Localization of GFP in frozen sections from unfixed mouse tissues: immobilization of a highly soluble marker protein by formaldehyde vapor.

Green fluorescent protein (GFP) and its variants, such as enhanced GFP (EGFP), have been introduced into mammalian cells by transgenes, e.g., to distinguish donor from host cells after transplantation. Free GFP is extremely soluble and leaks out from liquid-covered cryostat sections so that fixation of whole organs before sectioning has been mandatory. This precludes the analysis of serial sections with respect to fixation-sensitive enzyme activities and antigens. We describe here a vapor fixation for sections from unfixed cryostat blocks of tissue that allows unrestricted enzyme and immunohistochemistry on adjacent sections, as demonstrated for cross-striated muscle and other tissues from EGFP transgenic "green mice" and for a transplantation experiment.     

Influenza A virus infection inhibits the efficient recruitment of Th2 cells into the airways and the development of airway eosinophilia

Most infections with respiratory viruses induce Th1 responses characterized by the generation of Th1 and CD8(+) T cells secreting IFN-gamma, which in turn have been shown to inhibit the development of Th2 cells. Therefore, it could be expected that respiratory viral infections mediate protection against asthma. However, the opposite seems to be true, because viral infections are often associated with the exacerbation of asthma. For this reason, we investigated what effect an influenza A (flu) virus infection has on the development of asthma. We found that flu infection 1, 3, 6, or 9 wk before allergen airway challenge resulted in a strong suppression of allergen-induced airway eosinophilia. This effect was associated with strongly reduced numbers of Th2 cells in the airways and was not observed in IFN-gamma- or IL-12 p35-deficient mice. Mice infected with flu virus and immunized with OVA showed decreased IL-5 and increased IFN-gamma, eotaxin/CC chemokine ligand (CCL)11, RANTES/CCL5, and monocyte chemoattractant protein-1/CCL2 levels in the bronchoalveolar lavage fluid, and increased airway hyperreactivity compared with OVA-immunized mice. These results suggest that the flu virus infection reduced airway eosinophilia by inducing Th1 responses, which lead to the inefficient recruitment of Th2 cells into the airways. However, OVA-specific IgE and IgG1 serum levels, blood eosinophilia, and goblet cell metaplasia in the lung were not reduced by the flu infection. Flu virus infection also directly induced AHR and goblet cell metaplasia. Taken together, our results show that flu virus infections can induce, exacerbate, and suppress features of asthmatic disease in mice.     

Phage as agents of lateral gene transfer

When establishing lysogeny, temperate phages integrate their genome as a prophage into the bacterial chromosome. Prophages thus constitute in many bacteria a substantial part of laterally acquired DNA. Some prophages contribute lysogenic conversion genes that are of selective advantage to the bacterial host. Occasionally, phages are also involved in the lateral transfer of other mobile DNA elements or bacterial DNA. Recent advances in the field of genomics have revealed a major impact by phages on bacterial chromosome evolution.     

Different sensitivities of mutants and chimeric forms of human muscle and liver fructose-1,6-bisphosphatases towards AMP

AMP is an allosteric inhibitor of human muscle and liver fructose-1,6-bisphosphatase (FBPase). Despite strong similarity of the nucleotide binding domains, the muscle enzyme is inhibited by AMP approximately 35 times stronger than liver FBPase: I0.5 for muscle and for liver FBPase are 0.14 microM and 4.8 microM, respectively. Chimeric human muscle (L50M288) and chimeric human liver enzymes (M50L288), in which the N-terminal residues (1-50) were derived from the human liver and human muscle FBPases, respectively, were inhibited by AMP 2-3 times stronger than the wild-type liver enzyme. An amino acid exchange within the N-terminal region of the muscle enzyme towards liver FBPase (Lys20-->Glu) resulted in 13-fold increased I0.5 values compared to the wild-type muscle enzyme. However, the opposite exchanges in the liver enzyme (Glu20-->Lys and double mutation Glu19-->Asp/Glu20-->Lys) did not change the sensitivity for AMP inhibition of the liver mutant (I0.5 value of 4.9 microM). The decrease of sensitivity for AMP of the muscle mutant Lys20-->Glu, as well as the lack of changes in the inhibition by AMP of liver mutants Glu20-->Lys and Glu19-->Asp/Glu20-->Lys, suggest a different mechanism of AMP binding to the muscle and liver enzyme.     

Reconstitution of gamma-secretase activity

gamma-Secretase is a membrane protein complex with an unusual aspartyl protease activity that catalyses the regulated intramembranous cleavage of the beta-amyloid precursor protein (APP) to release the Alzheimer's disease (AD)-associated amyloid beta-peptide (Abeta) and the APP intracellular domain (AICD). Here we show the reconstitution of gamma-secretase activity in the yeast Saccharomyces cerevisiae, which lacks endogenous gamma-secretase activity. Reconstituted gamma-secretase activity depends on the presence of four complex components including presenilin (PS), nicastrin (Nct), APH-1 (refs 3-6) and PEN-2 (refs 4, 7), is associated with endoproteolysis of PS, and produces Abeta and AICD in vitro. Thus, the biological activity of gamma-secretase is reconstituted by the co-expression of human PS, Nct, APH-1 and PEN-2 in yeast.     

The E3 ubiquitin ligase AIP4 mediates ubiquitination and sorting of the G protein-coupled receptor CXCR4

Ubiquitination of the chemokine receptor CXCR4 serves as a targeting signal for lysosomal degradation, but the mechanisms mediating ubiquitination and lysosomal sorting remain poorly understood. Here we report that the Nedd4-like E3 ubiquitin ligase AIP4 mediates ubiquitination of CXCR4 at the plasma membrane, and of the ubiquitin binding protein Hrs on endosomes. CXCR4 activation promotes CXCR4 colocalization with AIP4 and Hrs within the same region of endosomes. Endosomal sorting of CXCR4 is dependent on Hrs as well as the AAA ATPase Vps4, the latter involved in regulating the ubiquitination status of both CXCR4 and Hrs. We propose a model whereby AIP4, Hrs, and Vps4 coordinate a cascade of ubiquitination and deubiquitination events that sort CXCR4 to the degradative pathway.     

RNomics in Archaea reveals a further link between splicing of archaeal introns and rRNA processing

The bulge–helix–bulge (BHB) motif recognised by the archaeal splicing endonuclease is also found in the long processing stems of archaeal rRNA precursors in which it is cleaved to generate pre-16S and pre-23S rRNAs. We show that in two species, Archaeoglobus fulgidus and Sulfolobus solfataricus, representatives from the two major archaeal kingdoms Euryarchaeota and Crenarchaeota, respectively, the pre-rRNA spacers cleaved at the BHB motifs surrounding pre-16S and pre-23S rRNAs subsequently become ligated. In addition, we present evidence that this is accompanied by circularisation of ribosomal pre-16S and pre-23S rRNAs in both species. These data reveal a further link between intron splicing and pre-rRNA processing in Archaea, which might reflect a common evolutionary origin of the two processes. One spliced RNA species designated 16S-D RNA, resulting from religation at the BHB motif of 16S pre-rRNA, is a highly abundant and stable RNA which folds into a three-stem structure interrupted by two single-stranded regions as assessed by chemical probing. It spans a region of the pre-rRNA 5′ external transcribed spacer exhibiting a highly conserved folding pattern in Archaea. Surprisingly, 16S-D RNA contains structural motifs found in archaeal C/D box small RNAs and binds to the L7Ae protein, a core component of archaeal C/D box RNPs. This supports the notion that it might have an important but still unknown role in pre-rRNA biogenesis or might even target RNA molecules other than rRNA.     

Transfer RNA-mediated antitermination in vitro

The threonyl-tRNA synthetase gene (thrS) is a member of the T-box family of ~250 genes, found essentially in Gram-positive bacteria, regulated by a tRNA-dependent antitermination mechanism in response to starvation for the cognate amino acid. While interaction between uncharged tRNA and the untranslated leader region of these genes has been firmly established by genetic means, attempts to show this interaction or to reconstitute the antitermination mechanism in vitro using purified tRNAs have so far failed. In addition, a number of conserved sequences have been identified in the T-box leaders, for which no function has yet been assigned. This suggests that factors other than the tRNA are important for this type of control. Here we demonstrate tRNA-mediated antitermination for the first time in vitro, using the regulatory tRNA^Thr isoacceptor isolated from Bacillus subtilis and a partially purified protein fraction. As predicted by the model, aminoacylation of tRNA^Thr(GGU) with threonine completely abolishes its ability to act as an effector. The role of the partially purified protein fraction can be functionally substituted by high concentrations of spermidine. However, this polyamine does not play a significant role in the induction of thrS expression in vivo, suggesting that it is specific protein co-factors that promote T-box gene regulation in conjunction with uncharged tRNA.     

Protein kinase C induces actin reorganization via a Src- and Rho-dependent pathway

We have investigated the mechanism of PKC-induced actin reorganization in A7r5 vascular smooth muscle cells. PKC activation by 12-O-tetradecanoylphorbol-13-acetate induces the disassembly of actin stress fibers concomitant with the appearance of membrane ruffles. PKC also induces rapid tyrosine phosphorylation in these cells. As we could show, utilizing the Src-specific inhibitor PP2 and a kinase-deficient c-Src mutant, actin reorganization is dependent on PKC-induced Src activation. Subsequently, the activity of the small G-protein RhoA is decreased, whereas Rac and Cdc42 activities remain unchanged. Disassembly of actin stress fibers could also be observed using the Rho kinase-specific inhibitor Y-27632, indicating that the decrease in RhoA activity on its own is responsible for actin reorganization. In addition, we show that tyrosine phosphorylation of p190RhoGAP is increased upon 12-O-tetradecanoylphorbol-13-acetate stimulation, directly linking Src activation to a decrease in RhoA activity. Our data provide substantial evidence for a model elucidating the molecular mechanisms of PKC-induced actin rearrangements.     

Modulation of the catalytic activity of neutrophil collagenase MMP-8 on bovine collagen I. Role of the activation cleavage and of the hemopexin-like domain

The cleavage of bovine collagen I by neutrophil collagenase MMP-8 has been followed at pH 7.4, 37 degrees C. The behavior of the whole enzyme molecule (whMMP-8), displaying both the catalytic domain and the hemopexin-like domain, has been compared under the same experimental conditions with that of the catalytic domain only. The main observation is that whMMP-8 cleaves bovine collagen I only at a single specific site, as already reported by many others (Mallya, S. K., Mookhtiar, K. A., Gao, Y., Brew, K., Dioszegi, M., Birkedal-Hansen, H., and van Wart, H. E. (1990) Biochemistry 29, 10628-10634; Kn?uper, V., Osthues, A., DeClerk, Y. A., Langley, K. A., Bl?ser, J., and Tschesche, H. (1993) Biochem. J. 291, 847-854; Marini, S., Fasciglione, G. F., De Sanctis, G., D'Alessio, S., Politi, V., and Coletta, M. (2000) J. Biol. Chem. 275, 18657-18663), whereas the catalytic domain lacks this specificity and cleaves the collagen molecule at multiple sites. Furthermore, a meaningful difference is observed for the cleavage features displayed by two forms of the catalytic domain, which differ for the N terminus resulting from the activation process (i.e. the former Met(80) of the proenzyme (MetMMP-8) and the former Phe(79) of the proenzyme (PheMMP-8)). Thus, the PheMMP-8 species is characterized by a much faster k(cat)/K(m), fully attributable to a lower K(m), suggesting that the conformation of the catalytic domain, induced by the insertion of this N-terminal residue in a specific pocket (Reinemer, P., Grams, F., Huber, R., Kleine, T., Schnierer, S., Piper, M., Tschesche, H., and Bode, W. (1994) FEBS Lett. 338, 227-233), brings about a better, although less discriminatory, recognition process of cleavage site(s) on bovine collagen I.