Recent studies implicating dendritic protein synthesis in synaptic plasticity have focused attention on identifying components of the molecular machinery involved in processing dendritic RNA. Although Translin was originally identified as a protein capable of binding single-stranded DNA, subsequent studies have demonstrated that it also binds RNA in vitro. Because previous studies indicated that Translin-containing RNA/singlestranded DNA binding complexes are highly enriched in brain, we and others have proposed that it may be involved in dendritic RNA processing. To assess this possibility, we have conducted studies aimed at defining the localization of Translin and its partner protein, Trax, in brain. In situ hybridization studies demonstrated that both Translin and Trax are expressed in neurons with prominent staining apparent in cerebellar Purkinje cells and neuronal layers of the hippocampus. Subcellular fractionation studies demonstrated that both Translin and Trax are highly enriched in the cytoplasmic fraction compared with nuclear extracts. Furthermore, immunohistochemical studies with Translin antibodies revealed prominent staining in Purkinje neuron cell bodies that extends into proximal and distal dendrites. A similar pattern of somatodendritic localization was observed in hippocampal and neocortical pyramidal neurons. These findings demonstrate that Translin is expressed in neuronal dendrites and therefore support the hypothesis that the Translin/Trax complex may be involved in dendritic RNA processing. Key Words: Dendritic protein synthesisPurkinje cells-Synaptic plasticity-GS1 complex-Pyramidal neurons.

The cardiotoxicity of adriamycin limits its clinical use as a powerful drug for solid tumors and malignant hematological disease. Although the precise mechanism by which it causes cardiac damage is not yet known, it has been suggested that apoptosis is the principal process in adriamycin-induced cardiomyopathy, which involves DNA fragmentation, cytochrome C release, and caspase activation. However, there has been no direct evidence for the critical involvement of caspase-3 in adriamycin-induced apoptosis. To determine the requirements for the activation of caspase-3 in adriamycin-treated cardiac cells, the effect of a caspase inhibitor on the survival of and apoptotic changes in H9c2 cells was examined. Exposure of H9c2 cells to adriamycin resulted in a time- and dose-dependent cell death, and the cleavage of pro-caspase-3 and of the nuclear protein poly (ADP'ribose) polymerase (PARP). However, neither the reduction of cell viability nor the characteristic morphological changes induced by adriamycin were prevented by pretreatment with the general caspase inhibitor z-VAD.FMK. In contrast, caspase inhibition effectively blocked the apoptosis induced by H202 in H9c2 cells, as determined by an MTT assay or microscopy. We also observed that p53 expression was increased by adriamycin, and this increase was not affected by the inhibition of caspase activity, suggesting a role for p53 in adriamycin-induced caspase-independent apoptosis in cardiac toxicity.

Translin is a nucleic acid binding protein that has been implicated in regulating the targeting and translation of dendritic RNA. In previous studies, we found that Translin and its partner protein, Trax, are components of a gel-shift complex that is highly enriched in brain extracts. In those studies, we employed a DNA oligonucleotide, GS1, as a probe to label the complex. Translin has also been identified as a component of a gel-shift complex detected using an RNA oligonucleotide probe, derived from the 3¢ UTR of protamine-2 mRNA. Although we had assumed that these probes labeled the same complex, recent studies indicate that association of Trax with Translin suppresses its RNA binding activity. As these findings challenge this assumption and suggest that the native RNA binding complex does not contain Trax, we have re-examined this issue. We have found that the gel-shift complexes labeled with either GS1 or protamine-2 probes are 'supershifted' by addition of Trax antibodies, indicating that both are heteromeric Translin/Trax complexes. In addition, cross-competition studies provide additional evidence that these probes label the same complex. Furthermore, analysis of recombinant Translin/Trax complexes generated by co-transfection of Trax with Translin in hEK293T demonstrates that they are labeled with either probe. Although recombinant Translin forms a homomeric nucleic acid binding complex in vitro, our findings indicate that both Trax and Translin are components of the native gel-shift complex labeled with either GS1 or protamine-2 probes.

An AU-rich element (ARE) in the 3-untranslated region (UTR) of bcl-2 mRNA has previously been shown to be responsible for destabilizing bcl-2 mRNA during apoptosis through increasing AUF1 binding. In the present study, we investigated the effect of the region upstream of the ARE on bcl-2 mRNA stability using serial deletion constructs of the 3-UTR of bcl-2. Deletion of 30 nucleotides mostly consisting of the CA repeats, located upstream of the ARE, resulted in the stabilization of bcl-2 mRNA abundance, in the absence or presence of the ARE. The specificity of the CA repeats in terms of destabilizing bcl-2 mRNA was proven by the substituting the CA repeats with other alternative repeats of purine/ pyriminine, but this had no effect on the stability of bcl-2 mRNA. CA repeats alone, however, failed to confer instability to bcl-2 or gfp reporter mRNAs, indicating a requirement for additional sequences in the upstream region of the 3-UTR. Serial deletion and replacement of a part of the region upstream of the CA repeats revealed that the entire 131-nucleotide upstream region is an essential prerequisite for the CA repeat-dependent destabilization of bcl-2 mRNA. Unlike the ARE, CA repeatmediated degradation of bcl-2 mRNA was not accelerated upon apoptotic stimulus. Moreover, the upstream sequences and CA repeats are conserved among mammals. Collectively, CA repeats contribute to the constitutive decay of bcl-2 mRNA in the steady states, thereby maintaining appropriate bcl-2 levels in mammalian cells.Apoptosis is a tightly controlled cellular suicide program that is critical for the successful development of multicellular organisms, the maintenance of normal tissue homeostasis, and removal of damaged cells (1). The protooncogene bcl-2, originally isolated from the chromosomal breakpoint of a t(14, 18)-bearing B cell lymphoma, serves as an important repressor of apoptosis in a variety of cell types (2, 3). In line with its significant role in altering susceptibility to apoptosis, investigations of the mechanisms by which bcl-2 expression is modulated may prove crucial for identifying therapeutic strategies for cancer and some neurodegenerative diseases and for defining the role of bcl-2 in the development of multiple tissues (4).Recent studies have indicated that bcl-2 is regulated at both the transcriptional and posttranscriptional levels. A number of negative transcriptional regulatory sites have been described in the bcl-2 promoter region (5, 6), and several transcription factors, including cAMP response element binding protein, AMyb and WT1, are known to be involved in the positive regulation of bcl-2 transcription (7-9). In addition to the promoter region, some sequences within the coding region, such as estrogen response elements, have also been demonstrated to mediate the transcriptional modulation of bcl-2, as was shown in breast cancer cell lines (10). The posttranscriptional modification of bcl-2 includes the phosphorylation of Bcl-2 at the putative mitogen-activated protein kinase sites, which confers resista...