A novel association between clustered NF-kappaB and C/EBP binding sites is required for immune regulation of mosquito Defensin genes.

A comparative analysis identified key cis-acting regulatory elements responsible for the temporal control of mosquito Defensin gene expression. The promoters of Anopheles gambiae Defensin 1 and two isoforms of Aedes aegypti Defensin A are up-regulated by immune challenge. This stimulated activity depends upon a cluster of three NF-kappaB binding sites and closely associated C/EBP-like motifs, which function as a unit for optimal promoter activity. Binding of NF-kappaB and C/EBP like transcription factors is confirmed by electrophoretic mobility shift assay, including supershifts with antibodies to C/EBP. KappaB-like motifs are abundant within antimicrobial peptide gene promoters and most are very closely associated with putative C/EBP binding sites. This novel association between NF-kappaB and C/EBP binding sites may, therefore, be of widespread significance.

Antioxidant gene expression in the blood-feeding fly Glossina morsitans morsitans.

We report the characterization of 11 antioxidant genes from the tsetse fly Glossina m. morsitans. Through similarity searches which detected homology we suggest that these genes consist of two superoxide dismutases (one with a putative signal peptide), three thioredoxin peroxidases (one with a putative signal peptide), three peroxiredoxins, one further signal peptide-containing peroxidase with its closest similarity to a glutathione peroxidase, one catalase and one thioredoxin reductase. We describe the changes occurring in the expression levels of these genes during fly development, in different adult tissues, in the adult midgut through the digestive cycle and following trypanosome infection. Overall, nine of the 11 genes studied showed responses to changes in physiological circumstance, with the peroxiredoxin group showing the smallest variations throughout.

Association of midgut defensin with a novel serine protease in the blood-sucking fly Stomoxys calcitrans.

Using ELISA we provide direct evidence that the midgut defensins of the blood-sucking fly Stomoxys calcitrans are secreted into the gut lumen. We show that midgut defensin peptide levels increase up to fortyfold in response to a blood meal but not to a sugar meal. The data suggests the midgut defensin genes are post-transcriptionally regulated and that their function is protection of the stored blood meal from bacterial attack while it awaits digestion. Using recombinant defensins produced in Pichia pastoris we demonstrate that while in the gut cells the midgut defensins are bound in an SDS-stable complex to proteins with an apparent molecular weight of > 26 kDa from which they are released when secreted into the gut lumen. This > 26 kDa protein (Ssp3) has been cloned and sequenced and is a member of the serine protease S1 family with homologies to multiple insect proteases and to vertebrate trypsins and elastases.

Regulation of midgut defensin production in the blood-sucking insect Stomoxys calcitrans.

The Stomoxys midgut defensin (Smd) family of genes are exclusively expressed in the anterior midgut of adult flies. Their putative function is protection of the stored bloodmeal from microbial attack. Smd genes are constitutively expressed, up-regulated in response to a bloodmeal and further up-regulated by immune stimulation per os but only in the presence of a bloodmeal not a sugar meal. Smd genes are down-regulated in response to a systemic immune challenge. Smd gene constructs transfected into l(2)mbn cells undertake constitutive expression but are not up-regulated by immune challenge. Electrophoretic mobility shift assays (EMSA) suggest the rel-like sites in the proximal promoter region of Smd genes do not bind midgut factors and so are non-functional.

Inhibition of cyclo-oxygenase 2 expression in colon cells by the chemopreventive agent curcumin involves inhibition of NF-kappaB activation via the NIK/IKK signalling complex.

Colorectal cancer is a major cause of cancer deaths in Western countries, but epidemiological data suggest that dietary modification might reduce these by as much as 90%. Cyclo-oxygenase 2 (COX2), an inducible isoform of prostaglandin H synthase, which mediates prostaglandin synthesis during inflammation, and which is selectively overexpressed in colon tumours, is thought to play an important role in colon carcinogenesis. Curcumin, a constituent of turmeric, possesses potent anti-inflammatory activity and prevents colon cancer in animal models. However, its mechanism of action is not fully understood. We found that in human colon epithelial cells, curcumin inhibits COX2 induction by the colon tumour promoters, tumour necrosis factor alpha or fecapentaene-12. Induction of COX2 by inflammatory cytokines or hypoxia-induced oxidative stress can be mediated by nuclear factor kappa B (NF-kappaB). Since curcumin inhibits NF-kappaB activation, we examined whether its chemopreventive activity is related to modulation of the signalling pathway which regulates the stability of the NF-kappaB-sequestering protein, IkappaB. Recently components of this pathway, NF-kappaB-inducing kinase and IkappaB kinases, IKKalpha and beta, which phosphorylate IkappaB to release NF-kappaB, have been characterised. Curcumin prevents phosphorylation of IkappaB by inhibiting the activity of the IKKs. This property, together with a long history of consumption without adverse health effects, makes curcumin an important candidate for consideration in colon cancer prevention.

Characterization of transcriptional regulation of gamma-glutamyl transpeptidase in rat liver involving both positive and negative regulatory elements.

gamma-Glutamyl transpeptidase is normally not present in adult rat hepatocytes, but its expression is induced by a range of xenobiotics, including carcinogens and chemopreventive agents. Synthesis of the enzyme is mediated by at least six mRNAs transcribed from tandemly arranged promoters on a single gene. We previously identified and partially characterized promoter III as being responsible for upregulation of gamma-glutamyl transpeptidase in rat liver in response to inducing agents. In this study, we examined response elements involved in the regulation of this promoter by using reporter gene assays and in vitro DNase I footprinting and electrophoretic mobility shift assays. Among the response elements was a region with negative regulatory activity upstream of a 240-bp basal regulatory region covering the transcriptional start site. This negative regulatory region, lying between nt -465 and -185, contained sequences with considerable homology to silencer elements in the glutathione s-transferase P gene. The region of basal regulation (nt -185 to +55) contained a CCAAT box, a TFIID binding site, and a GAGA box. A hepatocyte nuclear factor 3-like sequence was identified at nt -234 to -254 and had a DNase I hypersensitive site characteristic of binding of members of the hepatocyte nuclear factor 3/fork head family of proteins and may be involved in the regulation of this promoter.

Characterization of a promoter for gamma-glutamyl transpeptidase activated in rat liver in response to aflatoxin B1 and ethoxyquin.

gamma-Glutamyl transpeptidase (GGT) is normally absent from adult rat hepatocytes but is induced by a range of xenobiotics, including carcinogens and chemoprotective agents. As many as six mRNA species for this enzyme have been described in both rat and mouse, with various degrees of tissue specificity. These originate from one gene and have separate promoters within alternative 5' untranslated sequences. By using a cDNA-derived sequence specific for GGT mRNA III to screen a rat genomic library, a clone that contains the promoter region for this mRNA was isolated and characterized. The transcriptional start site lay some 3.5 kb upstream from that already characterized for mRNA II in rat kidney. Luciferase activity was obtained after transfection of rat hepatoma-derived cell lines with constructs containing the putative promoter III fused to a luc reporter. Although this promoter lacks a TATA box, a sequence close to the start site that binds the transcription factor TFIID in vitro was identified. By using PCR techniques, mRNA III (homologous to both mouse III and IV) and an mRNA (IV) with homology to VI in mouse were found in ethoxyquin- and aflatoxin B1-treated rat liver and kidney as well as in a hepatoma-derived cell line. No evidence was found for a product homologous to mRNA from promoter V described in the mouse.

Suppression of heat-shock protein synthesis by short-chain fatty acids and alcohols.

We have shown that ethanol, propanol and butanol (at 0.5-2%) and salts of butyric and propionic acids (at 8-40 mM) all cause a major reduction in heat-shock protein (hsp) synthesis when present in the growth medium of Drosophila cultured cells (Kc and SL2) subjected to either increased temperature or chemical stressors. Inhibition of normal protein synthesis in unstressed cells was comparatively slight, and the usual suppression of synthesis of non-heat-shock proteins in stressed cells was unaffected. Maximum suppression of hsp synthesis occurred only if inhibitors were added before initiation of the stress response, an observation that eliminates the possibility that these findings are due to non-specific, toxic effects. Suppression was accompanied by severely reduced levels of both hsp70 mRNA and active heat-shock factor (HSF). We conclude that the inhibitors act by suppressing the initiation of transcription of heat-shock genes.

Histone H4 acetylation in Drosophila. Frequency of acetylation at different sites defined by immunolabelling with site-specific antibodies.

Electrophoresis, Western blotting and immunostaining with antibodies specific for histone H4 acetylated at lysines 5, 8, 12, or 16, were used to define patterns of H4 acetylation in cell lines from humans (HL60) and the fruit fly Drosophila (S2, Kc). In human cells, the mono-acetylated isoform H4Ac1 is acetylated predominantly at just one of the four possible lysine residues, lysine 16. This is the first step in the progressive acetylation of H4. In contrast, in Drosophila, H4Ac1 is acetylated at lysines 5, 8, or 12 with approximately equal frequency. Fundamental differences appear to exist in control of H4 acetylation in different species, despite the evolutionary conservation of acetylation sites.