or heavy metal tolerance of bacteria but also in improving the growth of plants, and provides a theoretical basis for studying the interaction between plant growth-promoting rhizobacteria and plants.

  6. CD56brightCD16- NK Cells Produce Adenosine through a CD38-Mediated Pathway and Act as Regulatory Cells Inhibiting Autologous CD4+ T Cell Proliferation.

    PubMed

    Morandi, Fabio; Horenstein, Alberto L; Chillemi, Antonella; Quarona, Valeria; Chiesa, Sabrina; Imperatori, Andrea; Zanellato, Silvia; Mortara, Lorenzo; Gattorno, Marco; Pistoia, Vito; Malavasi, Fabio

    2015-08-01

    Recent studies suggested that human CD56(bright)CD16(-) NK cells may play a role in the regulation of the immune response. Since the mechanism(s) involved have not yet been elucidated, in the present study we have investigated the role of nucleotide-metabolizing enzymes that regulate the extracellular balance of nucleotides/nucleosides and produce the immunosuppressive molecule adenosine (ADO). Peripheral blood CD56(dim)CD16(+) and CD56(bright)CD16(-) NK cells expressed similar levels of CD38. CD39, CD73, and CD157 expression was higher in CD56(bright)CD16(-) than in CD56(dim)CD16(+) NK cells. CD57 was mostly expressed by CD56(dim)CD16(+) NK cells. CD203a/PC-1 expression was restricted to CD56(bright)CD16(-) NK cells. CD56(bright)CD16(-) NK cells produce ADO and inhibit autologous CD4(+) T cell proliferation. Such inhibition was 1) reverted pretreating CD56(bright)CD16(-) NK cells with a CD38 inhibitor and 2) increased pretreating CD56(bright)CD16(-) NK cells with a nucleoside transporter inhibitor, which increase extracellular ADO concentration. CD56(bright)CD16(-) NK cells isolated from the synovial fluid of juvenile idiopathic arthritis patients failed to inhibit autologous CD4(+) T cell proliferation. Such functional impairment could be related to 1) the observed reduced CD38/CD73 expression, 2) a peculiar ADO production kinetics, and 3) a different expression of ADO receptors. In contrast, CD56(bright)CD16(-) NK cells isolated from inflammatory pleural effusions display a potent regulatory activity. In conclusion, CD56(bright)CD16(-) NK cells act as "regulatory cells" through ADO produced by an ectoenzymes network, with a pivotal role of CD38. This function may be relevant for the modulation of the immune response in physiological and pathological conditions, and it could be impaired during autoimmune/inflammatory diseases. PMID:26091716

  7. Molecular association of CD98, CD29, and CD147 critically mediates monocytic U937 cell adhesion.

    PubMed

    Kim, Mi-Yeon; Cho, Jae Youl

    2016-09-01

    Adhesion events of monocytes represent an important step in inflammatory responses induced by chemokines. The β1-integrin CD29 is a major adhesion molecule regulating leukocyte migration and extravasation. Although several adhesion molecules have been known as regulators of CD29, the molecular interactions between CD29 and its regulatory adhesion molecules (such as CD98 and CD147) have not been fully elucidated. Therefore, in this study, we examined whether these molecules are functionally, biochemically, and cell-biologically associated using monocytic U937 cells treated with aggregation-stimulating and blocking antibodies, as well as enzyme inhibitors. The surface levels of CD29, CD98, and CD147 (but not CD43, CD44, and CD82) were increased. The activation of CD29, CD98, and CD147 by ligation of them with aggregation-activating antibodies triggered the induction of cell-cell adhesion, and sensitivity to various enzyme inhibitors and aggregation-blocking antibodies was similar for CD29-, CD98-, and CD147-induced U937 cell aggregation. Molecular association between these molecules and the actin cytoskeleton was confirmed by confocal microscopy and immunoprecipitation. These results strongly suggest that CD29 might be modulated by its biochemical and cellular regulators, including CD98 and CD147, via the actin cytoskeleton. PMID:27610038

  8. Molecular association of CD98, CD29, and CD147 critically mediates monocytic U937 cell adhesion

    PubMed Central

    Kim, Mi-Yeon

    2016-01-01

    Adhesion events of monocytes represent an important step in inflammatory responses induced by chemokines. The β1-integrin CD29 is a major adhesion molecule regulating leukocyte migration and extravasation. Although several adhesion molecules have been known as regulators of CD29, the molecular interactions between CD29 and its regulatory adhesion molecules (such as CD98 and CD147) have not been fully elucidated. Therefore, in this study, we examined whether these molecules are functionally, biochemically, and cell-biologically associated using monocytic U937 cells treated with aggregation-stimulating and blocking antibodies, as well as enzyme inhibitors. The surface levels of CD29, CD98, and CD147 (but not CD43, CD44, and CD82) were increased. The activation of CD29, CD98, and CD147 by ligation of them with aggregation-activating antibodies triggered the induction of cell-cell adhesion, and sensitivity to various enzyme inhibitors and aggregation-blocking antibodies was similar for CD29-, CD98-, and CD147-induced U937 cell aggregation. Molecular association between these molecules and the actin cytoskeleton was confirmed by confocal microscopy and immunoprecipitation. These results strongly suggest that CD29 might be modulated by its biochemical and cellular regulators, including CD98 and CD147, via the actin cytoskeleton. PMID:27610038

  9. Molecular association of CD98, CD29, and CD147 critically mediates monocytic U937 cell adhesion

    PubMed Central

    Kim, Mi-Yeon

    2016-01-01

    Adhesion events of monocytes represent an important step in inflammatory responses induced by chemokines. The β1-integrin CD29 is a major adhesion molecule regulating leukocyte migration and extravasation. Although several adhesion molecules have been known as regulators of CD29, the molecular interactions between CD29 and its regulatory adhesion molecules (such as CD98 and CD147) have not been fully elucidated. Therefore, in this study, we examined whether these molecules are functionally, biochemically, and cell-biologically associated using monocytic U937 cells treated with aggregation-stimulating and blocking antibodies, as well as enzyme inhibitors. The surface levels of CD29, CD98, and CD147 (but not CD43, CD44, and CD82) were increased. The activation of CD29, CD98, and CD147 by ligation of them with aggregation-activating antibodies triggered the induction of cell-cell adhesion, and sensitivity to various enzyme inhibitors and aggregation-blocking antibodies was similar for CD29-, CD98-, and CD147-induced U937 cell aggregation. Molecular association between these molecules and the actin cytoskeleton was confirmed by confocal microscopy and immunoprecipitation. These results strongly suggest that CD29 might be modulated by its biochemical and cellular regulators, including CD98 and CD147, via the actin cytoskeleton.

  10. Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human

    SciTech Connect

    Palmer, T.D.; Hock, R.A.; Osborne, W.R.A.; Miller, A.D.

    1987-02-01

    Skin fibroblasts might be considered suitable recipients for therapeutic genes to cure several human genetic diseases; however, these cells are resistant to gene transfer by most methods. The authors studied the ability of retroviral vectors to transfer genes into normal human diploid skin fibroblasts. Retroviruses carrying genes for neomycin or hygromycin B resistance conferred drug resistance to greater than 50% of the human fibroblasts after a single exposure to virus-containing medium. This represents at least a 500-fold increase in efficiency over other methods. Transfer was achieved in the absence of helper virus by using amphotropic retrovirus-packaging cells. A retrovirus vector containing a human adenosine deaminase (ADA) cDNA was constructed and used to infect ADA/sup -/ fibroblasts from a patient with ADA deficiency. The infected cells produced 12-fold more ADA enzyme than fibroblasts from normal individuals and were able to rapidly metabolize exogenous deoxyadenosine and adenosine, metabolites that accumulate in plasma in ADA-deficient patients and are responsible for the severe combined immunodeficiency in these patients. These experiments indicate the potential of retrovirus-mediated gene transfer into human fibroblasts for gene therapy.

  11. Transition state structure of E. coli tRNA-specific adenosine deaminase.

    PubMed

    Luo, Minkui; Schramm, Vern L

    2008-02-27

    Bacterial tRNA-specific adenosine deaminase (TadA) catalyzes the essential deamination of adenosine to inosine at the wobble position of tRNAs and is necessary to permit a single tRNA species to recognize multiple codons. The transition state structure of Escherichia coli TadA was characterized by kinetic isotope effects (KIEs) and quantum chemical calculations. A stem loop of E. coli tRNA(Arg2) was used as a minimized TadA substrate, and its adenylate editing site was isotopically labeled as [1'-(3)H], [5'-(3)H2], [1'-(14)C], [6-(13)C], [6-(15)N], [6-(13)C, 6-(15)N] and [1-(15)N]. The intrinsic KIEs of 1.014, 1.022, 0.994, 1.014 and 0.963 were obtained for [6-(13)C]-, [6-(15)N]-, [1-(15)N]-, [1'-(3)H]-, [5'-(3)H2]-labeled substrates, respectively. The suite of KIEs are consistent with a late SNAr transition state with a complete, pro-S-face hydroxyl attack and nearly complete N1 protonation. A significant N6-C6 dissociation at the transition state of TadA is indicated by the large [6-(15)N] KIE of 1.022 and corresponds to an N6-C6 distance of 2.0 A in the transition state structure. Another remarkable feature of the E. coli TadA transition state structure is the Glu70-mediated, partial proton transfer from the hydroxyl nucleophile to the N6 leaving group. KIEs correspond to H-O and H-N distances of 2.02 and 1.60 A, respectively. The large inverse [5'-(3)H] KIE of -3.7% and modest normal [1'-(3)H] KIE of 1.4% indicate that significant ribosyl 5'-reconfiguration and purine rotation occur on the path to the transition state. The late SNAr transition-state established here for E. coli TadA is similar to the late transition state reported for cytidine deaminase. It differs from the early SNAr transition states described recently for the adenosine deaminases from human, bovine, and Plasmodium falciparum sources. The ecTadA transition state structure reveals the detailed architecture for enzymatic catalysis. This approach should be readily transferable for transition

  12. Non-viral delivery of the porphobilinogen deaminase cDNA into a mouse model of acute intermittent porphyria.

    PubMed

    Johansson, Annika; Nowak, Grzegorz; Möller, Christer; Harper, Pauline

    2004-05-01

    Acute intermittent porphyria (AIP), an inborn error of metabolism, results from the deficient activity of the third enzyme in the heme biosynthetic pathway, porphobilinogen deaminase (PBGD). Clinical symptoms of this autosomal dominant hepatic porphyria include episodic acute attacks of abdominal pain, neuropathy, and psychiatric disturbances. Current therapy based on intravenous heme administration is palliative and there is no way to prevent the attacks. Thus, efforts are focused on methods to replace the deficient activity in the liver to prevent the acute attacks of this hepatic porphyria. Here we explore the efficiency of a non-viral gene delivery to obtain PBGD expression in the liver of AIP transgenic mice. Four vectors were evaluated: naked DNA and DNA complexed to liposomes, polyethylenimine (PEI), and PEI-galactose, using a luciferase construct as reporter gene. The vectors were administered intravenously or directly into the portal vein with transient blood flow blockage. After tail vein injection of the DNA complexes, the liposome vector had the highest luciferase expression in lung and less in liver. When injected into the portal vein, the naked DNA had considerably higher hepatic reporter gene expression; 100 microg of naked DNA had the highest hepatic luciferase expression 24h after portal vein injection. When these vectors were used to deliver the PBGD gene into the AIP mouse model no enhancement of the endogenous PBGD activity in liver was detectable, despite the presence of the PBGD-plasmids as verified by PCR. Thus, more efficient non-viral vectors are needed to express sufficient PBGD activity over the endogenous hepatic level (approximately 30% of normal) in this murine system.

  13. Opposing Activity Changes in AMP Deaminase and AMP-Activated Protein Kinase in the Hibernating Ground Squirrel

    PubMed Central

    Cicerchi, Christina; Garcia, Gabriela E.; Roncal-Jimenez, Carlos A.; Trostel, Jessica; Jain, Swati; Mant, Colin T.; Rivard, Christopher J.; Ishimoto, Takuji; Shimada, Michiko; Sanchez-Lozada, Laura Gabriela; Nakagawa, Takahiko; Jani, Alkesh; Stenvinkel, Peter; Martin, Sandra L.; Johnson, Richard J.

    2015-01-01

    Hibernating animals develop fatty liver when active in summertime and undergo a switch to a fat oxidation state in the winter. We hypothesized that this switch might be determined by AMP and the dominance of opposing effects: metabolism through AMP deaminase (AMPD2) (summer) and activation of AMP-activated protein kinase (AMPK) (winter). Liver samples were obtained from 13-lined ground squirrels at different times during the year, including summer and multiples stages of winter hibernation, and fat synthesis and β-fatty acid oxidation were evaluated. Changes in fat metabolism were correlated with changes in AMPD2 activity and intrahepatic uric acid (downstream product of AMPD2), as well as changes in AMPK and intrahepatic β-hydroxybutyrate (a marker of fat oxidation). Hepatic fat accumulation occurred during the summer with relatively increased enzymes associated with fat synthesis (FAS, ACL and ACC) and decreased enoyl CoA hydratase (ECH1) and carnitine palmitoyltransferase 1A (CPT1A), rate limiting enzymes of fat oxidation. In summer, AMPD2 activity and intrahepatic uric acid levels were high and hepatic AMPK activity was low. In contrast, the active phosphorylated form of AMPK and β-hydroxybutyrate both increased during winter hibernation. Therefore, changes in AMPD2 and AMPK activity were paralleled with changes in fat synthesis and fat oxidation rates during the summer-winter cycle. These data illuminate the opposing forces of metabolism of AMP by AMPD2 and its availability to activate AMPK as a switch that governs fat metabolism in the liver of hibernating ground squirrel. PMID:25856396

  14. Opposing activity changes in AMP deaminase and AMP-activated protein kinase in the hibernating ground squirrel.

    PubMed

    Lanaspa, Miguel A; Epperson, L Elaine; Li, Nanxing; Cicerchi, Christina; Garcia, Gabriela E; Roncal-Jimenez, Carlos A; Trostel, Jessica; Jain, Swati; Mant, Colin T; Rivard, Christopher J; Ishimoto, Takuji; Shimada, Michiko; Sanchez-Lozada, Laura Gabriela; Nakagawa, Takahiko; Jani, Alkesh; Stenvinkel, Peter; Martin, Sandra L; Johnson, Richard J

    2015-01-01

    Hibernating animals develop fatty liver when active in summertime and undergo a switch to a fat oxidation state in the winter. We hypothesized that this switch might be determined by AMP and the dominance of opposing effects: metabolism through AMP deaminase (AMPD2) (summer) and activation of AMP-activated protein kinase (AMPK) (winter). Liver samples were obtained from 13-lined ground squirrels at different times during the year, including summer and multiples stages of winter hibernation, and fat synthesis and β-fatty acid oxidation were evaluated. Changes in fat metabolism were correlated with changes in AMPD2 activity and intrahepatic uric acid (downstream product of AMPD2), as well as changes in AMPK and intrahepatic β-hydroxybutyrate (a marker of fat oxidation). Hepatic fat accumulation occurred during the summer with relatively increased enzymes associated with fat synthesis (FAS, ACL and ACC) and decreased enoyl CoA hydratase (ECH1) and carnitine palmitoyltransferase 1A (CPT1A), rate limiting enzymes of fat oxidation. In summer, AMPD2 activity and intrahepatic uric acid levels were high and hepatic AMPK activity was low. In contrast, the active phosphorylated form of AMPK and β-hydroxybutyrate both increased during winter hibernation. Therefore, changes in AMPD2 and AMPK activity were paralleled with changes in fat synthesis and fat oxidation rates during the summer-winter cycle. These data illuminate the opposing forces of metabolism of AMP by AMPD2 and its availability to activate AMPK as a switch that governs fat metabolism in the liver of hibernating ground squirrel.

  15. Evaluation of toluene exposure via drinking water on levels of regional brain biogenic monoamines and their metabolites in CD-1 mice

    SciTech Connect

    Hsieh, G.C.; Sharma, R.P.; Parker, R.D.; Coulombe, R.A. Jr. )

    1990-10-01

    Toluene, a potentially neurotoxic substance, is found in trace amounts in groundwater. Adult male CD-1 mice were continuously fed drinking water ad libitum containing 0, 17, 80, and 405 mg/liter toluene. After a 28-day treatment, animals were tested for endogenous levels of the biogenic monoamines norepinephrine (NE), dopamine (DA), and serotonin (5-HT) and their respective metabolites, 3-methoxy-4-hydroxymandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA), in six discrete brain regions. The maximum toluene-induced increases of biogenic amines and their metabolites generally occurred at a toluene concentration of 80 mg/liter. In the hypothalamus, a major NE-containing compartment, the concentrations of NE significantly increased by 51, 63, and 34% in groups dosed with 17, 80, and 405 mg/liter, respectively. Significant increases of NE were also observed in the medulla oblongata and midbrain. Concomitantly, concentrations of VMA increased in various brain regions. Concentrations of DA were significantly higher in the corpus striatum and hypothalamus. Alterations in levels of DA metabolites, DOPAC and HVA, were marginal. Toluene significantly increased concentrations of 5-HT in all dissected brain regions, except cerebellum, and increased the 5-HIAA levels in the hypothalamus, corpus striatum, and cerebral cortex.

  16. Mycoplasma hyorhinis-encoded cytidine deaminase efficiently inactivates cytosine-based anticancer drugs

    PubMed Central

    Vande Voorde, Johan; Vervaeke, Peter; Liekens, Sandra; Balzarini, Jan

    2015-01-01

    Mycoplasmas may colonize tumor tissue in patients. The cytostatic activity of gemcitabine was dramatically decreased in Mycoplasma hyorhinis-infected tumor cell cultures compared with non-infected tumor cell cultures. This mycoplasma-driven drug deamination could be prevented by exogenous administration of the cytidine deaminase (CDA) inhibitor tetrahydrouridine, but also by the natural nucleosides or by a purine nucleoside phosphorylase inhibitor. The M. hyorhinis-encoded CDAHyor gene was cloned, expressed as a recombinant protein and purified. CDAHyor was found to be more catalytically active than its human equivalent and efficiently deaminates (inactivates) cytosine-based anticancer drugs. CDAHyor expression at the tumor site may result in selective drug inactivation and suboptimal therapeutic efficiency. PMID:26322268

  17. Epigenetic Function of Activation-Induced Cytidine Deaminase and Its Link to Lymphomagenesis

    PubMed Central

    Dominguez, Pilar M.; Shaknovich, Rita

    2014-01-01

    Activation-induced cytidine deaminase (AID) is essential for somatic hypermutation and class switch recombination of immunoglobulin (Ig) genes during B cell maturation and immune response. Expression of AID is tightly regulated due to its mutagenic and recombinogenic potential, which is known to target not only Ig genes, but also non-Ig genes, contributing to lymphomagenesis. In recent years, a new epigenetic function of AID and its link to DNA demethylation came to light in several developmental systems. In this review, we summarize existing evidence linking deamination of unmodified and modified cytidine by AID to base-excision repair and mismatch repair machinery resulting in passive or active removal of DNA methylation mark, with the focus on B cell biology. We also discuss potential contribution of AID-dependent DNA hypomethylation to lymphomagenesis. PMID:25566255

  18. Nonimmunoglobulin target loci of activation-induced cytidine deaminase (AID) share unique features with immunoglobulin genes

    PubMed Central

    Kato, Lucia; Begum, Nasim A.; Burroughs, A. Maxwell; Doi, Tomomitsu; Kawai, Jun; Daub, Carsten O.; Kawaguchi, Takahisa; Matsuda, Fumihiko; Hayashizaki, Yoshihide; Honjo, Tasuku

    2012-01-01

    Activation-induced cytidine deaminase (AID) is required for both somatic hypermutation and class-switch recombination in activated B cells. AID is also known to target nonimmunoglobulin genes and introduce mutations or chromosomal translocations, eventually causing tumors. To identify as-yet-unknown AID targets, we screened early AID-induced DNA breaks by using two independent genome-wide approaches. Along with known AID targets, this screen identified a set of unique genes (SNHG3, MALAT1, BCL7A, and CUX1) and confirmed that these loci accumulated mutations as frequently as Ig locus after AID activation. Moreover, these genes share three important characteristics with the Ig gene: translocations in tumors, repetitive sequences, and the epigenetic modification of chromatin by H3K4 trimethylation in the vicinity of cleavage sites. PMID:22308462

  19. Adenosine monophosphate deaminase 3 activation shortens erythrocyte half-life and provides malaria resistance in mice.

    PubMed

    Hortle, Elinor; Nijagal, Brunda; Bauer, Denis C; Jensen, Lora M; Ahn, Seong Beom; Cockburn, Ian A; Lampkin, Shelley; Tull, Dedreia; McConville, Malcolm J; McMorran, Brendan J; Foote, Simon J; Burgio, Gaetan

    2016-09-01

    The factors that determine red blood cell (RBC) lifespan and the rate of RBC aging have not been fully elucidated. In several genetic conditions, including sickle cell disease, thalassemia, and G6PD deficiency, erythrocyte lifespan is significantly shortened. Many of these diseases are also associated with protection from severe malaria, suggesting a role for accelerated RBC senescence and clearance in malaria resistance. Here, we report a novel, N-ethyl-N-nitrosourea-induced mutation that causes a gain of function in adenosine 5'-monophosphate deaminase (AMPD3). Mice carrying the mutation exhibit rapid RBC turnover, with increased erythropoiesis, dramatically shortened RBC lifespan, and signs of increased RBC senescence/eryptosis, suggesting a key role for AMPD3 in determining RBC half-life. Mice were also found to be resistant to infection with the rodent malaria Plasmodium chabaudi. We propose that resistance to P. chabaudi is mediated by increased RBC turnover and higher rates of erythropoiesis during infection. PMID:27465915

  20. Myoadenylate deaminase deficiency. Functional and metabolic abnormalities associated with disruption of the purine nucleotide cycle.

    PubMed Central

    Sabina, R L; Swain, J L; Olanow, C W; Bradley, W G; Fishbein, W N; DiMauro, S; Holmes, E W

    1984-01-01

    To assess the role of the purine nucleotide cycle in human skeletal muscle function, we evaluated 10 patients with AMP deaminase deficiency (myoadenylate deaminase deficiency; MDD). 4 MDD and 19 non-MDD controls participated in an exercise protocol. The latter group was composed of a patient cohort (n = 8) exhibiting a constellation of symptoms similar to those of the MDD patients, i.e., postexertional aches, cramps, and pains; as well as a cohort of normal, unconditioned volunteers (n = 11). The individuals with MDD fatigued after performing only 28% as much work as their non-MDD counterparts. Muscle biopsies were obtained from the four MDD patients and the eight non-MDD patients at rest and following exercise to the point of fatigue. Creatine phosphate content fell to a comparable extent in the MDD (69%) and non-MDD (52%) patients at the onset of fatigue. Following exercise the 34% decrease in ATP content of muscle from the non-MDD subjects was significantly greater than the 6% decrease in ATP noted in muscle from the MDD patients (P = 0.048). Only one of four MDD patients had a measurable drop in ATP compared with seven of eight non-MDD patients. At end-exercise the muscle content of inosine 5'-monophosphate (IMP), a product of AMP deaminase, was 13-fold greater in the non-MDD patients than that observed in the MDD group (P = 0.008). Adenosine content of muscle from the MDD patients increased 16-fold following exercise, while there was only a twofold increase in adenosine content of muscle from the non-MDD patients (P = 0.028). Those non-MDD patients in whom the decrease in ATP content following exercise was measurable exhibited a stoichiometric increase in IMP, and total purine content of the muscle did not change significantly. The one MDD patient in whom the decrease in ATP was measurable, did not exhibit a stoichiometric increase in IMP. Although the adenosine content increased 13-fold in this patient, only 48% of the ATP catabolized could be accounted for

  1. Markerless Gene Deletion with Cytosine Deaminase in Thermus thermophilus Strain HB27

    PubMed Central

    Wang, Lei; Hoffmann, Jana; Watzlawick, Hildegard

    2015-01-01

    We developed a counterselectable deletion system for Thermus thermophilus HB27 based on cytosine deaminase (encoded by codA) from Thermaerobacter marianensis DSM 12885 and the sensitivity of T. thermophilus HB27 to the antimetabolite 5-fluorocytosine (5-FC). The deletion vector comprises the pUC18 origin of replication, a thermostable kanamycin resistance marker functional in T. thermophilus HB27, and codA under the control of a constitutive putative trehalose promoter from T. thermophilus HB27. The functionality of the system was demonstrated by deletion of the bglT gene, encoding a β-glycosidase, and three carotenoid biosynthesis genes, CYP175A1, crtY, and crtI, from the genome of T. thermophilus HB27. PMID:26655764

  2. Nonimmunoglobulin target loci of activation-induced cytidine deaminase (AID) share unique features with immunoglobulin genes.

    PubMed

    Kato, Lucia; Begum, Nasim A; Burroughs, A Maxwell; Doi, Tomomitsu; Kawai, Jun; Daub, Carsten O; Kawaguchi, Takahisa; Matsuda, Fumihiko; Hayashizaki, Yoshihide; Honjo, Tasuku

    2012-02-14

    Activation-induced cytidine deaminase (AID) is required for both somatic hypermutation and class-switch recombination in activated B cells. AID is also known to target nonimmunoglobulin genes and introduce mutations or chromosomal translocations, eventually causing tumors. To identify as-yet-unknown AID targets, we screened early AID-induced DNA breaks by using two independent genome-wide approaches. Along with known AID targets, this screen identified a set of unique genes (SNHG3, MALAT1, BCL7A, and CUX1) and confirmed that these loci accumulated mutations as frequently as Ig locus after AID activation. Moreover, these genes share three important characteristics with the Ig gene: translocations in tumors, repetitive sequences, and the epigenetic modification of chromatin by H3K4 trimethylation in the vicinity of cleavage sites.

  3. Activation-Induced Cytidine Deaminase Links Ovulation-Induced Inflammation and Serous Carcinogenesis.

    PubMed

    Sapoznik, Stav; Bahar-Shany, Keren; Brand, Hadar; Pinto, Yishay; Gabay, Orshay; Glick-Saar, Efrat; Dor, Chen; Zadok, Oranit; Barshack, Iris; Zundelevich, Adi; Gal-Yam, Einav Nili; Yung, Yuval; Hourvitz, Ariel; Korach, Jacob; Beiner, Mario; Jacob, Jasmine; Levanon, Erez Y; Barak, Michal; Aviel-Ronen, Sarit; Levanon, Keren

    2016-02-01

    In recent years, the notion that ovarian carcinoma results from ovulation-induced inflammation of the fallopian tube epithelial cells (FTECs) has gained evidence. However, the mechanistic pathway for this process has not been revealed yet. In the current study, we propose the mutator protein activation-induced cytidine deaminase (AID) as a link between ovulation-induced inflammation in FTECs and genotoxic damage leading to ovarian carcinogenesis. We show that AID, previously shown to be functional only in B lymphocytes, is expressed in FTECs under physiological conditions, and is induced in vitro upon ovulatory-like stimulation and in vivo in carcinoma-associated FTECs. We also report that AID activity results in epigenetic, genetic and genomic damage in FTECs. Overall, our data provides new insights into the etiology of ovarian carcinogenesis and may set the ground for innovative approaches aimed at prevention and early detection. PMID:26936395

  4. High levels of CD34+CD38low/−CD123+ blasts are predictive of an adverse outcome in acute myeloid leukemia: a Groupe Ouest-Est des Leucémies Aiguës et Maladies du Sang (GOELAMS) study

    PubMed Central

    Vergez, François; Green, Alexa S.; Tamburini, Jerome; Sarry, Jean-Emmanuel; Gaillard, Baptiste; Cornillet-Lefebvre, Pascale; Pannetier, Melanie; Neyret, Aymeric; Chapuis, Nicolas; Ifrah, Norbert; Dreyfus, François; Manenti, Stéphane; Demur, Cecile; Delabesse, Eric; Lacombe, Catherine; Mayeux, Patrick; Bouscary, Didier; Recher, Christian; Bardet, Valerie

    2011-01-01

    Background Acute myeloid leukemias arise from a rare population of leukemic cells, known as leukemic stem cells, which initiate the disease and contribute to frequent relapses. Although the phenotype of these cells remains unclear in most patients, these cells are enriched within the CD34+CD38low/− compartment expressing the interleukin-3 alpha chain receptor, CD123. The aim of this study was to determine the prognostic value of the percentage of blasts with the CD34+CD38low/−CD123+ phenotype. Design and Methods The percentage of CD34+CD38low/−CD123+ cells in the blast population was determined at diagnosis using flow cytometry. One hundred and eleven patients under 65 years of age with de novo acute myeloid leukemia and treated with intensive chemotherapy were retrospectively included in the study. Correlations with complete response, disease-free survival and overall survival were evaluated with univariate and multivariate analyses. Results A proportion of CD34+CD38low/−CD123+ cells greater than 15% at diagnosis and an unfavorable karyotype were significantly correlated with a lack of complete response. By logistic regression analysis, a percentage of CD34+CD38low/−CD123+ higher than 15% retained significance with an odds ratio of 0.33 (0.1–0.97; P=0.044). A greater than 1% population of CD34+CD38low/−CD123+ cells negatively affected disease-free survival (0.9 versus 4.7 years; P<0.0001) and overall survival (1.25 years versus median not reached; P<0.0001). A greater than 1% population of CD34+CD38low/−CD123+ cells retained prognostic significance for both parameters after multivariate analysis. Conclusions The percentage of CD34+CD38low/−CD123+ leukemic cells at diagnosis was significantly correlated with response to treatment and survival. This prognostic marker might be easily adopted in clinical practice to rapidly identify patients at risk of treatment failure. PMID:21933861

  5. Characterization of a novel resistance-related deoxycytidine deaminase from Brassica oleracea var. capitata.

    PubMed

    Shibu, Marthandam Asokan; Yang, Hsueh-Hui; Lo, Chaur-Tsuen; Lin, Hong-Shin; Liu, Shu-Ying; Peng, Kou-Cheng

    2014-02-26

    Brassica oleracea deoxycytidine deaminase (BoDCD), a deoxycytidine deaminase (DCD, EC 3.5.4.14) enzyme, is known to play an important role in the Trichoderma harzianum ETS 323 mediated resistance mechanism in young leaves of B. oleracea var. capitata during Rhizoctonia solani infection. BoDCD potentially neutralizes cytotoxic products of host lipoxygenase activity, and thereby BoDCD restricts the hypersensitivity-related programmed cell death induced in plants during the initial stages of infection. To determine the biochemical characteristics and to partially elucidate the designated functional properties of BoDCD, the enzyme was cloned into an Escherichia coli expression system, and its potential to neutralize the toxic analogues of 2'-deoxycytidine (dC) was examined. BoDCD transformants of E. coli cells were found to be resistant to 2'-deoxycytidine analogues at all of the concentrations tested. The BoDCD enzyme was also overexpressed as a histidine-tagged protein and purified using nickel chelating affinity chromatography. The molecular weight of BoDCD was determined to be 20.8 kDa as visualized by SDS-PAGE. The substrate specificity and other kinetic properties show that BoDCD is more active in neutralizing cytotoxic cytosine β-d-arabinofuranoside than in deaminating 2'-deoxycytinde to 2'-deoxyuridine in nucleic acids or in metabolizing cytidine to uridine. The optimal temperature and pH of the enzyme were 27 °C and 7.5. The Km and Vmax values of BoDCD were, respectively, 91.3 μM and 1.475 mM for its natural substrate 2'-deoxycytidine and 63 μM and 2.072 mM for cytosine β-d-arabinofuranoside. The phenomenon of neutralization of cytotoxic dC analogues by BoDCD is discussed in detail on the basis of enzyme biochemical properties.

  6. Expression of a functional human adenosine deaminase in transgenic tobacco plants.

    PubMed

    Singhabahu, Sanjeewa; George, John; Bringloe, David

    2013-06-01

    An inherited disorder, adenosine deaminase deficiency is a form of severe combined immunodeficiency, which is ultimately caused by an absence of adenosine deaminase (ADA), a key enzyme of the purine salvage pathway. The absence of ADA-activity in sufferers eventually results in a dysfunctional immune system due to the build-up of toxic metabolites. To date, this has been treated with mixed success, using PEG-ADA, made from purified bovine ADA coupled to polyethylene glycol. It is likely, however, that an enzyme replacement therapy protocol based on recombinant human ADA would be a more effective treatment for this disease. Therefore, as a preliminary step to produce biologically active human ADA in transgenic tobacco plants a human ADA cDNA has been inserted into a plant expression vector under the control of the CaMV 35S promoter and both human and TMV 5' UTR control regions. Plant vector expression constructs have been used to transform tobacco plants via Agrobacterium-mediated transformation. Genomic DNA, RNA and protein blot analyses have demonstrated the integration of the cDNA construct into the plant nuclear genome and the expression of recombinant ADA mRNA and protein in transgenic tobacco leaves. Western blot analysis has also revealed that human and recombinant ADA have a similar size of approximately 41 kDa. ADA-specific activities of between 0.001 and 0.003 units per mg total soluble protein were measured in crude extracts isolated from transformed tobacco plant leaves. PMID:23264022

  7. CD3-T cell receptor modulation is selectively induced in CD8 but not CD4 lymphocytes cultured in agar.

    PubMed

    Oudrhiri, N; Farcet, J P; Gourdin, M F; M'Bemba, E; Gaulard, P; Katz, A; Divine, M; Galazka, A; Reyes, F

    1990-11-01

    The CD3-T cell receptor (TcR) complex is central to the immune response. Upon binding by specific ligands, internalized CD3-TcR molecules increase, and either T cell response or unresponsiveness may ensue depending on the triggering conditions. Using semi-solid agar culture, we have shown previously that quiescent CD4 but not CD8 lymphocytes generate clonal colonies under phytohaemagglutinin stimulation. Here we have demonstrated that the agar induces selective CD3-TcR modulation in the CD8 and not in the CD4 subset. CD8 lymphocytes preactivated in liquid culture and recultured in agar with exogenous recombinant interleukin-2 generate colonies with a modulated CD3-TcR surface expression. The peptides composing the CD3-TcR complex are synthesized in CD8 colonies as well as in CD4; however, the CD3 gamma chain is phosphorylated at a higher level in CD8 colonies. A component of the agar polymer, absent in agarose, appears to be the ligand that induces differential CD3-TcR modulation in the CD8 subset. In contrast to agar culture, CD8 colonies can be derived from quiescent CD8 lymphocytes in agarose. These CD8 colonies express unmodulated CD-TcR. CD3-TcR modulation with anti-CD3 monoclonal antibody prior to culturing in agarose inhibits the colony formation. We conclude that given triggering conditions can result in both CD3-TcR modulation and inhibition of the proliferative response selectively in the CD8 lymphocyte subset and not in the CD4. PMID:2146997

  8. CD3-T cell receptor modulation is selectively induced in CD8 but not CD4 lymphocytes cultured in agar.

    PubMed Central

    Oudrhiri, N; Farcet, J P; Gourdin, M F; M'Bemba, E; Gaulard, P; Katz, A; Divine, M; Galazka, A; Reyes, F

    1990-01-01

    The CD3-T cell receptor (TcR) complex is central to the immune response. Upon binding by specific ligands, internalized CD3-TcR molecules increase, and either T cell response or unresponsiveness may ensue depending on the triggering conditions. Using semi-solid agar culture, we have shown previously that quiescent CD4 but not CD8 lymphocytes generate clonal colonies under phytohaemagglutinin stimulation. Here we have demonstrated that the agar induces selective CD3-TcR modulation in the CD8 and not in the CD4 subset. CD8 lymphocytes preactivated in liquid culture and recultured in agar with exogenous recombinant interleukin-2 generate colonies with a modulated CD3-TcR surface expression. The peptides composing the CD3-TcR complex are synthesized in CD8 colonies as well as in CD4; however, the CD3 gamma chain is phosphorylated at a higher level in CD8 colonies. A component of the agar polymer, absent in agarose, appears to be the ligand that induces differential CD3-TcR modulation in the CD8 subset. In contrast to agar culture, CD8 colonies can be derived from quiescent CD8 lymphocytes in agarose. These CD8 colonies express unmodulated CD-TcR. CD3-TcR modulation with anti-CD3 monoclonal antibody prior to culturing in agarose inhibits the colony formation. We conclude that given triggering conditions can result in both CD3-TcR modulation and inhibition of the proliferative response selectively in the CD8 lymphocyte subset and not in the CD4. Images Fig. 3 Fig. 4 Fig. 5 PMID:2146997

  9. Elevated IFN-alpha/beta levels in a streptozotocin-induced type I diabetic mouse model promote oxidative stress and mediate depletion of spleen-homing CD8+ T cells by apoptosis through impaired CCL21/CCR7 axis and IL-7/CD127 signaling.

    PubMed

    Mahmoud, Mohamed H; Badr, Gamal; Badr, Badr Mohamed; Kassem, Ahmad Usama; Mohamed, Mahmoud Shaaban

    2015-10-01

    Type 1 diabetes mellitus (T1D) is associated with increased type 1 interferon (IFN) levels and subsequent severe defects in lymphocyte function, which increase susceptibility to infections. The blockade of type 1 IFN receptor 1 (IFNAR1) in non-obese diabetic mice has been shown to delay T1D onset and decrease T1D incidence by enhancing spleen CD4+ T cells and restoring B cell function. However, the effect of type 1 IFN blockade during T1D on splenic CD8+ T cells has not previously been studied. Therefore, we investigated, for the first time, the effect of IFNAR1 blockade on the survival and architecture of spleen-homing CD8+ T cells in a streptozotocin-induced T1D mouse model. Three groups of mice were examined: a non-diabetic control group; a diabetic group; and a diabetic group treated with an anti-IFNAR1 blocking antibody. We observed that T1D induction was accompanied by a marked destruction of β cells followed by a marked reduction in insulin levels and increased IFN-α and IFN-β levels in the diabetic group. The diabetic mice also exhibited many abnormal changes including an elevation in blood and spleen free radical (reactive oxygen species and nitric oxide) and pro-inflammatory cytokine (IL-6 and TNF-α) levels, a significant decrease in IL-7 levels, and subsequently, a significant decrease in the numbers of spleen-homing CD8+ T cells. This decrease in spleen-homing CD8+ T cells resulted from a marked reduction in the CCL21-mediated entry of CD8+ T cells into the spleen and from increased apoptosis due to a marked reduction in IL-7-mediated STAT5 and AKT phosphorylation. Interestingly, type 1 IFN signaling blockade in diabetic mice significantly restored the numbers of splenic CD8+ T cells by restoring free radical, pro-inflammatory cytokine and IL-7 levels. These effects subsequently rescued splenic CD8+ T cells from apoptosis through a mechanism that was dependent upon CCL21- and IL-7-mediated signaling. Our data suggest that type 1 IFN is an essential

  10. CD Recorders.

    ERIC Educational Resources Information Center

    Falk, Howard

    1998-01-01

    Discussion of CD (compact disc) recorders describes recording applications, including storing large graphic files, creating audio CDs, and storing material downloaded from the Internet; backing up files; lifespan; CD recording formats; continuous recording; recording software; recorder media; vulnerability of CDs; basic computer requirements; and…

  11. CD Rainbows

    ERIC Educational Resources Information Center

    Ouseph, P. J.

    2007-01-01

    Several papers have been published on the use of a CD as a grating for undergraduate laboratories and/or for high school and college class demonstrations. Four years ago "The Physics Teacher" had a spectacular cover picture showing emission spectrum as viewed through a CD with no coating. That picture gave the impetus to develop a system that can…

  12. A label-free fluorescent molecular beacon based on DNA-templated silver nanoclusters for detection of adenosine and adenosine deaminase.

    PubMed

    Zhang, Min; Guo, Su-Miao; Li, Ying-Ru; Zuo, Peng; Ye, Bang-Ce

    2012-06-01

    A simple and reliable fluorescent molecular beacon is developed utilizing DNA-templated silver nanoclusters as a signal indicator and adenosine triphosphate (ATP) and adenosine deaminase as mechanical activators.

  13. Elevated Plasma Level of Interferon-λ1 in Chronic Spontaneous Urticaria: Upregulated Expression in CD8+ and Epithelial Cells and Induction of Inflammatory Cell Accumulation

    PubMed Central

    Wang, S. F.; Gao, X. Q.; Xu, Y. N.; Li, D. N.; Wang, H. Y.

    2016-01-01

    Interferon- (IFN-) λ1 is regarded as a potent bio-active molecule in innate immunity. However, little is known about its role in chronic spontaneous urticaria (CSU). We therefore investigated expression of IFN-λ1 in CSU, its cellular location, and its influence on inflammatory cell accumulation by using flow cytometry analysis, skin tissue dispersion, immunohistochemical stain, and a mouse peritoneal inflammation model. The results showed that level of IFN-λ1 was 2.0-fold higher in plasma of the patients with CSU than the level in healthy control (HC) subjects. Among leukocytes examined, only CD8+ T cells expressed more IFN-λ1 in CSU blood. Double labeling immunohistochemical staining revealed that IFN-λ1+ inflammatory cells such as mast cells, eosinophils, B cells, neutrophils, and macrophages were mainly located in dermis, whereas epidermis tissue highly expressed IFN-λ1. IFN-λ1 induced a dose-dependent increase in number of eosinophils, lymphocytes, mast cells, macrophages, and neutrophils in the peritoneum of mice at 6 h following injection, which was inhibited by pretreatment of the animals with anti-intercellular adhesion molecule- (ICAM-) 1 and/or anti-L-selectin antibodies. In conclusion, IFN-λ1 is likely to play a role in the pathogenesis of CSU. Blocking IFN-λ1 production may help to reduce the accumulation of inflammatory cells in the involved CSU skin. PMID:27445435

  14. Proteomic analysis of CD44(+) and CD44(-) gastric cancer cells.

    PubMed

    Yu, Dayeon; Shin, Hyun-Soo; Choi, Go; Lee, Yong Chan

    2014-11-01

    CD44 is a cell surface protein and it is widely used as a cancer stem cell marker in various cancer types including gastric cancer. We conducted proteomic analysis in CD44(+) and CD44(-) gastric cancer cells to understand characteristics of CD44(+) and CD44(-) cells. In the present study, we sorted cells from the gastric cancer cell line MKN45 according to CD44 expression to separate out CD44(+) and CD44(-) cells. And we conducted RT-PCR to identify mRNA expression of cancer stem cell markers in CD44(+) and CD44(-) cells. Cancer stem cell markers showed upregulated expression in CD44(+) cells. Next, we performed two-dimensional electrophoresis analysis to determine the differential expression pattern of proteins in each group; control, CD44(+), and CD44(-) MKN45 cells. We found a total of 113 spots that varied in expression between CD44(+) and CD44(-) cells, and subjected 20 of those protein spots to MALDI-MS. We selected the three proteins (HSPA8; heat shock cognate 71 kDa protein isoform 1, ezrin, α-enolase) upregulated in CD44(+) cells than CD44(-) cells and one protein (prohibitin) showed increased expression in CD44(-) cells. We validated the protein expression levels of four selected proteins by Western blot. We suggest that our study could be a helpful background to study CD44(+) cancer stem-like cells and differences between CD44(+) and CD44(-) cells in gastric cancer. PMID:25081334

  15. Protein preparation and preliminary X-ray crystallographic analysis of a putative glucosamine 6-phosphate deaminase from Streptococcus mutants

    SciTech Connect

    Hu, Guan-Jing; Li, Lan-Fen; Li, Dan; Liu, Cong; Wei, Shi-Cheng; Liang, Yu-He Su, Xiao-Dong

    2007-09-01

    A glucosamine 6-phosphate deaminase homologue from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.4 Å resolution. The SMU.636 protein from Streptococcus mutans is a putative glucosamine 6-phosphate deaminase with 233 residues. The smu.636 gene was PCR-amplified from S. mutans genomic DNA and cloned into the expression vector pET-28a(+). The resultant His-tagged fusion protein was expressed in Escherichia coli and purified to homogeneity in two steps. Crystals of the fusion protein were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 Å resolution and belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.83, b = 82.13, c = 134.70 Å.

  16. Crystal Structure of Staphylococcus aureus tRNA Adenosine Deaminase TadA in Complex with RNA

    SciTech Connect

    Losey,H.; Ruthenburg, A.; Verdine, G.

    2006-01-01

    Bacterial tRNA adenosine deaminases (TadAs) catalyze the hydrolytic deamination of adenosine to inosine at the wobble position of tRNA(Arg2), a process that enables this single tRNA to recognize three different arginine codons in mRNA. In addition, inosine is also introduced at the wobble position of multiple eukaryotic tRNAs. The genes encoding these deaminases are essential in bacteria and yeast, demonstrating the importance of their biological activity. Here we report the crystallization and structure determination to 2.0 A of Staphylococcus aureus TadA bound to the anticodon stem-loop of tRNA(Arg2) bearing nebularine, a non-hydrolyzable adenosine analog, at the wobble position. The cocrystal structure reveals the basis for both sequence and structure specificity in the interactions of TadA with RNA, and it additionally provides insight into the active site architecture that promotes efficient hydrolytic deamination.

  17. Characterization of ACC deaminase from the biocontrol and plant growth-promoting agent Trichoderma asperellum T203.

    PubMed

    Viterbo, Ada; Landau, Udi; Kim, Sofia; Chernin, Leonid; Chet, Ilan

    2010-04-01

    1-aminocyclopropane-1-carboxylate (ACC) deaminase activity was evaluated in the biocontrol and plant growth-promoting fungus Trichoderma asperellum T203. Fungal cultures grown with ACC as the sole nitrogen source showed high enzymatic activity. The enzyme encoding gene (Tas-acdS) was isolated, and an average 3.5-fold induction of the gene by 3 mM ACC was detected by real-time PCR. Escherichia coli bacteria carrying the intron-free cDNA of Tas-acdS cloned into the vector pAlter-EX1 under the control of the tac promoter revealed specific ACC deaminase (ACCD) activity and the ability to promote canola (Brassica napus) root elongation in pouch assays. RNAi silencing of the ACCD gene in T. asperellum showed decreased ability of the mutants to promote root elongation of canola seedlings. These data suggest a role for ACCD in the plant root growth-promotion effect by T. asperellum.

  18. High levels of the adhesion molecule CD44 on leukemic cells generate acute myeloid leukemia relapse after withdrawal of the initial transforming event.

    PubMed

    Quéré, R; Andradottir, S; Brun, A C M; Zubarev, R A; Karlsson, G; Olsson, K; Magnusson, M; Cammenga, J; Karlsson, S

    2011-03-01

    Multiple genetic hits are detected in patients with acute myeloid leukemia (AML). To investigate this further, we developed a tetracycline-inducible mouse model of AML, in which the initial transforming event, overexpression of HOXA10, can be eliminated. Continuous overexpression of HOXA10 is required to generate AML in primary recipient mice, but is not essential for maintenance of the leukemia. Transplantation of AML to secondary recipients showed that in established leukemias, ∼80% of the leukemia-initiating cells (LICs) in bone marrow stopped proliferating upon withdrawal of HOXA10 overexpression. However, the population of LICs in primary recipients is heterogeneous, as ∼20% of the LICs induce leukemia in secondary recipients despite elimination of HOXA10-induced overexpression. Intrinsic genetic activation of several proto-oncogenes was observed in leukemic cells resistant to inactivation of the initial transformation event. Interestingly, high levels of the adhesion molecule CD44 on leukemic cells are essential to generate leukemia after removal of the primary event. This suggests that extrinsic niche-dependent factors are also involved in the host-dependent outgrowth of leukemias after withdrawal of HOXA10 overexpression event that initiates the leukemia.

  19. Genetic variation in CD36, HBA, NOS3 and VCAM1 is associated with chronic haemolysis level in sickle cell anaemia: a longitudinal study.

    PubMed

    Coelho, Andreia; Dias, Alexandra; Morais, Anabela; Nunes, Baltazar; Ferreira, Emanuel; Picanço, Isabel; Faustino, Paula; Lavinha, João

    2014-03-01

    Chronic haemolysis stands out as one of the hallmarks of sickle cell anaemia, a clinically heterogeneous autosomal recessive monogenic anaemia. However, the genetic architecture of this sub-phenotype is still poorly understood. Here, we report the results of an association study between haemolysis biomarkers (serum LDH, total bilirubin and reticulocyte count) and the inheritance of 41 genetic variants of ten candidate genes in a series of 99 paediatric SS patients (median current age of 9.9 yr) followed up in two general hospitals in Greater Lisboa area (median follow-up per patient of 5.0 yr). Although in a large number of tests a seemingly significant (i.e. P < 0.05) association was observed, the following ones were confirmed upon correction for multiple comparisons: (i) an increased serum LDH level was associated with haplotype 7 within VCAM1 gene; (ii) a lower total bilirubin was associated with the 3.7-kb deletion at HBA gene, rs2070744_T allele at NOS3 gene, and haplotype 9 within VCAM1 promoter; and (iii) a diminished reticulocyte count was associated with the 3.7-kb deletion at HBA, whereas an increased count was associated with rs1984112_G allele at CD36 gene. On the whole, our findings suggest a complex genetic architecture for the sickle cell anaemia haemolysis process involving multiple pathways, namely control of vascular cell adhesion, NO synthesis and erythrocyte volume and haemoglobinisation.

  20. Low Levels of CD36 in Peripheral Blood Monocytes in Subclinical Atherosclerosis in Rheumatoid Arthritis: A Cross-Sectional Study in a Mexican Population

    PubMed Central

    Gómez-Bañuelos, Eduardo; Martín-Márquez, Beatriz Teresita; Martínez-García, Erika Aurora; Figueroa-Sanchez, Mauricio; Nuñez-Atahualpa, Lourdes; Rocha-Muñoz, Alberto Daniel; Sánchez-Hernández, Pedro Ernesto; Navarro-Hernandez, Rosa Elena; Madrigal-Ruiz, Perla Monserrat; Saldaña-Millan, Adan Alberto; Duran-Barragan, Sergio; Gonzalez-Lopez, Laura; Gamez-Nava, Jorge Ivan; Vázquez-Del Mercado, Mónica

    2014-01-01

    Patients with rheumatoid arthritis (RA) have a higher risk for atherosclerosis. There is no clinical information about scavenger receptor CD36 and the development of subclinical atherosclerosis in patients with RA. The aim of this study was to evaluate the association between membrane expression of CD36 in peripheral blood mononuclear cells (PBMC) and carotid intima-media thickness (cIMT) in patients with RA. Methods. We included 67 patients with RA from the Rheumatology Department of Hospital Civil “Dr. Juan I. Menchaca,” Guadalajara, Jalisco, Mexico. We evaluated the cIMT, considering subclinical atherosclerosis when >0.6 mm. Since our main objective was to associate the membrane expression of CD36 with subclinical atherosclerosis, other molecules related with cardiovascular risk such as ox-LDL, IL-6, and TNFα were tested. Results. We found low CD36 membrane expression in PBMC from RA patients with subclinical atherosclerosis (P < 0.001). CD36 mean fluorescence intensity had negative correlations with cIMT (r = −0.578, P < 0.001), ox-LDL (r = −0.427, P = 0.05), TNFα (r = −0.729, P < 0.001), and IL-6 (r = −0.822, P < 0.001). Conclusion. RA patients with subclinical atherosclerosis showed low membrane expression of CD36 in PBMC and increased serum proinflammatory cytokines. Further studies are needed to clarify the regulation of CD36 in RA. PMID:25006585

  1. CD Rom.

    PubMed

    1996-02-01

    A new CD-Rom has been launched by Guy's and St Thomas' Trust's poisonous unit to help health professionals discover which species have been involved in cases of plant poisoning. The unit says thousands of people are poisoned every year by eating or touching plants - the majority of those people affected being under the age of seven. The CD-Rom covers several thousand species of plant, and has been jointly researched with Kew Gardens.

  2. Different responses of low grain-Cd-accumulating and high grain-Cd-accumulating rice cultivars to Cd stress.

    PubMed

    Wang, Feijuan; Wang, Min; Liu, Zhouping; Shi, Yan; Han, Tiqian; Ye, Yaoyao; Gong, Ning; Sun, Junwei; Zhu, Cheng

    2015-11-01

    Cadmium (Cd) is a major heavy metal pollutant which is highly toxic to plants and animals. The accumulation of Cd in rice grains is a major agricultural problem in regions with Cd pollution. A hydroponics experiment using low grain-Cd-accumulating rice (xiushui 11) and high grain-Cd-accumulating rice (xiushui 110) was carried out to characterize the different responses of rice cultivars to Cd stress. We found that xiushui 11 was more tolerant to Cd than xiushui 110, and xiushui 11 suffered less oxidative damage. Cell walls played an important role in limiting the amount of Cd that entered the protoplast, especially in xiushui 11. Cd stored in organelles as soluble fractions, leading to greater physiological stress of Cd detoxification. We found that Cd can disturb the ion homeostasis in rice roots because Cd(2+) and Ca(2+) may have a similar uptake route. Xiushui 11 had a faster root-to-shoot transport of Cd, and the expression level of OsPCR1 gene which was predicted related with Cd accumulation in rice was consist with the Cd transport of root-to-shoot in rice and maintain the greater Cd tolerance of xiushui 11. These results suggest there are different Cd detoxification and accumulation mechanisms in rice cultivars.

  3. Different responses of low grain-Cd-accumulating and high grain-Cd-accumulating rice cultivars to Cd stress.

    PubMed

    Wang, Feijuan; Wang, Min; Liu, Zhouping; Shi, Yan; Han, Tiqian; Ye, Yaoyao; Gong, Ning; Sun, Junwei; Zhu, Cheng

    2015-11-01

    Cadmium (Cd) is a major heavy metal pollutant which is highly toxic to plants and animals. The accumulation of Cd in rice grains is a major agricultural problem in regions with Cd pollution. A hydroponics experiment using low grain-Cd-accumulating rice (xiushui 11) and high grain-Cd-accumulating rice (xiushui 110) was carried out to characterize the different responses of rice cultivars to Cd stress. We found that xiushui 11 was more tolerant to Cd than xiushui 110, and xiushui 11 suffered less oxidative damage. Cell walls played an important role in limiting the amount of Cd that entered the protoplast, especially in xiushui 11. Cd stored in organelles as soluble fractions, leading to greater physiological stress of Cd detoxification. We found that Cd can disturb the ion homeostasis in rice roots because Cd(2+) and Ca(2+) may have a similar uptake route. Xiushui 11 had a faster root-to-shoot transport of Cd, and the expression level of OsPCR1 gene which was predicted related with Cd accumulation in rice was consist with the Cd transport of root-to-shoot in rice and maintain the greater Cd tolerance of xiushui 11. These results suggest there are different Cd detoxification and accumulation mechanisms in rice cultivars. PMID:26318143

  4. An age-related numerical and functional deficit in CD19(+) CD24(hi) CD38(hi) B cells is associated with an increase in systemic autoimmunity.

    PubMed

    Duggal, Niharika A; Upton, Jane; Phillips, Anna C; Sapey, Elizabeth; Lord, Janet M

    2013-10-01

    Autoimmunity increases with aging indicative of reduced immune tolerance, but the mechanisms involved are poorly defined. In recent years, subsets of B cells with immunoregulatory properties have been identified in murine models of autoimmune disorders, and these cells downregulate immune responses via secretion of IL10. In humans, immature transitional B cells with a CD19(+) CD24(hi) CD38(hi) phenotype have been reported to regulate immune responses via IL10 production. We found the frequency and numbers of CD19(+) CD24(hi) CD38(hi) cells were reduced in the PBMC pool with age. IL10 expression and secretion following activation via either CD40, or Toll-like receptors was also impaired in CD19(+) CD24(hi) CD38(hi) B cells from healthy older donors. When investigating the mechanisms involved, we found that CD19(+) CD24(hi) CD38(hi) B-cell function was compromised by age-related effects on both T cells and B cells: specifically, CD40 ligand expression was lower in CD4 T cells from older donors following CD3 stimulation, and signalling through CD40 was impaired in CD19(+) CD24(hi) CD38(hi) B cells from elders as evidenced by reduced phosphorylation (Y705) and activation of STAT3. However, there was no age-associated change in expression of costimulatory molecules CD80 and CD86 on CD19(+) CD24(hi) CD38(hi) cells, suggesting IL10-dependent immune suppression is impaired, but contact-dependent suppressive capacity is intact with age. Finally, we found a negative correlation between CD19(+) CD24(hi) CD38(hi) B-cell IL10 production and autoantibody (Rheumatoid factor) levels in older adults. We therefore propose that an age-related decline in CD19(+) CD24(hi) CD38(hi) B cell number and function may contribute towards the increased autoimmunity and reduced immune tolerance seen with aging.

  5. NTP pattern of avian embryonic red cells: role of RNA degradation and AMP deaminase/5'-nucleotidase activity.

    PubMed

    Baumann, Rosemarie; Gotz, Robert; Dragon, Stefanie

    2003-03-01

    During terminal erythroid differentiation, degradation of RNA is a potential source for nucleotide triphosphates (NTPs) that act as allosteric effectors of hemoglobin. In this investigation, we assessed the developmental profile of RNA and purine/pyrimidine trinucleotides in circulating embryonic chick red blood cells (RBC). Extensive changes of the NTP pattern are observed which differ significantly from what is observed for adult RBC. The biochemical mechanisms have not been identified yet. Therefore, we studied the role of AMP deaminase and IMP/GMP 5'-nucleotidase, which are key enzymes for the regulation of the purine nucleotide pool. Finally, we tested the effect of major NTPs on the oxygen affinity of embryonic/adult hemoglobin. The results are as follows. 1) Together with ATP, UTP and CTP serve as allosteric effectors of hemoglobin. 2) Degradation of erythroid RNA is apparently a major source for NTPs. 3) Developmental changes of nucleotide content depend on the activities of key enzymes (AMP deaminase, IMP/GMP 5'-nucleotidase, and pyrimidine 5'-nucleotidase). 4) Oxygen-dependent hormonal regulation of AMP deaminase adjusts the red cell ATP concentration and therefore the hemoglobin oxygen affinity.

  6. Active RNAP pre-initiation sites are highly mutated by cytidine deaminases in yeast, with AID targeting small RNA genes

    PubMed Central

    Taylor, Benjamin JM; Wu, Yee Ling; Rada, Cristina

    2014-01-01

    Cytidine deaminases are single stranded DNA mutators diversifying antibodies and restricting viral infection. Improper access to the genome leads to translocations and mutations in B cells and contributes to the mutation landscape in cancer, such as kataegis. It remains unclear how deaminases access double stranded genomes and whether off-target mutations favor certain loci, although transcription and opportunistic access during DNA repair are thought to play a role. In yeast, AID and the catalytic domain of APOBEC3G preferentially mutate transcriptionally active genes within narrow regions, 110 base pairs in width, fixed at RNA polymerase initiation sites. Unlike APOBEC3G, AID shows enhanced mutational preference for small RNA genes (tRNAs, snoRNAs and snRNAs) suggesting a putative role for RNA in its recruitment. We uncover the high affinity of the deaminases for the single stranded DNA exposed by initiating RNA polymerases (a DNA configuration reproduced at stalled polymerases) without a requirement for specific cofactors. DOI: http://dx.doi.org/10.7554/eLife.03553.001 PMID:25237741

  7. Inhibition of 5-aminoimidazole-4-carboxamide ribotide transformylase, adenosine deaminase and 5'-adenylate deaminase by polyglutamates of methotrexate and oxidized folates and by 5-aminoimidazole-4-carboxamide riboside and ribotide.

    PubMed Central

    Baggott, J E; Vaughn, W H; Hudson, B B

    1986-01-01

    With the use of a continuous spectrophotometric assay and initial rates determined by the method of Waley [Biochem. J. (1981) 193, 1009-1012] methotrexate was found to be a non-competitive inhibitor, with Ki(intercept) = 72 microM and Ki(slope) = 41 microM, of 5-aminoimidazole-4-carboxamide ribotide transformylase, whereas a polyglutamate of methotrexate containing three gamma-linked glutamate residues was a competitive inhibitor, with Ki = 3.15 microM. Pentaglutamates of folic acid and 10-formylfolic acid were also competitive inhibitors of the transformylase, with Ki values of 0.088 and 1.37 microM respectively. Unexpectedly, the pentaglutamate of 10-formyldihydrofolic acid was a good substrate for the transformylase, with a Km of 0.51 microM and a relative Vmax. of 0.72, which compared favourably with a Km of 0.23 microM and relative Vmax. of 1.0 for the tetrahydro analogue. An analysis of the progress curve of the transformylase-catalysed reaction with the above dihydro coenzyme revealed that the pentaglutamate of dihydrofolic acid was a competitive product inhibitor, with Ki = 0.14 microM. The continuous spectrophotometric assay for adenosine deaminase based on change in the absorbance at 265 nm was shown to be valid with adenosine concentrations above 100 microM, which contradicts a previous report [Murphy, Baker, Behling & Turner (1982) Anal. Biochem. 122, 328-337] that this assay was invalid above this concentration. With the spectrophotometric assay, 5-aminoimidazole-4-carboxamide riboside was found to be a competitive inhibitor of adenosine deaminase, with (Ki = 362 microM), whereas the ribotide was a competitive inhibitor of 5'-adenylate deaminase, with Ki = 1.01 mM. Methotrexate treatment of susceptible cells results in (1) its conversion into polyglutamates, (2) the accumulation of oxidized folate polyglutamates, and (3) the accumulation of 5-aminoimidazole-4-carboxamide riboside and ribotide. The above metabolic events may be integral elements

  8. CD-ROM-aided Databases

    NASA Astrophysics Data System (ADS)

    Sano, Tomoyuki; Suzuki, Masataka; Nishida, Hideo

    The Development of CAI system using CD-ROM and NAPLPS (North American Presentation Level Protocol Syntax) was taken place by Himeji Dokkyo University. The characteristics of CAI using CD-ROM as information processing series for the department of liberal arts student are described. The system is that the computer program, vast amount of voice data and graphics data are stored in a CD-ROM. It is very effective to improve learning ability of student.

  9. Quantitative analysis of beta-actin, beta-2-microglobulin and porphobilinogen deaminase mRNA and their comparison as control transcripts for RT-PCR.

    PubMed

    Lupberger, J; Kreuzer, K A; Baskaynak, G; Peters, U R; le Coutre, P; Schmidt, C A

    2002-02-01

    Quantitation of target mRNAs using the reverse-transcription polymerase chain reaction found a widespread field of application in diverse biomedical diagnostic assays. However, the problem of varying sample quality has to be solved by correcting target molecule amounts through detection of an endogenous control template. The choice of an appropriate reference gene is still object of debate as pseudogene co-amplification and expression level variations may limit the usefulness of some currently used reference reactions. We compared quantitative expression levels of the commonly used endogenous reference genes beta-actin (beta-actin), beta-2-microglobulin (beta2-MG) and porphobilinogen deaminase (PBDG) using the TaqMan chemistry. With these assays we investigated the respective expression patterns in K562 cells and leucocytes of normal individuals as well as of malignoma patients. In K562 cells 1544+246 beta-actin, 65+30 beta2-MG and 22+/-8 PBDG copies/cell were detected. In normal leucocytes 491+/-97 beta-actin, 40+/-17 beta2-MG and <1 PBDG copies/cell were quantified. Leucocytes of various malignancies exhibited 84+/-51 beta-actin, 106+/-8 beta2-MG and <1 PBDG copies/cell. We conclude that beta2-MG is the most suitable reference gene tested as its variation between different sample origins and within distinct cell types was acceptable low.

  10. Heterologous expression of ACC deaminase from Trichoderma asperellum improves the growth performance of Arabidopsis thaliana under normal and salt stress conditions.

    PubMed

    Zhang, Fuli; Zhang, Ju; Chen, Long; Shi, Xiaoying; Lui, Zhihua; Li, Chengwei

    2015-09-01

    Transgenic Arabidopsis thaliana plants expressing the 1-aminocyclopropane-1-carboxylate deaminase gene (ACCD) of Trichoderma asperellum ACCC30536 (TaACCD) were created and their growth performance was assessed under normal and salt stress conditions. In order to characterize their growth, root length, root number, fresh weight (FW), relative water content (RWC), seed production, and seed number were measured. Under normal growing condition, all growth parameters except for dry weight (DW) of the transgenic plants increased significantly compared to WT plants. Furthermore, the transgenic line also exhibited higher tolerance and faster growth than WT plants in the presence of 150 mM NaCl. The increased salt stress tolerance of the transgenic plants is attributed to a greater RWC, root weight, root length, root number and FW under salt stress, and to reduced reactive oxygen species (ROS) level, cell death and electrolyte leakage compared to WT plants. The reduction in ROS levels could be explained by increased activity of several antioxidant enzymes, including peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT). Thus, we propose that heterologous expression of TaACCD could be used to improve salt stress tolerance in plants. PMID:26004912

  11. Heterologous expression of ACC deaminase from Trichoderma asperellum improves the growth performance of Arabidopsis thaliana under normal and salt stress conditions.

    PubMed

    Zhang, Fuli; Zhang, Ju; Chen, Long; Shi, Xiaoying; Lui, Zhihua; Li, Chengwei

    2015-09-01

    Transgenic Arabidopsis thaliana plants expressing the 1-aminocyclopropane-1-carboxylate deaminase gene (ACCD) of Trichoderma asperellum ACCC30536 (TaACCD) were created and their growth performance was assessed under normal and salt stress conditions. In order to characterize their growth, root length, root number, fresh weight (FW), relative water content (RWC), seed production, and seed number were measured. Under normal growing condition, all growth parameters except for dry weight (DW) of the transgenic plants increased significantly compared to WT plants. Furthermore, the transgenic line also exhibited higher tolerance and faster growth than WT plants in the presence of 150 mM NaCl. The increased salt stress tolerance of the transgenic plants is attributed to a greater RWC, root weight, root length, root number and FW under salt stress, and to reduced reactive oxygen species (ROS) level, cell death and electrolyte leakage compared to WT plants. The reduction in ROS levels could be explained by increased activity of several antioxidant enzymes, including peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT). Thus, we propose that heterologous expression of TaACCD could be used to improve salt stress tolerance in plants.

  12. Characterization of immunoglobulin gene somatic hypermutation in the absence of activation-induced cytidine deaminase

    PubMed Central

    Longo, Nancy S.; Satorius, Colleen L.; Plebani, Alessandro; Durandy, Anne; Lipsky, Peter E.

    2008-01-01

    Somatic hypermutation (SHM) of Ig genes depends upon the deamination of C nucleotides in WRCY (W=A/T, R=A/G, Y=C/T) motifs by activation-induced cytidine deaminase (AICDA). Despite this, a large number of mutations occur in WA motifs that can be accounted for by the activity of polymerase eta (POL η). To determine whether there are AICDA-independent mutations and to characterize the relationship between AICDA- and POL η-mediated mutations, 1,470 H chain and 1,313 kappa and lambda chain rearrangements from three AICDA−/− patients were analyzed. The Ig mutation frequency of all VH genes from AICDA−/− patients was 40-fold less than that of normal donors whereas the mutation frequency of mutated VH sequences from AICDA−/− patients was 6.8-fold less than normal donors. AICDA−/− B cells lack mutations in WRCY/RGYW motifs as well as replacement mutations and mutational targeting in complementarity determining regions. A significantly reduced mutation frequency in WA motifs compared to normal donors and an increased percentage of transitions, which may relate to reduced uracil DNA-glycosylase (UNG) activity, suggest a role for AICDA in regulating POL η and UNG activity. Similar results were observed in VL rearrangements. The residual mutations were predominantly G:C substitutions, indicating that AICDA-independent cytidine deamination was a likely, yet inefficient, mechanism for mutating Ig genes. PMID:18606684

  13. Adaptive evolution of threonine deaminase in plant defense against insect herbivores

    SciTech Connect

    Gonzales-Vigil, Eliana; Bianchetti, Christopher M.; Phillips, Jr., George N.; Howe, Gregg A.

    2011-11-07

    Gene duplication is a major source of plant chemical diversity that mediates plant-herbivore interactions. There is little direct evidence, however, that novel chemical traits arising from gene duplication reduce herbivory. Higher plants use threonine deaminase (TD) to catalyze the dehydration of threonine (Thr) to {alpha}-ketobutyrate and ammonia as the committed step in the biosynthesis of isoleucine (Ile). Cultivated tomato and related Solanum species contain a duplicated TD paralog (TD2) that is coexpressed with a suite of genes involved in herbivore resistance. Analysis of TD2-deficient tomato lines showed that TD2 has a defensive function related to Thr catabolism in the gut of lepidopteran herbivores. During herbivory, the regulatory domain of TD2 is removed by proteolysis to generate a truncated protein (pTD2) that efficiently degrades Thr without being inhibited by Ile. We show that this proteolytic activation step occurs in the gut of lepidopteran but not coleopteran herbivores, and is catalyzed by a chymotrypsin-like protease of insect origin. Analysis of purified recombinant enzymes showed that TD2 is remarkably more resistant to proteolysis and high temperature than the ancestral TD1 isoform. The crystal structure of pTD2 provided evidence that electrostatic interactions constitute a stabilizing feature associated with adaptation of TD2 to the extreme environment of the lepidopteran gut. These findings demonstrate a role for gene duplication in the evolution of a plant defense that targets and co-opts herbivore digestive physiology.

  14. Involvement of activation-induced cytidine deaminase in skin cancer development

    PubMed Central

    Toda, Yoshinobu; Hiai, Hiroshi; Uemura, Munehiro; Nakamura, Motonobu; Hattori, Yukari; Bessho, Kazuhisa; Minato, Nagahiro

    2016-01-01

    Most skin cancers develop as the result of UV light–induced DNA damage; however, a substantial number of cases appear to occur independently of UV damage. A causal link between UV-independent skin cancers and chronic inflammation has been suspected, although the precise mechanism underlying this association is unclear. Here, we have proposed that activation-induced cytidine deaminase (AID, encoded by AICDA) links chronic inflammation and skin cancer. We demonstrated that Tg mice expressing AID in the skin spontaneously developed skin squamous cell carcinoma with Hras and Trp53 mutations. Furthermore, genetic deletion of Aicda reduced tumor incidence in a murine model of chemical-induced skin carcinogenesis. AID was expressed in human primary keratinocytes in an inflammatory stimulus–dependent manner and was detectable in human skin cancers. Together, the results of this study indicate that inflammation-induced AID expression promotes skin cancer development independently of UV damage and suggest AID as a potential target for skin cancer therapeutics. PMID:26974156

  15. Modulatory effect of iron chelators on adenosine deaminase activity and gene expression in Trichomonas vaginalis.

    PubMed

    Primon-Barros, Muriel; Rigo, Graziela Vargas; Frasson, Amanda Piccoli; Santos, Odelta dos; Smiderle, Lisiane; Almeida, Silvana; Macedo, Alexandre José; Tasca, Tiana

    2015-11-01

    Trichomonas vaginalis is a flagellate protozoan that parasitises the urogenital human tract and causes trichomoniasis. During the infection, the acquisition of nutrients, such as iron and purine and pyrimidine nucleosides, is essential for the survival of the parasite. The enzymes for purinergic signalling, including adenosine deaminase (ADA), which degrades adenosine to inosine, have been characterised in T. vaginalis. In the evaluation of the ADA profile in different T. vaginalis isolates treated with different iron sources or with limited iron availability, a decrease in activity and an increase in ADA gene expression after iron limitation by 2,2-bipyridyl and ferrozine chelators were observed. This supported the hypothesis that iron can modulate the activity of the enzymes involved in purinergic signalling. Under bovine serum limitation conditions, no significant differences were observed. The results obtained in this study allow for the assessment of important aspects of ADA and contribute to a better understanding of the purinergic system in T. vaginalis and the role of iron in establishing infection and parasite survival.

  16. Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution

    NASA Astrophysics Data System (ADS)

    Senavirathne, Gayan; Bertram, Jeffrey G.; Jaszczur, Malgorzata; Chaurasiya, Kathy R.; Pham, Phuong; Mak, Chi H.; Goodman, Myron F.; Rueda, David

    2015-12-01

    Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ~5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer.

  17. Adenosine Deaminase Acting on RNA-1 (ADAR1) Inhibits HIV-1 Replication in Human Alveolar Macrophages

    PubMed Central

    Levy, David N.; Li, Yonghua; Kumar, Rajnish; Burke, Sean A.; Dawson, Rodney; Hioe, Catarina E.; Borkowsky, William; Rom, William N.; Hoshino, Yoshihiko

    2014-01-01

    While exploring the effects of aerosol IFN-γ treatment in HIV-1/tuberculosis co-infected patients, we observed A to G mutations in HIV-1 envelope sequences derived from bronchoalveolar lavage (BAL) of aerosol IFN-γ-treated patients and induction of adenosine deaminase acting on RNA 1 (ADAR1) in the BAL cells. IFN-γ induced ADAR1 expression in monocyte-derived macrophages (MDM) but not T cells. ADAR1 siRNA knockdown induced HIV-1 expression in BAL cells of four HIV-1 infected patients on antiretroviral therapy. Similar results were obtained in MDM that were HIV-1 infected in vitro. Over-expression of ADAR1 in transformed macrophages inhibited HIV-1 viral replication but not viral transcription measured by nuclear run-on, suggesting that ADAR1 acts post-transcriptionally. The A to G hyper-mutation pattern observed in ADAR1 over-expressing cells in vitro was similar to that found in the lungs of HIV-1 infected patients treated with aerosol IFN-γ suggesting the model accurately represented alveolar macrophages. Together, these results indicate that ADAR1 restricts HIV-1 replication post-transcriptionally in macrophages harboring HIV-1 provirus. ADAR1 may therefore contribute to viral latency in macrophages. PMID:25272020

  18. A Role for Host Activation-Induced Cytidine Deaminase in Innate Immune Defense against KSHV

    PubMed Central

    Bekerman, Elena; Jeon, Diana; Ardolino, Michele; Coscoy, Laurent

    2013-01-01

    Activation-induced cytidine deaminase (AID) is specifically induced in germinal center B cells to carry out somatic hypermutation and class-switch recombination, two processes responsible for antibody diversification. Because of its mutagenic potential, AID expression and activity are tightly regulated to minimize unwanted DNA damage. Surprisingly, AID expression has been observed ectopically during pathogenic infections. However, the function of AID outside of the germinal centers remains largely uncharacterized. In this study, we demonstrate that infection of human primary naïve B cells with Kaposi's sarcoma-associated herpesvirus (KSHV) rapidly induces AID expression in a cell intrinsic manner. We find that infected cells are marked for elimination by Natural Killer cells through upregulation of NKG2D ligands via the DNA damage pathway, a pathway triggered by AID. Moreover, without having a measurable effect on KSHV latency, AID impinges directly on the viral fitness by inhibiting lytic reactivation and reducing infectivity of KSHV virions. Importantly, we uncover two KSHV-encoded microRNAs that directly regulate AID abundance, further reinforcing the role for AID in the antiviral response. Together our findings reveal additional functions for AID in innate immune defense against KSHV with implications for a broader involvement in innate immunity to other pathogens. PMID:24244169

  19. Platelet aggregation and serum adenosine deaminase (ADA) activity in pregnancy associated with diabetes, hypertension and HIV.

    PubMed

    Leal, Claudio A M; Leal, Daniela B R; Adefegha, Stephen A; Morsch, Vera M; da Silva, José E P; Rezer, João F P; Schrekker, Clarissa M L; Abdalla, Faida H; Schetinger, Maria R C

    2016-07-01

    Platelet aggregation and adenosine deaminase (ADA) activity were evaluated in pregnant women living with some disease conditions including hypertension, diabetes mellitus and human immunodeficiency virus infection. The subject population is consisted of 15 non-pregnant healthy women [control group (CG)], 15 women with normal pregnancy (NP), 7 women with hypertensive pregnancy (HP), 10 women with gestational diabetes mellitus (GDM) and 12 women with human immunodeficiency virus-infected pregnancy (HIP) groups. The aggregation of platelets was checked using an optical aggregometer, and serum ADA activity was determined using the colorimetric method. After the addition of 5 µM of agonist adenosine diphosphate, the percentage of platelet aggregation was significantly (p < 0·05) increased in NP, HP, GDM and HIP groups when compared with the CG, while the addition of 10 µM of the same agonist caused significant (p < 0·05) elevations in HP, GDM and HIP groups when compared with CG. Furthermore, ADA activity was significantly (p < 0·05) enhanced in NP, HP, GDM and HIP groups when compared with CG. In this study, the increased platelet aggregation and ADA activity in pregnancy and pregnancy-associated diseases suggest that platelet aggregation and ADA activity could serve as peripheral markers for the development of effective therapy in the maintenance of homeostasis and some inflammatory process in these pathophysiological conditions. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27273565

  20. Modulatory effect of iron chelators on adenosine deaminase activity and gene expression in Trichomonas vaginalis

    PubMed Central

    Primon-Barros, Muriel; Rigo, Graziela Vargas; Frasson, Amanda Piccoli; dos Santos, Odelta; Smiderle, Lisiane; Almeida, Silvana; Macedo, Alexandre José; Tasca, Tiana

    2015-01-01

    Trichomonas vaginalis is a flagellate protozoan that parasitises the urogenital human tract and causes trichomoniasis. During the infection, the acquisition of nutrients, such as iron and purine and pyrimidine nucleosides, is essential for the survival of the parasite. The enzymes for purinergic signalling, including adenosine deaminase (ADA), which degrades adenosine to inosine, have been characterised in T. vaginalis. In the evaluation of the ADA profile in different T. vaginalis isolates treated with different iron sources or with limited iron availability, a decrease in activity and an increase in ADA gene expression after iron limitation by 2,2-bipyridyl and ferrozine chelators were observed. This supported the hypothesis that iron can modulate the activity of the enzymes involved in purinergic signalling. Under bovine serum limitation conditions, no significant differences were observed. The results obtained in this study allow for the assessment of important aspects of ADA and contribute to a better understanding of the purinergic system in T. vaginalis and the role of iron in establishing infection and parasite survival. PMID:26517498

  1. Involvement of activation-induced cytidine deaminase in skin cancer development.

    PubMed

    Nonaka, Taichiro; Toda, Yoshinobu; Hiai, Hiroshi; Uemura, Munehiro; Nakamura, Motonobu; Yamamoto, Norio; Asato, Ryo; Hattori, Yukari; Bessho, Kazuhisa; Minato, Nagahiro; Kinoshita, Kazuo

    2016-04-01

    Most skin cancers develop as the result of UV light-induced DNA damage; however, a substantial number of cases appear to occur independently of UV damage. A causal link between UV-independent skin cancers and chronic inflammation has been suspected, although the precise mechanism underlying this association is unclear. Here, we have proposed that activation-induced cytidine deaminase (AID, encoded by AICDA) links chronic inflammation and skin cancer. We demonstrated that Tg mice expressing AID in the skin spontaneously developed skin squamous cell carcinoma with Hras and Trp53 mutations. Furthermore, genetic deletion of Aicda reduced tumor incidence in a murine model of chemical-induced skin carcinogenesis. AID was expressed in human primary keratinocytes in an inflammatory stimulus-dependent manner and was detectable in human skin cancers. Together, the results of this study indicate that inflammation-induced AID expression promotes skin cancer development independently of UV damage and suggest AID as a potential target for skin cancer therapeutics.

  2. Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution.

    PubMed

    Senavirathne, Gayan; Bertram, Jeffrey G; Jaszczur, Malgorzata; Chaurasiya, Kathy R; Pham, Phuong; Mak, Chi H; Goodman, Myron F; Rueda, David

    2015-01-01

    Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ∼ 5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer. PMID:26681117

  3. Evolution of class switch recombination function in fish activation-induced cytidine deaminase, AID.

    PubMed

    Wakae, Koshou; Magor, Brad G; Saunders, Holly; Nagaoka, Hitoshi; Kawamura, Akemi; Kinoshita, Kazuo; Honjo, Tasuku; Muramatsu, Masamichi

    2006-01-01

    Following activation of mammalian B cells, class switch recombination (CSR) and somatic hypermutation (SHM) of the Ig heavy chain (IgH) gene can improve the functions of the expressed antibodies. Activation-induced cytidine deaminase (AID) is the only known B cell-specific protein required for inducing CSR and SHM in mammals. Lower vertebrates have an AID homologue, and there is some evidence of SHM in vivo. However there is no evidence of CSR in the cartilaginous or bony fishes, and this may be due in part to a lack of cis-elements in the IgH gene that are the normal targets of AID-mediated recombination. We have tested whether bony fish (zebrafish and catfish) AID can mediate CSR and SHM in mammalian cells. As expected, ectopic expression of fish AID in mouse fibroblasts resulted in mutations in an introduced SHM reporter gene, indicating that fish AID can mediate SHM. Unexpectedly, expression of fish AID in mouse AID-/- B cells induced surface IgG expression as well as switched transcripts from Ig gene loci, clearly indicating that the fish AID protein can mediate CSR, at least in mouse cells. These results suggest that the AID protein acquired the ability to mediate CSR before the IgH locus evolved the additional exon clusters and switch regions that are the targets of recombination. We discuss how pleiotropic functions of specific domains within the AID protein may have facilitated the early evolution of CSR in lower vertebrates.

  4. Target DNA sequence directly regulates the frequency of activation-induced deaminase-dependent mutations.

    PubMed

    Chen, Zhangguo; Viboolsittiseri, Sawanee S; O'Connor, Brian P; Wang, Jing H

    2012-10-15

    Activation-induced deaminase (AID) catalyses class switch recombination (CSR) and somatic hypermutation (SHM) in B lymphocytes to enhance Ab diversity. CSR involves breaking and rejoining highly repetitive switch (S) regions in the IgH (Igh) locus. S regions appear to be preferential targets of AID. To determine whether S region sequence per se, independent of Igh cis regulatory elements, can influence AID targeting efficiency and mutation frequency, we established a knock-in mouse model by inserting a core Sγ1 region into the first intron of proto-oncogene Bcl6, which is a non-Ig target of SHM. We found that the mutation frequency of the inserted Sγ1 region was dramatically higher than that of the adjacent Bcl6 endogenous sequence. Mechanistically, S region-enhanced SHM was associated with increased recruitment of AID and RNA polymerase II, together with Spt5, albeit to a lesser extent. Our studies demonstrate that target DNA sequences influence mutation frequency via regulating AID recruitment. We propose that the nucleotide sequence preference may serve as an additional layer of AID regulation by restricting its mutagenic activity to specific sequences despite the observation that AID has the potential to access the genome widely.

  5. The Role of Histidine-Proline-Rich Glycoprotein as Zinc Chaperone for Skeletal Muscle AMP Deaminase

    PubMed Central

    Ranieri-Raggi, Maria; Moir, Arthur J. G.; Raggi, Antonio

    2014-01-01

    Metallochaperones function as intracellular shuttles for metal ions. At present, no evidence for the existence of any eukaryotic zinc-chaperone has been provided although metallochaperones could be critical for the physiological functions of Zn2+ metalloenzymes. We propose that the complex formed in skeletal muscle by the Zn2+ metalloenzyme AMP deaminase (AMPD) and the metal binding protein histidine-proline-rich glycoprotein (HPRG) acts in this manner. HPRG is a major plasma protein. Recent investigations have reported that skeletal muscle cells do not synthesize HPRG but instead actively internalize plasma HPRG. X-ray absorption spectroscopy (XAS) performed on fresh preparations of rabbit skeletal muscle AMPD provided evidence for a dinuclear zinc site in the enzyme compatible with a (μ-aqua)(μ-carboxylato)dizinc(II) core with two histidine residues at each metal site. XAS on HPRG isolated from the AMPD complex showed that zinc is bound to the protein in a dinuclear cluster where each Zn2+ ion is coordinated by three histidine and one heavier ligand, likely sulfur from cysteine. We describe the existence in mammalian HPRG of a specific zinc binding site distinct from the His-Pro-rich region. The participation of HPRG in the assembly and maintenance of skeletal muscle AMPD by acting as a zinc chaperone is also demonstrated. PMID:24970226

  6. CD74 in Kidney Disease

    PubMed Central

    Valiño-Rivas, Lara; Baeza-Bermejillo, Ciro; Gonzalez-Lafuente, Laura; Sanz, Ana Belen; Ortiz, Alberto; Sanchez-Niño, Maria Dolores

    2015-01-01

    CD74 (invariant MHC class II) regulates protein trafficking and is a receptor for macrophage migration inhibitory factor (MIF) and d-dopachrome tautomerase (d-DT/MIF-2). CD74 expression is increased in tubular cells and/or glomerular podocytes and parietal cells in human metabolic nephropathies, polycystic kidney disease, graft rejection and kidney cancer and in experimental diabetic nephropathy and glomerulonephritis. Stressors like abnormal metabolite (glucose, lyso-Gb3) levels and inflammatory cytokines increase kidney cell CD74. MIF activates CD74 to increase inflammatory cytokines in podocytes and tubular cells and proliferation in glomerular parietal epithelial cells and cyst cells. MIF overexpression promotes while MIF targeting protects from experimental glomerular injury and kidney cysts, and interference with MIF/CD74 signaling or CD74 deficiency protected from crescentic glomerulonephritis. However, CD74 may protect from interstitial kidney fibrosis. Furthermore, CD74 expression by stressed kidney cells raises questions about the kidney safety of cancer therapy strategies delivering lethal immunoconjugates to CD74-expressing cells. Thus, understanding CD74 biology in kidney cells is relevant for kidney therapeutics. PMID:26441987

  7. Altered CD45 isoform expression affects lymphocyte function in CD45 Tg mice.

    PubMed

    Tchilian, Elma Z; Dawes, Ritu; Hyland, Lisa; Montoya, Maria; Le Bon, Agnes; Borrow, Persephone; Hou, Sam; Tough, David; Beverley, Peter C L

    2004-09-01

    Transgenic mice have been constructed expressing high (CD45RABC) and low (CD45R0) molecular weight CD45 isoforms on a CD45-/- background. Phenotypic analysis and in vivo challenge of these mice with influenza and lymphocytic choriomeningitis viruses shows that T cell differentiation and peripheral T cell function are related to the level of CD45 expression but not to which CD45 isoform is expressed. In contrast, B cell differentiation is not restored, irrespective of the level of expression of a single isoform. All CD45 trangenic mice have T cells with an activated phenotype and increased T cell turnover. These effects are more prominent in CD8 than CD4 cells. The transgenic mice share several properties with humans expressing variant CD45 alleles and provide a model to understand immune function in variant individuals. PMID:15302847

  8. Role of caffeic acid phenethyl ester on mitomycin C induced clastogenesis: analysis of chromosome aberrations, micronucleus, mitotic index and adenosine deaminase activity in vivo.

    PubMed

    Sulaiman, Ghassan Mohammad

    2012-05-01

    The aim of the present investigation is to determine whether the caffeic acid phenethyl ester (CAPE) in combination with mitomycine-C (MMC) can ameliorate MMC-induced clastogenesis in the bone marrow cells of mice. The scoring of chromosomal aberrations, mitotic activity and micronuclei were undertaken in the current study as markers of clastogenicity. The action of CAPE in adenosine deaminase enzyme (ADA) activities of serum, thymus and spleen were also investigated. The animals were orally administered CAPE alone at the doses 5 or 10 mg kg b.wt.(-1) for 5 days then sacrificed 24 hours after the CAPE administration. MMC was administered to mice either alone at a single dose (2 mg kg b.wt.(-1)) by intraperitoneal injection, before or after CAPE treatment. Pre or post - treatment with two doses of CAPE significantly decreased the number of chromosomal aberrations, micronuclei and adapted the mitotic activity reduction in the bone marrow cells of mice induced by MMC when compared with only MMC given group. In addition, combination treatment with MMC caused a significant decrease in the activities of ADA in serum, thymus and spleen. The results of this study showed that ADA activity probably related to high levels of reactive oxygen species. This study concluded that the protective effect of CAPE against MMC clastogenesis resides at least in part, in its antioxidant effects.

  9. Deficiency in L-serine deaminase interferes with one-carbon metabolism and cell wall synthesis in Escherichia coli K-12.

    PubMed

    Zhang, Xiao; El-Hajj, Ziad W; Newman, Elaine

    2010-10-01

    Escherichia coli K-12 provided with glucose and a mixture of amino acids depletes L-serine more quickly than any other amino acid even in the presence of ammonium sulfate. A mutant without three 4Fe4S L-serine deaminases (SdaA, SdaB, and TdcG) of E. coli K-12 is unable to do this. The high level of L-serine that accumulates when such a mutant is exposed to amino acid mixtures starves the cells for C(1) units and interferes with cell wall synthesis. We suggest that at high concentrations, L-serine decreases synthesis of UDP-N-acetylmuramate-L-alanine by the murC-encoded ligase, weakening the cell wall and producing misshapen cells and lysis. The inhibition by high L-serine is overcome in several ways: by a large concentration of L-alanine, by overproducing MurC together with a low concentration of L-alanine, and by overproducing FtsW, thus promoting septal assembly and also by overexpression of the glycine cleavage operon. S-Adenosylmethionine reduces lysis and allows an extensive increase in biomass without improving cell division. This suggests that E. coli has a metabolic trigger for cell division. Without that reaction, if no other inhibition occurs, other metabolic functions can continue and cells can elongate and replicate their DNA, reaching at least 180 times their usual length, but cannot divide.

  10. Speckled-like Pattern in the Germinal Center (SLIP-GC), a Nuclear GTPase Expressed in Activation-induced Deaminase-expressing Lymphomas and Germinal Center B Cells*

    PubMed Central

    Richter, Kathleen; Brar, Sukhdev; Ray, Madhumita; Pisitkun, Prapaporn; Bolland, Silvia; Verkoczy, Laurent; Diaz, Marilyn

    2009-01-01

    We identified a novel GTPase, SLIP-GC, with expression limited to a few tissues, in particular germinal center B cells. It lacks homology to any known proteins, indicating that it may belong to a novel family of GTPases. SLIP-GC is expressed in germinal center B cells and in lymphomas derived from germinal center B cells such as large diffuse B cell lymphomas. In cell lines, SLIP-GC is expressed in lymphomas that express activation-induced deaminase (AID) and that likely undergo somatic hypermutation. SLIP-GC is a nuclear protein, and it localizes to replication factories. Reduction of SLIP-GC levels in the Burkitt lymphoma cell line Raji and in non-Hodgkin lymphoma cell lines resulted in an increase in DNA breaks and apoptosis that was AID-dependent, as simultaneous reduction of AID abrogated the deleterious effects of SLIP-GC reduction. These results strongly suggest that SLIP-GC is a replication-related protein in germinal center B cells whose reduction is toxic to cells through an AID-dependent mechanism. PMID:19734146

  11. Rapid identification of micro-organisms from urinary tract infections by beta-glucuronidase, phenylalanine deaminase, cytochrome oxidase and indole tests on isolation media.

    PubMed

    Giammanco, G; Pignato, S

    1994-12-01

    Two commercially available media recommended for the isolation and rapid identification of Escherichia coli from urinary tract infections were supplemented with L-phenylalanine and L-tryptophan. The non-selective medium proved suitable for the direct detection of lactose fermentation, beta-glucuronidase and phenylalanine deaminase activities, indole production and the oxidase test. It was highly efficient in making a presumptive identification at species level of the most common gram-negative urinary pathogens, E. coli, Proteus mirabilis and Pseudomonas aeruginosa, that account for c. 85% of all urinary isolates. Among the gram-positive isolates, most colonies were non-fluorescent and could be separated into staphylococci and enterococci on the basis of the catalase test. Fluorescent colonies were found to be Staphylococcus haemolyticus isolates, 61% of which were fluorescent. The selective medium proved suitable for the same biochemical tests, with the exception of indole, which was not visible against the red colour of the medium. Therefore, the differentiation of P. mirabilis from other Proteus-Providencia species was impossible on this medium. PMID:7966214

  12. Torsional energy levels of CH{sub 3}OH{sup +}/CH{sub 3}OD{sup +}/CD{sub 3}OD{sup +} studied by zero-kinetic energy photoelectron spectroscopy and theoretical calculations

    SciTech Connect

    Dai, Zuyang; Gao, Shuming; Wang, Jia; Mo, Yuxiang

    2014-10-14

    The torsional energy levels of CH{sub 3}OH{sup +}, CH{sub 3}OD{sup +}, and CD{sub 3}OD{sup +} have been determined for the first time using one-photon zero kinetic energy photoelectron spectroscopy. The adiabatic ionization energies for CH{sub 3}OH, CH{sub 3}OD, and CD{sub 3}OD are determined as 10.8396, 10.8455, and 10.8732 eV with uncertainties of 0.0005 eV, respectively. Theoretical calculations have also been performed to obtain the torsional energy levels for the three isotopologues using a one-dimensional model with approximate zero-point energy corrections of the torsional potential energy curves. The calculated values are in good agreement with the experimental data. The barrier height of the torsional potential energy without zero-point energy correction was calculated as 157 cm{sup −1}, which is about half of that of the neutral (340 cm{sup −1}). The calculations showed that the cation has eclipsed conformation at the energy minimum and staggered one at the saddle point, which is the opposite of what is observed in the neutral molecule. The fundamental C–O stretch vibrational energy level for CD{sub 3}OD{sup +} has also been determined. The energy levels for the combinational excitation of the torsional vibration and the fundamental C–O stretch vibration indicate a strong torsion-vibration coupling.

  13. Dependence of the electrical parameters of MBE-grown Cd{sub x}Hg{sub 1} {sub -} {sub x}Te films on the level of doping with indium

    SciTech Connect

    Varavin, V. S. Dvoretskii, S. A.; Ikusov, D. G.; Mikhailov, N. N.; Sidorov, Yu. G.; Sidorov, G. Yu.; Yakushev, M. V.

    2008-06-15

    Dependences of the minority-carrier lifetime and electron mobility in Cd{sub x}Hg{sub 1-x}Te films on their indium-doping level are studied. Films with x {approx} 0.22 grown by molecular-beam epitaxy on GaAs substrates were in situ doped with indium across their entire thickness. The temperature dependences of the lifetime were studied in the temperature range 77-300 K. The decrease in the lifetime, observed as the doping level increases, is governed by the mechanism of Auger recombination. As the doping level becomes higher, the mobility decreases in qualitative agreement with theoretical calculations.

  14. Peripheral canine CD4(+)CD8(+) double-positive T cells - unique amongst others.

    PubMed

    von Buttlar, Heiner; Bismarck, Doris; Alber, Gottfried

    2015-12-15

    T lymphocytes co-expressing CD4 and CD8 ("double-positive T cells") are commonly associated with a thymic developmental stage of T cells. Their first description in humans and pigs as extrathymic T cells with a memory phenotype almost 30 years ago came as a surprise. Meanwhile peripheral double-positive T cells have been described in a growing number of different species. In this review we highlight novel data from our very recent studies on canine peripheral double-positive T cells which point to unique features of double-positive T cells in the dog. In contrast to porcine CD4(+)CD8(+) T cells forming a homogenous cellular population based on their expression of CD4 and CD8α, canine CD4(+)CD8(+) T cells can be divided into three different cellular subsets with distinct expression levels of CD4 and CD8α. Double-positive T cells expressing CD8β are present in humans and dogs but absent in swine. Moreover, canine CD4(+)CD8(+) T cells can not only develop from CD4(+) single-positive T cells but also from CD8(+) single-positive T cells. Together, this places canine CD4(+)CD8(+) T cells closer to their human than porcine counterparts since human double-positive T cells also appear to be heterogeneous in their CD4 and CD8α expression and have both CD4(+) and CD8(+) T cells as progenitor cells. However, CD4(+) single-positive T cells are the more potent progenitors for canine double-positive T cells, whereas CD8(+) single-positive T cells are more potent progenitors for human double-positive T cells. Canine double-positive T cells have an activated phenotype and may have as yet unrecognized roles in vivo in immunity to infection or in inflammatory diseases such as chronic infection, autoimmunity, allergy, or cancer.

  15. Presepsin (soluble CD14 subtype) and procalcitonin levels for mortality prediction in sepsis: data from the Albumin Italian Outcome Sepsis trial

    PubMed Central

    2014-01-01

    Introduction Sepsis, a leading cause of death in critically ill patients, is the result of complex interactions between the infecting microorganisms and the host responses that influence clinical outcomes. We evaluated the prognostic value of presepsin (sCD14-ST), a novel biomarker of bacterial infection, and compared it with procalcitonin (PCT). Methods This is a retrospective, case–control study of a multicenter, randomized clinical trial enrolling patients with severe sepsis or septic shock in ICUs in Italy. We selected 50 survivors and 50 non-survivors at ICU discharge, matched for age, sex and time from sepsis diagnosis to enrollment. Plasma samples were collected 1, 2 and 7 days after enrollment to assay presepsin and PCT. Outcome was assessed 28 and 90 days after enrollment. Results Early presepsin (day 1) was higher in decedents (2,269 pg/ml, median (Q1 to Q3), 1,171 to 4,300 pg/ml) than in survivors (1,184 pg/ml (median, 875 to 2,113); P = 0.002), whereas PCT was not different (18.5 μg/L (median 3.4 to 45.2) and 10.8 μg/L (2.7 to 41.9); P = 0.31). The evolution of presepsin levels over time was significantly different in survivors compared to decedents (P for time-survival interaction = 0.03), whereas PCT decreased similarly in the two groups (P = 0.13). Presepsin was the only variable independently associated with ICU and 28-day mortality in Cox models adjusted for clinical characteristics. It showed better prognostic accuracy than PCT in the range of Sequential Organ Failure Assessment score (area under the curve (AUC) from 0.64 to 0.75 vs. AUC 0.53 to 0.65). Conclusions In this multicenter clinical trial, we provide the first evidence that presepsin measurements may have useful prognostic information for patients with severe sepsis or septic shock. These preliminary findings suggest that presepsin may be of clinical importance for early risk stratification. PMID:24393424

  16. MPA-capped CdTe quantum dots exposure causes neurotoxic effects in nematode Caenorhabditis elegans by affecting the transporters and receptors of glutamate, serotonin and dopamine at the genetic level, or by increasing ROS, or both

    NASA Astrophysics Data System (ADS)

    Wu, Tianshu; He, Keyu; Zhan, Qinglin; Ang, Shengjun; Ying, Jiali; Zhang, Shihan; Zhang, Ting; Xue, Yuying; Tang, Meng

    2015-12-01

    As quantum dots (QDs) are widely used in biomedical applications, the number of studies focusing on their biological properties is increasing. While several studies have attempted to evaluate the toxicity of QDs towards neural cells, the in vivo toxic effects on the nervous system and the molecular mechanisms are unclear. The aim of the present study was to investigate the neurotoxic effects and the underlying mechanisms of water-soluble cadmium telluride (CdTe) QDs capped with 3-mercaptopropionic acid (MPA) in Caenorhabditis elegans (C. elegans). Our results showed that exposure to MPA-capped CdTe QDs induced behavioral defects, including alterations to body bending, head thrashing, pharyngeal pumping and defecation intervals, as well as impaired learning and memory behavior plasticity, based on chemotaxis or thermotaxis, in a dose-, time- and size-dependent manner. Further investigations suggested that MPA-capped CdTe QDs exposure inhibited the transporters and receptors of glutamate, serotonin and dopamine in C. elegans at the genetic level within 24 h, while opposite results were observed after 72 h. Additionally, excessive reactive oxygen species (ROS) generation was observed in the CdTe QD-treated worms, which confirmed the common nanotoxicity mechanism of oxidative stress damage, and might overcome the increased gene expression of neurotransmitter transporters and receptors in C. elegans induced by long-term QD exposure, resulting in more severe behavioral impairments.

  17. MPA-capped CdTe quantum dots exposure causes neurotoxic effects in nematode Caenorhabditis elegans by affecting the transporters and receptors of glutamate, serotonin and dopamine at the genetic level, or by increasing ROS, or both.

    PubMed

    Wu, Tianshu; He, Keyu; Zhan, Qinglin; Ang, Shengjun; Ying, Jiali; Zhang, Shihan; Zhang, Ting; Xue, Yuying; Tang, Meng

    2015-12-28

    As quantum dots (QDs) are widely used in biomedical applications, the number of studies focusing on their biological properties is increasing. While several studies have attempted to evaluate the toxicity of QDs towards neural cells, the in vivo toxic effects on the nervous system and the molecular mechanisms are unclear. The aim of the present study was to investigate the neurotoxic effects and the underlying mechanisms of water-soluble cadmium telluride (CdTe) QDs capped with 3-mercaptopropionic acid (MPA) in Caenorhabditis elegans (C. elegans). Our results showed that exposure to MPA-capped CdTe QDs induced behavioral defects, including alterations to body bending, head thrashing, pharyngeal pumping and defecation intervals, as well as impaired learning and memory behavior plasticity, based on chemotaxis or thermotaxis, in a dose-, time- and size-dependent manner. Further investigations suggested that MPA-capped CdTe QDs exposure inhibited the transporters and receptors of glutamate, serotonin and dopamine in C. elegans at the genetic level within 24 h, while opposite results were observed after 72 h. Additionally, excessive reactive oxygen species (ROS) generation was observed in the CdTe QD-treated worms, which confirmed the common nanotoxicity mechanism of oxidative stress damage, and might overcome the increased gene expression of neurotransmitter transporters and receptors in C. elegans induced by long-term QD exposure, resulting in more severe behavioral impairments.

  18. Restriction of Porcine Endogenous Retrovirus by Porcine APOBEC3 Cytidine Deaminases

    PubMed Central

    Dörrschuck, Eva; Fischer, Nicole; Bravo, Ignacio G.; Hanschmann, Kay-Martin; Kuiper, Heidi; Spötter, Andreas; Möller, Ronny; Cichutek, Klaus; Münk, Carsten; Tönjes, Ralf R.

    2011-01-01

    Xenotransplantation of porcine cells, tissues, and organs shows promise to surmount the shortage of human donor materials. Among the barriers to pig-to-human xenotransplantation are porcine endogenous retroviruses (PERV) since functional representatives of the two polytropic classes, PERV-A and PERV-B, are able to infect human embryonic kidney cells in vitro, suggesting that a xenozoonosis in vivo could occur. To assess the capacity of human and porcine cells to counteract PERV infections, we analyzed human and porcine APOBEC3 (A3) proteins. This multigene family of cytidine deaminases contributes to the cellular intrinsic immunity and act as potent inhibitors of retroviruses and retrotransposons. Our data show that the porcine A3 gene locus on chromosome 5 consists of the two single-domain genes A3Z2 and A3Z3. The evolutionary relationships of the A3Z3 genes reflect the evolutionary history of mammals. The two A3 genes encode at least four different mRNAs: A3Z2, A3Z3, A3Z2-Z3, and A3Z2-Z3 splice variant A (SVA). Porcine and human A3s have been tested toward their antiretroviral activity against PERV and murine leukemia virus (MuLV) using novel single-round reporter viruses. The porcine A3Z2, A3Z3 and A3Z2-Z3 were packaged into PERV particles and inhibited PERV replication in a dose-dependent manner. The antiretroviral effect correlated with editing by the porcine A3s with a trinucleotide preference for 5′ TGC for A3Z2 and A3Z2-Z3 and 5′ CAC for A3Z3. These results strongly imply that human and porcine A3s could inhibit PERV replication in vivo, thereby reducing the risk of infection of human cells by PERV in the context of pig-to-human xenotransplantation. PMID:21307203

  19. Investigation into effects of antipsychotics on ectonucleotidase and adenosine deaminase in zebrafish brain.

    PubMed

    Seibt, Kelly Juliana; Oliveira, Renata da Luz; Bogo, Mauricio Reis; Senger, Mario Roberto; Bonan, Carla Denise

    2015-12-01

    Antipsychotic agents are used for the treatment of psychotic symptoms in patients with several brain disorders, such as schizophrenia. Atypical and typical antipsychotics differ regarding their clinical and side-effects profile. Haloperidol is a representative typical antipsychotic drug and has potent dopamine receptor antagonistic functions; however, atypical antipsychotics have been developed and characterized an important advance in the treatment of schizophrenia and other psychotic disorders. Purine nucleotides and nucleosides, such as ATP and adenosine, constitute a ubiquitous class of extracellular signaling molecules crucial for normal functioning of the nervous system. Indirect findings suggest that changes in the purinergic system, more specifically in adenosinergic activity, could be involved in the pathophysiology of schizophrenia. We investigated the effects of typical and atypical antipsychotics on ectonucleotidase and adenosine deaminase (ADA) activities, followed by an analysis of gene expression patterns in zebrafish brain. Haloperidol treatment (9 µM) was able to decrease ATP hydrolysis (35%), whereas there were no changes in hydrolysis of ADP and AMP in brain membranes after antipsychotic exposure. Adenosine deamination in membrane fractions was inhibited (38%) after haloperidol treatment when compared to the control; however, no changes were observed in ADA soluble fractions after haloperidol exposure. Sulpiride (250 µM) and olanzapine (100 µM) did not alter ectonucleotidase and ADA activities. Haloperidol also led to a decrease in entpd2_mq, entpd3 and adal mRNA transcripts. These findings demonstrate that haloperidol is an inhibitor of NTPDase and ADA activities in zebrafish brain, suggesting that purinergic signaling may also be a target of pharmacological effects promoted by this drug.

  20. Crystal structure of a membrane-bound l-amino acid deaminase from Proteus vulgaris.

    PubMed

    Ju, Yingchen; Tong, Shuilong; Gao, Yongxiang; Zhao, Wei; Liu, Qi; Gu, Qiong; Xu, Jun; Niu, Liwen; Teng, Maikun; Zhou, Huihao

    2016-09-01

    l-amino acid oxidases/deaminases (LAAOs/LAADs) are a class of oxidoreductases catalyzing the oxidative deamination of l-amino acids to α-keto acids. They are widely distributed in eukaryotic and prokaryotic organisms, and exhibit diverse substrate specificity, post-translational modifications and cellular localization. While LAAOs isolated from snake venom have been extensively characterized, the structures and functions of LAAOs from other species are largely unknown. Here, we reported crystal structure of a bacterial membrane-bound LAAD from Proteus vulgaris (pvLAAD) in complex with flavin adenine dinucleotide (FAD). We found that the overall fold of pvLAAD does not resemble typical LAAOs. Instead it, is similar to d-amino acid oxidases (DAAOs) with an additional hydrophobic insertion module on protein surface. Structural analysis and liposome-binding assays suggested that the hydrophobic module serves as an extra membrane-binding site for LAADs. Bacteria from genera Proteus and Providencia were found to encode two classes of membrane-bound LAADs. Based on our structure, the key roles of residues Q278 and L317 in substrate selectivity were proposed and biochemically analyzed. While LAADs on the membrane were proposed to transfer electrons to respiratory chain for FAD re-oxidization, we observed that the purified pvLAAD could generate a significant amount of hydrogen peroxide in vitro, suggesting it could use dioxygen to directly re-oxidize FADH2 as what typical LAAOs usually do. These findings provide a novel insights for a better understanding this class of enzymes and will help developing biocatalysts for industrial applications. PMID:27422658

  1. Adenosine deaminase activity in serum and lymphocytes of rats infected with Sporothrix schenckii.

    PubMed

    Castro, Verônica S P; Pimentel, Victor C; Da Silva, Aleksandro S; Thomé, Gustavo R; Wolkmer, Patrícia; Castro, Jorge L C; Costa, Márcio M; da Silva, Cássia B; Oliveira, Daniele C; Alves, Sydney H; Schetinger, Maria R C; Lopes, Sonia T A; Mazzanti, Cinthia M

    2012-07-01

    Sporotrichosis is a fungal infection of subcutaneous or chronic evolution, inflammatory lesions characterized by their pyogranulomatous aspect, caused by the dimorphic fungus Sporothrix schenckii. Adenosine deaminase (ADA) is a "key" enzyme in the purine metabolism, promoting the deamination of adenosine, an important anti-inflammatory molecule. The increase in ADA activity has been demonstrated in several inflammatory conditions; however, there are no data in the literature associated with this fungal infection. The objective of this study was to evaluate the activity of serum ADA (S-ADA) and lymphocytes (L-ADA) of rats infected with S. schenckii. We used seventy-eight rats divided into two groups. In the first experiment, rats were infected subcutaneously and in the second experiment, infected intraperitoneally. Blood samples for hematologic evaluation and activities of S-ADA and L-ADA were performed at days 15, 30, and 40 post-infection (PI) to assess disease progression. In the second experiment, it was observed an acute decrease in activity of S-ADA and L-ADA (P < 0.05), suggesting a compensatory mechanism in an attempt to protect the host from excessive tissue damage. With chronicity of disease the rats in the first and second experiment at 30 days PI showed an increased activity of L-ADA (P < 0.05), promoting an inflammatory response in an attempt to combat the spread of the agent. Thus, it is suggested that infection with S. schenckii alters the activities of S-ADA in experimentally infected rats, demonstrating the involvement of this enzyme in the pathogenesis of sporotrichosis.

  2. Activation-Induced Cytidine Deaminase Contributes to Pancreatic Tumorigenesis by Inducing Tumor-Related Gene Mutations.

    PubMed

    Sawai, Yugo; Kodama, Yuzo; Shimizu, Takahiro; Ota, Yuji; Maruno, Takahisa; Eso, Yuji; Kurita, Akira; Shiokawa, Masahiro; Tsuji, Yoshihisa; Uza, Norimitsu; Matsumoto, Yuko; Masui, Toshihiko; Uemoto, Shinji; Marusawa, Hiroyuki; Chiba, Tsutomu

    2015-08-15

    Pancreatic ductal adenocarcinoma (PDAC) develops via an accumulation of various gene mutations. The mechanism underlying the mutations in PDAC development, however, is not fully understood. Recent insight into the close association between the mutation pattern of various cancers and specific mutagens led us to investigate the possible involvement of activation-induced cytidine deaminase (AID), a DNA editing enzyme, in pancreatic tumorigenesis. Our immunohistochemical findings revealed AID protein expression in human acinar ductal metaplasia, pancreatic intraepithelial neoplasia, and PDAC. Both the amount and intensity of the AID protein expression increased with the progression from precancerous to cancerous lesions in human PDAC tissues. To further assess the significance of ectopic epithelial AID expression in pancreatic tumorigenesis, we analyzed the phenotype of AID transgenic (AID Tg) mice. Consistent with our hypothesis that AID is involved in the mechanism of the mutations underlying pancreatic tumorigenesis, we found precancerous lesions developing in the pancreas of AID Tg mice. Using deep sequencing, we also detected Kras and c-Myc mutations in our analysis of the whole pancreas of AID Tg mice. In addition, Sanger sequencing confirmed the presence of Kras, c-Myc, and Smad4 mutations, with the typical mutational footprint of AID in precancerous lesions in AID Tg mice separated by laser capture microdissection. Taken together, our findings suggest that AID contributes to the development of pancreatic precancerous lesions by inducing tumor-related gene mutations. Our new mouse model without intentional manipulation of specific tumor-related genes provides a powerful system for analyzing the mutations involved in PDAC.

  3. Adenosine deaminase in CSF and pleural fluid for diagnosis of tubercular meningitis and pulmonary tuberculosis.

    PubMed

    Nepal, A K; Gyawali, N; Poudel, B; Mahato, R V; Lamsal, M; Gurung, R; Baral, N; Majhi, S

    2012-12-01

    Tuberculosis (TB) is one of the most common infectious diseases in developing countries including Nepal. Delay in diagnosis and treatment of tuberculosis results in poor prognosis of the disease. This study was conducted to estimate diagnostic cut off values of Adenosine Deaminase (ADA) in cerebrospinal fluid (CSF) and pleural fluid and to evaluate the sensitivity, specificity, positive and negative predictive values ofADA in pleural fluid and CSF from patients with tuberculous and non-tuberculous disease. A total of 98 body fluid (CSF: 24, Pleural fluid: 74) specimens were received for the estimation of ADA. ADA activity was measured at 37 degrees C by spectrophotometric method of Guisti and Galanti, 1984 at 625nm wavelength. Among the patients enrolled for the study subjects for which CSF were received (n = 24) included 8 tuberculous meningitis (TBM), and 16 non-tubercular meningitis (NTM). Pleural fluid samples (n = 74) were received from 19 pulmonary TB with pleural effusion, 17 PTB without pleural effusion and 37 of non-tuberculous disease patients. CSF ADA activity were (11. 1 +/- 2.03 IU/L) and (5.3 +/- +1.89 IU/L) (p <00001) in TM and non-NTM groups and Pleural fluid ADA activity were (10 +/- 22.18 IU/L) and (23.79 +/- 11.62 IU/L) (p < 0.001) in PTB and non-TB groups respectively. ADA test in body fluids, which is simple, cost-effective and sensitive, specific for the tubercular disease is recommended to perform before forwarding the cumbersome and expensive procedures like culture and PCR for TB diagnosis. PMID:24579533

  4. Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B

    PubMed Central

    Quist, Jelmar S.; Temiz, Nuri A.; Tutt, Andrew N. J.; Grigoriadis, Anita; Harris, Reuben S.

    2016-01-01

    Molecular, cellular, and clinical studies have combined to demonstrate a contribution from the DNA cytosine deaminase APOBEC3B (A3B) to the overall mutation load in breast, head/neck, lung, bladder, cervical, ovarian, and other cancer types. However, the complete landscape of mutations attributable to this enzyme has yet to be determined in a controlled human cell system. We report a conditional and isogenic system for A3B induction, genomic DNA deamination, and mutagenesis. Human 293-derived cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs. Cells were subjected to 10 rounds of A3B-eGFP exposure that each caused 80–90% cell death. Control pools were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools. Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing. A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations. Surprisingly, both control and A3B-eGFP clones also elicited strong mutator phenotypes characteristic of defective mismatch repair. Despite this additional mutational process, the 293-based system characterized here still yielded a genome-wide view of A3B-catalyzed mutagenesis in human cells and a system for additional studies on the compounded effects of simultaneous mutation mechanisms in cancer cells. PMID:27163364

  5. Mutations in the human adenosine deaminase gene that affect protein structure and RNA splicing

    SciTech Connect

    Akeson, A.L.; Wiginton, D.A.; States, C.J.; Perme, C.M.; Dusing, M.R.; Hutton, J.J.

    1987-08-01

    Adenosine deaminase deficiency is one cause of the genetic disease severe combined immunodeficiency. To identify mutations responsible for ADA deficiency, the authors synthesized cDNAs to ADA mRNAs from two cell lines, GM2756 and GM2825A, derived from ADA-deficient immunodeficient patients. Sequence analysis of GM2756 cDNA clones revealed a different point mutation in each allele that causes amino acid changes of alanine to valine and arginine to histidine. One allele of GM2825A also has a point mutation that causes an alanine to valine substitution. The other allele of GM2825A was found to produce an mRNA in which exon 4 had been spliced out but had no other detrimental mutations. S1 nuclease mapping of GM2825A mRNA showed equal abundance of the full-length ADA mRNA and the ADA mRNA that was missing exon 4. Several of the ADA cDNA clones extended 5' of the major initiation start site, indicating multiple start sites for ADA transcription. The point mutations in GM2756 and GM2825A and the absence of exon 4 in GM2825A appear to be directly responsible for the ADA deficiency. Comparison of a number of normal and mutant ADA cDNA sequences showed a number of changes in the third base of codons. These change do not affect the amino acid sequence. Analyses of ADA cDNAs from different cell lines detected aberrant RNA species that either included intron 7 or excluded exon 7. Their presence is a result of aberrant splicing of pre-mRNAs and is not related to mutations that cause ADA deficiency.

  6. Ascitic fluid gamma interferon concentrations and adenosine deaminase activity in tuberculous peritonitis.

    PubMed Central

    Sathar, M A; Simjee, A E; Coovadia, Y M; Soni, P N; Moola, S A; Insam, B; Makumbi, F

    1995-01-01

    The gamma interferon (gamma-IFN) concentration and the adenosine deaminase (ADA) activity were evaluated in 30 patients with tuberculous peritonitis, 21 patients with ascites due to a malignant disorder, and 41 patients with cirrhosis. The gamma-IFN concentrations were significantly higher (p < 0.0001) in tuberculous peritonitis patients (mean: 6.70 U/ml) than in the malignant (mean: 3.10 U/ml) and cirrhotic (mean: 3.08 U/ml) groups. Use of a cut off value of > or = 3.2 U/ml gave the assay a sensitivity of 93% (25 of 27), a specificity of 98% (54 of 55), positive (P+) and negative (P-) predictive values of 96% and a test accuracy of 96%. The ADA activity was significantly (p < 0.0001) higher in the tuberculous peritonitis group (mean: 101.84 U/l) than in the control groups (cirrhosis (mean: 13.49 U/l) and malignancy (mean: 19.35 U/l)). A cut off value of > 30 U/l gave the ADA test a sensitivity of 93% (26 of 28) a specificity of 96% (51 of 53), a (P+) value of 93%, a (P-) value of 96%, and a test accuracy of 95%. There was a significant (p < 0.0001) correlation (r = 0.72) between ADA activity and gamma-IFN values in patients with tuberculous peritonitis. These results show that a high concentration of gamma-IFN in ascitic fluid is as valuable as the ADA activity in the diagnosis of tuberculous peritonitis. Both are rapid non-invasive diagnostic tests for tuberculous peritonitis. PMID:7698702

  7. Structural flexibility, an essential component of the allosteric activation in Escherichia coli glucosamine-6-phosphate deaminase.

    PubMed

    Rudiño-Piñera, E; Morales-Arrieta, S; Rojas-Trejo, S P; Horjales, E

    2002-01-01

    A new crystallographic structure of the free active-site R conformer of the allosteric enzyme glucosamine-6-phosphate deaminase from Escherichia coli, coupled with previously reported structures of the T and R conformers, generates a detailed description of the heterotropic allosteric transition in which structural flexibility plays a central role. The T conformer's external zone [Horjales et al. (1999), Structure, 7, 527-536] presents higher B values than in the R conformers. The ligand-free enzyme (T conformer) undergoes an allosteric transition to the free active-site R conformer upon binding of the allosteric activator. This structure shows three alternate conformations of the mobile section of the active-site lid (residues 163-182), in comparison to the high B values for the unique conformation of the T conformer. One of these alternate R conformations corresponds to the active-site lid found when the substrate is bound. The disorder associated with the three alternate conformations can be related to the biological regulation of the K(m) of the enzyme for the reaction, which is metabolically required to maintain adequate concentrations of the activator, which holds the enzyme in its R state. Seven alternate conformations for the active-site lid and three for the C-terminus were refined for the T structure using isotropic B factors. Some of these conformers approach that of the R conformer in geometry. Furthermore, the direction of the atomic vibrations obtained with anisotropic B refinement supports the hypothesis of an oscillating rather than a tense T state. The concerted character of the allosteric transition is also analysed in view of the apparent dynamics of the conformers.

  8. Iron inhibits activation-induced cytidine deaminase enzymatic activity and modulates immunoglobulin class switch DNA recombination.

    PubMed

    Li, Guideng; Pone, Egest J; Tran, Daniel C; Patel, Pina J; Dao, Lisa; Xu, Zhenming; Casali, Paolo

    2012-06-15

    Immunoglobulin (Ig) class switch DNA recombination (CSR) and somatic hypermutation (SHM) are critical for the maturation of the antibody response. Activation-induced cytidine deaminase (AID) initiates CSR and SHM by deaminating deoxycytidines (dCs) in switch (S) and V(D)J region DNA, respectively, to generate deoxyuracils (dUs). Processing of dUs by uracil DNA glycosylase (UNG) yields abasic sites, which are excised by apurinic/apyrimidinic endonucleases, eventually generating double strand DNA breaks, the obligatory intermediates of CSR. Here, we found that the bivalent iron ion (Fe(2+), ferrous) suppressed CSR, leading to decreased number of switched B cells, decreased postrecombination Iμ-C(H) transcripts, and reduced titers of secreted class-switched IgG1, IgG3, and IgA antibodies, without alterations in critical CSR factors, such as AID, 14-3-3γ, or PTIP, or in general germline I(H)-S-C(H) transcription. Fe(2+) did not affect B cell proliferation or plasmacytoid differentiation. Rather, it inhibited AID-mediated dC deamination in a dose-dependent fashion. The inhibition of intrinsic AID enzymatic activity by Fe(2+) was specific, as shown by lack of inhibition of AID-mediated dC deamination by other bivalent metal ions, such as Zn(2+), Mn(2+), Mg(2+), or Ni(2+), and the inability of Fe(2+) to inhibit UNG-mediated dU excision. Overall, our findings have outlined a novel role of iron in modulating a B cell differentiation process that is critical to the generation of effective antibody responses to microbial pathogens and tumoral cells. They also suggest a possible role of iron in dampening AID-dependent autoimmunity and neoplastic transformation.

  9. The PurR mutation of Drosophila melanogaster confers resistance to purine and 2,6-diaminopurine by elevating adenosine deaminase activity.

    PubMed

    Dutton, F L; Chovnick, A

    1990-01-01

    Media supplemented with purine (7H-imidazo[4,5-d]pyrimidine) or the purine analogue 2,6-diaminopurine (DAP) can be employed to select several classes of purine-resistant variants from mutagenized cultures of Drosophila. One class results in elevated resistance to purine and diaminopurine which is correlated with elevated activity of the enzyme adenosine deaminase (adenosine aminohydrolase = EC 3.5.4.4). The first member of this class, Pur R, maps to position 82 +/- in the right arm of the second chromosome. The Pur R mutation causes an elevation of adenosine deaminase (ADA) enzyme activity, apparently by altering a thermolabile, ADA-specific repressor. Pur R may thus encode a negative regulator of adenosine deaminase activity similar to the ADA-binding protein found in mammalian systems.

  10. Crystallization and preliminary X-ray crystallographic analysis of biodegradative threonine deaminase (TdcB) from Salmonella typhimurium

    SciTech Connect

    Simanshu, Dhirendra K.; Chittori, Sagar; Savithri, H. S.; Murthy, M. R. N.

    2006-03-01

    S. typhimurium biodegradative threonine deaminase (TdcB), a member of the β-family of PLP-dependent enzymes, has been overexpressed, purified and crystallized in three different crystal forms using the hanging-drop vapour-diffusion method. Biodegradative threonine deaminase (TdcB) catalyzes the deamination of l-threonine to α-ketobutyrate, the first reaction in the anaerobic breakdown of l-threonine to propionate. Unlike the biosynthetic threonine deaminase, TdcB is insensitive to l-isoleucine and is activated by AMP. Here, the cloning of TdcB (molecular weight 36 kDa) from Salmonella typhimurium with an N-terminal hexahistidine affinity tag and its overexpression in Escherichia coli is reported. TdcB was purified to homogeneity using Ni–NTA affinity column chromatography and crystallized using the hanging-drop vapour-diffusion technique in three different crystal forms. Crystal forms I (unit-cell parameters a = 46.32, b = 55.30, c = 67.24 Å, α = 103.09, β = 94.70, γ = 112.94°) and II (a = 56.68, b = 76.83, c = 78.50 Å, α = 66.12, β = 89.16, γ = 77.08°) belong to space group P1 and contain two and four molecules of TdcB, respectively, in the asymmetric unit. Poorly diffracting form III crystals were obtained in space group C2 and based on the unit-cell volume are most likely to contain one molecule per asymmetric unit. Two complete data sets of resolutions 2.2 Å (crystal form I) and 1.7 Å (crystal form II) were collected at 100 K using an in-house X-ray source.

  11. Crystallization and preliminary X-ray characterization of the tetrapyrrole-biosynthetic enzyme porphobilinogen deaminase from Bacillus megaterium

    PubMed Central

    Azim, N.; Deery, E.; Warren, M. J.; Erskine, P.; Cooper, J. B.; Wood, S. P.; Akhtar, M.

    2013-01-01

    The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses an early step of the tetrapyrrole-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor which is covalently linked by a thioether bridge to an invariant cysteine residue. Expression in Escherichia coli of a His-tagged form of Bacillus megaterium PBGD permitted the crystallization and preliminary X-ray analysis of the enzyme from this species at high resolution. PMID:23908040

  12. Hyperbilirubinemia and rapid fatal hepatic failure in severe combined immunodeficiency caused by adenosine deaminase deficiency (ADA-SCID).

    PubMed

    Kühl, J S; Schwarz, K; Münch, A; Schmugge, M; Pekrun, A; Meisel, C; Wahn, V; Ebell, W; von Bernuth, H

    2011-03-01

    Adenosin deaminase (ADA) deficiency is the cause for Severe Combined Immunodeficiency (SCID) in about 15% of patients with SCID, often presenting as T (-)B (-)NK (-)SCID. Treatment options for ADA-SCID are enzyme replacement, bone marrow transplantation or gene therapy. We here describe the first patient with ADA-SCID and fatal hepatic failure despite bone marrow transplantation from a 10/10 HLA identical related donor. As patients with ADA-SCID may be at yet underestimated increased risk for rapid hepatic failure we speculate whether hepatitis in ADA-SCID should lead to the immediate treatment with enzyme replacement by pegylated ADA.

  13. Adenosine Deaminase Acts as a Natural Antagonist for Dipeptidyl Peptidase 4-Mediated Entry of the Middle East Respiratory Syndrome Coronavirus

    PubMed Central

    Raj, V. Stalin; Smits, Saskia L.; Provacia, Lisette B.; van den Brand, Judith M. A.; Wiersma, Lidewij; Ouwendijk, Werner J. D.; Bestebroer, Theo M.; Spronken, Monique I.; van Amerongen, Geert; Rottier, Peter J. M.; Fouchier, Ron A. M.; Bosch, Berend Jan; Osterhaus, Albert D.M.E.

    2014-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) replicates in cells of different species using dipeptidyl peptidase 4 (DPP4) as a functional receptor. Here we show the resistance of ferrets to MERS-CoV infection and inability of ferret DDP4 to bind MERS-CoV. Site-directed mutagenesis of amino acids variable in ferret DPP4 thus revealed the functional human DPP4 virus binding site. Adenosine deaminase (ADA), a DPP4 binding protein, competed for virus binding, acting as a natural antagonist for MERS-CoV infection. PMID:24257613

  14. Adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the Middle East respiratory syndrome coronavirus.

    PubMed

    Raj, V Stalin; Smits, Saskia L; Provacia, Lisette B; van den Brand, Judith M A; Wiersma, Lidewij; Ouwendijk, Werner J D; Bestebroer, Theo M; Spronken, Monique I; van Amerongen, Geert; Rottier, Peter J M; Fouchier, Ron A M; Bosch, Berend Jan; Osterhaus, Albert D M E; Haagmans, Bart L

    2014-02-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) replicates in cells of different species using dipeptidyl peptidase 4 (DPP4) as a functional receptor. Here we show the resistance of ferrets to MERS-CoV infection and inability of ferret DDP4 to bind MERS-CoV. Site-directed mutagenesis of amino acids variable in ferret DPP4 thus revealed the functional human DPP4 virus binding site. Adenosine deaminase (ADA), a DPP4 binding protein, competed for virus binding, acting as a natural antagonist for MERS-CoV infection. PMID:24257613

  15. Studies on Plant Growth Promoting Properties of Fruit-Associated Bacteria from Elettaria cardamomum and Molecular Analysis of ACC Deaminase Gene.

    PubMed

    Jasim, B; Anish, Mathew Chacko; Shimil, Vellakudiyan; Jyothis, Mathew; Radhakrishnan, E K

    2015-09-01

    Endophytic microorganisms have been reported to have diverse plant growth promoting mechanisms including phosphate solubilization, N2 fixation, production of phyto-hormones and ACC (1-aminocyclopropane-1-carboxylate) deaminase and antiphyto-pathogenic properties. Among these, ACC deaminase production is very important because of its regulatory effect on ethylene which is a stress hormone with precise role in the control of fruit development and ripening. However, distribution of these properties among various endophytic bacteria associated with fruit tissue and its genetic basis is least investigated. In the current study, 11 endophytic bacteria were isolated and identified from the fruit tissue of Elettaria cardamomum and were studied in detail for various plant growth promoting properties especially ACC deaminase activity using both culture-based and PCR-based methods. PCR-based screening identified the isolates EcB 2 (Pantoea sp.), EcB 7 (Polaromonas sp.), EcB 9 (Pseudomonas sp.), EcB 10 (Pseudomonas sp.) and EcB 11 (Ralstonia sp.) as positive for ACC deaminase. The PCR products were further subjected to sequence analysis which proved the similarity of the sequences identified in the study with ACC deaminase sequences reported from other sources. The detailed bioinformatic analysis of the sequence including homology-based modelling and molecular docking confirmed the sequences to have ACC deaminase activity. The docking of the modelled proteins was done using patch dock, and the detailed scrutiny of the protein ligand interaction revealed conservation of key amino acids like Lys51, Ser78, Tyr268 and Tyr294 which play important role in the enzyme activity. These suggest the possible regulatory effect of these isolates on fruit physiology. PMID:26164855

  16. A conserved glutamate residue in the C-terminal deaminase domain of pentatricopeptide repeat proteins is required for RNA editing activity.

    PubMed

    Hayes, Michael L; Dang, Kim N; Diaz, Michael F; Mulligan, R Michael

    2015-04-17

    Many transcripts expressed from plant organelle genomes are modified by C-to-U RNA editing. Nuclear encoded pentatricopeptide repeat (PPR) proteins include an RNA binding domain that provides site specificity. In addition, many PPR proteins include a C-terminal DYW deaminase domain with characteristic zinc binding motifs (CXXC, HXE) and has recently been shown to bind zinc ions. The glutamate residue of the HXE motif is catalytically required in the reaction catalyzed by cytidine deaminase. In this work, we examine the activity of the DYW deaminase domain through truncation or mutagenesis of the HXE motif. OTP84 is required for editing three chloroplast sites, and transgenes expressing OTP84 with C-terminal truncations were capable of editing only one of the three cognate sites at high efficiency. These results suggest that the deaminase domain of OTP84 is required for editing two of the sites, but another deaminase is able to supply the deamination activity for the third site. OTP84 and CREF7 transgenes were mutagenized to replace the glutamate residue of the HXE motif, and transgenic plants expressing OTP84-E824A and CREF7-E554A were unable to efficiently edit the cognate editing sites for these genes. In addition, plants expressing CREF7-E554A exhibited substantially reduced capacity to edit a non-cognate site, rpoA C200. These results indicate that the DYW deaminase domains of PPR proteins are involved in editing their cognate editing sites, and in some cases may participate in editing additional sites in the chloroplast. PMID:25739442

  17. Studies on Plant Growth Promoting Properties of Fruit-Associated Bacteria from Elettaria cardamomum and Molecular Analysis of ACC Deaminase Gene.

    PubMed

    Jasim, B; Anish, Mathew Chacko; Shimil, Vellakudiyan; Jyothis, Mathew; Radhakrishnan, E K

    2015-09-01

    Endophytic microorganisms have been reported to have diverse plant growth promoting mechanisms including phosphate solubilization, N2 fixation, production of phyto-hormones and ACC (1-aminocyclopropane-1-carboxylate) deaminase and antiphyto-pathogenic properties. Among these, ACC deaminase production is very important because of its regulatory effect on ethylene which is a stress hormone with precise role in the control of fruit development and ripening. However, distribution of these properties among various endophytic bacteria associated with fruit tissue and its genetic basis is least investigated. In the current study, 11 endophytic bacteria were isolated and identified from the fruit tissue of Elettaria cardamomum and were studied in detail for various plant growth promoting properties especially ACC deaminase activity using both culture-based and PCR-based methods. PCR-based screening identified the isolates EcB 2 (Pantoea sp.), EcB 7 (Polaromonas sp.), EcB 9 (Pseudomonas sp.), EcB 10 (Pseudomonas sp.) and EcB 11 (Ralstonia sp.) as positive for ACC deaminase. The PCR products were further subjected to sequence analysis which proved the similarity of the sequences identified in the study with ACC deaminase sequences reported from other sources. The detailed bioinformatic analysis of the sequence including homology-based modelling and molecular docking confirmed the sequences to have ACC deaminase activity. The docking of the modelled proteins was done using patch dock, and the detailed scrutiny of the protein ligand interaction revealed conservation of key amino acids like Lys51, Ser78, Tyr268 and Tyr294 which play important role in the enzyme activity. These suggest the possible regulatory effect of these isolates on fruit physiology.

  18. Real-Time PCR in HIV/Trypanosoma cruzi Coinfection with and without Chagas Disease Reactivation: Association with HIV Viral Load and CD4+ Level

    PubMed Central

    de Freitas, Vera Lúcia Teixeira; da Silva, Sheila Cristina Vicente; Sartori, Ana Marli; Bezerra, Rita Cristina; Westphalen, Elizabeth Visone Nunes; Molina, Tatiane Decaris; Teixeira, Antonio R. L.; Ibrahim, Karim Yaqub; Shikanai-Yasuda, Maria Aparecida

    2011-01-01

    Background Reactivation of chronic Chagas disease, which occurs in approximately 20% of patients coinfected with HIV/Trypanosoma cruzi (T. cruzi), is commonly characterized by severe meningoencephalitis and myocarditis. The use of quantitative molecular tests to monitor Chagas disease reactivation was analyzed. Methodology Polymerase chain reaction (PCR) of kDNA sequences, competitive (C-) PCR and real-time quantitative (q) PCR were compared with blood cultures and xenodiagnosis in samples from 91 patients (57 patients with chronic Chagas disease and 34 with HIV/T. cruzi coinfection), of whom 5 had reactivation of Chagas disease and 29 did not. Principal Findings qRT-PCR showed significant differences between groups; the highest parasitemia was observed in patients infected with HIV/T. cruzi with Chagas disease reactivation (median 1428.90 T. cruzi/mL), followed by patients with HIV/T. cruzi infection without reactivation (median 1.57 T. cruzi/mL) and patients with Chagas disease without HIV (median 0.00 T. cruzi/mL). Spearman's correlation coefficient showed that xenodiagnosis was correlated with blood culture, C-PCR and qRT-PCR. A stronger Spearman correlation index was found between C-PCR and qRT-PCR, the number of parasites and the HIV viral load, expressed as the number of CD4+ cells or the CD4+/CD8+ ratio. Conclusions qRT-PCR distinguished the groups of HIV/T. cruzi coinfected patients with and without reactivation. Therefore, this new method of qRT-PCR is proposed as a tool for prospective studies to analyze the importance of parasitemia (persistent and/or increased) as a criterion for recommending pre-emptive therapy in patients with chronic Chagas disease with HIV infection or immunosuppression. As seen in this study, an increase in HIV viral load and decreases in the number of CD4+ cells/mm3 and the CD4+/CD8+ ratio were identified as cofactors for increased parasitemia that can be used to target the introduction of early, pre-emptive therapy. PMID

  19. Differentiation and stability of T helper 1 and 2 cells derived from naive human neonatal CD4+ T cells, analyzed at the single-cell level

    PubMed Central

    1996-01-01

    The development of CD4+ T helper (Th) type 1 and 2 cells is essential for the eradication of pathogens, but can also be responsible for various pathological disorders. Therefore, modulation of Th cell differentiation may have clinical utility in the treatment of human disease. Here, we show that interleukin (IL) 12 and IL-4 directly induce human neonatal CD4- T cells, activated via CD3 and CD28, to differentiate into Th1 and Th2 subsets. In contrast, IL-13, which shares many biological activities with IL-4, failed to induce T cell differentiation, consistent with the observation that human T cells do not express IL-13 receptors. Both the IL-12-induced Th1 subset and the IL-4-induced Th2 subset produce large quantities of IL-10, confirming that human IL-10 is not a typical human Th2 cytokine. Interestingly, IL- 4-driven Th2 cell differentiation was completely prevented by an IL-4 mutant protein (IL-4.Y124D), indicating that this molecule acts as a strong IL-4 receptor antagonist. Analysis of single T cells producing interferon gamma or IL-4 revealed that induction of Th1 cell differentiation occurred rapidly and required only 4 d of priming of the neonatal CD4+ T cells in the presence of IL-12. The IL-12-induced Th1 cell phenotype was stable and was not significantly affected when repeatedly stimulated in the presence of recombinant IL-4. In contrast, the differentiation of Th2 cells occurred slowly and required not only 6 d of priming, but also additional restimulation of the primed CD4+ T cells in the presence of IL-4. Moreover, IL-4-induced Th2 cell phenotypes were not stable and could rapidly be reverted into a population predominantly containing Th0 and Th1 cells, after a single restimulation in the presence of IL-12. The observed differences in stability of IL-12- and IL-4-induced human Th1 and Th2 subsets, respectively, may have implications for cytokine-based therapies of chronic disease. PMID:8760801

  20. Attrition of TCR Vα7.2+ CD161++ MAIT Cells in HIV-Tuberculosis Co-Infection Is Associated with Elevated Levels of PD-1 Expression

    PubMed Central

    Saeidi, Alireza; Tien Tien, Vicky L.; Al-Batran, Rami; Al-Darraji, Haider A.; Tan, Hong Y.; Yong, Yean K.; Ponnampalavanar, Sasheela; Barathan, Muttiah; Rukumani, Devi V.; Ansari, Abdul W.; Velu, Vijayakumar; Kamarulzaman, Adeeba; Larsson, Marie; Shankar, Esaki M.

    2015-01-01

    Mucosal-associated invariant T (MAIT) cells are evolutionarily conserved antimicrobial MR1-restricted CD8+ T cells co-expressing the semi-invariant TCR Vα7.2, and are numerous in the blood and mucosal tissues of humans. MAIT cells appear to undergo exhaustion in chronic viral infections. However, their role in human immunodeficiency virus type 1 (HIV-1) mono-infection and HIV/tuberculosis (TB) co-infection have seldom been elaborately investigated. We conducted a cross-sectional study to investigate the frequencies and phenotypes of CD161++CD8+ T cells among anti-retroviral therapy (ART)/anti-TB therapy (ATT) treatment-naïve HIV/TB co-infected, ART/TB treated HIV/TB co-infected, ART naïve HIV-infected, ART-treated HIV-infected patients, and HIV negative healthy controls (HCs) by flow cytometry. Our data revealed that the frequency of MAIT cells was severely depleted in HIV mono- and HIV/TB co-infections. Further, PD-1 expression on MAIT cells was significantly increased in HIV mono- and HIV-TB co-infected patients. The frequency of MAIT cells did not show any significant increase despite the initiation of ART and/or ATT. Majority of the MAIT cells in HCs showed a significant increase in CCR6 expression as compared to HIV/TB co-infections. No marked difference was seen with expressions of chemokine co-receptor CCR5 and CD103 among the study groups. Decrease of CCR6 expression appears to explain why HIV-infected patients display weakened mucosal immune responses. PMID:25894562

  1. CD4 cell count and CD4/CD8 ratio increase during rituximab maintenance in granulomatosis with polyangiitis patients

    PubMed Central

    Nossent, Johannes C.

    2016-01-01

    Introduction Rituximab (RTX) is a B cell-depleting agent approved for the treatment of granulomatosis with polyangiitis (GPA). RTX reduces antibody producing precursor plasma cells and inhibits B and T cells interaction. Infections related to T cell immunodeficiency are not infrequent during RTX treatment. Our study investigated CD4 cell count and CD4/CD8 ratio in GPA patients during the first two years of long-term RTX treatment. Methods A single centre cohort study of 35 patients who received median total cumulative dose of cyclophosphamide (CYC) of 15 g and were treated with RTX 2 g followed by retreatment with either 2 g once annually or 1 g biannually. Serum levels of total immunoglobulin (Ig) and lymphocytes subsets were recorded at RTX initiation and at 3, 6, 12, 18 and 24 months. Low CD4 count and inverted CD4/CD8 ratio were defined as CD4 < 0.3 × 109/l and ratio < 1. Results The CD4 cell count and CD4/CD8 ratio decreased slightly following the initial RTX treatment and then increased gradually during maintenance treatment. While the proportion of patients with low CD4 cell count decreased from 43% at baseline to 18% at 24 months, the ratio remained inverted in 40%. Oral daily prednisolone dose at baseline, CYC exposure and the maintenance regimen did not influence the CD4 cell count and ratio. Being older (p = 0.012) and having a higher CRP (p = 0.044) and ESR (p = 0.024) at baseline significantly increased the risk of inverted CD4/CD8 ratio at 24 months. Inverted ratio at baseline associated with lower total Ig levels during the study. Conclusions Overall, the CD4 and CD4/CD8 ratio increased during maintenance RTX therapy in GPA with no discernible impact of other immunosuppressive therapy. However the increase in CD4 was not followed by an increase in the CD4/CD8 ratio, especially in older patients. Inverted CD4/CD8 ratio associated with lower Ig levels, suggesting a more profound B cell depleting effect of RTX with a relative increase in CD8

  2. CD4 cell count and CD4/CD8 ratio increase during rituximab maintenance in granulomatosis with polyangiitis patients

    PubMed Central

    Nossent, Johannes C.

    2016-01-01

    Introduction Rituximab (RTX) is a B cell-depleting agent approved for the treatment of granulomatosis with polyangiitis (GPA). RTX reduces antibody producing precursor plasma cells and inhibits B and T cells interaction. Infections related to T cell immunodeficiency are not infrequent during RTX treatment. Our study investigated CD4 cell count and CD4/CD8 ratio in GPA patients during the first two years of long-term RTX treatment. Methods A single centre cohort study of 35 patients who received median total cumulative dose of cyclophosphamide (CYC) of 15 g and were treated with RTX 2 g followed by retreatment with either 2 g once annually or 1 g biannually. Serum levels of total immunoglobulin (Ig) and lymphocytes subsets were recorded at RTX initiation and at 3, 6, 12, 18 and 24 months. Low CD4 count and inverted CD4/CD8 ratio were defined as CD4 < 0.3 × 109/l and ratio < 1. Results The CD4 cell count and CD4/CD8 ratio decreased slightly following the initial RTX treatment and then increased gradually during maintenance treatment. While the proportion of patients with low CD4 cell count decreased from 43% at baseline to 18% at 24 months, the ratio remained inverted in 40%. Oral daily prednisolone dose at baseline, CYC exposure and the maintenance regimen did not influence the CD4 cell count and ratio. Being older (p = 0.012) and having a higher CRP (p = 0.044) and ESR (p = 0.024) at baseline significantly increased the risk of inverted CD4/CD8 ratio at 24 months. Inverted ratio at baseline associated with lower total Ig levels during the study. Conclusions Overall, the CD4 and CD4/CD8 ratio increased during maintenance RTX therapy in GPA with no discernible impact of other immunosuppressive therapy. However the increase in CD4 was not followed by an increase in the CD4/CD8 ratio, especially in older patients. Inverted CD4/CD8 ratio associated with lower Ig levels, suggesting a more profound B cell depleting effect of RTX with a relative increase in CD8

  3. Cellular HIV-1 Inhibition by Truncated Old World Primate APOBEC3A Proteins Lacking a Complete Deaminase Domain

    PubMed Central

    Katuwal, Miki; Wang, Yaqiong; Schmitt, Kimberly; Guo, Kejun; Halemano, Kalani; Santiago, Mario L.; Stephens, Edward B.

    2014-01-01

    The APOBEC3 (A3) deaminases are retrovirus restriction factors that were proposed as inhibitory components of HIV-1 gene therapy vectors. However, A3 mutational activity may induce undesired genomic damage and enable HIV-1 to evade drugs and immune responses. Here, we show that A3A protein from Colobus guereza (colA3A) can restrict HIV-1 replication in producer cells in a deaminase-independent manner without inducing DNA damage. Neither HIV-1 reverse transcription nor integration were significantly affected by colA3A, but capsid protein synthesis was inhibited. The determinants for colA3A restriction mapped to the N-terminal region. These properties extend to A3A from mandrills and De Brazza’s monkeys. Surprisingly, truncated colA3A proteins expressing only the N-terminal 100 amino acids effectively exclude critical catalytic regions but retained potent cellular restriction activity. These highlight a unique mechanism of cellular HIV-1 restriction by several Old World monkey A3A proteins that may be exploited for functional HIV-1 cure strategies. PMID:25262471

  4. Crystallization and preliminary X-ray crystallographic analysis of the tRNA-specific adenosine deaminase from Streptococcus pyogenes

    SciTech Connect

    Ku, Min-Je; Lee, Won-Ho; Nam, Ki-hyun; Rhee, Kyeong-hee; Lee, Ki-Seog; Kim, Eunice EunKyung; Yu, Myung-Hee; Hwang, Kwang Yeon

    2005-04-01

    The tRNA-specific adenosine deaminase from the pathogenic bacteria S. pyogenes has been overexpressed and crystallized. The tRNA-specific adenosine deaminase from the pathogenic bacteria Streptococcus pyogenes (spTAD) has been overexpressed in Escherichia coli and crystallized in the presence of Zn{sup 2+} ion at 295 K using ammonium sulfate as a precipitant. Flash-cooled crystals of spTAD diffracted to 2.0 Å using 30%(v/v) glycerol as a cryoprotectant. X-ray diffraction data have been collected to 2.0 Å using synchrotron radiation. The crystal belongs to the tetragonal space group P4{sub 2}2{sub 1}2, with unit-cell parameters a = b = 81.042, c = 81.270 Å. The asymmetric unit contains one subunit of spTAD, with a corresponding crystal volume per protein weight (V{sub M}) of 3.3 Å{sup 3} Da{sup −1} and a solvent content of 62.7%.

  5. First-In-Class Small Molecule Inhibitors of the Single-Strand DNA Cytosine Deaminase APOBEC3G

    PubMed Central

    Li, Ming; Shandilya, Shivender M.D.; Carpenter, Michael A.; Rathore, Anurag; Brown, William L.; Perkins, Angela L.; Harki, Daniel A.; Solberg, Jonathan; Hook, Derek J.; Pandey, Krishan K.; Parniak, Michael A.; Johnson, Jeffrey R.; Krogan, Nevan J.; Somasundaran, Mohan; Ali, Akbar; Schiffer, Celia A.; Harris, Reuben S.

    2012-01-01

    APOBEC3G is a single-stranded DNA cytosine deaminase that comprises part of the innate immune response to viruses and transposons. Although APOBEC3G is the prototype for understanding the larger mammalian polynucleotide deaminase family, no specific chemical inhibitors exist to modulate its activity. High-throughput screening identified 34 compounds that inhibit APOBEC3G catalytic activity. 20/34 small molecules contained catechol moieties, which are known to be sulfhydryl reactive following oxidation to the orthoquinone. Located proximal to the active site, C321 was identified as the binding site for the inhibitors by a combination of mutational screening, structural analysis, and mass spectrometry. Bulkier substitutions C321-to-L, F, Y, or W mimicked chemical inhibition. A strong specificity for APOBEC3G was evident, as most compounds failed to inhibit the related APOBEC3A enzyme or the unrelated enzymes E. coli uracil DNA glycosylase, HIV-1 RNase H, or HIV-1 integrase. Partial, but not complete, sensitivity could be conferred to APOBEC3A by introducing the entire C321 loop from APOBEC3G. Thus, a structural model is presented in which the mechanism of inhibition is both specific and competitive, by binding a pocket adjacent to the APOBEC3G active site, reacting with C321, and blocking access substrate DNA cytosines. PMID:22181350

  6. 1-aminocyclopropane-1-carboxylate deaminase from Pseudomonas fluorescens promoting the growth of Chinese cabbage and its polyclonal antibody.

    PubMed

    Soh, Byoung Yul; Lee, Gun Woong; Go, Eun Byeul; Kim, Byeo-Ri; Lee, Kui-Jae; Chae, Jong-Chan

    2014-05-01

    Bacterial 1-aminocyclopropane-1-carboxlyate (ACC) deaminase (AcdS) is an enzyme that cleaves ACC, a precursor of the plant hormone ethylene, into α-ketobutyrate and ammonia. The acdS gene was cloned from Pseudomonas fluorescens, which was capable of improving the seedling of Chinese cabbage under salinity condition. The recombinant AcdS (rAcdS) exhibited optimal activity at pH 8.5 and 30°C. Strong activity was sustained at up to 100 mM NaCl. The polyclonal anti-P. fluorescens AcdS antibody was produced in a rabbit that had been immunized with the purified rAcdS. This antibody successfully recognized the homologous antigens derived from the total proteins of isolated plant growth-promoting microorganisms. A statistically significant correlation was observed between the intensity of hybridization signal and AcdS activity measured by a biochemical method, suggesting its application as a useful indicator for active deaminases.

  7. First-In-Class Small Molecule Inhibitors of the Single-Strand DNA Cytosine Deaminase APOBEC3G

    SciTech Connect

    Li, Ming; Shandilya, Shivender M.D.; Carpenter, Michael A.; Rathore, Anurag; Brown, William L.; Perkins, Angela L.; Harki, Daniel A.; Solberg, Jonathan; Hook, Derek J.; Pandey, Krishan K.; Parniak, Michael A.; Johnson, Jeffrey R.; Krogan, Nevan J.; Somasundaran, Mohan; Ali, Akbar; Schiffer, Celia A.; Harris, Reuben S.

    2012-04-04

    APOBEC3G is a single-stranded DNA cytosine deaminase that comprises part of the innate immune response to viruses and transposons. Although APOBEC3G is the prototype for understanding the larger mammalian polynucleotide deaminase family, no specific chemical inhibitors exist to modulate its activity. High-throughput screening identified 34 compounds that inhibit APOBEC3G catalytic activity. Twenty of 34 small molecules contained catechol moieties, which are known to be sulfhydryl reactive following oxidation to the orthoquinone. Located proximal to the active site, C321 was identified as the binding site for the inhibitors by a combination of mutational screening, structural analysis, and mass spectrometry. Bulkier substitutions C321-to-L, F, Y, or W mimicked chemical inhibition. A strong specificity for APOBEC3G was evident, as most compounds failed to inhibit the related APOBEC3A enzyme or the unrelated enzymes E. coli uracil DNA glycosylase, HIV-1 RNase H, or HIV-1 integrase. Partial, but not complete, sensitivity could be conferred to APOBEC3A by introducing the entire C321 loop from APOBEC3G. Thus, a structural model is presented in which the mechanism of inhibition is both specific and competitive, by binding a pocket adjacent to the APOBEC3G active site, reacting with C321, and blocking access to substrate DNA cytosines.

  8. The effect of adenosine deaminase inhibition on the A1 adenosinergic and M2 muscarinergic control of contractility in eu- and hyperthyroid guinea pig atria.

    PubMed

    Pak, Krisztian; Zsuga, Judit; Kepes, Zita; Erdei, Tamas; Varga, Balazs; Juhasz, Bela; Szentmiklosi, Andras Jozsef; Gesztelyi, Rudolf

    2015-08-01

    The A1 adenosine and M2 muscarinic receptors exert protective (including energy consumption limiting) effects in the heart. We investigated the influence of adenosine deaminase (ADA) inhibition on a representative energy consumption limiting function, the direct negative inotropic effect elicited by the A1 adenosinergic and M2 muscarinergic systems, in eu- and hyperthyroid atria. Furthermore, we compared the change in the interstitial adenosine level caused by ADA inhibition and nucleoside transport blockade, two well-established processes to stimulate the cell surface A1 adenosine receptors, in both thyroid states. A classical isolated organ technique was applied supplemented with the receptorial responsiveness method (RRM), a concentration estimating procedure. Via measuring the contractile force, the direct negative inotropic capacity of N(6)-cyclopentyladenosine, a selective A1 receptor agonist, and methacholine, a muscarinic receptor agonist, was determined on the left atria isolated from 8-day solvent- and thyroxine-treated guinea pigs in the presence and absence of 2'-deoxycoformycin, a selective ADA inhibitor, and NBTI, a selective nucleoside transporter inhibitor. We found that ADA inhibition (but not nucleoside transport blockade) increased the signal amplification of the A1 adenosinergic (but not M2 muscarinergic) system. This action of ADA inhibition developed in both thyroid states, but it was greater in hyperthyroidism. Nevertheless, ADA inhibition produced a smaller rise in the interstitial adenosine concentration than nucleoside transport blockade did in both thyroid states. Our results indicate that ADA inhibition, besides increasing the interstitial adenosine level, intensifies the atrial A1 adenosinergic function in another (thyroid hormone-sensitive) way, suggesting a new mechanism of action of ADA inhibition. PMID:25877465

  9. Estrogen receptors bind to and activate the HOXC4/HoxC4 promoter to potentiate HoxC4-mediated activation-induced cytosine deaminase induction, immunoglobulin class switch DNA recombination, and somatic hypermutation.

    PubMed

    Mai, Thach; Zan, Hong; Zhang, Jinsong; Hawkins, J Seth; Xu, Zhenming; Casali, Paolo

    2010-11-26

    Estrogen enhances antibody and autoantibody responses through yet to be defined mechanisms. It has been suggested that estrogen up-regulates the expression of activation-induced cytosine deaminase (AID), which is critical for antibody class switch DNA recombination (CSR) and somatic hypermutation (SHM), through direct activation of this gene. AID, as we have shown, is induced by the HoxC4 homeodomain transcription factor, which binds to a conserved HoxC4/Oct site in the AICDA/Aicda promoter. Here we show that estrogen-estrogen receptor (ER) complexes do not directly activate the AID gene promoter in B cells undergoing CSR. Rather, they bind to three evolutionarily conserved and cooperative estrogen response elements (EREs) we identified in the HOXC4/HoxC4 promoter. By binding to these EREs, ERs synergized with CD154 or LPS and IL-4 signaling to up-regulate HoxC4 expression, thereby inducing AID and CSR without affecting B cell proliferation or plasmacytoid differentiation. Estrogen administration in vivo significantly potentiated CSR and SHM in the specific antibody response to the 4-hydroxy-3-nitrophenylacetyl hapten conjugated with chicken γ-globulin. Ablation of HoxC4 (HoxC4(-/-)) abrogated the estrogen-mediated enhancement of AID gene expression and decreased CSR and SHM. Thus, estrogen enhances AID expression by activating the HOXC4/HoxC4 promoter and inducing the critical AID gene activator, HoxC4.

  10. CD27(-)CD45(+) γδ T cells can be divided into two populations, CD27(-)CD45(int) and CD27(-)