The heme oxygenase 1 (HO-1) gene is rapidly activated in the liver after lipopolysaccharide (LPS) treatment. Ninety minutes after LPS treatment (0.1 mg/kg, intraperitoneally) hepatic HO-1 messenger RNA (mRNA) of mice was 40 times the control value. To investigate the hepatic cellular source of the increased HO-1 transcript, we treated mice with LPS and galactosamine (700 mg/kg, intraperitoneally), a selective transcriptional inhibitor of hepatocytes. Galactosamine prevented the LPS-mediated increase of HO-1 mRNA in the liver, indicating that hepatocytes are the main cell type in which HO-1 mRNA accumulates after LPS treatment. We then tested in vitro and in vivo the hypothesis that LPS-mediated hepatic accumulation of HO-1 mRNA is caused by intercellular communication between Kupffer cells and hepatocytes. Isolated rat hepatocytes showed an increase in HO-1 mRNA compared with controls after 90 minutes of exposure to a LPS stimulated Kupffer cell-conditioned medium. This suggests that soluble mediators from Kupffer cells were responsible for this effect. To study the role of Kupffer cells in vivo, we treated mice with Kupffer cell-inactivating or -depleting agents and LPS. Gadolinium chloride and liposome-encapsulated dichloromethylene diphosphonate lowered LPS-mediated HO-1 mRNA accumulation (by about 50%); in these groups hepatic levels of interleukin (IL)-1beta were decreased, by more than 75%. Methylpalmitate hardly affected hepatic HO-1 mRNA accumulation or IL-1beta content after LPS treatment. There was no relationship between HO-1 mRNA and serum TNF or IL-6 levels. These results suggest that LPS-mediated hepatic HO-1 mRNA accumulation is a hepatocyte response partly caused by soluble mediators, particularly IL-1beta, released from Kupffer cells.

Bacteriophage P4's superinfection immunity mechanism is unique among those of other known bacteriophages in several respects: (i) the P4 immunity factor is not a protein but a short, stable RNA (CI RNA); (ii) in the prophage the expression of the replication operon is prevented by premature transcription termination rather than by repression of transcription initiation; (iii) transcription termination is controlled via RNA-RNA interactions between the CI RNA and two complementary target sequences on the nascent transcript; and (iv) the CI RNA is produced by processing of the same transcript it controls. It was thought that several host-encoded factors may participate in the molecular events required for P4 immunity expression, i.e., RNA processing, RNA-RNA interactions, and transcription termination. To identify such factors we searched for Escherichia coli mutations that affect P4 lysogenization. One such mutation, bfl-1, severely reduced P4's lysogenization frequency and delayed both the disappearance of the long transcripts that cover the entire replication operon and the appearance of the CI RNA. By physical mapping and genetic analysis we show that bfl-1 is allelic to pnp, which codes for polynucleotide phosphorylase, a 3-to-5 exonucleolytic enzyme. A previously isolated pnp null mutant (pnp-7) exhibited a phenotype similar to that of bfl-1. These results indicate that the polynucleotide phosphorylase of E. coli is involved with the maturation pathway of bacteriophage P4's RNA immunity factor.P4 is a satellite phage of Escherichia coli that depends on the genes of a helper phage, such as P2, for capsid and tail morphogenesis and cell lysis. Autonomous P4 replication requires the expression of the P4 ␣ gene, encoded in the distal part of the P4 left operon (Fig. 1A), and leads either to the lytic cycle (when a helper phage genome is present in the host cell) or to the multicopy-plasmid state (in the absence of the helper). The P4 genome may also integrate in the host cell chromosome and establish superinfection immunity that prevents expression of the left operon (for a review, see references 15 and 26).The P4 superinfection immunity mechanism is unique among the known bacteriophages in several aspects. First, the promoter (P LE ) responsible for the early expression of the left operon is not repressed in the prophage; thus, in order to prevent expression of P4 replication genes, premature transcription termination of the left operon is established at a Rho-dependent terminator about 480 nucleotides (nt) downstream of P LE (Fig. 1B) (5,10,16). Second, the P4 immunity factor is a small RNA, 69 nt long (CI RNA), which is produced by processing of the leader region of the left operon itself; RNase P is required for the correct maturation of the CI RNA 5Ј end (10,14,16). Third, transcription termination depends on interactions between the CI RNA and two complementary regions on the leader transcript of the left operon (10,16,36).To understand in detail the P4 immunity mechanism, several events should be clarifie...

Sox2 is one of the earliest known transcription factors expressed in the developing neural tube. Although it is expressed throughout the early neuroepithelium, we show that its later expression must depend on the activity of more than one regionally restricted enhancer element. Thus, by using transgenic assays and by homologous recombination-mediated deletion, we identify a region upstream of Sox2 (−5.7 to −3.3 kb) which can not only drive expression of a (beta)-geo transgene to the developing dorsal telencephalon, but which is required to do so in the context of the endogenous gene. The critical enhancer can be further delimited to an 800 bp fragment of DNA surrounding a nuclease hypersensitive site within this region, as this is sufficient to confer telencephalic expression to a 3.3 kb fragment including the Sox2 promoter, which is otherwise inactive in the CNS. Expression of the 5.7 kb Sox2(beta)-geo transgene localizes to the neural plate and later to the telencephalic ventricular zone. We show, by in vitro clonogenic assays, that transgene-expressing (and thus G418-resistant) ventricular zone cells include cells displaying functional properties of stem cells, i.e. self-renewal and multipotentiality. We further show that the majority of telencephalic stem cells express the transgene, and this expression is largely maintained over two months in culture (more than 40 cell divisions) in the absence of G418 selective pressure. In contrast, stem cells grown in parallel from the spinal cord never express the transgene, and die in G418. Expression of endogenous telencephalic genes was similarly observed in long-term cultures derived from the dorsal telencephalon, but not in spinal cord-derived cultures. Thus, neural stem cells of the midgestation embryo are endowed with region-specific gene expression (at least with respect to some networks of transcription factors, such as that driving telencephalic expression of the Sox2 transgene), which can be inherited through multiple divisions outside the embryonic environment.

Bacteriophage P4 autonomous replication may result in the lytic cycle or in plasmid maintenance, depending, respectively, on the presence or absence of the helper phage P2 genome in the Escherichia coli host cell. Alternatively, P4 may lysogenize the bacterial host and be maintained in an immune-integrated condition. A key step in the choice between the lytic/plasmid vs. the lysogenic condition is the regulation of P4 alpha operon. This operon may be transcribed from two promoters, PLE and PLL, and encodes both immunity (promoter proximal) and replication (promoter distal) functions. PLE is a constitutive promoter and transcription of the downstream replication genes is regulated by transcription termination. The trans-acting immunity factor that controls premature transcription termination is a short RNA encoded in the PLE proximal part of the operon. Expression of the replication functions in the lytic/plasmid condition is achieved by activation of the PLL promoter. Transcription from PLL is insensitive to the termination mechanism that acts on transcription starting from PLE.PLL is also negatively regulated by P4 orf88, the first gene downstream of PLL. An additional control on P4 DNA replication is exerted by the P4 cnr gene product.