Detection Of Terminal Deoxynucleotidyl Transferase Research Articles

Photocatalytic colorimetry and fluorescence dual-signal biosensor for instant determination of terminal deoxynucleotidyl transferase

Terminal deoxynucleotidyl transferase (TdT) is a crucial biomarker for diseases such as lymphoblastic leukemia. Herein, a photocatalytic colorimetry and fluorescence dual-signal biosensor (PhoCoF-sensor) for simple, low-cost and sensitive detection of TdT was developed, which integrated with the TdT-triggered polymerization reaction and the fluorescence-photocatalytic dual performance of double-strand DNA/SYBR Green-I (dsDNA/SG-I) complex. The target TdT catalyzed the elongation of single-stranded DNA in the dTTP substrate pool, and then hybridized with complementary DNA to form dsDNA/SG-I complex that exhibited obvious fluorescent and excellent photocatalytic activity. Reactive oxygen species were generated from photosensitization of the dsDNA/SG-I complex acted as the visual colorimetric readout. The PhoCoF-sensor was successfully used to detect TdT activity with a series of color and fluorescence changes in the concentration range from 0.5 U/mL to 20.0 U/mL. The PhoCoF-sensor further improves the assay simplicity and accuracy and reduces the time and cost, which overcomes the limitations of traditional detection TdT methods and single-signal sensors. Additionally, this PhoCoF-sensor can readily be integrated with centrifugal microfluidic device for one-step test and achieve quantitative determination without sophisticated instruments. Moreover, the PhoCoF-sensor has good sensitivity and good practical reliability and is suitable for instant determination in early disease diagnosis and drug discovery.

Pulmonary oxidative stress and apoptosis in mice chronically exposed to hydrothermal volcanic emissions.

Recent studies have shown that exposure to hydrothermal emissions has a negative impact on the respiratory system. Still, volcanogenic air pollution studies are still outnumbered when compared to anthropogenic studies which can result in an unknown risk to the human populations living near volcanically active areas. This study was carried out in São Miguel Island, with noneruptive volcanically active environments, such as the Furnas volcano caldera. Its noneruptive volcanism presents itself as hydrothermal emissions, mainly by the release of nearly 1000 T d-1 of CO2 along with H2S, and the radioactive gas radon; metals [e.g., mercury (Hg), cadmium (Cd), copper (Cu), and zinc (Zn)] and particulate matter are also released in a daily basis. We test the hypothesis whether chronic exposure to hydrothermal emissions causes pulmonary oxidative stress, using Mus musculus as a surrogate species. Mus musculus was live-captured in two villages with hydrothermal emissions and one village without any type of volcanic activity. The level of pulmonary oxidative stress was immunohistochemically assessed by using an OxyIHCTM Oxidative stress detection kit, and the detection of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) was used to evaluate apoptosis in lung tissues. Mice chronically exposed to hydrothermal emissions presented increased levels of oxidative stress and amount of apoptotic cells. We demonstrate, for the first time, the high oxidative stress potential in the lungs of mice chronically exposed to hydrothermal emissions. This study highlights the usefulness of M. musculus as a bioindicator species and enforces the necessity of regularly biomonitor the inhabitants of hydrothermal areas to prevent respiratory pathologies.

Signal-on electrochemical assay for label-free detection of TdT and BamHI activity based on grown DNA nanowire-templated copper nanoclusters.

Electrochemical methods allow fast and inexpensive analysis of enzymatic activity. Here, a simple and yet efficient "signal-on" electrochemical assay for sensitive, label-free detection of DNA-related enzyme activity was established on the basis of terminal deoxynucleotidyl transferase (TdT)-mediated extension strategy. TdT, which is a template-independent DNA polymerase, can catalyze the sequential addition of deoxythymidine triphosphate (dTTP) at the 3'-OH terminus of single-stranded DNA (ssDNA); then, the TdT-yield T-rich DNA nanowires can be employed as the synthetic template of copper nanoclusters (CuNCs). Grown DNA nanowires-templated CuNCs (noted as DNA-CuNCs) were attached onto graphene oxide (GO) surface and exhibited unique electrocatalytic activity to H2O2 reduction. Under optimal conditions, the proposed biosensor was utilized for quantitatively monitoring TdT activity, with the observed LOD of 0.1 U/mL. It also displayed high selectivity to TdT with excellent stability, and offered a facile, convenient electrochemical method for TdT-relevant inhibitors screening. Moreover, the proposed sensor was successfully used for BamHI activity detection, in which a new 3'-OH terminal was exposed by the digestion of a phosphate group. Ultimately, it has good prospects in DNA-related enzyme-based biochemical studies, disease diagnosis, and drug discovery. Graphical Abstract Extraordinary TdT-generated DNA-CuNCs are synthesized and act as a novel electrochemical sensing platform for sensitive detection of TdT and BamHI activity in biological environments.

Single quantum dot-based nanosensor for rapid and sensitive detection of terminal deoxynucleotidyl transferase.

We developed a simple and rapid method for terminal deoxynucleotidyl transferase (TdT) assay on the basis of the polymerization-directed exonuclease-assisted construction of a single quantum dot (QD)-based fluorescence resonance energy transfer (FRET) nanosensor. This method is very sensitive with a detection limit as low as 1 × 10-6 U μL-1, and it can be used for the screening of TDT inhibitors and accurate quantification of TdT activity even in 5 cancer cells.

Endogenous κ-Opioid Peptide Mediates the Cardioprotection Induced by Ischemic Postconditioning

The aim of this study was to investigate the underlying mechanism that dynorphin, an endogenous kappa opioid receptor (κ-OR) agonist, triggers antiapoptotic effect of postconditioning (Postcon). In addition to vehicle treatment, Sprague Dawley rats (n = 6) underwent a 30-minute left anterior descending occlusion followed by 2 hours of reperfusion with or without a Postcon stimulus. The selective κ-OR antagonist nor-binaltorphimine (Nor-BNI) was administered intravenously 5 minutes before reperfusion. Infarct size was determined by using 2,3,5-triphenyltetrazolium chloride staining. Blood plasma concentrations of creatine kinase (CK) and lactate dehydrogenase (LDH) and myocardial caspase-3 activity were analyzed spectrophotometrically. Myocardial apoptosis was analyzed by the detection of terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling. Immunoreactive dynorphin in blood serum and myocardium was measured by means of an antigen-competitive enzyme-linked immunosorbent assay. Infarction size, caspase-3 activity, apoptotic index, and CK and LDH levels were significantly higher in the ischemic/reperfusion group than in the vehicle group (P < 0.01). Postcon significantly reduced infarction size, caspase-3 activity, apoptotic index, CK and LDH levels (P < 0.01 vs. ischemic/reperfusion). Dynorphin content significantly increased after Postcon (P < 0.01). All the effects described above were abolished by Nor-BNI, with the exception of dynorphin content. We found that cardiac protection and antiapoptotic effect of Postcon is mediated by the activation of κ-OR. Effect of Postcon is mediated, at least partially, by enhanced dynorphin expression.

Blast crisis in chronic myeloid leukemia. Results with vindesine and prednisolone

Nine patients with blast crises of chronic myeloid leukaemia were treated with a combination of vindesine and prednisolone. Vindesine, 2 mg/m2, was administered intravenously on two successive days each week and prednisolone, 60 mg/m2, orally once daily. Blast crises were divided into myeloblastic and lymphoblastic ones using cytochemical parameters as well as detection of terminal deoxynucleotidyl transferase. Complete remission was achieved in four patients, partial remission in one patient; in four patients treatment was unsuccessful. According to cytochemical findings, a therapeutic success was obtained in three of four patients with lymphoblastic and two of four patients with myeloblastic crises whereas no response to the treatment was seen in one patient with an undifferentiated type. Side effects of the therapy were frequent, but of only low degree and never led to interruption of treatment. On the basis of these results and from experience reported in the literature, the combination of vindesine and prednisolone can be recommended as the therapy of choice in blast crises of chronic myeloid leukaemia.

Detection of Terminal Deoxynucleotidyl Transferase (TdT) in Nonhematopoietic Small Round Cell Tumors of Children

Detection of Terminal Deoxynucleotidyl Transferase (TdT) in Nonhematopoietic Small Round Cell Tumors of Children

Terminal deoxynucleotidyl transferase staining of malignant lymphomas in paraffin sections: a useful method for the diagnosis of lymphoblastic lymphoma.

Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase located in the cell nucleus which catalyses the polymerization of deoxynucleotides at the 3'hydroxyl ends of oligo- or polydeoxynucleotide initiators without a template. TdT is known as a useful marker for the diagnosis of acute lymphoblastic leukaemia/lymphoma, but its detection usually requires fresh tissue specimens or cell suspensions, using either an enzyme analysis or immuno-fluorescence or -peroxidase staining. Until the recent development of the use of microwave-treated paraffin sections for immunoperoxidase staining, detection of TdT in paraffin sections required rather complicated processes. This new simple technique was applied to paraffin sections from the tumour tissue specimens of 16 patients with lymphoblastic lymphoma and of seven patients with non-endemic Burkitt's lymphoma, which is sometimes difficult to differentiate from lymphoblastic lymphoma because of their similar clinicopathological characteristics. In addition, as a control, ten cases each were examined of adult T-cell leukaemia/lymphoma (ATLL) and angioimmunoblastic lymphoma (AILD), which are both peripheral T-cell lymphomas. The tumour cells from 15 of the 16 (94 per cent) patients with lymphoblastic lymphoma were found to be TdT-positive. The specificity of the anti-TdT antibody used was confirmed by immunoblot and the specific 60 kD band was detected only in a specimen of lymphoblastic lymphoma. These results show that the immunostaining of TdT on paraffin-embedded sections is a useful method for differentiating lymphoblastic lymphoma from other lymphomas. This method is applicable to a routine diagnostic service.

TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE STAINING OF MALIGNANT LYMPHOMAS IN PARAFFIN SECTIONS: A USEFUL METHOD FOR THE DIAGNOSIS OF LYMPHOBLASTIC LYMPHOMA

Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase located in the cell nucleus which catalyses the polymerization of deoxynucleotides at the 3′ hydroxyl ends of oligo- or polydeoxynucleotide initiators without a template. TdT is known as a useful marker for the diagnosis of acute lymphoblastic leukaemia/lymphoma, but its detection usually requires fresh tissue specimens or cell suspensions, using either an enzyme analysis or immuno-fluorescence or -peroxidase staining. Until the recent development of the use of microwave-treated paraffin sections for immunoperoxidase staining, detection of TdT in paraffin sections required rather complicated processes. This new simple technique was applied to paraffin sections from the tumour tissue specimens of 16 patients with lymphoblastic lymphoma and of seven patients with non-endemic Burkitt's lymphoma, which is sometimes difficult to differentiate from lymphoblastic lymphoma because of their similar clinicopathological characteristics. In addition, as a control, ten cases each were examined of adult T-cell leukaemia/lymphoma (ATLL) and angioimmunoblastic lymphoma (AILD), which are both peripheral T-cell lymphomas. The tumour cells from 15 of the 16 (94 per cent) patients with lymphoblastic lymphoma were found to be TdT-positive. The specificity of the anti-TdT antibody used was confirmed by immunoblot and the specific 60 kD band was detected only in a specimen of lymphoblastic lymphoma. These results show that the immunostaining of TdT on paraffin-embedded sections is a useful method for differentiating lymphoblastic lymphoma from other lymphomas. This method is applicable to a routine diagnostic service. © 1997 John Wiley & Sons, Ltd.

Detection of Terminal Deoxynucleotidyl Transferase (TdT) in Nonhematopoietic Small Round Cell Tumors of Children

Detection of Terminal Deoxynucleotidyl Transferase (TdT) in Nonhematopoietic Small Round Cell Tumors of Children

Detection of terminal deoxynucleotidyl transferase by flow cytometry: a three color method.

This short communication describes a three-color flow cytometric analysis procedure for terminal deoxynucleotidyl transferase (TdT). The method combines CD45-gating principles and a new permeabilization method based on a commercially prepared lysing reagent. While traditional permeabilizing fixatives are often cumbersome to use, distort cell light scatter, and fail to retain the fluorochrome peridin chlorophyll alpha protein (perCP), this simple method retains light scatter and does not affect perCP, making it ideal for three-color analysis. The procedure simply involves staining whole blood or bone marrow with CD45 perCP and an appropriate phycoerythrin (PE) conjugated monoclonal antibody (MoAb) and lysing with FACS lysing solution (Becton Dickinson, San Jose, CA). After washing the cells, fluorescein isothiocyanate (FITC) conjugated anti-TdT is added. The cells are washed, fixed with formaldehyde, and acquired on a flow cytometer compensated for three-color analysis. Display of CD45 perCP vs. right angle light scatter (RALS) allows identification of blasts. Dual color expression of anti-TdT FITC and a PE conjugated MoAb identifies TdT expression on blast populations defined by specific lineage associated antigens. This method is not only useful for TdT analysis but may also prove to be a valuable tool for looking at expression of other cytoplasmic antigens in combination with surface antigens on CD45-defined blast populations.

Flow cytometric analysis of terminal deoxynucleotidyl transferase. A simplified method.

The authors evaluated a new cell membrane permeabilization method for the flow cytometric detection of terminal deoxynucleotidyl transferase (TdT). In this method, gradient-separated leukocytes or unseparated blood or bone marrow cells were incubated in a commercially available diethylene glycol-based red blood cell lysing solution, which not only lyses red blood cells, but also permeabilizes leukocyte cell membranes; the light scattering properties of the cells are retained. The validity of the current method was demonstrated by the good concordance of the findings with previously published data as follows: (1) practically identical results were obtained when an established method for cell permeabilization was used in parallel on the same samples; (2) the proportion of TdT-positive cells in normal peripheral blood was negligible; (3) the proportion of TdT-positive cells in normal bone marrow averaged 1%, and a significant portion of TdT-positive cells in normal bone marrow expressed CD10 and CD34; and (4) TdT-positive cell populations were seen with the expected frequencies in various types of leukemia. This method for TdT flow cytometry provides significant advantages over previously used methods and is especially suitable for TdT detection in routine laboratories.

Flow-cytometric detection of terminal deoxynucleotidyl transferase and other intracellular antigens in combination with membrane antigens in acute lymphatic leukemias

Development of a new fixation procedure allowed flow-cytometric analysis of nuclear and other intracellular antigens in acute lymphatic leukemia (ALL). A short fixation of the cells with buffered formaldehyde acetone (BFA) rendered the cell membrane permeable, allowing the monoclonal antibodies (MoAbs) to penetrate the cell. Through this method, a rapid analysis of intracellular antigens, specific for acute lymphatic leukemia [such as terminal deoxynucleotidyl transferase (TdT), immunoglobulin M (IgM) heavy chain, and antigens recognized by the CD22 or CD3 MoAbs) was performed by flow cytometry. The surface antigens remained intact after this fixation procedure, enabling simultaneous detection of membrane and intracellular antigens. The binding of biotinylated antibodies against several B- and T-lymphoid membrane antigens was detected with streptavidin-phycoerythrin (red fluorescence), whereas the intracellular antigens were stained with FITC-labeled polyclonal antibodies, or indirectly with FITC-labeled goat anti-mouse IgG (green fluorescence). Through this combination of markers, minor cell populations can be detected and a rapid and quantitative immunodiagnosis can be performed.

Flow-Cytometric Detection of Terminal Deoxynucleotidyl Transferase and Other Intracellular Antigens in Combination With Membrane Antigens in Acute Lymphatic Leukemias

Development of a new fixation procedure allowed flow-cytometric analysis of nuclear and other intracellular antigens in acute lymphatic leukemia (ALL). A short fixation of the cells with buffered formaldehyde acetone (BFA) rendered the cell membrane permeable, allowing the monoclonal antibodies (MoAbs) to penetrate the cell. Through this method, a rapid analysis of intracellular antigens, specific for acute lymphatic leukemia [such as terminal deoxynucleotidyl transferase (TdT), immunoglobulin M (IgM) heavy chain, and antigens recognized by the CD22 or CD3 MoAbs) was performed by flow cytometry. The surface antigens remained intact after this fixation procedure, enabling simultaneous detection of membrane and intracellular antigens. The binding of biotinylated antibodies against several B- and T-lymphoid membrane antigens was detected with streptavidin-phycoerythrin (red fluorescence), whereas the intracellular antigens were stained with FITC-labeled polyclonal antibodies, or indirectly with FITC-labeled goat anti-mouse IgG (green fluorescence). Through this combination of markers, minor cell populations can be detected and a rapid and quantitative immunodiagnosis can be performed.

Spurious Detection of Terminal Deoxynucleotidyl Transferase in Phytohemagglutinin-Stimulated Lymphocytes

Expression of terminal transferase (TdT) is believed to be restricted to primitive lymphoid cells; recently, however, indirect immunofluorescent (IF) assays have been used to demonstrate the apparent presence of TdT in phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes and in various nonlymphoid malignancies. Using an IF assay, we found that a heteroantiserum to TdT reacted with cultured and PHA-stimulated human peripheral blood mononuclear cells, but we were unable to confirm the presence of TdT in these cells using immunoblotting and biochemical assays. We conclude that the IF results are spurious and most likely represent recognition by the heteroantiserum of inducible protein(s) other than TdT.

Spurious detection of terminal deoxynucleotidyl transferase in phytohemagglutinin-stimulated lymphocytes

Expression of terminal transferase (TdT) is believed to be restricted to primitive lymphoid cells; recently, however, indirect immunofluorescent (IF) assays have been used to demonstrate the apparent presence of TdT in phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes and in various nonlymphoid malignancies. Using an IF assay, we found that a heteroantiserum to TdT reacted with cultured and PHA-stimulated human peripheral blood mononuclear cells, but we were unable to confirm the presence of TdT in these cells using immunoblotting and biochemical assays. We conclude that the IF results are spurious and most likely represent recognition by the heteroantiserum of inducible protein(s) other than TdT.

Detection of terminal deoxynucleotidyl transferase in acute leukemias using monoclonal antibodies directed against native and denatured sites.

Monoclonal antibodies (MoAb) directed against human terminal deoxynucleotidyl transferase (TdT) have been developed recently. The authors evaluated the reactivity of two TdT MoAb, one directed against a native site and the other against a denatured site, in bone marrow samples from patients with acute lymphoblastic leukemia (ALL) and acute myelogenous leukemia (AML). Results were correlated with the immunophenotype and compared with those obtained with an anti-TdT polyclonal antibody (PoAb). The authors found that 39 of the 45 children (87%) with ALL were positive with the anti-TdT PoAb, while only 25 of 45 (56%) were positive using MoAb. In the 41 of 45 cases of ALL for which marker studies were available, there was no relationship between immunophenotype and reactivity with the PoAb or either MoAb. Five cases of AML were studied and two were positive using the PoAb, but none showed staining with the MoAb. The authors' findings demonstrate that, although MoAb may be used to detect TdT in acute leukemia, the two MoAb used do not correlate with immunophenotype and are less sensitive than the PoAb. However, the MoAb appears to demonstrate more specificity for ALL than the PoAb, since it was not reactive in PoAb+ AML.

Evaluation of a new enzyme-linked immunoassay for terminal deoxynucleotidyl transferase in haematologic malignancies.

We have evaluated a new solid phase enzyme immunoassay (EIA) for detection of terminal deoxynucleotidyl transferase (TDT). The EIA is greater than 100 times more sensitive than previously used tests for enzyme activity. In 284 clinical specimens of human peripheral blood and bone marrow, the EIA detected TdT antigen in 97% of peripheral blood and 100% of bone marrow samples that were positive for enzymatic activity. The excellent sensitivity and specificity of this new test suggests that it can be used in clinical situations where quantitative TdT measurements are desired.

Detection of Terminal Deoxynucleotidyl Transferase

Detection of Terminal Deoxynucleotidyl Transferase

Terminal Transferase Activity and Lymphoblastic Neoplasms

Terminal deoxynucleotidyl transferase (TdT), an enzyme normally present in the human thymus and in a subpopulation of marrow cells, has the unique ability to catalyze the addition of deoxynucleoside triphosphates onto the single-strand, free end of DNA in a template-independent fashion.1 The presence of TdT within the lymphoblasts of a child with acute lymphoblastic leukemia was first noted 10 years ago.2 Since then, the detection of TdT has become a conventional laboratory aid to morphologic studies for the classification of leukemia, the enzyme being present in nearly every case of acute lymphoblastic leukemia examined.1 , 3 , 4 Several years ago, Donlon and co-workers . . .