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Cells respond to a range of cytokines and other inflammatory stimuli by selectively expressing a wide range of genes. These proinflammatory signals bind to receptors and initiate intracellular signalling cascades. This results in the activation of proinflammatory deoxyribonucleic acid (DNA)-binding proteins or transcription factors such as activator protein-1 (AP-1) or nuclear factor-kappa B (NF-kappa B). Following activation, these factors bind to specific recognition sequences in the control regions (promoters) of target genes causing modulation of gene transcription. Furthermore, numerous sites for regulation of cytokine and cytokine receptor genes by these transcription factors are found in their promoter regions. Many factors affect the formation and activity of AP-1 dimers (Fos and Jun heterodimers) through protein specific interactions or by the modulation of pre-existing complexes by phosphorylation. These complexes can vary markedly in their ability to stimulate gene transcription. Thus, induction of AP-1 activity is regulated by stimuli that either induce the de novo synthesis of AP-1 subunits or increase the activity of previously formed AP-1 dimers. NF-kappa B is activated by a number of agents including tumour necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), lipopolysaccharide (LPS) and viruses. NF-kappa B plays a central role in a range of immunological responses due to its ability to switch on inflammatory genes which lead to further activation of NF-kappa B is a heterodimer of proteins which vary in their ability to bind to DNA and/or to activate gene transcription. NF-kappa B activation is primarily regulated by sequestration of heterodimers within the cytoplasm as inactive complexes with inhibitory molecules (inhibitory-kappa B (I-kappa B)). Treatment of cells with inducing agents results in the phosphorylation of the I-kappa B molecule which is targeted for rapid degradation. This causes a rapid dissociation of the cytoplasmic NF-kappa B/I-kappa B complexes and allows translocation of the active NF-kappa B to the nucleus. Cross-coupling between NF-kappa B and AP-1 has been described which results in a synergistic increase in activity at both AP-1 and NF-kappa B sites. Elevation of both AP-1 and NF-kappa B, as reported in asthma, may therefore lead to far greater inflammation than would be present if either transcription factor alone were activated.

Prostaglandin (PG) release, which is increased in vivo by inflammatory conditions and in vitro by pro-inflammatory cytokines, is decreased by glucocorticoids. Two phospholipase A2 isoforms, secretory (sPLA2) and cytosolic (cPLA2,), have been implicated in inflammation. These enzymes catalyse the release of arachidonic acid which is then converted to prostaglandins by the cyclooxygenases (COX-1 and COX-2). Regulation of these events at the mRNA level is poorly characterised in epithelial cells. We have used a human epithelial-like cell line (A549) as a model system to study mRNA expression of sPLA2, cPLA2, COX-1 and COX-2. Following treatment of cells and extraction of RNA, semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to examine expression of these genes. We show a coordinate induction of both cPLA2 and COX-2 mRNA by pro-inflammatory cytokines which correlated with increased PGE2 release. By contrast, sPLA2 mRNA was undetectable and COX-1 was found to be expressed at a constant low level. In addition dexamethasone pretreatment significantly reduced both cPLA2 and COX-2 mRNA levels as well as PGE2 release following cytokine stimulation. These data indicate a major role for control of prostaglandin synthesis at the mRNA level of key synthetic genes in epithelial cells. Furthermore we show that a major mechanism of glucocorticoid action in preventing prostaglandin release occurs by suppression of cPLA2 and COX-2 mRNA levels.

Background: Eicosanoids such as leukotrienes, prostaglandins, lipoxins, and 15-hydroperoxyeicosatetraenoic acid (15-HETE) cause bronchoconstriction, increased microvascular permeability, mucus secretion, and polymorph chemotaxis. These pro-inflammatory effects are important in diseases such as asthma and cystic fibrosis where the levels of mediators are increased both in the stable and acute state. A study was conducted to examine the expression of the mRNA for the enzymes of the eicosanoid pathways (5-lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP), cyclo-oxygenases 1 and 2 (COX-1, COX-2), and 15-lipoxygenase (15-LO)) in normal subjects and in patients with stable atopic asthma and stable cystic fibrosis., Methods: Reverse transcription polymerase chain reaction (RT-PCR) was used to examine the expression of total RNA for 5-LO, FLAP, COX-1, COX-2, and 15-LO in peripheral blood polymorphonuclear cells and mononuclear cells from the three subjects groups., Results: The expression of mRNA for 5-LO and FLAP was similar in normal subjects and in patients with asthma and cystic fibrosis. COX-1 was increased in both cell types in asthmatic patients. COX-2 and 15-LO were increased in polymorphs of patients with atopic asthma but not in mononuclear cells. COX-2 and 15-LO were undetectable in either cell type in patients with cystic fibrosis whereas COX-1 levels in polymorphs were similar to those in patients with asthma., Conclusions: The increased leukotriene production in asthma and cystic fibrosis is not explained by an increase in transcription of 5-LO and FLAP. Transcription of 15-LO and COX-2 is increased in atopic asthma. Transcription of COX-1 is increased in both atopic asthma and cystic fibrosis.

The localization and distribution of the human glucocorticoid receptor (GR) mRNA and protein was investigated in human lung obtained from transplant donors and recipients by in situ hybridization, RNA blot analysis, immunolocalization, and Western analysis. Subjects were either nonasthmatic or had mild asthma requiring only beta(2)-agonists. No difference in amount of GR mRNA was found in total RNA isolated from nonasthmatic or asthmatic donor lung. In situ hybridization showed the highest concentration of GR mRNA in the alveolar walls and vascular endothelium and smooth muscle, with lesser amounts in the airway epithelium and smooth muscle. There was no change in the level or sites of expression of GR mRNA between normal and asthmatic subjects. Immunolocalization of GR confirmed the in situ hybridization data. There was no change in the level or sites of expression of GR, in either the lung or airway, between normal and asthmatic subjects. Immunolocalization of GR in bronchial biopsies from two normal and asthmatic subjects confirmed the localization and distribution of GR. Western analysis and mobility shift assays confirmed no differences in GR levels between the two subject groups. The localization of GR mRNA and protein to specific cell types within lung and airway will make it possible to study the cellular targets of glucocorticoid therapy in inflammatory lung diseases such as asthma.

Glucocorticosteroids are the most effective therapy for the suppression of airway inflammation. Classically, ligand binding to the inactive cytosolic receptor (GR) causes a conformational change enabling nuclear translocation of the active GR and alteration in the transcriptional response. We have investigated GR-transcription factor interactions within the cytoplasm where many transcription factors are localized prior to activation. Active DNA binding GR complexes were found within the cytoplasm of human lung epithelial cells (A549). Stimulation by dexamethasone (1 microM) caused translocation of active GR into the nucleus and inhibition of RANTES release. Coincubation of cells with dexamethasone and TNF alpha (1 ng/ml) or PMA (0.1 microM) prevented RANTES release and the translocation of activated GR to the nucleus without affecting GR levels as detected by western blotting. These results suggest that a major site of pro-inflammatory cytokine action may be within the cytoplasm of steroid-responsive cells acting via prevention of the GR translocation into the nucleus.

Objective: Endotoxin and cytokines have been reported to have both stimulatory and inhibitory effects on endothelial cell-derived nitric oxide release. The discrepancy may be explained by differential regulation of the endothelial and inducible type of nitric oxide synthase gene expression. This study aimed to investigate the differential effect of lipopolysaccharide treatment in vivo on the three isoforms (endothelial, brain-type, and inducible) of nitric oxide synthase gene expression in the rat., Design: Prospective, controlled, animal trial., Setting: Experimental laboratory of a postgraduate medical research institution., Subjects: Normal, anesthetized rats., Interventions: Animals were treated with lipopolysaccharide (15 mg/kg iP), saline (1 mL/kg ip), or lipopolysaccharide plus dexamethasone (3 mg/kg ip, 50 mins before lipopolysaccharide administration) in vivo 4 hrs before experimentation., Measurements and Main Results: The expression of endothelial, brain-type, and inducible nitric oxide synthase mRNAs was quantified by Northern blot analysis using bovine, rat, and mouse cDNA probes, respectively. An endothelial nitric oxide synthase mRNA was detected at 4.3 kilobase in the heart, lung, and aorta, and a 10-kilobase brain-type nitric oxide synthase mRNA was detected in the brain. The endothelial and brain-type signals were strong in tissues from animals treated with saline, but were reduced by three- to four-fold in tissues from lipopolysaccharide-treated rats as estimated by optical density ratio. The 4.4-kilobase inducible nitric oxide synthase mRNA detected using the murine cDNA probe was absent or negligible in the heart, lung, and brain from saline-treated rats, but was markedly increased in the same tissues from lipopolysaccharide-treated animals. Dexamethasone significantly inhibited lipopolysaccharide-induced inducible nitric oxide synthase mRNA expression, but had no effect on the down-regulation of endothelial and brain nitric oxide synthase mRNAs., Conclusions: Rats treated with lipopolysaccharide in vivo display down-regulation of endothelial nitric oxide mRNA in the heart, lung, and aorta, and brain-type nitric oxide synthase mRNA in the brain There was a parallel up-regulation of inducible nitric oxide synthase mRNA in all tissues except in the aorta. Dexamethasone prevents the induction of inducible nitric oxide synthase mRNA. but has no effect on the down-regulation of endothelial and brain-type nitric oxide synthase mRNAs induced by lipopolysaccharide. Thus, endotoxin regulates constitutive and inducible nitric oxide synthase mRNA differentially.

Epithelial cells are actively involved in inflammation and play a role in inflammatory diseases such as asthma. Numerous proinflammatory genes, expressed in the airway epithelium, are regulated by the transcription factor NF-kappa B. We show that the proinflammatory cytokines, IL-1 beta and TNF alpha, as well as a protein kinase C activator cause NF-kappa B activation in A549 epithelial cells. This observation is consistent with a major role for NF-kappa B in inflammation. We also demonstrate that IL-1 beta costimulation with a protein synthesis inhibitor, cycloheximide, or a transcription blocker, actinomycin D, results in superinduction of NF-kappa B but not the transcription factors Oct 1, AP-1, and Sp-1. We speculate that this may be due to lack of de novo synthesis of the NF-kappa B inhibitor, I kappa B alpha, and suggest that this phenomena may help explain the widely observed effect of mRNA superinduction of primary response genes in response to translational blockers.

Beta 2-adrenoreceptor agonists and glucocorticosteroids are the two most effective treatments for asthma and are often used in combination. Glucocorticoids mediate their anti-inflammatory effects through the action of activated glucocorticoid receptors (GRs). Many of the effects of GRs on the synthesis of cytokines and other inflammatory mediators are due to a direct interaction with other deoxyribonucleic acid (DNA)-binding proteins belonging to the basic leucine zipper (bZIP) group of transcription factors, such as activator protein-1 (AP-1) and nuclear factor-kappa B (NF-kappa B). Beta 2-agonists are potent bronchodilators at low doses and at high doses can activate gene transcription via a bZIP protein, cyclic adenosine monophosphate (cAMP) response element binding protein (CREB). Activated GRs and CREB can interact with each other within the nucleus to modulate both DNA-binding and gene transcription in either a positive or inhibitory manner, depending on cell type. In lung cells, high doses of beta 2-agonists reduce the ability of GR to bind DNA, a process which is mediated by CREB activation. Inhibition of GR DNA-binding by CREB raises the possibility that high-dose beta 2-agonists could have functional antiglucocorticoid activity and may be a basis for the reported increase in asthma morbidity and mortality in industrialized countries, which have increasing per capita beta 2-agonist use.

Background: Recruitment of inflammatory cells in the lungs may contribute to tissue injury as a result of mediators released from these cells. Interleukin 1 beta (IL-1 beta) is a potent inducer of neutrophil accumulation, a process that may require local protein biosynthesis. Macrophage inflammatory protein 2 (MIP-2) is a approximately 6 kD heparin binding protein and is a member of the C-X-C superfamily that causes significant neutrophil chemotaxis and activation in vitro. A study was performed to determine whether IL-1 beta could induce the expression of MIP-2 in the lungs of Brown-Norway rats., Methods: rhIL-1 beta (500 U) or 0.9% NaCl was injected intratracheally and bronchoalveolar lavage (BAL) cells and lung tissues were evaluated for MIP-2 mRNA expression after RNA extraction by Northern blot analysis. MIP-2 probe was prepared from cDNA obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) of BAL cells obtained from a rat treated with lipopolysaccharide., Results: There was no detectable MIP-2 mRNA in the lungs of control rats but a marked enhancement of the expression at four hours with no expression at 12 hours and a slight expression at 24 hours. IL-1 beta induced a significant influx of neutrophils into BAL fluid with a transient increase in macrophages. In situ hybridisation of lungs using MIP-2 cDNA probe labelled with digoxigenin showed expression of MIP-2 mRNA in airway mononuclear cells and airway epithelium at four hours after IL-1 beta; at 24 hours the signal had nearly gone., Conclusion: IL-1 beta induces the expression of MIP-2 mRNA in rat lung. MIP-2 may be one chemokine that could contribute to IL-1 beta induced neutrophil influx.

Background: The importance of cytokines in the asthmatic inflammatory response is becoming apparent. The aim of this study was to determine whether the non-invasive method of induced sputum combined with the polymerase chain reaction would allow the detection of messenger RNA (mRNA) encoding a range of cytokines on a qualitative basis., Methods: Four groups were studied comprising 10 normal subjects, six atopic, 10 mild and five moderately severe asthmatic subjects. Sputum was induced by the inhalation of nebulised 3.5% saline and total RNA extracted from the expectorated cells. Expression of cytokine message within induced sputum was examined by reverse transcription and the polymerase chain reaction (PCR) using primers specific for a range of cytokines (IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, RANTES, TNF alpha, IFN alpha, IFN gamma). Presence or absence of the signal was determined at 35 and 70 cycles of PCR by electrophoretic size fractionation on ethidium bromide stained agarose gels., Results: Cytokine message was detectable in sputum by this method. All samples showed a positive result for actin control. Analysis of signal for the cytokines in all subjects showed that, at 70 cycles, IL-1, IL-5, IL-8, and TNF alpha were detected in more subjects than would be expected by chance. IL-5 mRNA was detected in more of the asthmatic patients (moderate 80%, mild 40%) than in the atopic subjects (33%), who in turn showed expression of this cytokine in more individuals than nonatopic subjects (10%)., Conclusions: The combination of sputum induction and PCR appears to be a useful, non-invasive tool to explore the chronic inflammation of asthma and possibly other lung disorders. It should enable differences between normal and asthmatic subjects to be identified for future confirmation by quantitative techniques.

Resistance to the anti-inflammatory effects of glucocorticoids in asthma and other inflammatory and immune diseases is uncommon, but presents a management problem. Understanding the mechanisms of steroid resistance provides new insights into the mechanism of steroid action as well as the underlying chronic disease process. In patients with primary steroid-resistant (SR) asthma there is no abnormality in the pharmacokinetics of the exogenous steroid and no significant defect in steroid binding to the glucocorticoid receptor (GR). Recent studies have demonstrated a marked reduction in the binding of GR to DNA; this appears to be due to increased binding of GR to the transcription factor activator protein-1 (AP-1). Secondary steroid resistance in asthma may arise in response to the release of cytokines that activate AP-1 and other transcription factors that bind directly to GR. A similar effect may also be seen with high concentrations of beta 2-agonists that activate another GR binding transcription factor, CREB. Several existing and novel treatment strategies are possible in the management of SR asthma.

Inhaled beta 2-adrenoceptor agonists are the most effective bronchodilator treatment in asthma, yet paradoxically high doses may be associated with increased asthma morbidity and mortality. Steroids are the most effective therapy in controlling asthmatic inflammation and act by binding to specific sequences of DNA (GRE), thus modulating gene transcription. We report that in rat lung, the beta 2-adrenoceptor agonists, salbutamol and fenoterol, decrease the binding of glucocorticoid receptors to GRE, by 46 +/- 4% although it has no effect on the affinity or number of glucocorticoid receptors. The inhibition of GRE binding by salbutamol is concentration-dependent, can be blocked by propranolol and is seen following forskolin treatment. This effect appears to be due to an interaction between the glucocorticoid receptor and the transcription factor, cAMP response element binding protein (CREB), which is activated by high concentrations of beta 2-adrenoceptor agonists. We suggest that by this mechanism high doses of inhaled beta 2-adrenoceptor agonists may inhibit the anti-inflammatory effects of endogenous glucocorticoids and exogenous corticosteroids used for asthma therapy.

Glucocorticosteroids have a wide variety of effects which result in the long-term dampening of inflammatory responses. An important site of steroid action may be on the control of the activator protein-1 (AP-1) binding to deoxyribonucleic acid (DNA). AP-1 is a proinflammatory transcription factor composed of a heterodimer of Fos and Jun proto-oncogenes, which can be induced by phorbol esters and various cytokines. We have examined the hypothesis that dexamethasone may inhibit inflammation via an effect on AP-1 activation in human lung tissue. The effect of dexamethasone on the phorbol ester and cytokine activation of AP-1 and its monomers was examined in human lung tissue obtained from transplantation donors. AP-1 activation was measured by its ability to bind DNA, its localization in the nucleus by Western blotting, and the levels of fos and jun messenger ribonucleic acids (mRNAs) using Northern blotting. The phorbol ester, phorbol myristate acetate (PMA), caused a significant 2-3 fold increase in AP-1 DNA binding, which was sustained for 24 h and completely attenuated by co-incubation with dexamethasone. Dexamethasone alone caused a 40% decrease in AP-1 DNA binding. Dexamethasone modulated the expression of both c-jun and c-fos mRNA and produced long-term (24 h) 40% reduction in both mRNAs when compared to control tissues. PMA induced a rapid and prolonged increase in c-Fos and c-Jun nuclear localization, which was not attenuated by co-incubation with dexamethasone.(ABSTRACT TRUNCATED AT 250 WORDS)

Eotaxin was recently identified as the major eosinophil chemoattractant in bronchoalveolar lavage fluid obtained 3h after allergen challenge of sensitised guinea-pigs. We now report the cDNA cloning of this C-C chemokine. The 777 base-pair clone, pEo3122, consists of a 40 base 5' untranslated region, an open reading frame of 288 bases predicting a 73 amino acid mature protein plus a 23 amino acid signal peptide, and a 3' untranslated region of 449 bases containing a poly A tail. Northern blot analysis showed eotaxin mRNA in the lungs of naive and sensitised guinea-pigs, which was considerably increased after allergen challenge. Eotaxin may be an important mediator of eosinophil accumulation and activation in allergic reactions. As eotaxin stimulates human eosinophils, this chemokine and related molecules may be involved in human diseases such as asthma where eosinophil accumulation is a prominent feature.

Platelet-activating factor (PAF) is a potent inflammatory mediator and it actions are mediated via specific cell surface receptors which are coupled to G-proteins. PAF stimulates several functions in monocytes and may modulate the expression of its own receptor. To investigate the possible modulation of PAF receptor mRNA expression Northern blot analysis of total RNA from human monocytes was performed using the cDNA of human leukocyte PAF receptor as a probe. Following the addition of 100 nM PAF, there was a 2.0-fold increase in PAF receptor mRNA at 60 minutes after the stimulation, which was inhibited by pretreatment with the PAF receptor antagonist WEB 2086. This increase returned to control level at 120 and 180 min. The increase of PAF receptor mRNA was statistically significant for 10 nM to 1 microM of PAF, while 100 nM of lysoPAF did not increase PAF receptor mRNA levels. These results suggest that PAF receptor expression can be regulated by PAF itself at the transcriptional level.

Platelet-activating factor (PAF) has been implicated in the pathogenesis of several inflammatory pulmonary diseases, and specific binding sites have been demonstrated in human and guinea pig lung membranes by radioligand binding experiments. Both human and guinea pig PAF receptors (PAFR) have recently been cloned. We have used molecular probes to study the gene expression of PAFR in human and animal lung and in situ hybridization to determine the distribution of PAFR mRNA in peripheral lung. Northern blot analysis of total lung RNA from human lung parenchyma, using a 1.1-kb SmaI-EcoRI fragment of human PAFR cDNA or a 0.9-kb SmaI-SmaI fragment of guinea pig PAFR cDNA, demonstrated the expression of PAFR mRNA in human lung, with a single transcript of 4 kb. There was a significant increase in PAFR mRNA in the lungs of asthmatic patients and a significant decrease in PAFR mRNA in the lungs of cigarette smokers compared with normal patients. Similarly, the expression of PAFR mRNA on guinea pig and rat lung was detected as a single transcript of 3 kb, using both guinea pig and human PAFR cDNA probes. A full-length 1.8-kb human leukocyte PAFR cDNA probe was used as the DNA template for producing 35S-labeled antisense and sense cRNA probes for use in in situ hybridization studies of human peripheral lung. These studies revealed high levels of PAFR mRNA hybridization in blood vessels, moderate levels of hybridization in alveolar walls and peripheral airway smooth muscle, but no specific signal in airway epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Free radicals generated by a partial reduction of O2 pose a serious threat to tissues and vital organs and cells. The major site of interaction between the lung and inhaled oxidants is the epithelium. We have examined the effect of pyrogallol, an O2- generator, on the ability of human epithelial cells to produce active DNA binding proteins and inducible nitric oxide synthase (iNOS) mRNA in cultured A549 epithelial cells. NF kappa B binding in the nuclei of these cells was determined by electrophoretic mobility shift assays. iNOS mRNA was measured using reverse transcription of PCR. There was a time- and concentration-dependent induction of NF kappa B binding, followed by a time and dose dependent increase in iNOS mRNA levels. These results suggest that in airways the initial response to oxidative stress may be to induce NF kappa B-responsive genes, such as iNOS, which may play an important role in defending the airway against oxidative stress.

Interleukin-8 (IL-8) is a neutrophil chemotactic factor expressed in many cell types, including human airway epithelial cells (HAEC). Inhaled corticosteroids are now used increasingly early in the treatment of airway inflammation such as in asthma, and directly interact with HAEC at relatively high concentrations. We have investigated the effect of dexamethasone on IL-8 expression in primary cultured HAEC obtained from transplantation donors. Northern blot analysis was used to measure IL-8 mRNA levels in HAEC, and radioimmunoassay was used to measure IL-8 protein in culture supernatant fluids. We demonstrated that IL-8 was expressed by primary cultured HAEC and that this was enhanced by IL-1 beta and tumour necrosis factor-alpha stimulation, but not by IL-6 or lipopolysaccharide. Dexamethasone suppressed IL-8 mRNA expression and protein synthesis dose-dependently in both resting and stimulated HAEC. The half-life of IL-8 mRNA determined in the presence of actinomycin D was less than 1 hr, and dexamethasone preincubation had no effect on mRNA stability. These results support the view that HAEC may play an important role in the pathogenesis of airway inflammatory diseases, and that glucocorticosteroids may exert their anti-inflammatory effects by blocking IL-8 gene expression and generation in these cells.

Nitric oxide (NO) is detectable in exhaled air. To elucidate whether airway epithelial cells could be a source of NO, we investigated the expression of inducible nitric oxide synthase (iNOS) by the murine lung epithelial cell line, LA-4, in response to cytokine stimulation and the ability of corticosteroids to modulate this effect. Stimulation with cytomix, a combination of interleukin-1 beta, tumor necrosis factor-alpha, and interferon-gamma, elevated nitrite levels by 873% in the culture supernatants and enhanced the conversion of arginine to citrulline by 273% at 24 h. An increased number of cells stained for iNOS and an increase in iNOS mRNA was also observed. Dexamethasone decreased the cytokine-induced increase in nitrite levels, NOS activity, iNOS immunoreactivity, and mRNA but did not change the half life of iNOS mRNA. These results show that lung epithelial cells can release NO, a process which can be inhibited by dexamethasone.

Glucocorticosteroids are highly effective in controlling inflammation and the molecular mechanisms involved are now becoming clear. Activation of glucocorticoid receptors results in increased or decreased transcription of a number of genes involved in the inflammatory process. Of particular importance is the repression of cytokine gene transcription and the direct interaction between the glucocorticoid receptor and other transcription factors activated in chronic inflammation. In this review, Peter Barnes and Ian Adcock discuss recent studies that have increased our understanding of these mechanisms and that may lead to improved anti-inflammatory therapies in the future.

Nitric oxide (NO) may mediate the hypotension of septic shock, but the effect of endotoxin on inducible NO synthase (iNOS) mRNA expression remains unclear. We studied the effects of lipopolysaccharide (LPS) treatment in vivo on iNOS mRNA expression using reverse transcription and polymerase chain reaction. The iNOS mRNA was absent or negligible in any tissue studied from control rats, but was markedly increased in lung, liver, spleen, skeletal muscle and kidney from LPS-treated rats. The LPS-induced increase in iNOS mRNA was prevented by dexamethasone. Our results indicate that LPS treatment in vivo induces the expression of an iNOS mRNA via a dexamethasone-sensitive mechanism, and thus provide direct molecular evidence for the involvement of NO in septic shock.

Strategies for the expression of precursors of eukaryotic secreted proteins as part of fused proteins in Escherichia coli have been explored. A fusion protein with beta-galactosidase at the N-terminal end and honeybee prepromelittin at the C-terminal end (beta-gal-pM) was expressed in low amounts as a cleaved polypeptide, from which the promelittin portion had been removed. Inclusion in the induction culture of 10 mM MgCl2 or 8.3% (v/v) ethanol, inhibitors of signal peptidase, gave rise to the full-length beta-gal-pM fusion protein. The results suggest that a soluble recombinant fusion protein with a signal peptide in an internal location 660 residues from the N-terminus is recognized by the E. coli translocation apparatus in the inner membrane and by leader peptidase. High-level production (about 45% of total cellular proteins) of prepromelittin was achieved when it was part of a fusion protein at the C-terminus of a truncated insoluble polypeptide from bacteriophage gene 10. This fusion protein separated into inclusion bodies in an aggregated form. In contrast, attempts to express prepromelittin by itself or at the N-terminal end of a fusion with mouse dihydrofolate reductase (pM-DHFR) proved unsuccessful.

We describe a simple, rapid, and inexpensive procedure for the isolation of plasmid DNA in high yields from Escherichia coli cultures. The procedure entails two main steps, which involve treating intact bacterial cells with phenol/chloroform in the presence of Triton X-100 and LiCl followed by polyethylene glycol precipitation. Plasmid DNA preparations isolated by this method are highly pure and virtually devoid of RNA. The DNA is suitable substrate for restriction mapping, DNA-modifying enzymes, and in vitro transcription with SP6 and T7 RNA polymerases.

There was no visible thymus in ageing rats of 18 months, and 7 days after orchidectomy there was still no evidence of a thymus. By 30 days after the operation, however, there was a well-defined and well developed bilobular thymus overlying the heart, although it was smaller than those observed in 10-week-old rats. Histologically, the tissue appeared normal, was well vascularized, filled with lymphocytes and several mitotic figures were also seen. When compared with sham-operated animals, blood from these animals had a significantly higher lymphocyte count. These results have important implications for the possible enhancement of the immune system with associated improvement of health during ageing.