The neuronal apoptosis inhibitory protein (NAIP) was identified as a candidate gene for the inherited neurodegenerative disorder spinal muscular atrophy. NAIP is the founding member of a human protein family that is characterized by highly conserved N-terminal motifs called baculovirus inhibitor of apoptosis repeats (BIR). Five members of the human family of inhibitor of apoptosis proteins including NAIP have been shown to be antiapoptotic in various systems. To date, a mechanism for the antiapoptotic effect of NAIP has not been elucidated. To investigate NAIP function, we found cytoprotection of NAIP-expressing primary cortical neurons treated to undergo caspase-3-dependent apoptosis. The additional treatment of these neurons with the pancaspase inhibitor boc-aspartyl(OMe)-fluoromethylketone did not result in increased survival. Similar cytoprotective effects were obtained using HeLa cells transiently transfected with a NAIP N-terminal construct and treated to undergo a caspase-3-dependent cell death. To examine whether NAIP inhibits caspases directly, recombinant N-terminal NAIP protein containing BIR domains was overexpressed, purified, and tested for caspase inhibition potential. Our results demonstrate that inhibition of caspases is selective and restricted to the effector group of caspases, with K i values as low as ϳ14 nM for caspase-3 and ϳ45 nM for caspase-7. Additional investigations with NAIP fragments containing either one or two NAIP BIRs revealed that the second BIR and to a lesser extent the third BIR alone are sufficient to mediate full caspase inhibition.

In Bacillus subtilis, two genes, thrS and thrZ, encode distinct threonyl‐tRNA synthetase enzymes. Normally, only the thrS gene is expressed. Here we show that either gene, thrS or thrZ, is sufficient for normal cell growth and sporulation. Reducing the intracellular ThrS protein concentration induces thrZ expression in a dose‐compensatory manner. Starvation for threonine simultaneously induces thrZ and stimulates thrS expression. The 5′‐leader sequences of thrS and thrZ contain, respectively, one and three transcription terminators preceded by a conserved sequence. We show that this sequence is essential for the regulation of thrS via a transcriptional antitermination mechanism. We propose that both genes, thrS and thrZ, are regulated by the same mechanism such that the additional regulatory domains present before thrZ account for its non‐expression. In contrast to Escherichia coli, structurally similar regulatory domains, i.e. the consensus sequence preceding a terminator structure, are found in the leader regions of most aminoacyl‐tRNA synthetase genes of Gram‐positive bacteria. This suggests that they are regulated by a common mechanism.

The "housekeeping" threonyl-tRNA synthetase gene (thrS) of BaciUlus subtilis is shown to be transcribed in vivo and in vitro from a single promoter. In vitro, 85% of all messages transcribed from the thrS promoter are terminated at a strong factor-independent terminator localized upstream of the thrS Shine-Dalgarno sequence, within the 305-nucleotide-long leader region. Overexpression of thrS represses transcriptional and translational thrS-lacZ fusions to a similar extent, suggesting that thrS is autoregulated at the transcriptional level. We show that autogenous control does not act at the level of transcription initiation but involves antitermination of the transcription mechanism. thrZ, the second threonyl-tRNA synthetase gene, is also autogenously regulated. However, the ability of the ThrS synthetase to repress thrS as well as thrZ expression is much greater than that of the ThrZ synthetase.In Bacillus species, most of the known aminoacyl-tRNA synthetase genes seem to be regulated by a similar mechanism involving antitermination of transcription. This assumption is based on the presence of strongly conserved structural elements in their regulatory regions (12, 14, 22; for a review, see reference 23); an 18-nucleotide (nt) consensus sequence called the T-box, followed by a stem-and-loop structure upstream of the Shine-Dalgarno sequence. Upstream leader sequences comprise domains of conserved secondary structure, with a codon specific for the appropriate amino acid at the identical position in each sequence. In the case of tyrS, this codon has been shown to be sufficient for the specificity of derepression following tyrosine starvation (12). Of the 12 aminoacyl-tRNA synthetase genes cloned and sequenced so far, only 3 do not present the conserved elements in their leader region: Bacillus stearothermophilus metS (17), Bacillus subtilis metS (which nevertheless contains a stem-loop structure upstream of the Shine-Dalgarno sequence [18]), and B. subtilis gltX (4). Furthermore, certain amino acid biosynthesis operons in B. subtilis, ilv-leu (10) and cysE-cysS (4), and in Lactobacillus species, the trpDCFBA operon of Lactobacillus casei (19) and the trpEGDCFBA and his operons of Lactococcus lactis (2, 7), also carry these conserved elements. This situation is in contrast to that in Escherichia coli, for which there is no evidence for a general mechanism of regulation (11, 23).Two independent genes, thrS and thrZ, encode two isozymes of threonyl-tRNA synthetase, ThrS and ThrZ, respectively, in B. subtilis (21). Expression of both genes has been shown to be regulated by antitermination of transcription, but only thrS is expressed under normal growth conditions (22 derepression of B. subtilis tyrS after tyrosine starvation (14). The terminator structure itself and the T-box are functionally related. Destruction of the T-box has no effect on thrS expression in the absence of a functional terminator (22).Here we show that expression of the B. subtilis thrS gene influences not only thrZ expression but also the expressi...

Multinucleated giant cells (MGC) are a hallmark of granulomatous reactions but the mechanisms that regulate their formation are unknown. To address this issue, we cultured resident alveolar macrophages (AM) from rat lung and examined the effects of defined cytokines on AM differentiation and MGC formation. The presence of MGC was found after 3 days in culture with maximal numbers obtained at 7 days and thereafter (up to 21 days). Macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (25-75 U/mL) stimulated the formation of MGC (up to 4-fold), whereas interleukin (IL) -3, IL-10, and interferon-gamma (IFN-gamma) had no stimulatory effect. Interestingly, MGC with distinct phenotypes were observed in AM cultures: (1) spherical MGC with 3-16 nuclei, dense cytoplasm, and lower expression of beta3 integrin (Type 1) and (2) irregular MGC with 3-30 nuclei, thin and vacuolated cytoplasm, and higher expression of beta3 integrin (Type 2). Furthermore, the actions of M-CSF and GM-CSF on AM were found to be different. GM-CSF promoted, in AM cultures, the appearance of an elongated fibroblastoid phenotype and stimulated mostly the formation of Type 2 MGC. In contrast, M-CSF did not cause significant change in the general morphology of regular AM but stimulated the appearance of both Type 1 and Type 2 MGC. Reverse transcriptase-polymerase chain reaction analysis demonstrated that, under these conditions, M-CSF induced GM-CSF gene expression in AM. In addition, neutralizing antibodies against M-CSF selectively decreased the formation of Type 1 MGC, whereas neutralizing anti-GM-CSF inhibited Type 2 formation. These data suggest that M-CSF promotes AM differentiation into Type 1 MGC, whereas GM-CSF stimulates the formation of Type 2 and that M-CSF and GM-CSF may selectively regulate in an autocrine fashion AM differentiation into distinct MGC.