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Recent evidence indicates that nerve growth factor (NGF) produces its effects through signaling contributions from both TrkA and the p75 receptor. In contrast to its trophic actions through TrkA, NGF binding to p75 has been shown to activate programmed cell death through a mechanism involving the stress kinase JNK. However, this receptor also activates nuclear factor kappaB (NF-kappaB), the role of which has yet to be determined. We investigated the function of p75-mediated NF-kappaB stimulation in regulating cell survival in the rat schwannoma cell line RN22, which expresses p75, but not TrkA. Gel shift assays demonstrated activation of NF-kappaB in response to NGF within 30 min and lasting at least 4 h. NGF also stimulated JNK in the cells (detected by in vitro kinase assays) with a similar time course. Preventing activation of NF-kappaB with the specific inhibitor SN50 resulted in NGF-induced cell loss. Similarly, transfection of the cells with a mutant form of the endogenous NF-kappaB inhibitor (IkappaBalphaDeltaN), which cannot be degraded and therefore remains bound to NF-kappaB, preventing its activation, resulted in a significant increase in the number of apoptotic cells following NGF treatment. These results suggest that NGF activation of NF-kappaB through the p75 receptor promotes survival, counterbalancing the pro-apoptotic signal.

Neurotrophins activate the Trk tyrosine kinase receptors, which subsequently initiate signaling pathways that have yet to be fully resolved, resulting in neuronal survival and differentiation. The ability of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) to activate GTP binding to p21ras was investigated using cultured embryonic chick neurons. In both sympathetic and sensory neurons, the addition of NGF markedly increased the formation of Ras-GTP. The magnitude of the effect was found to depend upon the developmental stage, peaking at embryonic day 11 in sympathetic neurons and at embryonic day 9 in sensory neurons, times when large numbers of neurons depend on NGF for survival. Surprisingly, following the addition of BDNF, no formation of Ras-GTP could be observed in neurons cultured with BDNF. When sensory neurons were cultured with NGF alone, both NGF and BDNF stimulated GTP binding to Ras. In rat cerebellar granule cells, while the acute exposure of these cells to BDNF resulted in the formation Ras-GTP, no response was observed following previous exposure of the cells to BDNF, as was observed with sensory neurons. However, this desensitization was not observed in a transformed cell line expressing TrkB. In neurons, the mechanism underlying the loss of the BDNF response appeared to involve a dramatic loss of binding to cell-surface receptors, as determined by cross-linking with radiolabeled BDNF. Receptor degradation could not account for the desensitization since cell lysates from neurons pretreated with BDNF revealed that the levels of TrkB were comparable to those in untreated cells. These results indicate that in neurons, the pathways activated by NGF and BDNF are differentially regulated and that prolonged exposure to BDNF results in the inability of TrkB to bind its ligand.

We have recently identified a mouse enzyme termed gamma-glutamyl leukotrienase (GGL) that converts leukotriene C4 (LTC4) to leukotriene D4 (LTD4). It also cleaves some other glutathione (GSH) conjugates, but not GSH itself (Carter, B. Z., Wiseman, A. L., Orkiszewski, R., Ballard, K. D., Ou, C.-N., and Lieberman, M. W. (1997) J. Biol. Chem. 272, 12305-12310). We have now cloned a full-length mouse cDNA coding for GGL activity and the corresponding gene. GGL and gamma-glutamyl transpeptidase constitute a small gene family. The two cDNAs share a 57% nucleotide identity and 41% predicted amino acid sequence identity. Their corresponding genes have a similar intron-exon organization and are located 3 kilobases apart. A search of Genbank and reverse transcription-polymerase chain reaction analysis failed to identify additional family members. Mapping of the GGL transcription start site revealed that the GGL promoter is TATA-less but contains an initiator, a control element for transcription initiation. Northern blots for GGL expression were negative. As judged by ribonuclease protection, in situ hybridization, and measurement of enzyme activity, spleen had the highest level of GGL expression. GGL is also expressed in thymic lymphocytes, bronchiolar epithelial cells, pulmonary interstitial cells, renal proximal convoluted tubular cells, and crypt cells of the small intestine as well as in cerebral, cerebellar, and brain stem neurons but not in glial cells. GGL is widely distributed in mice, suggesting an important role for this enzyme.

We have investigated the metabolism of leukotriene C4 (LTC4) in gamma-glutamyl transpeptidase (GGT)-deficient mice (Lieberman, M. W., Wiseman, A. L., Shi, Z-Z., Carter, B. Z., Barrios, R., Ou, C-N., Chevez-Barrios, P., Wang, Y., Habib, G. M., Goodman, J. C., Huang, S. L., Lebovitz, R. M., and Matzuk, M. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7923-7926) and have found substantial conversion of LTC4 to leukotriene D4 by high performance liquid chromatography and continuous flow fast atom bombardment-tandem mass spectrometric analyses. LTC4-converting activity has a tissue distribution different from GGT with highest activity in spleen followed by small intestine, kidney, and pancreas and lower activity in liver and lung. The activity is membrane-bound and is inhibited by acivicin, a known inhibitor of GGT. The enzyme was partially purified from the small intestine of GGT-deficient mice by papain treatment and gel filtration chromatography. The partially purified fragment released by papain has an apparent molecular mass of 65-70 kDa and the same substrate specificity as the tissue homogenate. In addition to LTC4, S-decyl-GSH is also cleaved. GSH itself, oxidized GSH, and the synthetic substrates used to analyze GGT activity (gamma-glutamyl-p-nitroanilide and gamma-glutamyl-4-methoxy-2-naphthylamide) are not substrates for this newly discovered enzyme. These data demonstrate that in addition to GGT at least one other enzyme cleaves LTC4 in mice. To reflect this enzyme's preferred substrate, we suggest that it be named gamma-glutamyl leukotrienase.

Continued passage of the human parvovirus, adeno-associated virus (AAV), at high multiplicity of infection in human cells results in the accumulation of AAV particles containing variant genomes. We have analyzed the structure of individual variant AAV genomes by molecular cloning in the Escherichia coli plasmid, pBR328. Each of the AAV inserts in six individual recombinant plasmids contained a single internal deletion but in contrast to a previous model, the locations of the deletions were nonrandom. The molecular cloning protocol also generated recombinant plasmids containing the entire AAV2 DNA sequence which yielded infectious AAV particles when transfected into human 293 cells in the presence of helper adenovirus using a DEAE-transfection procedure. Infectious AAV genomes were also generated by recombination when cells were jointly transfected with a mixture of plasmids containing two different mutant AAV genomes. The efficiency of this recombination appear to be influenced by the degree of homology between the mutant AAV genomes.

We have previously identified five promoters in the 5'-flanking region of the mouse gamma-glutamyl transpeptidase (gamma GT) gene. We now report the localization of a sixth promoter that supports the transcription of type III RNA, the major gamma GT RNA in fetal liver. We made a fetal liver cDNA library enriched for gamma GT RNA and obtained 12 gamma GT type III-specific clones. The longest clone is consistent with a transcription start site for type III RNA at a position 5' to the type IV promoter and about 5 kilobase(s) (kb) 5' to the first coding exon. We estimated by ribonuclease protection assay that about 80% of the gamma GT mRNA in fetal liver was type III. Primer extension and nuclease protection analyses mapped the 5' end of type III mRNA in fetal liver and kidney to a single cluster of potential major and minor transcription start sites. Deletion analysis using transient expression of chloramphenicol acetyltransferase constructs of the type III promoter region revealed the greatest activity with a 1-kb 5'-flanking fragment in mouse kidney proximal tubular cells and no detectable activity in NIH-3T3 fibroblasts. These studies demonstrate that the type III 5' region of the mouse gamma GT gene is organized into two distinct exons (IIIa and IIIb) and that type III RNA is expressed under the control of its own promoter.

A complex which is active in in vitro synthesis of adeno-associated virus (AAV) DNA was solubilized from Vero cells that were co-infected with AAV and either adenovirus (Ad5) or a herpes simplex virus type 1 (HSV-1) as the helper virus. The complexes from the Ad5 and HSV-1-infected cells sedimented at 23 S and 28 S, respectively. The optimal conditions for in vitro DNA synthesis for the two types of complex using the endogenous AAV template and the endogenous DNA polymerase, differed with respect to the effect of KCl and K2SO4 concentration. In addition the complex from HSV-1-infected cells, but not that from Ad5-infected cells, was inhibited by phosphonoacetic acid. Thus, the two complexes appear to contain different DNA polymerase activities. This was verified by phosphocellulose chromatography of the DNA polymerases solubilized from the isolated complexes. The major activity in the complex from HSV-1 infected cells was the HSV-induced DNA polymerase with lesser amounts of cellular DNA polymerase alpha and gamma or both. The complex from the Ad5-infected cells contained mainly a cellular DNA polymerase gamma.