AML-1B is a hematopoietic transcription factor that is functionally inactivated by multiple chromosomal translocations in human acute myeloblastic and B-cell lymphocytic leukemias. The t(8;21)(q22;q22) translocation replaces the C terminus, including the transactivation domain of AML-1B, with ETO, a nuclear protein of unknown function. We previously showed that AML-1-ETO is a dominant inhibitor of AML-1B-dependent transcriptional activation. Here we demonstrate that AML-1-ETO also inhibits C/EBP-␣-dependent activation of the myeloid cell-specific, rat defensin NP-3 promoter. AML-1B bound the core enhancer motifs present in the NP- AML1 is one of the most frequent targets of chromosomal abnormalities in acute leukemias and is involved in multiple translocations. The t(8;21)(q22;q22) translocation fuses residues 1 to 177 of AML1, including the DNA binding domain, to ETO (MTG8), a gene of unknown function that is homologous to the Drosophila gene nervy (8,9,13,41,42,44,48). It is the second most common chromosomal abnormality in acute myeloblastic leukemias (AML) (42). A second translocation, t(3; 21), is rare in de novo AML and is detected in therapy-related AML and during blast crisis of chronic myelogenous leukemias (40,46,49,59). It fuses the first five or six exons of AML1 to three different exons of EviI, a gene encoding a transcription factor on chromosome 3 (43, 47, 60). A third translocation, t(12;21), fuses the first 333 amino acids of TEL, an ets-like protein (17), to nearly all of AML-1B, the largest AML1 product (38), and has been detected in approximately 30% of pediatric B-cell acute lymphoblastic leukemia cases (16,57,58,63). Unlike AML-1-ETO, TEL-AML-1B contains the entire carboxy terminus of AML-1B, including the nuclear matrix targeting signal (NMTS) and transactivation (TA) domain (76). A fourth alteration, inv (16) (4,14,19,27,36,38,45,55,66,68,78). The core binding motif is necessary but not sufficient for tissue-specific activation of myeloid promoters and lymphoid enhancers; therefore, it is possible that AML-1B functions as a promoter organizer. We previously showed that the t(8;21) and t(12;21) translocations convert AML-1B from a transcriptional activator to a repressor (25,38). Because only one allele of AML1 is altered in leukemic cells expressing t(8;21) and because substoichiometric levels of AML-1-ETO efficiently repressed AML-1B-dependent transcriptional activation, we hypothesized that the t(8;21) product is a dominant inhibitor of AML-1B function (14,36,38). AML-1-ETO also repressed transcriptional activation induced by AML-2 (CBF-␣3 or PEBP-2␣C) and the murine homolog of AML-3 (mAML-3, CBF-␣1, or PEBP-2␣A) (1,33,39,51). Thus, AML-1-ETO is able to repress transcription mediated by all core binding factors in hemato-* Corresponding author. Mailing address:
Short-term exposure (43 C for 15 min) of the rat testis to mild heat results within 6 h in stage- and cell-specific activation of germ cell apoptosis. Initiation of apoptosis was preceded by a redistribution of Bax from a cytoplasmic to paranuclear localization in heat-susceptible germ cells. Here we show that the relocation of Bax is accompanied by cytosolic translocation of cytochrome c and is associated with activation of the initiator caspase 9 and the executioner caspases 3, 6, and 7 and cleavage of poly(ADP) ribose polymerase. Furthermore, early in apoptosis, a significant amount of Bax also accumulates in endoplasmic reticulum, as assessed by Western blot analyses of fractionated testicular lysates. In additional studies using the FasL-defective gld mice, we have shown that heat-induced germ cell apoptosis is not blocked, thus providing evidence that the Fas signaling system may be dispensable for heat-induced germ cell apoptosis in the testis. Taken together, these results demonstrate that the mitochondria- and possibly also endoplasmic reticulum-dependent pathways are the key apoptotic pathways for heat-induced germ cell death in the testis.
Aim: To develop antibody–aptamer functionalized fibre‐optic biosensor for specific detection of Listeria monocytogenes from food products. Methods and Results: Aptamer, a single‐stranded oligonucleotide ligand that displays affinity for the target molecule, was used in the assay to provide sensor specificity. Aptamer‐A8, specific for internalin A, an invasin protein of L. monocytogenes, was used in the fibre‐optic sensor together with antibody in a sandwich format for detection of L. monocytogenes from food. Biotinylated polyclonal anti‐Listeria antibody, P66, was immobilized on streptavidin‐coated optical waveguide surface for capturing bacteria, and Alexa Fluor 647‐conjugated A8 was used as a reporter. The biosensor was able to selectively detect pathogenic Listeria in pure culture and in mixture with other bacteria at a concentration of approx. 103 CFU ml−1. This sensor also successfully detected L. monocytogenes cells from artificially contaminated (initial inoculation of 102 CFU 25 g−1) ready‐to‐eat meat products such as sliced beef, chicken and turkey after 18 h of enrichment. Conclusion: Based on the data presented in this study, the antibody–aptamer functionalized fibre‐optic biosensor could be used as a detection tool for sensitive and specific detection of L. monocytogenes from foods. Significance and Impact of the Study: The study demonstrates feasibility and novel application of aptamer on fibre‐optic biosensor platform for the sensitive detection of L. monocytogenes from food products.
Programmed cell death occurs spontaneously during spermatogenesis and can be induced in a cell- and stage-specific manner by mild testicular hyperthermia. Studies using transgenic mice suggest the involvement of Bcl-2 proteins in regulating germ cell apoptosis. To delineate further the pathways involved, we examined the temporal changes in proapoptotic Bax and antiapoptotic Bcl-2 in rat testes after transient exposure to heat (43 degrees C for 15 min). Germ cell apoptosis, involving exclusively early (I-IV) and late (XII-XIV) stages, was activated within 6 h. Initiation of apoptosis was preceded by a redistribution of Bax from a cytoplasmic to perinuclear localization within 0.5 h of heating as assessed by immunocytochemical methods. In contrast, Bcl-2 is distributed both in the cytoplasm and nucleus in those cell types susceptible to heat-induced apoptosis. Despite the striking redistribution, Bax levels remained unchanged as determined by Western analysis; Bcl-2 levels increased significantly by 6 h after heat exposure. Reverse transcription-polymerase chain reaction analysis indicated no change in either Bax or Bcl-2 mRNA levels in response to heat, suggesting the involvement of post-transcriptional rather than transcriptional mechanisms mediating their activity. The marked subcellular redistribution of Bax prior to activation of apoptosis and the increase in Bcl-2 suggest an involvement of Bcl-2 family members in heat-induced apoptotic death of germ cells.
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