A cyclophilin-like peptidyl-prolyl cis/trans isomerase from Legionella pneumophila – characterization, molecular cloning and overexpression

Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires’disease). A major virulence factor, the Mip protein (FK506-binding protein, FKBP25mem), belongs to the enzyme family of peptidyl-prolyl cis/trans isomerases (PPIases). Here we show that L. pneumophila Philadelphia I possesses an additional cytoplasmic PPiase at a level of enzyme activity comparable to that of FKBP25mem. The N-terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPIases. The Icy gene (Legionella cycophn) was cloned and sequenced. It encodes a putative 164-amino-acid protein with a molecular mass of 17 968 Da called L. pneumophila cyclophilin 18 (L. p. Cyp18). Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coll with 60.5% and 51.5% identity, respectively. The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins. An L. pneumophila Cyp18 derivative with a 19-amino-acid polypeptide extension including a 6-histi-dine tag and an enterokinase cleavage site exhibits     

Immunohistochemical detection of secretoneurin, a novel neuropeptide endoproteolytically processed from secretogranin II, in normal human endocrine and neuronal tissues

Summary An antiserum raised against a synthetic peptide derived from the primary amino sequence of rat secretogranin II (chromogranin C) was used for immunological (quantitative radioimmunoassay analysis) and immunohistochemical studies of normal human endocrine and nervous tissues. This antibody recognized a novel and biologically active neuropeptide which was coined as secretoneurin. In endocrine tissues, secretoneurin was mainly co-localized with chromogranin A and B with some exceptions (e.g., parathyroid gland). Secretoneurin was demonstrated immunohistochemically in the adrenal medulla, thyroid C cells, TSH- and FSH/LH-produting cells of the anterior pituitary, A and B cells of pancreatic islets, in endocrine cells of the gastrointestinal tract and the bronchial mucosa, and the prostate. Immunoreactivity determined by radioimmunoassay analysis revealed high secretoneurin levels in the anterior and posterior pituitary and lower levels in pancreatic and thyroid tissue. A strong secretoneurin immunoreactivity was also found in ganglion cells of the submucdsal and myenteric plexus of the gastrointestinal tract, and in ganglionic cells of dorsal root ganglia, peripheral nerves, and ganglion cells of the adrenal medulla. Thus, secretoneurin may serve as a useful marker of gangliocytic/neuronal differentiation.     

MOLECULAR SYSTEMATICS OF ECTOCARPUS AND KUCKUCKIA (ECTOCARPALES, PHAEOPHYCEAE) INFERRED FROM PHYLOGENETIC ANALYSIS OF NUCLEAR- AND PIASTTD-ENCODED DNA SEQUENCES

The phylogeny of Ectocarpus and Kuckuckia strains representing widely separated populations from both hemispheres was inferred from sequence analysis of the internal transcribed spacers of the nuclear ribosomal DNA (ITS 1—5.8S-ITS 2) and the spacer region in the plastid-encoded ribulose- bis -phosphate-carboxylase (RUBISCO) cistron (partial rbc L-spacer-partial rbc S ). Both sequences resulted in matching phylogenies, with the RUBISCO spacer region being most informative at the level of genera and species and the internal transcribed spacer sequences at the level of species and populations. Three major clades were formed by strains previously described by morphology and physiology as Kuckuckia, E. fasciculatus, and E. siliculosus, confirming the validity of these taxa . Ectocarpus and Kuckuckia are regarded as sibling taxa with respect to the outgroup species Feldmannia simplex, Hincksia mitchelliae, and Pilayella littoralis. The clade formed by sexual E. siliculosus strains and most asexual Ectocarpus strains was subdivided into several clades that are consistent with geographical races within E. siliculosus. The inferred phylogeny of Ectocarpus corresponds generally with results from cross-fertilization experiments, morphology, and lipid analysis. A hypothesis on the origin and dispersal of E. siliculosus suggests several natural dispersal events during periods of global cooling as well as recent and possibly anthropogenic dispersal events .     

Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco

cDNAs encoding three proteins from barley ( Hordeum vulgare ), a class-II chitinase (CHI), a class-II β-1,3-glucanase (GLU) and a Type-I ribosome-inactivating protein (RIP) were expressed in tobacco plants under the control of the CaMV 35S-promoter. High-level expression of the transferred genes was detected in the transgenic plants by Northern and Western blot analysis. The leader peptides in CHI and GLU led to accumulation of these proteins in the intercellular space of tobacco leaves. RIP, which is naturally deposited in the cytosol of barley endosperm cells, was expressed either in its original cytosolic form or fused to a plant secretion peptide (spRIP). Fungal infection assays revealed that expression of the individual genes in each case resulted in an increased protection against the soilborne fungal pathogen Rhizoctonia solani , which infects a range of plant species including tobacco. To create a situation similar to 'multi-gene' tolerance, which traditional breeding experience has shown to provide crops with a longer-lasting protection, several of these antifungal genes were combined and protection against fungal attack resulting from their co-expression in planta was evaluated. Transgenic tobacco lines were generated with tandemly arranged genes coding for RIP and CHI as well as GLU and CHI. The performance of tobacco plants co-expressing the barley transgenes GLU/ CHI or CHI/RIP in a Rhizoctonia solani infection assay revealed significantly enhanced protection against fungal attack when compared with the protection levels obtained with corresponding isogenic lines expressing a single barley transgene to a similar level. The data indicate synergistic protective interaction of the co-expressed anti-fungal proteins in vivo .     

Reserve carbohydrate in maize stem : [C]glucose and [C]sucrose uptake characteristics

Maize (Zea mays L.) stem is thought to function alternately as a net importing and net exporting organ during ontogeny, depending on whole plant photosynthetic source and sink status. The [¹⁴C]sucrose and [¹⁴C]glucose uptake capacity of stem tissues was investigated to increase our understanding of the transport factors which may influence sink status.     

Functional Morphology of the Olfactory Organ in Spinachia spinachia (L.) (Teleostei,Gasterosteidae)

The functional morphology of the olfactory organ in Spinachia spinachia (L.), which has only a single nare, was studied by light microscopy, scanning electron microscopy, and experimental investigations. It was shown that only the incoming water passes over the olfactory epithelium. The device for ventilating this olfactory organ is an accessory ventilation sac activated by respiratory pressure changes in the buccal cavity. This one-way water current over the olfactory epithelium in a monotrematous olfactory organ was found to be possible because of the morphology of the olfactory organ combined with movements of the lateral wall of the olfactory organ and the nasal tube during respiration. The olfactory epithelium is divided into irregular islets. Both ciliated receptor cells and microvillous receptor cells are present.