Antibody-aptamer functionalized fibre-optic biosensor for specific detection of Listeria monocytogenes from food.

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 invasive 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. 10(3) CFU ml(-1). This sensor also successfully detected L. monocytogenes cells from artificially contaminated (initial inoculation of 10(2) 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.

Impairment of spermatogenesis in transgenic mice with selective overexpression of Bcl-2 in the somatic cells of the testis.

To explore the functional role of Bcl-2 in germ cell development, transgenic mice carrying 6 kilobases of the inhibin-alpha promoter were generated to express human bcl-2 gene product in the gonads. Although female transgenic mice demonstrated decreased follicle apoptosis, enhanced folliculogenesis, and increased germ cell tumorigenesis, the adult males exhibited variable impairment of spermatogenesis. The degree of damage ranged from tubules with intraepithelial vacuoles of varying sizes to near atrophied tubules consisting of Sertoli cells and a few spermatogonia. Although there was no significant change in body weight, an approximately 34% decrease in testicular weights was noted in transgenic animals compared with wild-type mice. Gamete maturation, assessed by determining the percentage of tubules with advanced (steps 13-16) spermatids, was decreased to 44.4% of the values measured in the wild-type animals. The incidence of germ cell apoptosis increased 3.8-fold in the transgenic animals and was associated with a marked loss of germ cells. Electron microscopy of the testes further revealed large vacuoles in the Sertoli cell cytoplasm and dilations of the intracellular spaces between adjacent Sertoli cells, spermatid malformations, and increased germ cell apoptosis in the transgenic animals. There was no evidence of Sertoli cell death either by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay or electron microscopy. Leydig cell ultrastructure, cell size and numbers, and plasma levels of testosterone were not different between normal and the transgenic animals. Collectively, these results support the critical role of Bcl-2 in male germ cell development and are consistent with the gender-specific role of the Bcl-2 family members in reproduction.

Redistribution of Bax is an early step in an apoptotic pathway leading to germ cell death in rats, triggered by mild testicular hyperthermia.

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.

The t(8;21) fusion product, AML-1-ETO, associates with C/EBP-alpha, inhibits C/EBP-alpha-dependent transcription, and blocks granulocytic differentiation.

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-alpha-dependent activation of the myeloid cell-specific, rat defensin NP-3 promoter. AML-1B bound the core enhancer motifs present in the NP-3 promoter and activated transcription approximately sixfold. Similarly, C/EBP-alpha bound NP-3 promoter sequences and activated transcription approximately sixfold. Coexpression of C/EBP-alpha with AML-1B or its family members, AML-2 and murine AML-3, synergistically activated the NP-3 promoter up to 60-fold. The t(8;21) product, AML-1-ETO, repressed AML-1B-dependent activation of NP-3 and completely inhibited C/EBP-alpha-dependent activity as well as the synergistic activation. In contrast, the inv(16) product, which indirectly targets AML family members by fusing their heterodimeric DNA binding partner, CBF-beta, to the myosin heavy chain, inhibited AML-1B but not C/EBP-alpha activation or the synergistic activation. AML-1-ETO and C/EBP-alpha were coimmunoprecipitated and thus physically interact in vivo. Deletion mutants demonstrated that the C terminus of ETO was required for AML-1-ETO-mediated repression of the synergistic activation but not for association with C/EBP-alpha. Finally, overexpression of AML-1-ETO in myeloid progenitor cells prevented granulocyte colony-stimulating factor-induced differentiation. Thus, AML-1-ETO may contribute to leukemogenesis by specifically inhibiting C/EBP-alpha- and AML-1B-dependent activation of myeloid promoters and blocking differentiation.