Comparison of an oligo-chip based assay with PCR method to measure the prevalence of tick-borne pathogenic bacteria in central Slovakia

Ticks are well-known vectors for a wide range of pathogenic microorganisms. We examined the presence of Rickettsia spp., Anaplasma spp., Borrelia spp., Coxiella burnetii and Francisella tularensis in Ixodes ricinus ticks collected in central Slovakia using oligo-chip based assay. Rickettsiae were detected in 5.6% of examined ticks. Borreliae and anaplasmae were identified in 2.1% and 2.8% ticks, respectively. All tested samples were negative for presence of Coxiella burnetii and Francisella tularensis. All these results were compared with those obtained by PCR analysis, and a close correlation between them was found. In addition, rickettsiae of spotted fever group (SFG), Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato were found in ticks using genera or species-specific PCR methods. They are circulating in 10 out of 18 studied localities.     

A novel tubular array associated with protein bodies in the rough endoplasmic reticulum ofopaque-2 maize

Summary The seed storage proteins of maize (Zea mays L.) are synthesized during endosperm development on membrane-bound polyribosomes. Protein body formation in normal genotypes occurs via a sequential deposition of the various types of zeins, and leads to the formation of spherical structures with a diameter of about l m. In the endosperm mutantopaque-2 the level of one zein class is reduced; these kernels exhibit an opaque phenotype instead of the vitreous phenotype displayed in normal genotypes, presumably due to the decrease in total zein protein at the time of desiccation. Previous microscopic examination ofopaque-2 protein bodies at 22 DAP (days after pollination) showed that the protein bodies were morphologically similar to those of normal genotypes. However, the endosperm ofopaque-2 maize at 14 DAP contains tubular arrays within the rough endoplasmic reticulum. These tubular arrays are tightly associated with the developing protein bodies. Long strands of tubules, sometimes 10 m in length, are observed in the endosperm, and partially formed protein bodies often seem to be forming directly from these tubular arrays. No immunostaining is associated with this tubular material when any of the anti-zein antibodies are used.Abbreviations BSA bovine serum albumin - DAP days after pollination - IgG immunoglobulin G Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Immunological detection and localization of a calsequestrin-like protein in red beet and cucumber cells

Summary Calsequestrin is a calcium binding protein present in the sarcoplasmic reticulum (SR) of animal muscle cells and is thought to be essential for the rapid uptake and release of Ca²⁺, and thus for the regulation of Ca²⁺-dependent cellular functions. Higher plant cells of red beet (Beta vulgaris L.) and cucumber (Cucumis sativus L.) contain a polypeptide of about M_r 55000 that cross-reacts with a monoclonal antibody raised against calsequestrin from rabbit skeletal muscle SR. In beet this protein changes its apparent molecular weight with pH as indicated in Western immunoblotting. Although this protein bound calcium it was not the dominant calcium-binding protein in red beet. Washing of beet root tissue leads to a slight increase of this polypeptide in microsomal fractions as indicated by immunoblotting. After immunoblotting to partially purified cell membrane fractions this polypeptide appeared to be predominantly associated with endoplasmic reticulum-enriched fractions. Immunogold labelling of ultrathin sections of cucumber hypocotyl using the anti-calsequestrin antibody showed that gold particles were very largely confined to the cytosol and often in close proximity to the ER. Clusters of up to nine gold particles were observed, often over small vesicular areas, as observed in some animal tissues. These results indicate that red beet and cucumber cells contain a protein which may be related to animal calsequestrin. It appears to be associated with the ER and could be involved in cellular calcium regulation.     

Vicinity analysis: a methodology for the identification of similar protein active sites

Vicinity analysis (VA) is a new methodology developed to identify similarities between protein binding sites based on their three-dimensional structure and the chemical similarity of matching residues. The major objective is to enable searching of the Protein Data Bank (PDB) for similar sub-pockets, especially in proteins from different structural and biochemical series. Inspection of the ligands bound in these pockets should allow ligand functionality to be identified, thus suggesting novel monomers for use in library synthesis. VA has been developed initially using the ATP binding site in kinases, an important class of protein targets involved in cell signalling and growth regulation. This paper defines the VA procedure and describes matches to the phosphate binding sub-pocket of cyclin-dependent protein kinase 2 that were found by searching a small test database that has also been used to parameterise the methodology.     

A Zea mays GTP-binding protein of the ARF family complements an Escherichia coli mutant with a temperature-sensitive malonyl-coenzyme A:acyl carrier protein transacylase

In an attempt to isolate a plant malonyl-coenzyme A:acyl carrier protein transacylase cDNA clone, by direct genetic selection in an Escherichia coli fabD mutant (LA2-89) with a maize cDNA expression library, a Zea mays cDNA clone encoding a GTP-binding protein of the ARF family was isolated. Complementation of a mutation affecting bacterial membrane lipid biosynthesis by a plant ARF protein, could indicate the existence of as yet unidentified bacterial equivalents of this ubiquitous eucaryotic GTP-binding protein.     

Identification of a protein kinase gene associated with pistillody,homeotic transformation of stamens into pistil-like structures,in alloplasmic wheat

Homeotic transformation of stamens into pistil-like structures (called pistillody) has been reported in cytoplasmic substitution (alloplasmic) lines of bread wheat (Triticum aestivum) having the cytoplasm of a wild relative species, Aegilops crassa. Our previous studies indicated that pistillody is caused by alterations of the class B MADS-box gene expression pattern associated with mitochondrial gene(s) in the Ae. crassa cytoplasm. To elucidate the nuclear gene involved in the cross-talk between pistillody-related mitochondrial gene(s) and nuclear homeotic genes, we performed cDNA subtraction analysis using cDNAs derived from young spikes of a pistillody line and a normal line. As a result, we identified a protein kinase gene, WPPK1 (wheat pistillody-related protein kinase 1), which is upregulated in the young spikes of the pistillody line. RT-PCR analysis indicated that WPPK1 is strongly expressed in pistils and pistil-like stamens in the pistillody line, suggesting that it is involved in the formation of pistil-like stamens as well as pistils. The full-length cDNA sequence for WPPK1 showed high similarity with a flowering plant PVPK-1 protein kinase, and phylogenetic analysis indicated that it is a member of AGC group protein kinases. Furthermore, a phosphorylation assay indicated that it has protein kinase activity. In situ hybridization analysis revealed that WPPK1 is expressed in developing pistils and pistil-like stamens as well as in their primordia. These indicate that in the alloplasmic line, WPPK1 plays a role in formation and development of pistil-like stamens.     

Promiscuous and specific phospholipid binding by domains in ZAC,a membrane-associated Arabidopsis protein with an ARF GAP zinc finger and a C2 domain

Arabidopsis proteins were predicted which share an 80 residue zinc finger domain known from ADP-ribosylation factor GTPase-activating proteins (ARF GAPs). One of these is a 37 kDa protein, designated ZAC, which has a novel domain structure in which the N-terminal ARF GAP domain and a C-terminal C2 domain are separated by a region without homology to other known proteins. Zac promoter/-glucuronidase reporter assays revealed highest expression levels in flowering tissue, rosettes and roots. ZAC protein was immuno-detected mainly in association with membranes and fractionated with Golgi and plasma membrane marker proteins. ZAC membrane association was confirmed in assays by a fusion between ZAC and the green fluorescence protein and prompted an analysis of the in vitro phospholipid-binding ability of ZAC. Phospholipid dot-blot and liposome-binding assays indicated that fusion proteins containing the ZAC-C2 domain bind anionic phospholipids non-specifically, with some variance in Ca²⁺ and salt dependence. Similar assays demonstrated specific affinity of the ZAC N-terminal region (residues 1–174) for phosphatidylinositol 3-monophosphate (PI-3-P). Binding was dependent in part on an intact zinc finger motif, but proteins containing only the zinc finger domain (residues 1–105) did not bind PI-3-P. Recombinant ZAC possessed GTPase-activating activity on Arabidopsis ARF proteins. These data identify a novel PI-3-P-binding protein region and thereby provide evidence that this phosphoinositide is recognized as a signal in plants. A role for ZAC in the regulation of ARF-mediated vesicular transport in plants is discussed.     

Identification of a key functional region in harpins from <Emphasis Type="Italic">Xanthomonas</Emphasis> that suppresses protein aggregation and mediates harpin expression in <Emphasis Type="Italic">E. coli</Emphasis>

In the current study, we identified a key functional region in harpins from Xanthomonas that suppressed protein aggregation and mediated its expression in E. coli. Our data suggested that the presence of two common features in harpins [Wei et al. (1992) Science 257:85-88], namely, high glycine content and lack of cysteine residues, were not sufficient for Xanthomonas to elicit hypersensitive response (HR) activity or heat stability. Additionally, bioinformatic analyses revealed that the secondary structure of a conserved N-terminal region consisting of 12 highly hydrophilic amino acids (QGISEKQLDQLL) was α-helical. Following site-directed mutagenesis deletion of this region, the three mutated harpin proteins, in cultures induced at 37°C, failed to elicit a HR in tobacco leaves. However, at 24°C, two mutated harpins retained the ability to elicit HR, albeit with lower expression levels than that noted with the wild-type. SDS-PAGE and Western blot data suggested the HpaG mutant protein was found almost entirely in the inclusion body. These data demonstrated that these conserved amino acid residues played a critical role in protein aggregation and inclusion body formation in harpins from Xanthomonas.     

Crosslinking of microsomal proteins identifies P-9000, a protein that is co-transported with phaseolin and phytohemagglutinin in bean cotyledons

Developing cotyledons of the common bean, Phaseolus vulgaris L., transport within their secretory system (endoplasmic reticulum and Golgi apparatus) the abundant vacuolar proteins, phaseolin and phytohemagglutinin. To identify proteins that may play a role in vacuolar targeting, we treated cotyledon microsomal fractions with a bifunctional crosslinking reagent, dithiobis(succinimidyl propionate), isolated protein complexes with antibodies to phaseolin and phytohemagglutinin, and analysed the polypeptides by sodium dodecylsulfate polyacrylamide gel electrophoresis. This allowed us to identify a protein of M_r=9000 (P-9000) that was crosslinked to both phaseolin and phytohemagglutinin. P-900 is abundantly present in the endoplasmic reticulum. The aminoterminus of P-9000 shows extensive sequence identity with the amino-terminus of PA1 (M_r=11 000), a cysteine-rich albumin whose processing products accumulate in the vacuoles of pea (Pisum sativum L.) cotyledons. Like PA1, P-9000 is synthesized as a pre-proprotein that is posttranslationally processed into smaller polypeptides. The possible functions of P-9000 are discussed.Abbreviations DSP dithiobis(succinimidyl propionate) - EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - kDa kilodalton - M_r relative molecular mass - PHA phytohemagglutinin - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis     

Identification of a novel protein, PriB, in Klebsiella pneumoniae

PriB is a primosomal protein required for the reinitiation of replication in bacteria. Here, we report the identification and characterization of a novel PriB protein in Klebsiella pneumoniae (KPN_04595; KpPriB). Unlike the well-studied Escherichia coli PriB protein (EcPriB), which exists as a homodimer comprising 104-aa polypeptides, KpPriB forms a monomer of only 55 aa, due to the absence of the 49 aa N-terminus in KpPriB. Although this N-terminal region (1–49 aa) in EcPriB contains several important residues, such as K18, R34, and W47, which are crucial for ssDNA binding, we found that KpPriB binds ssDNA, but not ssRNA, with comparable affinity as that for EcPriB. Results from filter-binding assays demonstrate that the KpPriB–ssDNA interaction is cooperative and salt-sensitive. Substituting the residue K33 in KpPriB with alanine, the position corresponding to the classic ssDNA-binding residue K82 of EcPriB located in loop L₄₅, significantly reduced ssDNA-binding activity and cooperativity. These results reveal that the 1–49 aa region of the classical PriB protein is unnecessary for ssDNA binding. On the basis of these findings, the structure–function relationships of KpPriB are discussed.     

Antibody-free peptide substrate screening of serine/threonine kinase (protein kinase A) with a biotinylated detection probe

Being different from anti-phosphotyrosine antibodies, anti-phosphoserine- or anti-phosphothreonine-specific antibodies with high affinity for the detection of serine/threonine kinase substrates are not readily available. Therefore, chemical modification methods were developed for the detection of phosphoserine or threonine in the screening of protein kinase substrates based on β-elimination and Michael addition. We have developed a biotin-based detection probe for identification of the phosphorylated serine or threonine residue. A biotin derivative induced a color reaction using alkaline phosphate-conjugated streptavidin that amplified the signal. It was effective for the detection and separation of the target peptide on the resin. The detection probe was successfully used in identifying PKA substrates from peptide libraries on resin beads. The peptide library was prepared as a ladder-type, such that the active peptides on the colored resin beads were readily sequenced with the truncated peptide fragments by MALDI-TOF/MS analysis after releasing the peptides from the resin bead through photolysis.     

Crystal structure of Apis mellifera OBP14, a C-minus odorant-binding protein, and its complexes with odorant molecules

Apis mellifera (Amel) relies on its olfactory system to detect and identify new-sources of floral food. The Odorant-Binding Proteins (OBPs) are the first proteins involved in odorant recognition and interaction, before activation of the olfactory receptors. The Amel genome possess a set of 21 OBPs, much fewer compared to the 60-70 OBPs found in Diptera genomes. We have undertaken a structural proteomics study of Amel OBPs, alone or in complex with odorant or model compounds. We report here the first 3D structure of a member of the C-minus class OBPs, AmelOBP14, characterized by only two disulfide bridges of the three typical of classical OBPs. We show that AmelOBP14 possesses a core of 6 α-helices comparable to that of classical OBPs, and an extra exposed C-terminal helix. Its binding site is located within this core and is completely closed. Fluorescent experiments using 1-NPN displacement demonstrate that AmelOBP14 is able to bind several compounds with sub micromolar dissociation constants, among which citralva and eugenol exhibit the highest affinities. We have determined the structures of AmelOBP14 in complex with 1-NPN, eugenol and citralva, explaining their strong binding. Finally, by introducing a double cysteine mutant at positions 44 and 97, we show that a third disulfide bridge was formed in the same position as in classical OBPs without disturbing the fold of AmelOBP14.     

The NAM8 gene in Saccharomyces cerevisiae encodes a protein with putative RNA binding motifs and acts as a suppressor of mitochondrial splicing deficiencies when overexpressed.

Summary We have characterized the nuclear geneNAM8 inSaccharomyces cerevisiae. It acts as a suppressor of mitochondrial splicing deficiencies when present on a multicopy plasmid. The suppressed mutations affect RNA folding and are located in both group I and group II introns. The gene is weakly transcribed in wildtype strains, its overexpression is a prerequisite for the suppressor action. Inactivation of theNAM8 gene does not affect cell viability, mitochondrial function or mitochondrial genome stability. TheNAM8 gene encodes a protein of 523 amino acids which includes two conserved (RNP) motifs common to RNA-binding proteins from widely different organisms. This homology with RNA-binding proteins, together with the intronic location of the suppressed mitochondrial mutations, suggests that the NAM8 protein could be a non-essential component of the mitochondrial splicing machinery and, when present in increased amounts, it could convert a deficient intron RNA folding pattern into a productive one.     

Fusion with maltose-binding protein (MBP) affects neither RNA N-glycosidase activity nor immunogenicity of karasurin-A, a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica

A genomic clone encoding mature karasurin-A (KRNA), a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica, was efficiently expressed in E. coli using an expression cassette vector pMAL-c2. The resultant recombinant KRNA fused with maltose-binding protein (MBP) was recovered from the soluble fraction of the bacterial cells and purified to near homogeneity after one round of the affinity chromatography. Neither the karasurin precursor retaining both N- and C-terminal peptides, nor the protein with the N-terminal peptide was successufully produced even as a MBP-fusion. The protein with its C-terminal peptide was over-produced but was recovered in an insoluble fraction. Both the recombinant MBP-KRNA fusion protein and recombinant KRNA with MBP removed were as active as the native KRNA from root tubers. The immunogenicity of the recombinant KRNA was also unaffected by fusion with MBP.     

The 24kDa outer envelope membrane protein from spinach chloroplasts: molecular cloning,in vivo expression and import pathway of a protein with unusual properties

The 24 kDa outer envelope membrane protein of spinach chloroplasts (omp24) represents a major constituent of this membrane. Sequences of tryptic and endoprotease Glu-C peptides derived from omp24 allowed the design of oligonucleotides which were used to generate a DNA fragment by polymerase chain reaction using spinach cDNA as template. This fragment served as a probe to screen a cDNA library for a full-length clone of the omp24 coding sequence. The protein predicted from the complete sequence only has 148 amino acids and a molecular mass of 16294 Da. It is an acidic protein (calculated isoelectric point 4.8) with a high content of proline residues. Expression of the coding sequence in Escherichia coli and characterization of the purified recombinant protein produced revealed that the overestimation of its molecular mass by SDS-PAGE (ca. 25 kDa) is due to its abnormal amino acid composition. Despite its rather low hydrophobicity (polarity index 49%), omp24 appears to be deeply embedded in the outer membrane. Insertion of omp24 into the membrane proceeds almost independently of surface receptors or targeting sequence but, in contrast to other known outer envelope membrane proteins, is stimulated by ATP.Abbreviations CNBr cyanogen bromide - IP isoelectric point - NCS N-chlorosuccinimide - NTA nitrilotriacetic acid - omp24 outer envelope membrane protein of spinach chloroplasts - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - SV8 protease Staphylococcus aureus V8 protease (Endoprotease Glu-C) - TPT chloroplast triose phosphate/phosphate translocator