The neuropoietic cytokine cholinergic differentiation factor/leukemia nhibitory factor (CDF/LIF) acts as a trophic factor, enhancing neuronal survival, and as a differentiation factor, altering neuronal gene expression. There Is also evidence that It plays a role in the response of adult neural tissue to inJury. We have exaied this possibility further in rats by analyzing changes in the levels of mRNAs for CDF/LIF and its two receptor subunits in response to peripheral nerve damage in culture and in vivo. Using a quantitative RNase protection assay, we find that CDF/LIF mRNA increases dramatically (176-fold) A striking feature of the neuropeptides induced in cultured neonatal sympathetic neurons by CDF/LIF, CNTF, oncostatin M, and growth promoting factor is that several of these same neuropeptides are also induced in adult sympathetic neurons when the ganglia are damaged by explantation in short-term culture or by axotomy in vivo. For instance, substance P and vasoactive intestinal polypeptide (VIP) are induced both by nerve damage and by application of these neuropoietic cytokines to pure neuronal cultures (4, 6, 7). In response to peripheral nerve damage, VIP is induced in dorsal root ganglia (DRGs) as well (8,9). Moreover, CDF/ LIF elevates VIP in dissociated DRG neurons (10). Part of the in vivo response to injury could be mediated by nonneuronal cells in the ganglia, since these cells have been shown to release CDF/LIF (11,12). CDF/LIF involvement in the neuropeptide response to nerve damage is further indicated by results from mice in which the CDF/LIF gene was disrupted by homologous recombination. Ganglia from such mutant mice display a much reduced neuropeptide response to culturing or axotomy (13).To further investigate the role of CDF/LIF in the events surrounding nerve damage, we have determined whether the levels of CDF/LIF and its receptor subunits change after nerve and ganglion injury. The major sources of CDF/LIF mRNA under these conditions were also localized by in situ hybridization. Some of these findings have been reported (14-16). MATERIALS AND METHODSSurgical Procedures and Organ Culture. Adult male and female rats were anesthetized by intraperitoneal injection of sodium pentobarbitol (Nembutol, Abbott; 40 mg/kg). The sciatic nerve was exposed and transected. To ensure that regeneration did not occur, a 3-mm piece of the nerve was removed and the remaining ends were deflected. Care was taken so that surrounding muscles were not damaged. The wound was closed with clips. Each time point is composed of data from nerves of nine animals. At the appropriate time, animals were killed by CO2 inhalation and various regions of the sciatic nerve were dissected and frozen at -700C. Corresponding regions of nerve were removed from the contralateral side of each rat as control.Fifteen superior cervical ganglia (SCGs) from postnatal day 1 (PI) rats and 15 adult DRGs and 10 SCGs from adult Sprague-Dawley (Simonson Laboratories, Gilroy, CA) rats were dissected, desheathed, and placed in organ cu...

RNA-RNA recombination is thought to occur preferentially at certain selected sites and in only a few RNA viruses; the mechanism for these restrictions is unknown. In this paper we report the development of a recombination assay for coronavirus, using polymerase chain reaction, in the absence of selection pressure. Our results showed that within a 1-kb region of the peplomer gene, RNA recombination occurred at almost every potential crossover site. Thus, coronavirus RNA recombination appears to be more random than previously realized. However, after serial passages of the recombinant viruses in tissue culture, the recombination sites among the progeny viruses became clustered in the region which contains the previously reported "hot spot" for coronavirus recombination. These results suggest that RNA recombination is common and random in nature, but only certain recombinants can be selected. Thus, the presence of recombinational "hot spots" for coronavirus or other RNA viruses most likely resulted from selection of certain recombinant viruses and not restriction on the occurrence of RNA recombination. The failure to detect recombinants in other RNA viruses may therefore be due to unfavorable properties of recombinant viruses. This approach can be used to detect recombinants in these viruses.

The cytokine leukaemia inhibitory factor (LIF) is up-regulated in glial cells after injury to the peripheral and central nervous systems. In addition, LIF is required for the changes in neuropeptide expression that normally occur when the axons of sympathetic and sensory neurons are transected. We investigated whether LIF is also necessary for the initial inflammatory response that follows mechanical injury to the sciatic nerve and cerebral cortex of adult mice. We find that inflammatory cell infiltration into crushed sciatic nerve is significantly slower in LIF knock-out (KO) mice compared with wild-type (WT) mice. Similarly, the microglial and astroglial responses to surgical injury of the cortex are significantly slower in LIF KO mice compared with WT mice. Consistent with these in vivo results, LIF is chemotactic for peritoneal macrophages in a microchamber culture assay. Thus, LIF is a key regulator of neural injury in vivo, where it is produced by glia and can act directly on neurons, glia and inflammatory cells. We also find that the initial inflammatory response to cortical injury is diminished in interleukin (IL)-6 KO mice. Surprisingly, however, the inflammatory response in LIF-IL-6 double KO mice is very similar to that of the single KO mice, suggesting that these cytokines may act in series rather than in parallel in this response.

The hemagglutinin-esterase (HE) membrane glycoprotein is present only in some members of the coronavirus family, including some strains of mouse hepatitis virus (MHV). In the JHM strain of MHV, expression of the HE gene is variable and corresponds to the number of copies of a UCUAA pentanucleotide sequence present at the 3'-end of the leader RNA. This copy number varies among MHV strains, depending on their passage history. The JHM isolates with two copies of UCUAA in their leader RNA showed a high level of HE expression, whereas the JHM isolate with three copies had a low-level expression. In this study, the analysis of HE gene expression was extended to other MHV strains. The synthesis of HE mRNA in these viruses also correlates with the copy number of UCUAA in the leader RNA and the particular intergenic sequence preceding the HE gene. In one MHV strain, MHV-1, no detectable HE mRNA was synthesized, despite the presence of a proper transcription initiation signal. This lack of HE mRNA expression was consistent with a leader RNA containing three UCUAA copies. However, mutations and deletions within the coding region of the MHV-1 HE gene have generated a stretch of sequence which resembled the transcriptional initiation motif, and was shown to initiate the synthesis of a novel smaller mRNA. These findings strengthened the theory that interactions between leader RNA and transcriptional initiation sequences regulate MHV subgenomic mRNA transcription. Sequence analysis revealed that most MHV strains, through extensive mutations, deletions, or insertions, have lost the complete HE open reading frame, thus turning HE into a pseudogene. This high degree of variation is unusual as the other three structural proteins (spike, membrane, and nucleocapsid) are well-maintained. In contrast to bovine coronavirus, which apparently requires HE for viral replication, the HE protein in MHV may be only an accessory protein which is not necessary for viral replication. JHM and MHV-S, however, have preserved the expression of HE protein.