Two monoclonal antibodies, E3cD8 and E3cA10, were generated to the EBNA 3c nuclear protein from the B95.8 isolate of Epstein-Barr virus (EBV). Both antibodies efficiently precipitate EBNA 3c from B95.8-transformed lymphoblastoid cell lines, and E3cA10 also detects EBNA 3c on Western blots. Whereas E3cD8 reacts with all 11 Type-1 isolates of EBV tested, and E3cA10 reacts with 14 of 17 Type-1 isolates, neither antibody detects the EBNA 3c protein encoded by Type-2 isolates. E3cD8 recognizes a peptide sequence (PA/PPQAPYQGY) in a repeat region of the B95.8 EBNA 3c coding sequence which is not present in the prototype Type-2 AG876 sequence. The E3cA10 antibody epitope has been mapped to the minimal five amino acid B95.8 peptide, WAPSV, which has an alanine to valine substitution in the AG876 virus isolate. This substitution was also found in three Type-1 EBV isolates that expressed EBNA 3c proteins not detected by E3cA10. In immunoprecipitation studies E3cA10 additionally coprecipitated the EBNA 2 protein from Type-1 isolates of EBV. The possibility of a direct interaction between EBNA 2 and EBNA 3c was ruled out by the demonstration that the antibody precipitated EBNA 2 from the Raji cell line which carries a virus with a deleted EBNA 3c gene. Since the WAPSV epitope identified in EBNA 3c is not present in EBNA 2, and no EBNA 2 linear peptide reactivity was detected in ELISA, it seems likely that E3cA10 recognizes a conformational epitope on EBNA 2. However, from the present data we cannot exclude the possibility that the antibody reacts with a cellular protein that physically associates with EBNA 2.

We have constructed multicopy and integrative streptomycete promoter probe vectors that employ the promoterless tyrosinase (melC) operon of Streptomyces glaucescens as the chromogenic transcriptional reporter. Each vector contains the reporter cassette, RC3, which comprises part of the melC operon flanked by transcription terminators; RC3 may be easily inserted into any vector. We demonstrate the use of the pIJ101-based mel vector, pMT3010, for the isolation of mutations which alter expression of the S. coelicolor glycerol operon. The S. glaucescens mel reporter system is well-suited for the visual, non-selective identification of regulatory mutants and can be used efficiently for screening several thousand clones at high colony density.

Isolated hepatocytes incubated in the presence of the NO donors S-nitroso-N-acetylpenicillamine (SNAP) and 3-morpholino-sydnonimine (SIN-I) displayed a time- and dose-dependent inhibition of glucose synthesis from lactate plus pyruvate as the substrate which correlated with NO production, but not nitrite production. Neither the parent compound of SNAP, N-acetyl-DL-penicillamine (NAP), nor nitrite or nitrate had any significant effect on glucose output, indicating that the inhibition was due to the generation of NO within the incubation medium. The concentrations of NO required for this effect (< 800 nM) are within the range reported to occur in intact tissues and in vivo. The magnitude of the inhibitory effect of SNAP (~ 50%) was comparable with that of endotoxin treatment of the rat with lactate plus pyruvate as the substrate. When the effect of SNAP on glucose synthesis and lactate plus pyruvate synthesis from a number of different substrates was examined, this showed a pattern comparable with that observed after endotoxin treatment of the rat, suggesting that NO may be the inhibitory mediator of the effects of bacterial endotoxin on hepatic gluconeogenesis. The NO donor had no effect on the flux through 6-phosphofructo-1-kinase, supporting the concept that the primary site of inhibition of gluconeogenesis by both NO and endotoxin resides at the level of phosphoenolpyruvate formation.

Treatment of rats with bacterial endotoxin resulted in a significant induction of hepatic nitric oxide synthase within 3 hours. The response was maximal at 12 hours and was maintained over 18 hours. The induction of nitric oxide synthase correlated well with the increase in plasma nitrate plus nitrite concentrations and also with the inhibition of glucose synthesis in subsequently isolated hepatocytes. The decline in the rate of gluconeogenesis also correlated with an inhibition of flux through phosphoenolpyruvate carboxykinase but not with alterations in flux through either pyruvate kinase or 6-phosphofructo-1-kinase, suggesting that a nitric oxide-induced inhibition of phosphoenolpyruvate carboxykinase may underlie the decreased glucose production in sepsis.