ABSTRACT
The additive main effects and multiplicative interaction (AMMI) and the genotype main effects and genotype x environment interaction (GGE) models stand out among the linear-bilinear models used in genotype x environment interaction studies. Despite the advantages of their use to describe genotype x environment (AMMI) or genotype and genotype x environment (GGE) interactions, these methods have known limitations that are inherent to fixed effects models, including difficulty in treating variance heterogeneity and missing data. Traditional biplots include no measure of uncertainty regarding the principal components. The present study aimed to apply the Bayesian approach to GGE biplot models and assess the implications for selecting stable and adapted genotypes. Our results demonstrated that the Bayesian approach applied to GGE models with non-informative priors was consistent with the traditional GGE biplot analysis, although the credible region incorporated into the biplot enabled distinguishing, based on probability, the performance of genotypes, and their relationships with the environments in the biplot. Those regions also enabled the identification of groups of genotypes and environments with similar effects in terms of adaptability and stability. The relative position of genotypes and environments in biplots is highly affected by the experimental accuracy. Thus, incorporation of uncertainty in biplots is a key tool for breeders to make decisions regarding stability selection and adaptability and the definition of mega-environments.
Subject(s)
Models, Genetic , Zea mays/genetics , Bayes Theorem , EnvironmentABSTRACT
In order to obtain information on the accuracy of activity measurements in Brazilian hospitals, several intercomparison exercises have been organized since 1998. The first exercise consisted of two intercomparison runs of 131I and 99mTc and had the participation of 21 hospitals localized in Rio de Janeiro city and surroundings. The second, with 131I (47 hospitals) and 123I (12 hospitals), had the participation of hospitals localized in the whole country. The results were analyzed under the statistical point of view and conformity to the norms of Regulatory Authority. These results have shown that such exercises are necessary to improve the quality of the measurements and to identify those instruments that are producing incorrect values.
Subject(s)
Radioisotopes/analysis , Radioisotopes/standards , Radiometry/standards , Brazil , Hospitals , Humans , Iodine Radioisotopes/analysis , Iodine Radioisotopes/standards , Reference Standards , Technetium/analysis , Technetium/standards , Technology, RadiologicABSTRACT
In recent years, evidence has accumulated that NAD+ serves as a precursor of metabolites that are involved in a number of regulatory processes. In this work we show that extracellularly added NAD+ was rapidly degraded by intact human monocytes to nicotinamide and ADP-ribose. Besides these main products, minor amounts of AMP, ADP and cADP-ribose were formed. Expression of CD38, which has been identified as NAD+-glycohydrolase (EC 3.2.2.6) degrading NAD+ into nicotinamide and ADP-ribose, was determined on freshly isolated human monocytes by flow cytometry and RT-PCR. Upon ligation with anti-CD38 mAb, CD38 underwent internalization, shedding and new expression. As monocytes possess an intracellular CD38 pool, it could serve as a source for newly expressed CD38. Differentiation of monocytes to macrophages resulted in down-regulation of surface expression of CD38. This decrease correlates with a reduction in NADase activity, indicating that the amount of functional active CD38 molecules decrease during differentiation. As CD38 mRNA was found to be diminished in macrophages, regulation of the gene product seems to occur at the level of transcription or mRNA stability.
Subject(s)
Antigens, CD , Antigens, Differentiation/metabolism , Macrophages/metabolism , Monocytes/metabolism , NAD+ Nucleosidase/metabolism , NAD/metabolism , ADP-ribosyl Cyclase , ADP-ribosyl Cyclase 1 , Antibodies, Monoclonal/pharmacology , Antigens, Differentiation/drug effects , Antigens, Differentiation/immunology , Cell Differentiation/drug effects , Cells, Cultured , Cycloheximide/pharmacology , Down-Regulation , Humans , Membrane Glycoproteins , Monensin/pharmacology , NAD+ Nucleosidase/drug effects , NAD+ Nucleosidase/immunology , Niacinamide/metabolism , Protein Synthesis Inhibitors/pharmacologyABSTRACT
Ascariasis is the most common intestinal helminthiasis worldwide. Heavily infected individuals are prone to develop bowel obstruction or perforation as well as biliary disease. Nevertheless, the presence of roundworms in the biliary tree outside endemic areas is very uncommon. The migration of these worms to the biliary system can cause biliary colic, pancreatitis, or even acute suppurative cholangitis with hepatic abscesses and septicemia. We report here on 2 infants with 14 and 15 months and a 9-year-old boy who suffered from massive biliary ascariasis and who presented with acute suppurative cholangitis. All cases were successfully treated by endoscopic retrograde cholangiopancreatography with worm extraction and adjuvant medical therapy. Physicians should be aware of ascariasis in patients with pancreatobiliary symptoms who have traveled to endemic areas or in immigrants from these areas.
Subject(s)
Ascariasis/therapy , Ascaris lumbricoides , Cholangiopancreatography, Endoscopic Retrograde , Cholangitis/therapy , Cholestasis/therapy , Animals , Ascariasis/diagnostic imaging , Child , Cholangiography , Cholangitis/diagnostic imaging , Cholestasis/diagnostic imaging , Diagnosis, Differential , Female , Humans , Infant , MaleABSTRACT
The elevation of the cytosolic and nuclear Ca(2+) concentration is a fundamental signal transduction mechanism in almost all eukaryotic cells. Interestingly, three Ca(2+)-mobilising second messengers, D-myo-inositol 1,4,5-trisphosphate (InsP(3)), cyclic adenosine diphosphoribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP(+)) were identified in a phylogenetically wide range of different organisms. Moreover, in an as yet very limited number of cell types, sea urchin eggs, mouse pancreatic acinar cells, and human Jurkat T-lymphocytes, all three Ca(2+)-mobilising ligands have been shown to be involved in the generation of Ca(2+) signals. This situation raises the question why during evolution all three messengers have been conserved in the same cell type. From a theoretical point of view the following points may be considered: (i) redundant mechanisms ensuring intact Ca(2+) signalling even if one system does not work, (ii) the need for subcellularly localised Ca(2+) elevations to obtain a certain physiological response of the cell, and (iii) tight control of a physiological response of the cell by a temporal sequence of Ca(2+) signalling events. These theoretical considerations are compared to the current knowledge regarding the three messengers in sea urchin eggs, mouse pancreatic acinar cells, and human Jurkat T lymphocytes.
Subject(s)
Adenosine Diphosphate Ribose/analogs & derivatives , Adenosine Diphosphate Ribose/metabolism , Calcium/metabolism , Inositol 1,4,5-Trisphosphate/metabolism , NADP/analogs & derivatives , NADP/metabolism , Second Messenger Systems , Adenosine Diphosphate Ribose/genetics , Animals , Biological Evolution , Cell Line , Cyclic ADP-Ribose , Humans , Inositol 1,4,5-Trisphosphate/genetics , NADP/genetics , Signal TransductionABSTRACT
The authors report the case of a 45-year-old woman who suffered from epigastric pain and was operated on 3 years before for colon cancer. The clinical investigation revealed that the patient had colon cancer metastasis to the dorsal pancreas of a pancreas divisum.
Subject(s)
Colonic Neoplasms/surgery , Pancreas/abnormalities , Pancreatic Neoplasms/secondary , Cholangiopancreatography, Endoscopic Retrograde , Colonic Neoplasms/pathology , Female , Humans , Middle Aged , Pancreas/pathology , Pancreatectomy , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/surgeryABSTRACT
Cyclic ADP-ribose (cADPR) is a natural compound that mobilizes calcium ions in several eukaryotic cells. Although it can lead to the release of calcium ions in T lymphocytes, it has not been firmly established as a second messenger in these cells. Here, using high-performance liquid chromatography analysis, we show that stimulation of the T-cell receptor/CD3 (TCR/CD3) complex results in activation of a soluble ADP-ribosyl cyclase and a sustained increase in intracellular levels of cADPR. There is a causal relation between increased cADPR concentrations, sustained calcium signalling and activation of T cells, as shown by inhibition of TCR/CD3-stimulated calcium signalling, cell proliferation and expression of the early- and late-activation markers CD25 and HLA-DR by using cADPR antagonists. The molecular target for cADPR, the type-3 ryanodine receptor/calcium channel, is expressed in T cells. Increased cADPR significantly and specifically stimulates the apparent association of [3H]ryanodine with the type-3 ryanodine receptor, indicating a direct modulatory effect of cADPR on channel opening. Thus we show the presence, causal relation and biological significance of the major constituents of the cADPR/calcium-signalling pathway in human T cells.
Subject(s)
Adenosine Diphosphate Ribose/analogs & derivatives , Calcium Signaling , Calcium/metabolism , Second Messenger Systems , T-Lymphocytes/metabolism , Adenosine Diphosphate Ribose/antagonists & inhibitors , Adenosine Diphosphate Ribose/metabolism , Adenosine Diphosphate Ribose/pharmacology , CD3 Complex/immunology , CD3 Complex/metabolism , Chromatography, High Pressure Liquid , Cyclic ADP-Ribose , Enzyme Activation , Humans , Inositol 1,4,5-Trisphosphate/antagonists & inhibitors , Inositol 1,4,5-Trisphosphate/metabolism , Jurkat Cells , Lymphocyte Activation , Ryanodine/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , T-Lymphocytes/immunologyABSTRACT
Cyclic ADP-ribose (cADPR) is a natural metabolite of beta-NAD+ with a potent Ca2+-mobilizing activity in different cell types, including T-lymphocytes. We investigated (i) whether stimulation of T-lymphocytes with different agonists affects the intracellular concentration of cADPR, and (ii) whether the lymphocyte antigen CD38, through its ectocellular ADP-ribosyl cyclase and cADPR-hydrolase enzymatic activities, can account for the regulation of the intracellular levels of cADPR and the Ca2+-mobilizing effects of this nucleotide in Jurkat and HPB.ALL T-lymphocytes. The anti-CD3 antibody OKT3, the sphingolipid sphingosine and lysophosphatidic acid induced an increase in intracellular cADPR with concomitant increases in the intracellular Ca2+ concentration ([Ca2+]i). In contrast, activation of an ectocellular ADP-ribosyl cyclase by preincubation of cells with beta-NAD+ led to a dose-dependent increase in cADPR, but no changes in [Ca2+]i were observed. However, extensive washing of the cells following preincubation with NAD+ demonstrated that the increases in cADPR were not intracellular but due to cell surface-associated nucleotide. Accordingly, measurements of ADP-ribosyl cyclase activity in intact T-cells showed ectocellular synthesis of cADPR, but no evidence was obtained for a shift of this activity into the cells which could account for intracellular accumulation of cADPR. Taken together, the results indicate no direct involvement of the ADP-ribosyl cyclase activity of CD38 on the regulation of the cADPR-mediated intracellular Ca2+-signalling in T-lymphocytes.
Subject(s)
Adenosine Diphosphate Ribose/analogs & derivatives , Antigens, CD , Antigens, Differentiation/physiology , Calcium Signaling , NAD+ Nucleosidase/physiology , T-Lymphocytes/physiology , ADP-ribosyl Cyclase , ADP-ribosyl Cyclase 1 , Adenosine Diphosphate Ribose/metabolism , Adenosine Diphosphate Ribose/physiology , Antigens, Differentiation/metabolism , Biological Transport , Blotting, Western , Catalysis , Cyclic ADP-Ribose , Flow Cytometry , Humans , Jurkat Cells , Membrane Glycoproteins , NAD+ Nucleosidase/metabolism , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/metabolismABSTRACT
A combined two-step high-performance liquid chromatographic (HPLC) method was developed for the analysis of endogenous levels of cyclic adenosine diphosphoribose (cADPR) in cell extracts. The detection sensitivity for cADPR was about 10 pmol. Linearity of the HPLC detection system was demonstrated in the range of 10 pmol up to 2 nmol. The method was validated in terms of within-day and between-day reproducibility of retention times and peak areas of standard nucleotides. The method was applied to the analysis of endogenous cADPR in human T cell lines. Sequential separation of perchloric acid extracts from cells on strong anion-exchange and reversed-phase ion-pair HPLC resulted in a single symmetrical peak co-eluting with standard cADPR. The identity of this endogenous material was further confirmed by its ability to be converted to ADPR upon heating the cell samples at 80 degrees C for 2 h. Recoveries of the combined perchloric acid extraction-HPLC analysis procedures were 48.3 +/- 10.2%. The determined intracellular concentrations of cADPR in quiescent Jurkat and HPB. ALL human T cells were 198 +/- 41 and 28 +/- 9 pmol/10(8) cells, respectively. In conclusion, a non-radioactive HPLC method presenting a specificity and sensitivity suitable for precise quantification of cADPR in cell extracts was developed.
Subject(s)
Adenosine Diphosphate Ribose/analogs & derivatives , Adenosine Diphosphate Ribose/analysis , Cell Line , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Cyclic ADP-Ribose , Humans , Indicators and Reagents , Jurkat Cells , Perchlorates , Reproducibility of Results , T-Lymphocytes/chemistryABSTRACT
1-(5-Phospho-beta-D-ribosyl)2'-phosphoadenosine 5'-phosphate cyclic anhydride [2'-phospho-cyclic ADP-ribose, cAdo(2')P(5')PP-Rib] was prepared enzymatically from NADP+ using ADP-ribosyl-cyclase from Aplysia californica. The product was purified by HPLC and characterized by NMR and mass spectroscopy, by conversion to 1-(5-phospho-beta-D-ribosyl)adenosine 5'-phosphate cyclic anhydride (cADP-Rib) by alkaline phosphatase and by resistance to snake venom phosphodiesterase. cAdo-(2')P(5')PP-Rib dose-dependently released Ca2+ from an intracellular, non-endoplasmic reticular Ca2+ pool of permeabilized Jurkat and HPB. ALL T-lymphocytes. In contrast, the closely related compounds 1-(5-phospho-beta-D-ribosyl)3'phosphoadenosine 5'-phosphate cyclic anhydride and 1-(5-phospho-beta-D-ribosyl)cyclic 2',3'-phosphoadenosine 5'-phosphate cyclic anhydride did not induce Ca2+-release from permeabilized T cells. The Ca2+ pool sensitive to cAdo(2')P(5')PP-Rib partially overlapped with the Ca2+ pool sensitive to cADP-Rib recently described in T cells [Guse, A. H., da Silva, C. P., Emmrich, F., Ashamu, G. A., Potter, B. V. L. & Mayr, G. W. (1995) Characterization of cyclic adenosine diphosphate-ribose-induced Ca2+-release in T-lymphocyte cell lines, J. Immunol. 155, 3353-3359]. Control experiments suggest that the results were neither due to Ca2+ contaminations in the cADP-Rib preparation nor to catabolism of cAdo(2')P(5')PP-Rib to cADP-Rib.
Subject(s)
Antigens, CD , Calcium/metabolism , Cyclic AMP/analogs & derivatives , NADP/analogs & derivatives , T-Lymphocytes/metabolism , ADP-ribosyl Cyclase , ADP-ribosyl Cyclase 1 , Adenosine Diphosphate Ribose/analogs & derivatives , Adenosine Diphosphate Ribose/metabolism , Adenosine Diphosphate Ribose/pharmacology , Animals , Antigens, Differentiation/metabolism , Aplysia , Cell Compartmentation , Chromatography, High Pressure Liquid , Coloring Agents/pharmacology , Cyclic ADP-Ribose , Cyclic AMP/chemical synthesis , Cyclic AMP/metabolism , Cyclic AMP/pharmacology , Humans , Jurkat Cells , Magnetic Resonance Spectroscopy , Membrane Glycoproteins , N-Glycosyl Hydrolases/metabolism , Ruthenium Red/pharmacology , T-Lymphocytes/drug effects , Tumor Cells, CulturedABSTRACT
Cyclic ADP-ribose (cADPr) is a potent Ca2+-mobilizing natural compound (Lee, H. C., Walseth, T. F., Bratt, G. T., Hayes, R. N., and Clapper, D. L. (1989) J. Biol. Chem. 264, 1608-1615) which has been shown to release Ca2+ from an intracellular store of permeabilized T-lymphocytes (Guse, A. H., Silva, C. P., Emmrich, F., Ashamu, G., Potter, B. V. L., and Mayr, G. W. (1995) J. Immunol. 155, 3353-3359). Microinjection of cADPr into intact single T lymphocytes dose dependently induced repetitive but irregular Ca2+ spikes which were almost completely dependent on the presence of extracellular Ca2+. The Ca2+ spikes induced by cADPr could be blocked either by co-injection of cADPr with the specific antagonist 8-NH2-cADPr, by omission of Ca2+ from the medium, or by superfusion of the cells with Zn2+ or SK-F 96365. Ratiometric digital Ca2+ imaging revealed that single Ca2+ spikes were initiated at several sites ("hot spots") close to the plasma membrane. These hot spots then rapidly formed a circular zone of high Ca2+ concentration below the plasma membrane which subsequently propagated like a closing optical diaphragm into the center of the cell. Taken together these data indicate a role for cADPr in Ca2+ entry in T-lymphocytes.
Subject(s)
Adenosine Diphosphate Ribose/analogs & derivatives , Calcium/metabolism , T-Lymphocytes/metabolism , Adenosine Diphosphate Ribose/pharmacology , Cyclic ADP-Ribose , Fluorescent Dyes , Fura-2 , Humans , Jurkat Cells , Microinjections , Signal Transduction , T-Lymphocytes/drug effectsABSTRACT
Intracellular Ca(2+)-signals belong to the major events transducing extracellular signals into living cells. The discovery of (i) a caffeine-sensitive intracellular Ca(2+)-pool in Jurkat T-lymphocytes [1] and (ii) cyclic adenosine diphosphoribose (cADPR) as an agent that mobilizes Ca2+ from a caffeine- and ryanodine sensitive Ca(2+)-store in sea urchin egg homogenates [2] prompted us to investigate the potential role of this compound in T-lymphocyte Ca(2+)-signalling. cADPR, as well as its 2'-phosphorylated derivative, 2'-phospho-cADPR (2'-cADPR), released Ca2+ in a dose-dependent, specific manner from intracellular, non-endoplasmic reticular stores of permeabilized Jurkat and HPB. ALL T cells. In addition, attempts were made to prove the presence of endogenous cADPR and 2'-P-cADPR by HPLC. Several HPLC protocols, including microbore-HPLC were tested resulting in the detection of endogenous cADPR by sequential separation on strong-anion exchange HPLC and reverse-phase ion-pair HPLC.
Subject(s)
Adenosine Diphosphate Ribose/analogs & derivatives , Calcium/metabolism , Signal Transduction , T-Lymphocytes/metabolism , Adenosine Diphosphate Ribose/chemistry , Adenosine Diphosphate Ribose/isolation & purification , Adenosine Diphosphate Ribose/physiology , Animals , Chromatography, High Pressure Liquid/methods , Cyclic ADP-Ribose , Humans , Molecular Structure , T-Lymphocytes/cytologyABSTRACT
The involvement of apoptosis in the mechanism of cell death induced by six clinically relevant anticancer drugs [methotrexate (MTX), doxorubicin (ADR), daunorubicin (DNR), vincristine (VCR), 6-mercaptopurine (6MP), and prednisolone (PRD)] in human leukemic T-lymphocytes (CCRF-CEM and Jurkat) was investigated by analysing changes in cell size and morphology, changes in membrane integrity, alterations in [Ca2+]i and induction of DNA fragmentation. MTX, ADR, and DNR showed pronounced dose- and time-dependent cytotoxic effects on both cell lines, whereas cell viability was not considerably reduced by 6MP or PRD. On the other hand, the cytotoxic activity of VCR was much higher on Jurkat cells than on CEM cells. With the exception of 6MP and PRD, all the other compounds induced extensive chromatin condensation, nuclear fragmentation, plasma membrane blebbing, and formation of apoptotic bodies and fragmentation of DNA in both cell lines. Occurrence of DNA fragmentation always preceded loss of membrane integrity. These observation are consistent with cell death being mediated by apoptosis. Significant increases in [Ca2+]i were only observed in CEM cells preincubated with MTX or DNR (10 microM). In contrast, MTX as well as VCR induced a reduction in the basal intracellular Ca2+ concentration in Jurkat T-cells. Although the ability to induce changes in [Ca2+]i correlated with higher cytotoxic potency of the anticancer drugs, a causal relationship between increased [Ca2+] and induction of apoptosis could not be clearly established. These results, therefore, suggest no determinant role for Ca2+ in triggering the process of endonucleolytic cleavage of genomic DNA in these leukemic T-lymphocytes.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Death/drug effects , T-Lymphocytes/drug effects , Apoptosis , Cell Count/drug effects , Daunorubicin/pharmacology , Dose-Response Relationship, Drug , Doxorubicin/pharmacology , Humans , Methotrexate/pharmacology , Time FactorsABSTRACT
Ca2+ release from intracellular stores is one of the major events transducing extracellular signals into living cells. Recently, a metabolite of nicotinamide adenine dinucleotide+ (NAD+), termed "cyclic adenosine diphosphate-ribose" (cADPr), has been described to release Ca2+ from caffeine-sensitive internal stores of cells. Jurkat T cells possess intracellular Ca2+ stores sensitive to caffeine, so a potential involvement of cADPr in Ca2+ signaling was investigated. cADPr released Ca2+ in a dose-dependent manner from intracellular stores of permeabilized Jurkat T cells. Half maximal release was obtained at 2.25 microM cADPr. Prior addition of D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) or thapsigargin did not influence cADPr-induced Ca2+ release, indicating the presence of different Ca2+ pools sensitive to Ins(1,4,5)P3 and cADPr. The specificity of the response was confirmed using the inhibitors ruthenium red, 8-NH2-cADPr, and 8-Br-cADPr. All three compounds blocked cADPr-induced, but not Ins(1,4,5)P3-induced, Ca2+ release in a dose-dependent manner. Cyclic GMP (cGMP)-induced Ca2+ release was also partly antagonized by ruthenium red, indicating involvement of a cGMP-dependent step in the formation of cADPr. The presence of endogenous cADPr was analyzed directly by HPLC. Sequential separation on strong anion exchange HPLC and reverse-phase, ion-pair HPLC resulted in a single symmetric peak co-eluting with standard cADPr. The identity of this endogenous material was further confirmed by its ability to release Ca2+ in saponin-permeabilized Jurkat T cells.
Subject(s)
Adenosine Diphosphate Ribose/analogs & derivatives , Calcium/metabolism , T-Lymphocytes/metabolism , Adenosine Diphosphate Ribose/pharmacology , Cell Line , Chromatography, High Pressure Liquid , Cyclic ADP-Ribose , Humans , Signal Transduction , T-Lymphocytes/drug effectsABSTRACT
Effects of the cytostatic drug adriamycin on inositol polyphosphate metabolism were analyzed in a human T-cell line (Jurkat) using a recently developed anion-exchange high performance liquid chromatography/post-column complexometric dye system. Treatment of intact T-cells with adriamycin prior to stimulation with an anti-CD3 monoclonal antibody induced a dose- and time-dependent decrease in the intracellular level of inositol 1,3,4,5-tetrakisphosphate (complete inhibition after 2 h at 10 microM adriamycin) and an increase in the level of inositol 1,3,4-trisphosphate without significantly changing the levels of other inositol phosphates. A marked inhibition of the inositol 1,4,5-trisphosphate 3-kinase activity and a slight activation of the inositol 1,3,4,5-tetrakisphosphate 5-phosphatase activity were observed in cytosolic extracts in the presence of adriamycin, providing an explanation for the drug-induced metabolic effect. Adriamycin thus seems to be an extremely valuable tool for further dissecting inositol polyphosphate metabolism, as well as signaling pathways. Along these lines, we observed that adriamycin did not change the free cytosolic Ca2+ concentration of Jurkat T-lymphocytes and, in particular, did not modulate Ca2+ influx upon T-cell receptor stimulation. We conclude that (i) inositol phosphate signaling pathways constitute an as yet undescribed target for the action of adriamycin and that (ii) an increase of inositol 1,3,4,5-tetrakisphosphate is not necessary for sustained Ca(2+)-entry in stimulated T-cells.