Neutrophilia in LFA-1-deficient mice confers resistance to listeriosis: possible contribution of granulocyte-colony-stimulating factor and IL-17

LFA-1 (CD11a/CD18) plays a crucial role in various inflammatory responses. In this study, we show that LFA-1(-/-) mice are far more resistant to Listeria monocytogenes infection than LFA-1(+/-) mice. Consistent with this, we found the following: 1) the numbers of granulocytes infiltrating the liver were markedly higher in LFA-1(-/-) mice than in LFA-1(+/-) mice, 2) increased antilisterial resistance in LFA-1(-/-) mice was abrogated by depletion of granulocytes, and 3) the numbers of granulocytes in peripheral blood, and the serum levels of both G-CSF and IL-17 were higher in LFA-1(-/-) mice than in LFA-1(+/-) mice. Neither spontaneous apoptosis nor survival of granulocytes from LFA-1(-/-) mice were affected by physiological concentrations of G-CSF. Our data suggest regulatory effects of LFA-1 on G-CSF and IL-17 secretion, and as a corollary on neutrophilia. Consequently, we conclude that increased resistance of LFA-1(-/-) mice to listeriosis is due to neutrophilia facilitating liver infiltration by granulocytes promptly after L. monocytogenes infection, although it is LFA-1 independent.     

Searching sequence space for high-affinity binding peptides using ribosome display

We present the construction of a synthetic library based on the scaffold of bovine heart fatty acid-binding protein (FABP) with 1.1x10(14) independent members. Ribosome display was applied to select streptavidin-binding peptides in vitro from 2x10(13) molecules of the library each encoding FABP with 15 contiguous random amino acid residues at its N terminus. The selection yielded several different binding peptides. The best binder possessed a dissociation constant as low as 4nM and, in contrast to the previously isolated peptides, contained no HPQ motif. A substitution analysis enabled shortening of the 15-mer peptide and revealed a 9-mer variant with a dissociation constant of 17nM, which is a 1000-fold increase of affinity compared to the already known peptides of this size. This high-affinity binding peptide in combination with the whole set of streptavidin conjugates should be an extremely useful tool for the detection and purification of recombinant proteins.     

Unique and conserved features of genome and proteome of SARS-coronavirus,an early split-off from the coronavirus group 2 lineage

The genome organization and expression strategy of the newly identified severe acute respiratory syndrome coronavirus (SARS-CoV) were predicted using recently published genome sequences. Fourteen putative open reading frames were identified, 12 of which were predicted to be expressed from a nested set of eight subgenomic mRNAs. The synthesis of these mRNAs in SARS-CoV-infected cells was confirmed experimentally. The 4382- and 7073 amino acid residue SARS-CoV replicase polyproteins are predicted to be cleaved into 16 subunits by two viral proteinases (bringing the total number of SARS-CoV proteins to 28). A phylogenetic analysis of the replicase gene, using a distantly related torovirus as an outgroup, demonstrated that, despite a number of unique features, SARS-CoV is most closely related to group 2 coronaviruses. Distant homologs of cellular RNA processing enzymes were identified in group 2 coronaviruses, with four of them being conserved in SARS-CoV. These newly recognized viral enzymes place the mechanism of coronavirus RNA synthesis in a completely new perspective. Furthermore, together with previously described viral enzymes, they will be important targets for the design of antiviral strategies aimed at controlling the further spread of SARS-CoV.     

Specific nitration at tyrosine 430 revealed by high resolution mass spectrometry as basis for redox regulation of bovine prostacyclin synthase

Treatment of bovine aortic microsomes containing active prostacyclin synthase (PGI(2) synthase) with increasing concentrations of peroxynitrite (PN) up to 250 microm of PN yielded specific staining of this enzyme on Western blots with antibodies against 3-nitrotyrosine (3-NT), whereas above 500 microm PN staining of additional proteins was also observed. Following treatment of aortic microsomes with 25 microm PN, PGI(2) synthase was about half-maximally nitrated and about half-inhibited. It was then isolated by gel electrophoresis and subjected to proteolytic digestion with several proteases. Digestion with thermolysin for 24 h provided a single specific peptide that was isolated by high performance liquid chromatography and identified as a tetrapeptide Leu-Lys-Asn-Tyr(3-nitro)-COOH corresponding to positions 427-430 of PGI(2) synthase. Its structure was established by precise mass determination using Fourier transform-ion cyclotron resonance-nanoelectrospray mass spectrometry and Edman microsequencing and ascertained by synthesis and mass spectrometric characterization of the authentic Tyr-nitrated peptide. Complete digestion by Pronase to 3-nitrotyrosine was obtained only after 72 h, suggesting that the nitrated Tyr-430 residue may be embedded in a tight fold around the heme binding site. These results provide evidence for the specific inhibition of PGI(2) synthase by nitration at Tyr-430 that may occur already at low levels of PN as a consequence of endothelial co-generation of nitric oxide and superoxide.     

Quantitative identification of the protonation state of histidines in vitro and in vivo

The C[bond]N coupling constants centered at the C(epsilon 1) and C(delta 2) carbons in histidine residues depend on the protonation state and tautomeric form of the imidazole ring, making them excellent indicators of pH or pK(a), and the ratio of the tautomeric states. In this paper, we demonstrate that the intensity ratios for the C(epsilon 1)-H and C(delta 2)-H cross-peaks measured with a constant time HSQC experiment without and with J(C[bond]N) amplitude modulation are determined by the ratios of the protonated and deprotonated forms and tautomeric states. This allows one to investigate the tautomeric state of histidines as well as their pK(a) in situations where changing the pH value by titration is difficult, for example, for in-cell NMR experiments. We apply this technique to the investigation of the bacterial protein NmerA and determine that the intracellular pH in the Escherichia coli cytoplasm is 7.1 +/- 0.1.     

Thermophilic protease-producing Geobacillus from Buranga hot springs in Western Uganda

Two proteolytic thermophilic aerobic bacterial strains, PA-9 and PA-5, were isolated from Buranga hot springs in western Uganda. The cells were rods, approximately 10–12 μm in length and 3 μm in width. Isolate PA-9 grew at between 38°C and 68°C (optimum, 62°C), and PA-5 grew at between 37°C and 72°C (optimum, 60°C). Both isolates grew optimally at pH 7.5–8.5. Their 16S rRNA gene sequences indicated that they belong to the newly described genus Geobacillus. Zymogram analysis of the crude enzyme extracts revealed the presence of two extracellular proteases for isolate PA-5, and at least eight for isolate PA-9. The optimum temperature and pH for casein-degrading activity were 70°C, pH 6.5 for isolate PA-9, but caseinolytic activity could also be observed at 2°C. In the case of isolate PA-5, optimal activity was observed over a temperature and pH range of 50–70°C and pH 5–10, respectively. Received: 26 November 2001 / Accepted: 12 December 2001     

Cholera toxin B pretreatment of macrophages and monocytes diminishes their proinflammatory responsiveness to lipopolysaccharide

The cholera toxin B chain (CTB) has been reported to suppress T cell-dependent autoimmune diseases and to potentiate tolerance of the adaptive immune system. We have analyzed the effects of CTB on macrophages in vitro and have found that preincubation with CTB (10 microg/ml) suppresses the proinflammatory reaction to LPS challenge, as demonstrated by suppressed production of TNF-alpha, IL-6, IL-12(p70), and NO (p < 0.01) in cells of macrophage lines. Pre-exposure to CTB also suppresses LPS-induced TNF-alpha and IL-12(p70) formation in human PBMC. Both native and recombinant CTB exhibited suppressive activity, which was shared by intact cholera toxin. In cells of the human monocyte line Mono Mac 6, exposure to CTB failed to suppress the production of IL-10 in response to LPS. Control experiments excluded a role of possible contamination of CTB by endotoxin or intact cholera toxin. The suppression of TNF-alpha production occurred at the level of mRNA formation. Tolerance induction by CTB was dose and time dependent. The suppression of TNF-alpha and IL-6 production could be counteracted by the addition of Abs to IL-10 and TGF-beta. IFN-gamma also antagonized the actions of CTB on macrophages. In contrast to desensitization by low doses of LPS, tolerance induction by CTB occurred silently, i.e., in the absence of a measurable proinflammatory response. These findings identify immune-deviating properties of CTB at the level of innate immune cells and may be relevant to the use of CTB in modulating immune-mediated diseases.