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Morphine is a drug of choice for the treatment of severe and chronic pain, but tolerance to the antinociceptive effect limits its use. The development of tolerance to morphine has recently been associated with neuronal apoptosis. In this study, our aim was to investigate the effects of metformin on morphine-induced neuronal apoptosis and antinociceptive tolerance in diabetic rats. Three days of cumulative dosing were administered to establish morphine tolerance in rats. The antinociceptive effects of metformin (50 mg/kg) and test dose of morphine (5 mg/kg) were considered at 30-min intervals by thermal antinociceptive tests. To induce diabetic neuropathy, streptozotocin (STZ, 65 mg/kg) was injected intraperitoneally. ELISA kits were used to measure caspase-3, bax and bcl-2 levels from dorsal root ganglion (DRG) tissue. Semi-quantitative scoring system was used to evaluate apoptotic cells with the the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method. The findings suggest that co-administration of metformin with morphine to diabetic rats showed a significant increase in antinociceptive effect compared to morphine alone. The antinociceptive tests indicated that metformin significantly attenuated morphine antinociceptive tolerance in diabetic rats. In addition, metformin decreased the levels of apoptotic proteins caspase 3 and Bax in DRG neurons, while significantly increased the levels of antiapoptotic Bcl-2. Semiquantitative scoring showed that metformin provided a significant reduction in apoptotic cell counts in diabetic rats. These data revealed that metformin demonstrated antiapoptotic activity in diabetic rat DRG neurons and attenuated morphine tolerance. The antiapoptotic activity of metformin probably plays a significant role in reducing morphine tolerance.

Ultrasensitive and specific detection of cocaine is of great significance for monitoring cocaine abuse. Herein, a fluorescent aptasensor via coupling CRISPR-Cas12a, with magnetic nanoparticles (MNPs), split-aptamer, and terminal deoxynucleotidyl transferase (TdT), was developed for the detection of cocaine. In short, the complete cocaine aptamer is split into two parts, one is modified on magnetic nanoparticles (MNPs) and the other is free. The presence of cocaine will mediate the binding of these two segments. Then TdT will mediate the extension to form an ultra-long sequence that can bind with multiple CRISPR-Cas12a resulting in the trans-cleavage activity of CRISPR-Cas12a being triggered. Thence, the DNA reporter which is bi-labeled with fluorophore and quencher is cleaved resulting in the generation of a fluorescence signal. The developed fluorescent aptasensor realizes the detection of cocaine with excellent sensitivity and specificity. The detection limit is low down to 33 pM, and the linear range is from 330 to 1.65 × 10

Spermatogenesis accompanied by self-renewal and differentiation of spermatogonia under complicated regulation is crucial for male fertility. Our previous study demonstrated that the loss of the B-lymphoma Mo-MLV insertion region 1 (BMI1) could cause male infertility and found a potential interaction between BMI1 and proline-rich protein 11 (PRR11); however, the specific co-regulatory effects of BMI1/PRR11 on spermatogonia maintenance remain unclear.The expression of PRR11 was downregulated in a mouse spermatogonia cell line (GC-1) via transfection with PRR11-siRNAs, and PRR11 knockdown was verified by real-time reverse transcriptase polymerase chain reaction (RT-qPCR). The proliferative activity of GC-1 cells was determined using the cell counting kit (CCK-8), colony formation, and 5-ethynyl-2-deoxyuridine (EdU) incorporation assay. A Transwell assay was performed to evaluate the effects of PRR11 on GC-1 cell migration. A terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to measure GC-1 cell apoptosis. Furthermore, co-immunoprecipitation, RT-qPCR, and western blot analyses were used for investigating the regulatory mechanisms involved in this regulation. It was found that downregulation of PRR11 could cause a marked inhibition of proliferation and migration and induced apoptosis in GC-1 cells. Moreover, silencing of PRR11 obviously led to a reduction in the BMI1 protein level. PRR11 was found to interact with BMII at the endogenous protein level. PRR11 knockdown produced a decrease in BMI1 protein stability via an increase in BMI1 ubiquitination after which derepression in the transcription of protein tyrosine phosphatase receptor type M (Ptprm) occurred. Importantly, knockdown of Ptprm in PRR11-deficient GC-1 cells led to a reversal of proliferation and migration of GC-1 cells.This study uncovered a novel mechanism by which PRR11 cooperated with BMI1 to facilitate GC-1 maintenance through targeting Ptprm. Our findings may provide a better understanding of the regulatory network in spermatogonia maintenance.

Purpose This study assesses human biodistribution, radiation dosimetry, safety and tumour uptake of cell death indicator labelled with 68Ga ([68Ga]Ga-CDI), a novel radiopharmaceutical that can image multiple forms of cell death. Methods Five participants with at least one extracranial site of solid malignancy > 2 cm and no active cancer treatment in the 8 weeks prior to the study were enrolled. Participants were administered 205 ± 4.1 MBq (range, 200–211 MBq) of [68Ga]Ga-CDI and 8 serial PET scans acquired: the first commencing immediately and the last 3 h later. Participants were monitored for clinical, laboratory and electrocardiographic side effects and adverse events. Urine and blood radioactivity was measured. Spherical volumes of interest were drawn over tumour, blood pool and organs to determine biodistribution and calculate dosimetry. In one participant, tumour specimens were analysed for cell death using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining. Results [68Ga]Ga-CDI is safe and well-tolerated with no side effects or adverse events. [68Ga]Ga-CDI is renally excreted, demonstrates low levels of physiologic uptake in the other organs and has excellent imaging characteristics. The mean effective dose was 2.17E − 02 ± 4.61E − 03 mSv/MBq. It images constitutive tumour cell death and correlates with tumour cell death on histology. Conclusion [68Ga]Ga-CDI is a novel cell death imaging radiopharmaceutical that is safe, has low radiation dosimetry and excellent biodistribution and imaging characteristics. It has potential advantages over previously investigated radiopharmaceuticals for imaging of cell death and has progressed to a proof-of-concept trial. Trial registration ACTRN12621000641897 (28/5/2021, retrospectively registered)

Various DNA glycosylases involved in base excision repair may be associated with a wide disease spectrum that includes cancer, myocardial infarction, neurodegenerative disorders, etc. In this paper, we developed a sensitive method for simultaneous detection of multiple DNA glycosylases based on the target-initiated removal of damaged base and terminal deoxynucleotidyl transferase (TdT)-assisted labeling and signal amplification. We designed three specific stem-loop probes which contained specific targeting damaged bases in the stem for uracil DNA glycosylase (UDG), human alkyladenine DNA glycosylase (hAAG), and human 8-oxoguanine DNA glycosylase 1 (hOGG1), respectively. Target DNA glycosylase can initiate the recognition and clearance of damaged base on immobilized 3' blocked stem-loop probe, releasing apurine/apyrimidine (AP) site which can be hydrolyzed by AP endonuclease to produce 3'OH probe fragment for TdT extension. Numerous biotin-modified dUTPs were successively labeled on the 3' terminus of the probe fragments, and then reacted with streptavidin-phycoerythrin (SA-PE) for analysis by using the Luminex xMAP array platform. The amplification strategy based on TdT has been utilized to simultaneously and sensitively detect three different DNA glycosylases with detection limits of 10

Background It has been determined through extensive studies that autophagy, the Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome and apoptotic responses in macrophages jointly contribute to atherogenesis and its development in the presence of lipid abnormalities. Few studies have investigated in full-scale if the intervention time for lipids abnormality or NLRP3 activation have a significant effect on autophagy, NLRP3 or the apoptotic status in macrophages. Methods Human THP-1 monocyte-derived macrophages were established by challenging THP-1 monocytes with 80 µg/ml oxidized low-density lipoprotein (ox-LDL) for specific durations. Foam cell formation was observed by Oil Red O (ORO) staining. Western blots were employed to determine protein expression. Transmission electron microscope (TEM) and immunofluorescence microscopy were applied to observe the autophagic status of cells. Cell apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Results The cells were treated with ox-LDL for 12 h and 36 h, which were considered to represent early and advanced stages of atherogenesis for this study. The results showed that inhibition of ox-LDL phagocytosis by cytochalasin D in the early stage improved autophagic status, reduced NLRP3 activation and the apoptotic response significantly. In contrast, cytochalasin D had little effect on blocking the detrimental effect of ox-LDL at the advanced stage. Moreover, the changes in autophagy, apoptosis and NLRP3 expression after treatment with small interfering (si) RNA targeting NLRP3 in the early and advanced stages of atherogenesis were consistent with the above data. Conclusions Interventions against lipid disorders or inflammatory reactions in the early or advanced stages of atherogenesis may have different results depending on when they are applied during the process of atherosclerotic pathogenesis. These results may help improve therapeutic strategies for atherosclerosis prevention. Furthermore, a healthy lifestyle should still be recommended as the most important and inexpensive measure to prevent atherogenesis.

Sensitive and specific detection of nucleic acids or proteins, which act as biomarkers, is of great importance in disease diagnosis. By combing the concept and operation of an endonuclease-assisted target-responsive amplification method and peroxidase-mimic DNAzyme generated by terminal deoxynucleotidyl transferase (TdT), a novel and facile colorimetric biosensor was developed for DNA and protein. Target DNA and thrombin were chosen as representative biomolecules. The production of cleavage fragments can only be triggered by specific target binding and the following nicking process, which do not occur spontaneously. In the signal collection part, numerous guanine-rich DNA were produced through the prolongation of cleavage fragments by TdT and formed highly effective DNAzyme with hemin. In this novel amplification method, we succeeded in realizing sensitive and specific detection of target DNA and thrombin. Under optimal conditions, target DNA can be detected as low as 1 pM, and thrombin with a detection limit of 100 pM. The method also proves the potential versatility and feasibility of TdT-generated DNAzyme in various bio-analyses.

Early diagnosis of hepatitis C virus (HCV) infection is essential to prevent disease from spreading and progression. Herein, a novel electrochemical biosensor was developed for ultrasensitive detection of HCV core antigen (HCVcAg) based on terminal deoxynucleotidyl transferase (TdT) amplification and DNA nanowires (DNW). After sandwich-type antibody-antigen recognition, the antibody-conjugated DNA was pulled to the electrode surface and further extended into a long DNA sequence by robust TdT reaction. Then, large numbers of methylene blue-loaded DNW (MB@DNW) as signal labels are linked to the extended DNA sequence. This results in an amplified electrochemical signal for HCVcAg determination, typically measured at around -0.25 V (Ag/AgCl). Under the optimum conditions, the proposed biosensor achieved a wide linear range for HCVcAg from 0.1 to 312.5 pg/mL with a low limit of detection of 32 fg/mL. The good practicality of the biosensor was demonstrated by recovery experiment (recoveries from 98 to 104% with RSD of 2.5-4.4%) and comparison with enzyme-linked immunosorbent assay (ELISA). Given the highlighted performance, the biosensor is expected to act as a reliable sensing tool for HCVcAg determination in clinics. Schematic representation of the ultrasensitive electrochemical biosensor based on terminal deoxynucleotidyl transferase (TdT) amplification linked with methylene blue-loaded DNA nanowires (MB@DNW), which can be applied to the determination of hepatitis C virus core antigen (HCVcAg) in clinical samples. dTTPs, 2'-deoxythymidine 5'-triphosphate.

Prostate-specific antigen (PSA) is the only biomarker for the diagnosis of prostate cancer. So the PSA screening test is very important due to the high occurrence of prostate cancer in men. In this work, a label-free fluorescent method was developed based on terminal deoxynucleotidyl transferase (TdT) and G-quadruplex-thioflavin T complex for detecting PSA. In the absence of PSA, the PSA aptamer can be used as the primer for TdT extension reactions, resulting in the formation of G-quadruplexes and generation of strong fluorescent signals. After the addition of PSA, the PSA-aptamer complex prevented the TdT extension reaction due to steric hindrance, thus resulting in a poor fluorescent signal. The assay showed a wide linear range (0.1 to 80 pg/mL) and a detection limit of 0.086 pg/mL (S/N = 3). It also has good specificity for PSA determination and gives satisfactory results when applied to biological samples. Conceivably, its merits such as good selectivity and high sensitivity indicate that the proposed method has a promising application potential in the clinical diagnosis and treatment of prostate cancer. Graphical abstract.

New storage technologies are needed to keep up with the global demands of data generation. DNA is an ideal storage medium due to its stability, information density and ease-of-readout with advanced sequencing techniques. However, progress in writing DNA is stifled by the continued reliance on chemical synthesis methods. The enzymatic synthesis of DNA is a promising alternative, but thus far has not been well demonstrated in a parallelized manner. Here, we report a multiplexed enzymatic DNA synthesis method using maskless photolithography. Rapid uncaging of Co2+ ions by patterned UV light activates Terminal deoxynucleotidyl Transferase (TdT) for spatially-selective synthesis on an array surface. Spontaneous quenching of reactions by the diffusion of excess caging molecules confines synthesis to light patterns and controls the extension length. We show that our multiplexed synthesis method can be used to store digital data by encoding 12 unique DNA oligonucleotide sequences with video game music, which is equivalent to 84 trits or 110 bits of data., Writing data in DNA is still a bottleneck due to the reliance on chemical synthesis methods. Here the authors report multiplexed enzymatic DNA synthesis using maskless photolithography.

The article describes an on-chip amplification scheme initiated by a terminal deoxynucleotidyl transferase (TdT) for highly sensitive fluorometric determination of protein. Two thrombin-binding aptamers were designed to capture thrombin as they can form a sandwich structure for improved specificity. An amino-modified aptamer (TBA29) was first immobilized on a silicon chip. After capture of thrombin, a second aptamer (TBA15) was conjugated to the second binding site of thrombin. The 3'-terminal of aptamer TBA15 is exposed on the chip surface, and then fluorescein-labeled 12-dATP associates to the 3'-terminal with the help of TdT. This results in signal amplification, and eventually leads to highly sensitive detection. Under optimal conditions, fluorescence intensity is linearly related to the logarithm of thrombin concentration in the range of 100 fM - 0.1 μM, and the detection limit is as low as 2.0 fM. The assay is sensitive and selective even over potentially interfering proteins and in the presence of human serum. Graphical abstract Schematic strategy for thrombin detection. Two thrombin-binding aptamers were designed to capture thrombin to form a sandwich structure for improved specificity. The protein detection is based on TdT initiated on-chip fluorescent amplification.

Human terminal deoxynucleotidyl transferase (hTdT) is a DNA polymerase that functions to generate diversity in the adaptive immune system. Here, we focus on the function of naturally occurring single-nucleotide polymorphisms (SNPs) of hTdT to evaluate their role in genetic-generated immune variation. The data demonstrate that the genetic variations generated by the hTdT SNPs will vary the human immune repertoire and thus its responses. Human TdT catalyzes template-independent addition of nucleotides (N-additions) during coding joint formation in V(D)J recombination. Its activity is crucial to the diversity of the antigen receptors of B and T lymphocytes. We used in vitro polymerase assays and in vivo human cell V(D)J recombination assays to evaluate the activity and the N-addition levels of six natural (SNP) variants of hTdT. In vitro, the variants differed from wild-type hTdT in polymerization ability with four having significantly lower activity. In vivo, the presence of TdT varied both the efficiency of recombination and N-addition, with two variants generating coding joints with significantly fewer N-additions. Although likely heterozygous, individuals possessing these genetic changes may have less diverse B- and T-cell receptors that would particularly effect individuals prone to adaptive immune disorders, including autoimmunity.

Heterozygosity for mutations in ribosomal protein genes frequently leads to a dominant phenotype of retarded growth and small adult bristles in Drosophila (the Minute phenotype). Cells with Minute genotypes are subject to cell competition, characterized by their selective apoptosis and removal in mosaic tissues that contain wild-type cells. Competitive apoptosis was found to depend on the pro-apoptotic reaper, grim and head involution defective genes but was independent of p53. Rp/+ cells are protected by anti-apoptotic baculovirus p35 expression but lacked the usual hallmarks of ‘undead' cells. They lacked Dronc activity, and neither expression of dominant-negative Dronc nor dronc knockdown by dsRNA prevented competitive apoptosis, which also continued in dronc null mutant cells or in the absence of the initiator caspases dredd and dream/strica. Only simultaneous knockdown of dronc and dream/strica by dsRNA was sufficient to protect Rp/+ cells from competition. By contrast, Rp/Rp cells were also protected by baculovirus p35, but Rp/Rp death was dronc-dependent, and undead Rp/Rp cells exhibited typical dronc-dependent expression of Wingless. Independence of p53 and unusual dependence on Dream/Strica distinguish competitive cell death from noncompetitive apoptosis of Rp/Rp cells and from many other examples of cell death.

Reversible primer termination, enabled by polymerase–nucleotide conjugates, provides an enzymatic method for the de novo synthesis of oligonucleotides.

Considerable knowledge has been gained from temporal analyses of molecular events culminating in gene activation, but technical hurdles have hindered comparable studies of gene silencing. Here we describe the temporal assembly of silent chromatin at the mouse terminal transferase gene (Dntt), which is silenced and repositioned to pericentromeric heterochromatin during thymocyte maturation. Silencing was nucleated at the Dntt promoter by the ordered deacetylation of histone H3 at Lys9 (H3-Lys9), loss of methylation at H3-Lys4 and acquisition of methylation at H3-Lys9, followed by bidirectional spreading of each event. Deacetylation at H3-Lys9 coincided with pericentromeric repositioning, and neither of these early events required de novo protein synthesis. CpG methylation increased primarily in mature T cells that had left the thymus. A transformed thymocyte line supported reversible inactivation of Dntt without repositioning. In these cells, histone modification changes were nucleated at the promoter but did not spread. These results provide a foundation for elucidating the mechanisms of silent chromatin assembly during development.

The DNA polymerase (pol) X family is an ancient group of enzymes that function in DNA replication and repair (pol beta), translesion synthesis (pol lambda and pol micro) and terminal addition of non-templated nucleotides. This latter terminal deoxynucleotidyl transferase (TdT) activity performs the unique function of providing diversity at coding joins of immunoglobulin and T-cell receptor genes. The first isolated full-length TdT genes from shark and skate are reported here. Comparisons with the three-dimensional structure of mouse TdT indicate structural similarity with elasmobranch orthologues that supports both a template-independent mode of replication and a lack of strong nucleotide bias. The vertebrate TdTs appear more closely related to pol micro and fungal polymerases than to pol lambda and pol beta. Thus, unlike other molecules of adaptive immunity, TdT is a member of an ancient gene family with a clear gene phylogeny and a high degree of similarity, which implies the existence of TdT ancestors in jawless fishes and invertebrates.

Temporal control of DNA replication has been implicated in epigenetic regulation of gene expression on the basis of observations that certain tissue-specific genes replicate earlier in expressing than non-expressing cells. Here, we show evidence that several leukocyte-specific genes replicate early in lymphocytes regardless of their transcription and also in fibroblasts, where these genes are never normally expressed. Instead, the heritable silencing of some genes (Rag-1, TdT, CD8alpha and lambda5) and their spatial recruitment to heterochromatin domains within the nucleus of lymphocytes resulted in a markedly delayed resolution of sister chromatids into doublet signals discernable by 3D fluorescence in situ hybridization (FISH). Integration of transgenes within heterochromatin (in cis) did, however, confer late replication and this was reversed after variegated transgene expression. These findings emphasise that chromosomal location is important for defining the replication timing of genes and show that retarded sister-chromatid resolution is a novel feature of inactive chromatin.

The short splice variant of mouse terminal deoxynucleotidyl transferase (TdTS) catalyzes the addition of nontemplated nucleotides (N addition) at the coding joins of B cell and T cell antigen receptor genes. However, the activity and function of the long isoform of TdT (TdTL) have not been determined. We show here, in vitro and in vivo, that TdTL is a 3'--5' exonuclease that catalyzes the deletion of nucleotides at coding joins. These findings suggest that the two TdT isoforms may act in concert to preserve the integrity of the variable region of antigen receptors while generating diversity.

Exposure of some acute myeloid leukaemia (AML) cells to daunorubicin leads to rapid cell death, whereas other AML cells show natural drug resistance. This has been attributed to expression of functional P-glycoprotein resulting in reduced drug accumulation. However, it has also been proposed that P-glycoprotein-expressing multidrug-resistant (MDR) cells are inherently defective for apoptosis. To distinguish between these different possibilities, we have compared the cell death process in a human AML cell line (HL-60) with a MDR subline (HL-60/Vinc) at doses that yield either similar intracellular daunorubicin concentrations or comparable cytotoxicity. Adjustment of the dose to obtain the same intracellular drug accumulation in the two cell lines did not result in equal cytotoxicity, suggesting the presence of additional resistance mechanisms in the P-glycoprotein-expressing HL-60/Vinc cells. However, at equitoxic doses, similar cell death pathways were observed. In HL-60 cells, daunorubicin induced rapid apoptosis at 0.5–1 μM and delayed mitotic cell death at 0.1 μM. These concentrations are within the clinical dose range. Similarly, HL-60/Vinc cells underwent apoptosis at 50–100 μM daunorubicin and mitotic cell death at 10 μM. These results show, for the first time, that anthracyclines can induce cell death by a dual mechanism in both sensitive and MDR cells. Our results also show that not only the cytotoxicity, but also the kinetics and mechanism of cell death, are dose dependent. Interestingly, regrowth was observed only in association with delayed cell death and the formation of enlarged, often polyploid, cells with micronucleation, suggesting that morphological criteria may be useful to evaluate treatment efficacy in patients with myeloid leukaemias. © 1999 Cancer Research Campaign

Precursor lymphoblastic leukemia/lymphoblastic lymphoma (ALL/LBL) is a malignant neoplasm of precursor lymphocytes of T- or B-cell phenotype. We describe the unusual features of an ALL/LBL in an adolescent man in whom the disease presented with involvement of lymph nodes, but without bone marrow and peripheral blood involvement. Immunohistochemical studies revealed that the tumor cells were positive for CD3, CD34 class II, CD10, CD79a and CD99 but negative for TdT. Even though TdT was negative, he received ALL-therapy and is now in remission.

Terminal deoxynucleotidyl transferase (TdT) is a highly conserved vertebrate enzyme that possesses the unique ability to catalyze the random addition of deoxynucleoside 5'-triphosphates onto the 3'-hydroxyl group of a single-stranded DNA. It plays an important role in the generation of immunoglobin and T-cell receptor diversity. TdT is usually obtained from animal thymus gland or produced in a baculovirus system, but both procedures are rather tedious, and proteolysis occurs during purification. Attempts to overexpress TdT in bacteria have been unsuccessful or have yielded an enzyme with a lower specific activity. A dearth of TdT has thus hampered detailed structural and functional studies. In the present study, we report that by lowering growth temperature and overexpressing a rare arginyl tRNA, it is possible to boost the production in Escherichia coli of murine TdT with minimal proteolysis and high specific activity.

Nitrobenezene (NB) produces germ cell degeneration, especially of spermatocytes in rats. To examine the possible involvement of apoptosis in this process, the extent and nature of nuclear DNA fragmentation after NB dosing were assessed using both terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) and DNA gel electrophoresis, in addition to conventional histological and electron microscopic procedures. Adult Sprague Dawley rats were treated with a single oral dose of NB (250 mg/kg) and euthanized subsequently at 6, 12, and 24 h and 2, 3, 5, and 7 days. The earliest morphological signs of germ cell degeneration in testes were found in pachytene spermatocytes 24 h after dosing. Electron micrographs of degenerating spermatocytes showed marked nuclear chromatin condensation at the nuclear periphery and crowding of cytoplasmic constituents, which are characteristic of apoptosis. Coincident with the appearance of such morphological changes, degenerating spermatocytes contained fragmented DNA as revealed by TUNEL. The presence of DNA laddering, a hallmark of apoptosis on gel electrophoresis, was first apparent and most prominent at 24 h, gradually becoming less detectable. No such changes were observed up to 12 h after dosing or in control animals. These results demonstrated unequivocal involvement of apoptosis in the induction of germ cell degeneration caused by NB.

Catagen hair follicle involution has been reported to involve apoptosis, although the precise mechanism has not been satisfactorily resolved. Previous studies have involved solely morphological or electron microscopical methods. We report here studies on murine hair follicles during the first postnatal hair cycle conducted using the terminal deoxy-nucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method. Electrophoresis of DNA isolated from the hair follicles of the same animals was carried out in order to confirm the systematic fragmentation of DNA that typifies apoptosis. On day 10, when all the follicles were growing, there was no evidence of staining with TUNEL in the hair bulbs. Electrophoresis similarly did not show characteristic DNA ladders. By day 15, a few positive cells were observed in the hair bulbs and the numbers had increased by day 17 when many positive cells were seen, especially in the lower portions of the follicles. Electrophoresis demonstrated DNA ladders on days 15, 16 and 17, although the DNA ladder on day 15 was less prominent than that on day 17. These studies confirmed that apoptosis, as identified by techniques that measure DNA fragmentation, occurs in the lower regions of hair follicles towards the end of catagen.

Atm is part of a pathway that responds to DMA damage from ionizing radiation (IR). This pathway involves p53 as Atm-deficient cell lines and mice are defective in p53 induction after IR1–5. p53 is a multi-functional protein that simultaneously regulates distinct downstream pathways controlling cell-cycle progression and apoptosis6,7. However, the mechanisms by which p53 differentially activates downstream pathways are unknown. To determine the relationship between Atm and p53, we examined cell-cycle and apoptotic responses in Atm-, p53- (ref. 8) and p27-deficient9 mice after IR in the whole animal. As expected, p53 protein levels were not induced by IR in thymus of Atm-deiicient mice. IR-induced cell-cycle checkpoint function was also defective, and induction of p21 was attenuated in thymus from >U/7?-deficient mice. However, IR-induced apoptosis and Bax induction were completely normal; both of which are mediated by p53. IR-induced thymic apoptosis was suppressed in Atm/p53 double-mutant mice but not in Atm/p21 double mutants, demonstrating p53 dependence and Atm independence. Thus, Atm deficiency results in lack of p53 induction by IR, but only selective disruption of p53-dependent functions. Our results support a model in which upstream effectors such as Atm selectively activate p53 to regulate specific downstream pathways, providing a mechanism for controlling distinct cell-cycle and apoptotic responses.

We report a pilot study on the Fas receptor (APO-1, CD95) in vivo in 15 human squamous cell (non-small) carcinomas and ten normal bronchial specimens. The principal aim was to investigate whether the so-called death receptor, Fas, is present in these tumours. Ligation of Fas promptly induces apoptosis, particularly in T Jurkat cells in vitro, and expression of Fas on human cancer would thus theoretically be of great interest. The immunoreactivity for the anti-apoptotic protein Bcl-2 was also investigated, and the degree of apoptosis was evaluated by TdT dUTP nick end labelling (TUNEL) and conventional morphological criteria. Fas was present in all initial tumours but absent in control tissue, that is in the potential precursor cells of bronchial epithelium (P = 0.001). Fas was not detectable after radiotherapy (P = 0.03). We propose that radiotherapy induces an early selection of tumour cells rather than a down-regulation of Fas. Both Bcl-2 and apoptosis (TUNEL) were generally expressed at a modest level. In agreement with other studies, we did not find any significant correlation between Bcl-2 and prognosis, or between Bcl-2 and TUNEL. Hence, in this preliminary report, we have demonstrated Fas receptor in human squamous cell carcinomas in vivo. This is a novel finding, and the apparent absence of Fas after radiotherapy may have important therapeutic implications. Images Figure 1

Zinc (Zn) is a trace element in human cells and regarded as an essential nutrient with established deficiency states affecting multiple organs in the body. However, it has been reported that Zn uptake is associated with some serious harmful effects, such as inhibition of DNA synthesis and enhanced toxicity from reactive oxygen species. We have previously shown that in vivo administration of Zn2+ in C57/6J mice induces weight loss and massive hair loss where the normal course hair becomes replaced by fine vello hair, simulating the side effects from cancer chemotherapy where oxidative free radical damage is implicated in association with DNA fragmentation and programmed cell death (PCD). Here, in vitro flow cytometric studies on human Chang liver showed Zn2+ causing cell condensation with DNA fragmentation that occurred in a dose-dependent manner, an effect replicated by micrococcal nuclease digestion. Specific terminal deoxynucleotidyl transferase- (TdT) mediated labeling of 3′-OH ends of DNA nicks corroborated the flow cytometric profiles of propidium iodide-DNA binding where degradation of both 2 and 4N genomic DNA resulted in a solitary 1N peak presentation. DNA degradation concomitant with cell condensation is seen as an estabilished hallmark of PCD. We further showed that Zn2+ could enhance the generation of hydroxyl free radicals (OH•) by the transition metal vanadium. Glutathione, the cell's main reducing agent, underwent corresponding reduction. The results suggested that Zn supplementation could induce features resembling PCD.

Mice develop a fatal encephalomyelitis after infection with the Trinidad donkey strain of Venezuelan equine encephalitis (VEE) virus. Adult mice were inoculated intraperitoneally with VEE virus and the brains were examined at different time points. Morphological changes were assessed by histological staining. VEE virus antigen was detected with immunoperoxidase staining, and DNA fragmentation was evaluated in situ using the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) method. VEE antigen was found in many areas of the brain and it was prominent in neurons. There were mild associated inflammatory changes. DNA fragmentation was demonstrated in many of these areas using TUNEL. In areas with TUNEL staining, morphological neuronal changes ranged from nuclear chromatin condensations to nuclear and cellular fragmentation, which are characteristic of apoptosis. There is strong morphological and biochemical evidence of apoptotic cell death in this experimental model of VEE virus infection.

The ribonucleoprotein enzyme telomerase adds telomeric DNA onto chromosome ends and is normally regulated so that telomeric DNA lengths are kept within defined bounds. In the telomerase RNA gene from the yeast Kluyveromyces lactis, specific mutations that alter telomeric DNA sequences result in telomeres elongating to up to 100 times their normal length and impair cell growth. Some mutations cause immediate elongation whereas others behave like genetic time bombs, causing elongation only after a latent period of hundreds of generations.

Ameloblasts responsible for tooth enamel formation are classified into two different phases: secretion and maturation. At the transition between these secretion and maturation stages, a considerable number of cells die. In this study, we examined the morphology of degenerating ameloblasts by conventional electron microscopy, and DNA cleavage in degenerating ameloblast nuclei by the in situ terminal transferase assay. The results suggest that apoptosis (programmed cell death) in ameloblasts, including DNA ligation is induced at the transitional stage. The nuclear fragments, chromatin condensation and DNA relocation in apoptotic nuclei were examined quantitatively by post-embedding anti-DNA immunogold electron microscopy and the in situ terminal transferase assay combined with electron microscopy. Numerical analysis revealed that immunogold labeling density in the condensed chromatin of apoptotic nuclei was comparable on the average to that in the perinuclear heterochromatin of normal nuclei, and that individual apoptotic nuclear fragments exhibited highly variable gold particle density, from fragments with lower density to that of normal heterochromatin, to fragments with densities twice as high as that of normal heterochromatin. The in situ terminal transferase assay combined with electron microscopy detected DNA ends exposed by ultrathin sectioning as well as DNA cleavage by a putative endonuclease. In conclusion, the state of the DNA, including its ligation and degeneration, changes gradually during chromatin condensation and nuclear fragmentation of apoptosis.

The mouse lpr gene, which is an autosomal recessive gene causing autoimmune disease with features of human systemic lupus erythematosus and eventually death from severe immune-complex glomerulonephritis, has been mapped on chromosome 19. To determine its exact chromosomal location, a three-point backcross was carried out by mating (MRL/MpJ-lpr/lpr x MOL-MIT)F1 x MRL/MpJ-lpr/lpr using the genes Ly-44 (lymphocyte differentiation antigen-44) and Tdt (terminal deoxynucleotidyl transferase) as markers. The following order of genes is proposed, with the distances between genes given in parentheses: centromere-Ly-44 (19.3 cM)-lpr (6.1 cM)-Tdt-telomere. The Ly-44a and Tdta alleles are found in all laboratory strains and in the wild Western European subspecies, domesticus and brevirostris. In contrast, the Ly-44b and Tdtb alleles are found in some Asian subspecies, Chinese mice of wild origin, yamashinai and molossinus. Furthermore the third Tdt allele, Tdtc, is detected in castaneus.

Nuclear matrix extracted from KM-3, a human pre-B leukemia cell line, appears to have a site of linkage for terminal deoxynucleotidyl transferase (TdT). The immunocytochemical analysis of the distribution of TdT using a rabbit polyclonal antibody which recognizes human terminal transferase, shows that the nuclear framework of these cells contains sites of immunoreactivity that appear uniformly distributed on the matrix fibres, while the nucleolar region is unreactive. This evidence points out the possibility that TdT could reside in the proteinaceous scaffold of the nucleus defined as nuclear matrix, thus strengthening the evidence for the metabolic and regulatory roles ascribed to this nuclear framework.

Terminal deoxynucleotidyl transferase (TdT) contributes to the junctional diversity of immunoglobulin and T-cell receptors by incorporating nucleotides in a template-independent manner. A closely related enzyme, polymerase mu (polmu), a template-directed polymerase, plays a role in general end-joining double-strand break repair. We cloned zebrafish TdT and polmu and found them to be 43% identical in amino acid sequence. Comparisons with sequences of other species revealed conserved residues typical for TdT in the zebrafish sequence that support the template independence of this enzyme. Some but not all of these features were identified in zebrafish polmu. In adult fish, TdT expression was most prominent in thymus, pro- and mesonephros, the primary lymphoid organs in teleost fish and in spleen, intestine, and the tissue around the intestine. Polmu expression was detected not only in pro- and mesonephros, the major sites for B-lymphocyte development, but also in ovary and testis and in all tissue preparations to a low extent. TdT expression starts at 4 dpf and increases thereafter. Polmu is expressed at all times to a similar extent. In situ studies showed a strong expression of TdT and polmicro in the thymic cortex of 8-week-old fish. The characterization of zebrafish TdT and polmu provide new insights in fish lymphopoiesis and addresses the importance and evolution of TdT and polmu themselves.

Among site-directed DNA recombination systems, V(D)J recombination is noteworthy in that identical reactants yield different recombination products at the junction of joined segments. This variation is the basis for diversity at the base of antigen receptor binding pockets and corresponds to V-(D)-J DNA junctions. An abundance of certain junctions has been noted. It has been proposed that these junctions are favoured because they occur where short regions of homology in participating coding ends might align preferentially. Here we use a system that is entirely free from cellular selection to show that the diversity of coding joints can be severely restricted when the coding ends participating in the reaction have short regions of homology. This constraint on diversity is diminished but not eliminated by terminal deoxynucleotidyl transferase, a mechanistic feature that has implications for the establishment of the immune repertoire.

Telomeres define the ends of chromosomes; they consist of short tandemly repeated DNA sequences loosely conserved in eukaryotes (G1-8(T/A)1-4). Telomerase is a ribonucleoprotein which, in vitro, recognizes a single-stranded G-rich telomere primer and adds multiple telomeric repeats to its 3' end by using a template in the RNA moiety. In conjunction with other components, telomerase may balance the loss of telomeric repeats due to DNA replication. Another role of telomerase may be the de novo formation of telomeres. In eukaryotes like Tetrahymena, this process is an integral part of the formation of macronuclear chromosomes. In other eukaryotes this process stabilizes broken chromosomes. A case of human alpha-thalassaemia is caused by a truncation of chromosome 16 that has been healed by the addition of telomeric repeats (TTAGGG)n. Using an in vitro assay, I show here that human telomerase correctly recognizes the chromosome 16 breakpoint sequence and adds (TTAGGG)n repeats. The DNA sequence requirements are minimal and seem to define two modes of DNA recognition by telomerase.

The ends or telomeres of the linear chromosomes of eukaryotes are composed of tandem repeats of short DNA sequences, one strand being rich in guanine (G strand) and the complementary strand in cytosine. Telomere synthesis involves the addition of telomeric repeats to the G strand by telomere terminal transferase (telomerase). Telomeric G-strand DNAs from a variety of organisms adopt compact structures, the most stable of which is explained by the formation of G-quartets. Here we investigate the capacity of the different folded forms of telomeric DNA to serve as primers for the Oxytricha nova telomerase in vitro. Formation of the K(+)-stabilized G-quartet structure in a primer inhibits its use by telomerase. Furthermore, the octanucleotide T4G4, which does not fold, is a better primer than (T4G4)2, which can form a foldback structure. We conclude that telomerase does not require any folding of its DNA primer. Folding of telomeric DNA into G-quartet structures seems to influence the extent of telomere elongation in vitro and might therefore act as a negative regulator of elongation in vivo.

We have hypothesized that end-to-end chromosome fusions observed in some tumours could play a part in genetic instability associated with tumorigenesis and that fusion may result from the loss of the long stretches of G-rich repeats found at the ends of all linear chromosomes. We therefore asked whether there is telomere loss or reduction in common tumours. Here we show that in most of the colorectal carcinomas that we analysed, there is a reduction in the length of telomere repeat arrays relative to the normal colonic mucosa from the same patient. We speculate on the consequences of this loss for tumorigenesis. We also show that the telomere arrays are much smaller in colonic mucosa and blood than in fetal tissue and sperm, and that there is a reduction in average telomere length with age in blood and colon mucosa. We propose that the telomerase is inactive in somatic tissues, and that telomere length is an indicator of the number of cell divisions that it has taken to form a particular tissue and possibly to generate tumours.

The telomerase enzyme of Tetrahymena synthesizes repeats of the telomeric DNA sequence TTGGGG de novo in the absence of added template. The essential RNA component of this ribonucleoprotein enzyme has now been cloned and found to contain the sequence CAACCCCAA, which seems to be the template for the synthesis of TTGGGG repeats.

T lymphocytes of higher vertebrates are able to specifically recognize a seemingly unlimited number of foreign antigens via their receptors, the T cell antigen receptors (TCRs). T lymphocytes mature by passing through the thymus and acquire antigen specificity by expressing the TCR molecules on their cell surface. Genetic and somatic diversification mechanisms give rise to the enormous degree of TCR variability observed in mature T cells: germline and combinatorial diversity as well as junctional and the so-called N-region diversity. In contrast to the situation in immunoglobulin genes somatic hypermutation does not seem to play a significant role in TCR diversification. It is argued here that the enzyme terminal nucleotidyl-transferase is potentially a major factor in generating the immense diversity. We propose furthermore that this enzyme ensures the flexibility of T cell responses to novel antigens by random insertion of so-called N-region nucleotides. Apart from the physiological functions of TCR genes any involvement in the etiology of T cell neoplasia remains to be proven.

We have investigated glucocorticoid receptor (GR) level, terminal deoxynucleotidyl transferase (TdT) activity and the initial responsiveness to prednisone and vincristine in 31 patients with acute lymphatic leukemia (ALL) and with chronic myelogenous leukemia in blast transformation (CML/BT). All 11 patients with low levels of GR (5,000 binding sites per cell) were resistent to this initial treatment whereas 13 of the 20 patients with high levels of GR responded readily. The correlations between clinical responsiveness and GR-level, or between responsiveness and TdT activity were significant to thep

Of 112 patients with acute leukemia 13 exhibited terminal deoxyribonucleotidyl transferase activity (TdT) with a cell phenotype characterized by the presence of early or mature monocyte differentiation antigens detected by monoclonal antibodies and elevated serum lysozyme. These cells were either unclassifiable by cytochemical analysis or fell into the M2 or M5 categories by the French-American-British classification. These leukemias appear to form a distinct nosologic entity representing a malignant transformation among early cells in the monocyte lineage. Among 60 patients with acute myelogenous leukemia 87% expressed the polymorphic Ia antigen detected by monoclonal antibody 109d6 compared to an incidence of 40.5% in the control population, relative risk 9.2. Evidence was obtained that this antigen was present in healthy family members and remission B-cells and monocytes of patients, and that its presence was associated with a genetically defined susceptibility state for the development of acute myelogenous leukemia.

Terminal deoxynucleotidyl transferase (TdT) has become an important marker for the classification of acute leukemias. Originally detected by a biochemical assay, the presence of this enzyme in leukemia can now be detected by making use of a highly specific antibody and by applying the immunofluorescence method. In this study, an attempt was made to apply the unlabeled peroxidase-antiperoxidase (PAP) technique for the examination of TdT positive cells. We have optimized this method and have compared the different assays - biochemical, immunofluorescence, and immunocytochemical (PAP) - in ten patients with acute leukemia. The results obtained from all three methods are in accordance with one another, but the PAP method is a further simplification for the detection of TdT in the routine diagnostic of acute leukemias.

A micromethod for the determination of TdT in peripheral leukocytes and bone marrow cells has been developed that allows unequivocal identification and quantitation of TdT in less than 1 X 10(6) leukocytes from ALL patients, i.e., in 1 ml of peripheral blood and/or 0.5 ml of bone marrow obtained during routine clinical sampling. The method involves disruption of cell pellet with high salt and detergent followed by centrifugation of extracts at 12,000 X g and partial purification on phosphocellulose matrix by a batch elution technique using a standard laboratory microcentrifuge. Using this microassay, TdT activities have been determined in 500 samples of peripheral blood and bone marrow of 240 adult patients with acute leukemias (86 ALL, 108 ANLL, 44 blastic CML, two acute leukemias following P. vera). From an analysis of our data based on TdT activity, cell surface markers and growth patterns in soft agar and observations published in the literature, it can be concluded that the frequencies of TdT + phenotypes in the various clinical-morphological diagnostic groups are approximately 95% in ALL, 10% in ANLL, 50% in AUL, and 35% in blastic CML. Since the presence of high TdT activity is clearly associated with clinical response to specific forms of chemotherapy in blastic CML and most probably, also in ANLL, the determination of TdT should be considered in all cases of acute leukemias to objectively define prognostically important subgroups which can not be diagnosed by conventional means.

Optimized methods for extraction and enzyme assay in crude tissue preparations were used to determine the amounts of terminal deoxnucleotidyl transferase (TdT) in malignant lymphomas. The TdT concentration was increased only in lymphoblastic lymphomas (LL) and was as high in these tumours as in the white blood cells from untreated patients with acute lymphoblastic leukaemia (ALL). The enzymes extracted from such lymphomas and from the leukaemic lymphoblasts had the same properties. Moreover, forms of TdT with low and high mol. wt were found in the LL tumours, similar to other reports of TdT-positive leukaemias. The overall study points at some basic biochemical identity of certain lymphoblastic malignancies, irrespective of whether the transformed cells are in solid tumours or are disseminated in the blood.

A technique has been worked out for an easy detection of terminal transferase in individual cells by means of the unlabeled antibody enzyme (PAP) method. Terminal transferase is an important enzyme marker for the classification of acute lymphoblastic leukemias that show a variable extent of differentiation into the T-cell series. The incubation procedure giving the best results is described.

Malignant blast cells in the various forms of acute leukaemia seem to be ‘frozen’ at early stages of haematopoietic and lymphoid cell maturation. These blasts express relatively stable morphological, enzymatic and antigenic phenotypes which may largely represent the normal gene products of the corresponding precursor cells1–4. A number of findings support these suggestions. The phenotype of the common form of acute lymphoblastic leukaemia (cALL) corresponds to the phenotype of normal small non-T, non-B cells of lymphoid morphology detected in low numbers in the normal and regenerating infant bone marrow4,5. Also, the phenotype of blast cells in Thy-ALL corresponds approximately to that of cortical thymocytes or their immediate precursors6–9. Leukaemia-specific (virus-induced or mutant) changes or aberrant expression of normal gene products may also occur in leukaemia. To describe such phenomena, detailed single cell studies comparing leukaemic cells with their appropriate normal (frequently very rare) counterparts are needed. In this study we have performed such a comparative study and find that the human Thy-ALL blasts express unexpectedly high amounts of HLA-A, -B and -C antigens: the sensitive single cell assays used failed to find normal cells (in infant and fetal thymus, and in bone marrow) which express the exact phenotype of Thy-ALL blasts.

Thymosin fraction 5, a family of acidic polypeptides isolated from bovine thymus, contains several hormonal-like factors which have been shown to influence the maturation, differentiation and functions of T-cells. Some of these peptides have been chemically defined. Two of them, thymosin alpha 1 (M.W. 3108) and thymosin beta 4 (M.W. 4982) have been sequenced. In murine systems, terminal deoxynucleotidyl transferase (TdT) has been shown to be T-cell specific and to be present primarily in the cortisone sensitive immature T-cell populations. The daily injection of thymosin fraction 5 and two of its components, thymosin beta 3 and beta 4, significantly increases TdT activity in immune suppressed mice as compared to control groups. This study indicates that thymosin can act on prothymocytes and influence the early stages of T-cell differentiation. In an in vivo system, thymosin fraction 5 and the purified peptide, thymosin alpha 1, have high activities in decreasing TdT in normal murine thymocytes after a 22-h incubation. This effect suggests that thymosin can also act on thymocytes and regulate the later biochemical processes during T-cell differentiation.

CALF thymus terminal deoxynucleotidyl transferase (TdT) (EC 2.7.7.31) is a low molecular weight protein containing two peptides of MWs 24,000 and 8,000 in a native structure estimated to have a molecular weight of 32,000 (ref. 1). Use of antibody directed against homogeneous bovine transferase2 for immunofluorescent cytology showed the normal thymus localisation to be cytoplasmic and nuclear, whereas bone marrow localisation is only nuclear3. Several human lymphoblastoid cell lines that contain terminal transferase activity also exhibit the nuclear location by immunofluorescence4. As cultured lymphoblastoid cell lines are readily labelled with radioactive amino acids we surveyed the peptides present in the immuno-precipitated transferase from these lines by fluorography of SDS gels. The results presented here show that a single 58,000-MW peptide is precipitated by monospecific rabbit antibody against bovine transferase. No peptides as small as those peptides present in calf thymus transferase were detected.