Evaluation of C-reactive protein and its kinetics as a prognostic indicator in canine leptospirosis.

OBJECTIVE: To evaluate C-reactive protein at presentation and during hospitalisation in dogs with acute kidney injury resulting from leptospirosis to compare C-reactive protein at presentation in dogs with acute kidney injury of different aetiology and to study its correlation with markers of inflammation, azotaemia and survival. MATERIALS AND METHODS: Prospective observational study of 41 dogs with acute kidney injury secondary to leptospirosis and 15 control dogs with acute kidney injury of different aetiology. C-reactive protein was measured at presentation in both groups and daily for 7 days in a subgroup of 28 dogs with leptospirosis. The associations of C-reactive protein with neutrophil count, albumin, urea, creatinine and survival were analysed. RESULTS: C-reactive protein was increased at presentation in all dogs with leptospirosis but was not significantly different from dogs with acute kidney injury of different cause. It was associated with markers of inflammation (neutrophil count, albumin) but not with azotaemia (creatinine, urea). It decreased gradually from presentation to day 4, with significantly lower concentrations in survivors than non-survivors. Initial C-reactive protein was only weakly associated with outcome, but its average concentration from presentation to day 2 was more strongly associated. Absolute and relative changes in C-reactive protein during hospitalisation and creatinine at presentation were not associated with survival. CLINICAL SIGNIFICANCE: Serial assessment of C-reactive protein may improve outcome prediction in dogs with leptospirosis compared with a single measurement at presentation or with markers of renal function.

Single-cell analysis divides bovine monocyte-derived dendritic cells into subsets expressing either high or low levels of inducible nitric oxide synthase.

Dendritic cells (DC) are important cells at the interface between innate and adaptive immunity. DC have a key role in antigen processing and presentation to T cells. Effector functions of DC related to innate immunity have not been explored extensively. We show that bovine monocyte-derived DC (mDC) express inducible nitric oxide synthase (iNOS) mRNA and protein and produce NO upon triggering with interferon-gamma (IFN-gamma) and heat-killed Listeria monocytogenes (HKLM). An immunocytochemical analysis revealed that a sizeable subset (20-60%) copiously expresses iNOS (iNOShi) upon IFN-gamma/HKLM triggering, whereas the other subset expressed low levels of iNOS (iNOSlo). Monocyte-derived macrophages (mMphi) are more homogeneous with regard to iNOS expression. The number of cells within the iNOSlo mDC subset is considerably larger than the number of dead cells or cells unresponsive to IFN-gamma/HKLM. The large majority of cells translocated p65 to the nucleus upon triggering by IFN-gamma/HKLM. A contamination of mDC with iNOS-expressing mMphi was excluded as follows. (i) Cell surface marker analysis suggested that mDC were relatively homogeneous, and no evidence for a contaminating subset expressing macrophage markers (e.g. high levels of CD14) was obtained. (ii) iNOS expression was stronger in iNOShi mDC than in mMphi. The use of maturation-promoting stimuli revealed only subtle phenotypic differences between immature and mature DC in cattle. Nevertheless, these stimuli promoted development of considerably fewer iNOShi mDC upon triggering with IFN-gamma/HKLM. Immunocytochemical results showed that although a significant proportion of cells expressed iNOS only or TNF only upon triggering with IFN-gamma/HKLM, a significant number of cells expressed both iNOS and TNF, suggesting that TNF and iNOS producing (TIP) DC are present within bovine mDC populations obtained in vitro.

Precisely positioned nucleosomes are not essential for c-fos gene regulation in vivo.

Chromatin architecture plays a decisive role in many aspects of transcription regulation. We have tested the role of specific chromatin structures in c-fos gene regulation, using a gene transfer system based on episomes derived from the Epstein-Barr virus (EBV). This system reproduces in several respects the chromatin structure and regulation of the chromosomal c-fos gene. Using this approach, we first demonstrate that the pausing of RNA polymerase II downstream of the transcriptional start site does not require precisely positioned nucleosomes. Indeed, changing the pattern of MNase hypersensitive sites along the transcribed sequence does not perturb RNA polymerase II pausing or the regulation of the c-fos gene. Next, we show that a putative nucleosome positioned between the SIE/SRE elements (-300) and the CRE/TATA elements (-36) is not necessary for activation by a variety of inducers. Accordingly, total or partial deletion of the putative nucleosome sequence does not disturb c-fos regulation while the two regulatory sites flanking the nucleosome sequence remain hypersensitive to MNase. As described in this paper, EBV episomes are useful vectors to critically examine the role of the chromatin structure in gene transcription for human cells.

RNA polymerase II promoter-proximal pausing upregulates c-fos gene expression.

Transcription elongation regulates c-fos expression in mouse and human cells. In the inactive state of the gene, RNA polymerases are engaged only in the promoter-proximal region. Upon activation, RNA polymerases move efficiently along the complete gene. We have used Epstein-Barr virus (EBV) episomes as a gene transfer system to study the role of promoter-proximal pausing and transcript elongation in c-fos expression. We find that the sequence located immediately downstream of the transcriptional start site specifies pausing of RNA polymerases, dependent on both its orientation and position relative to the promoter. This sequence is, however, not necessary to maintain repression in the absence of a stimulus. As promoter-proximal pausing is therefore not a repression mechanism for the c-fos gene, the promoter and enhancer sequences are the main determinants of RNA polymerase elongation competence. Surprisingly, we find that promoter-proximal pausing further increases transcriptional levels from a variety of promoters. These observations lead us to hypothesize that promoter-proximal pausing of RNA polymerase II augments c-fos expression by allowing more efficient phosphorylation of the C-terminal domain of the large subunit.

Transition mutation in codon 248 of the p53 tumor suppressor gene induced by reactive oxygen species and a nitric oxide-releasing compound.

Exposing the human bronchial epithelial cell line BEAS-2B to the nitric oxide (NO) donor sodium 1-(N,N-diethylamino)diazen-1-ium-1, 2-diolate (DEA/NO) at an initial concentration of 0.6 mM while generating superoxide ion at the rate of 1 microM/min with the hypoxanthine/xanthine oxidase (HX/XO) system induced C:G-->T:A transition mutations in codon 248 of the p53 gene. This pattern of mutagenicity was not seen by 'fish-restriction fragment length polymorphism/polymerase chain reaction' (fish-RFLP/PCR) on exposure to DEA/NO alone, however, exposure to HX/XO led to various mutations, suggesting that co-generation of NO and superoxide was responsible for inducing the observed point mutation. DEA/NO potentiated the ability of HX/XO to induce lipid peroxidation as well as DNA single- and double-strand breaks under these conditions, while 0.6 mM DEA/NO in the absence of HX/XO had no significant effect on these parameters. The results show that a point mutation seen at high frequency in certain common human tumors can be induced by simultaneous exposure to reactive oxygen species and a NO source.

Effect of MALP-2, a lipopeptide from Mycoplasma fermentans, on bone resorption in vitro.

Mycoplasmas may be associated with rheumatoid arthritis in various animal hosts. In humans, mycoplasma arthritis has been recorded in association with hypogammaglobulinemia. Mycoplasma fermentans is one mycoplasma species considered to be involved in causing arthritis. To clarify which mycoplasmal compounds contribute to the inflammatory, bone-destructive processes in arthritis, we used a well-defined lipopeptide, 2-kDa macrophage-activating lipopeptide (MALP-2) from M. fermentans, as an example of a class of macrophage-activating compounds ubiquitous in mycoplasmas, to study its effects on bone resorption. MALP-2 stimulated osteoclast-mediated bone resorption in murine calvaria cultures, with a maximal effect at around 2 nM. Anti-inflammatory drugs inhibited MALP-2-mediated bone resorption by about 30%. This finding suggests that MALP-2 stimulates bone resorption partially by stimulating the formation of prostaglandins. Since interleukin-6 (IL-6) stimulates bone resorption, we investigated IL-6 production in cultured calvaria. MALP-2 stimulated the liberation of IL-6, while no tumor necrosis factor was detectable. Additionally, MALP-2 stimulated low levels of NO in calvaria cultures, an effect which was strongly increased in the presence of gamma interferon, causing an inhibition of bone resorption. MALP-2 stimulated the bone-resorbing activity of osteoclasts isolated from long bones of newborn rats and cultured on dentine slices without affecting their number. In bone marrow cultures, MALP-2 inhibited the formation of osteoclasts. It appears that MALP-2 has two opposing effects: it increases the bone resorption in bone tissue by stimulation of mature osteoclasts but inhibits the formation of new ones.

Regulation of c-fos expression by RNA polymerase elongation competence.

The molecular mechanisms underlying transcription elongation and their role in gene regulation are poorly characterized in eukaryotes. A number of genes, however, have been proposed to be regulated at the level of transcription elongation, including c-myc, c-fos and c-myb. Here, we analyze the control of transcription elongation at the mouse c-fos gene at the nucleotide level in intact cells. We find that RNA polymerases are engaged in the promoter-proximal part of the gene in the absence of gene activation signals and mRNA synthesis. Importantly, we determine that the engaged RNA polymerases originate from a continuous initiation of transcription which, in the absence of gene activation signals, terminate close to the promoter. We also observe that the c-fos gene presents an active chromatin conformation, with the promoter and upstream regulatory sequences constitutively occupied by proteins, accounting for the continuous initiation of RNA polymerase complexes. We propose that activation of c-fos gene expression results primarily from the assembly of elongation-competent RNA polymerases that can transcribe the complete gene. Our results suggest that the engaged RNA polymerases found downstream of a number of other eukaryotic promoters may be associated with transcription termination of elongation-incompetent polymerases in the absence of activating signals.

Tissue-specific chromatin structure at the hepatocyte growth factor/scatter factor gene promoter.

Hepatocyte growth factor/scatter factor (HGF/SF) is a recently characterised molecule with many remarkable functions. Its involvement in important processes such as cell proliferation, cell migration, morphogenesis and organ development implies that its activity should be tightly regulated. To understand the molecular mechanisms controlling HGF/SF transcription, we have analysed DNaseI hypersensitive sites (DHS) along rat and human HGF/SF genes in various tissues and cell types. We identified five DHS along the rat gene, two in the 5'-flanking region and three in the first intron. These sites are only found in rat tissues and rat cell lines, which express HGF/SF. The strongest hypersensitive site map to a region that corresponds to the promoter by start site analysis. A single tissue-specific DHS is present in human cell lines that express HGF/SF and corresponds to the promoter region. Our results suggest that chromatin accessibility plays a major role in the regulation of HGF/SF transcription regulation.

The role of chromatin in HIV-1 transcriptional regulation.

Regulation of transcription is a key feature of the HIV-1 life cycle. In recent years, various sequence elements and transcription factors have been shown to participate in HIV-1 transcription control. New evidence, however, has shown that chromatin organization plays a key role in the establishment of a transcriptionally regulated HIV-1 LTR. The present review discusses recent data obtained on reconstituted and genomic HIV-1 chromatin templates.

Nitric oxide and ethylnitrosourea: relative mutagenicity in the p53 tumor suppressor and hypoxanthine-phosphoribosyltransferase genes.

Nitric oxide (NO) is a cellular messenger which is mutagenic in bacteria and human TK6 cells and induces deamination of 5-methylcytosine (5meC) residues in vitro. The aims of this study were: (i) to investigate whether NO induces 5meC deamination in codon 248 of the p53 gene in cultured human bronchial epithelial cells (BEAS-2B); and (ii) to compare NO mutagenicity to that of ethylnitrosourea (ENU), a strong mutagen. Two approaches were used: (i) a novel genotypic assay, using RFLP/PCR technology on purified exon VII sequence of the p53 gene; and (ii) a phenotypic (HPRT) mutation assay using 6-thioguanine selection. BEAS-2B cells were either exposed to 4 mM DEA/NO (Et2N[N2O2]Na, an agent that spontaneously releases NO into the medium) or transfected with the inducible nitric oxide synthase (iNOS) gene. The genotypic mutation assay, which has a sensitivity of 1 x 10(-6), showed that 4 mM ENU induces detectable numbers of G --> A transitions in codon 248 of p53 while 5-methylcytosine deamination was not detected in either iNOS-transfected cells or cells exposed to 4 mM DEA/NO. Moreover, ENU was dose-responsively mutagenic in the phenotypic HPRT assay, reaching mutation frequencies of 24 and 96 times that of untreated control cells at ENU concentrations of 4 and 8 mM respectively; by contrast, 4 mM DEA/NO induced no detectable mutations in this assay, nor were any observed in cells transfected with murine iNOS. We conclude that if NO is at all promutagenic in these cells, it is significantly less so than the ethylating mutagen, ENU.

Tissue-specific regulation of mouse hepatocyte nuclear factor 4 expression.

Hepatocyte nuclear factor 4 (HNF-4) is a liver-enriched transcription factor and a member of the steroid hormone receptor superfamily. HNF-4 is required for the hepatoma-specific expression of HNF-1 alpha, another liver-enriched transcription factor, suggesting the early participation of HNF-4 in development. To prepare for further study of HNF-4 in development, the tissue-specific expression of the mouse HNF-4 gene was studied by analyzing the promoter region for required DNA elements. DNase-hypersensitive sites in the gene in liver and kidney tissues were found in regions both distal and proximal to the RNA start that were absent in tissues in which HNF-4 expression did not occur. By use of reporter constructs in transient-transfection assays and with transgenic mice, a region sufficient to drive liver-specific expression of HNF-4 was identified. While an HNF-1 binding site between bp -98 and -68 played an important role in the hepatoma-specific promoter activity of HNF-4 in transient-transfection assays, it was not sufficient for the liver-specific expression of a reporter gene in transgenic mice. Distal enhancer elements indicated by the presence of DNase I-hypersensitive sites at kb -5.5 and -6.5, while not functional in transient-transfection assays, were required for the correct expression of the mouse HNF-4 gene in animals.

Gene regulation in rodent hepatocytes during development, differentiation and disease.

The expression of genes in the liver is mostly controlled at the transcriptional level and depends on the regulatory interactions between cis-acting sequences and trans-acting molecules. Proximal promoters and distant enhancers in combination with a number of hepatocyte-enriched DNA-binding proteins and general transcription factors interact specifically with these elements and control the expression of liver-specific genes. Hepatocyte-enriched regulatory proteins have been isolated from liver nuclear extracts, characterized, and their corresponding genes have been cloned. These include the hepatocyte nuclear factors 1, 3, 4 (HNF-1,3,4), some members of the CAAAT/enhancer binding protein (C/EBP) family, and D site binding protein (DBP). These factors belong to larger families and are able to form heterodimers, perhaps with the exception of the HNF-3 family, with other members of the same family. Interestingly, the majority of the genes encoding such proteins are themselves regulated at the transcriptional level, although both transcriptional and post-transcriptional events modulate their expression during development, hepatocyte differentiation and disease, suggesting that a transcriptional cascade may play a critical role in mammalian liver development and differentiation.

Mapping of RNA polymerase on mammalian genes in cells and nuclei.

The assembly of an RNA polymerase II initiation complex at a promoter is associated with the melting of the DNA template to allow the polymerase to read the DNA sequence and synthesize the corresponding RNA. Using the specific single-stranded modifying reagent KMnO4 and a new genomic sequencing technique, we have explored the melted regions of specific genes in genomic DNA of whole cells or of isolated nuclei. We have demonstrated for the first time in vivo the melting in the promoter proximal transcribed region that is associated with the presence of RNA polymerase II complexes. An interferon-inducible gene, ISG-54, exhibited KMnO4 sensitivity over approximately 300 nucleotides downstream of the RNA initiation site in interferon-treated cells when the gene was actively transcribed but not in untreated cells where the gene was not transcribed. The extent of KMnO4 modification was proportional to transcription levels. The KMnO4 sensitivity was retained when nuclei were isolated from induced cells but was lost if the engaged polymerases were further allowed to elongate the nascent RNA chains ("run-on"). The sensitivity to KMnO4 in isolated nuclei was retained if the run-on incubation was performed in the presence of alpha-amanitin, which blocks progress of engaged polymerases. A similar analysis identified an open sequence of only approximately 30 bases just downstream of the start site of the transthyretin (TTR) gene in nuclei isolated from mouse liver, a tissue where TTR is actively transcribed. This abrupt boundary of KMnO4 sensitivity, which was removed completely by allowing engaged polymerases to elongate RNA chains, suggests that most polymerases transcribing this gene paused at about position +20. The possibility of mapping at the nucleotide level the position of actively transcribing RNA polymerases in whole cells or isolated nuclei opens new prospects in the study of transcription initiation and elongation.

Transiently and stably introduced CCAAT/enhancer-binding-protein genes are constitutively expressed in cultured cells.

CCAAT/enhancer-binding protein (C/EBP) is expressed in certain cell types including hepatocytes and adipocytes. In order to understand the mechanisms that control the expression of the mouse C/EBP gene in the liver as well as in adipocytes, we have studied both the endogenous gene and transfected C/EBP gene constructs. The initiation site of transcription was identified and a strong liver-specific DNase-I hypersensitive site located at -3 kb, which does not appear to contribute functionally to the regulation of the gene in a variety of either transiently or stably transfected cells with constructs which include sequences up to 6-kb upstream of the transcription start. C/EBP gene expression during the transition from preadipocytes to adipocytes was shown to be controlled at the level of transcription. However, adipocytes stably transfected with constructs that include -3.3 kb upstream of the C/EBP gene do not express the reporter genes in a differentiation-specific manner. We detected several DNA-binding proteins that interact with the upstream sites of the C/EBP gene. Those include two labile and two heat-stable site-specific DNA-binding proteins that are present in nuclear extracts from several tissues and cultured cell lines.

Interferon induction of gene transcription analyzed by in vivo footprinting.

The promoters of two interferon-induced genes (the ISG54 and guanylate-binding protein [GBP] genes) have been analyzed in whole cells and in isolated nuclei by using a new genomic sequencing technique. The ISG54 gene contains an interferon-simulating response element (ISRE), earlier shown to be necessary and sufficient for alpha interferon (IFN-alpha) induction, that appeared complexed with proteins in both transcribing and nontranscribing cells. However, the extent of protection and hypersensitivity to DNase I or dimethyl sulfate within the ISRE region was changed upon transcriptional induction, suggesting the binding of different factors in different transcriptional states. In addition to the ISRE, the GBP gene needs a newly recognized DNA element, called the GAS, that partly overlaps the ISRE for full induction by either IFN-alpha or IFN-gamma. This GAS element was transiently protected against DNase I in the nuclei of interferon-treated cells but was not protected at later times when transcription reached maximal levels. Thus, the GAS-binding activity may be necessary only transiently for the initial assembly of a transcription initiation complex on the GBP promoter. Dimethyl sulfate methylation of genomic DNA performed on intact cells showed a characteristic sensitivity over the GAS that correlated with transcription levels and that persisted longer than did DNase I protection over the GAS. These results demonstrate the involvement of the GAS in IFN-alpha and -gamma induction of GBP and suggest the presence of an altered DNA conformation or a small protein in the major groove of the GAS associated with ongoing GBP transcription.

Cytoplasmic activation of GAF, an IFN-gamma-regulated DNA-binding factor.

We have investigated events following treatment of cells with interferon-gamma (IFN-gamma) that lead to the immediate transcriptional activation of an inducible gene. A gamma-interferon activation factor (GAF) was activated in the cytoplasm of human fibroblasts immediately after IFN-gamma treatment and bound to a newly identified target DNA sequence, the gamma-interferon activation site (GAS). The time course of activation of GAF was different in fibroblasts and HeLa cells and correlated well with IFN-gamma-induced transcriptional activation in both cell types. IFN-gamma-dependent activation of GAF also occurred in enucleated cells (cytoplasts), showing that an inactive cytoplasmic precursor is converted to the active factor. These findings support the concept that ligand-specific signals originating at the cell surface are transmitted through latent cytoplasmic proteins which are activated to bind specific DNA sites and then move to the nucleus to activate the transcription of specific sets of genes.

Rapid in vivo footprinting technique identifies proteins bound to the TTR gene in the mouse liver.

In vivo examination of the occupancy of DNA elements that can regulate transcription is critical to reveal which proteins actually take part in establishing and maintaining gene expression. We describe a new genomic sequencing method involving the rapid purification of relevant DNA segments from the bulk of the genomic DNA using a biotinylated riboprobe. The purified sequences are revealed by a single primer extension using Taq DNA polymerase. We used this technique to study the promoter and the enhancer of mouse transthyretin (TTR), a gene highly expressed in the liver. Footprints showed high liver-specific occupancy of some, but not all, of the DNA sites that had been identified as important for expression by transfection studies in hepatoma cells. In addition, several previously undetected sites were observed that bound proteins specifically in liver. These results suggest that not all demonstrable binding sites are involved in ongoing transcription and that in vivo studies may reveal additional and probably more relevant sites.

Cell-specific transcriptional control of the mouse DNA-binding protein mC/EBP.

The mRNA encoding the mouse homolog of C/EBP, a rat DNA-binding protein that participates in activating a number of genes in hepatocytes, is present in liver cells at a far higher concentration than in most other cells, including spleen, kidney, muscle, and the majority of the brain. However, fat cells and intestinal cells contain 25-50% as much mRNA as liver cells. "Run-on" experiments show that the basis for the restricted cellular distribution of the mouse C/EBP mRNA is transcriptional regulation of the gene. We also show that disruption of cell-cell contacts incident to liver cell dispersion results in a prompt and extensive reduction in mouse C/EBP transcription as we had earlier shown to be the case for a group of 10 genes transcribed in a hepatocyte-specific fashion. In contrast, breaking cell contacts and plating the hepatocytes in culture leads to a prolonged increase in transcription of the Jun-B gene that encodes a widely distributed transcription factor. These results illustrate that the regulation of expression of a mammalian regulatory protein with limited tissue distribution is controlled at the level of transcription and depends on cell contacts.