Enteropathogens in adult patients with diarrhea and healthy control subjects: a 1-year prospective study in a Swedish clinic for infectious diseases.
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A 1-year prospective study was conducted to identify enteropathogens in adults with diarrhea (n=851) and in healthy control subjects (n=203) by use of conventional laboratory methods. Virulence factor genes for diarrheagenic Escherichia coli were detected by polymerase chain reaction. Enteropathogens were identified in 56% of patients and 16% of control subjects. The isolation rate was 65% for patients with symptoms for <1 week and for travelers; >1 pathogen was found in 11% of patients. The most frequent enteropathogens were Campylobacter (13% of patients), Clostridium difficile (13%), enterotoxigenic Escherichia coli (8%), Salmonella (7%), Shigella (4%), Blastocystis hominis (4%), calicivirus (3%), rotavirus (3%), enteroaggregative E. coli (2%), Aeromonas (2%), Giardia intestinalis (2%), Cryptosporidium (2%), and astrovirus (2%). Less frequently isolated (< or =1% of patients) were verotoxigenic E. coli, enteropathogenic E. coli, enteroinvasive E. coli, Entamoeba histolytica/Entamoeba dispar, microsporidia, and adenovirus. Fifty percent of the patients were hospitalized, and 43% needed intravenous fluids. The median duration of diarrhea was 14 days. Clinical features were not helpful for predicting the etiology of diarrhea. (+info)
Rhinosporidium seeberi: a human pathogen from a novel group of aquatic protistan parasites.
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Rhinosporidium seeberi, a microorganism that can infect the mucosal surfaces of humans and animals, has been classified as a fungus on the basis of morphologic and histochemical characteristics. Using consensus polymerase chain reaction (PCR), we amplified a portion of the R. seeberi 18S rRNA gene directly from infected tissue. Analysis of the aligned sequence and inference of phylogenetic relationships showed that R. seeberi is a protist from a novel clade of parasites that infect fish and amphibians. Fluorescence in situ hybridization and R. seeberi- specific PCR showed that this unique 18S rRNA sequence is also present in other tissues infected with R. seeberi. Our data support the R. seeberi phylogeny recently suggested by another group. R. seeberi is not a classic fungus, but rather the first known human pathogen from the DRIPs clade, a novel clade of aquatic protistan parasites (Ichthyosporea). (+info)
Cloning and characterization of a myosin from characean alga, the fastest motor protein in the world.
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In characean algae, very rapid cytoplasmic streaming is generated by sliding movement of an unconventional myosin on fixed actin cables. The speed of this sliding movement is the fastest among many molecular motors known so far. We have cloned a set of overlapping cDNAs encoding the heavy chain of this myosin by immunoscreening with antibody raised against characean myosin. The molecular mass of this heavy chain is 248 kDa, and the protein has a conserved motor domain, six IQ motifs, an extensive alpha-helical coiled-coil domain, and a C-terminal globular domain. Phylogenetic analysis suggested that this myosin belongs to class XI. (+info)
Comparison of conserved structural and regulatory domains within divergent 16S rRNA-23S rRNA spacer sequences of cyanobacteria.
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PCR amplification of the internal transcribed spacer (ITS) between the 16S rRNA and 23S rRNA genes of the cyanobacterium NOSTOC: PCC 7120 gave three products. Two represented true ITS regions of different sizes, while the third was a heteroduplex. The longer spacer (ITS-L) contained 512 nucleotides and carried tRNA(Ile) and tRNA(Ala) genes, separated by a large stem-loop structure (V2) composed of short tandemly repeated repetitive sequences. Both tRNA genes, and the 5' half of the intervening stem, were absent from the shorter spacer (ITS-S), of length 283 nucleotides, which was otherwise almost completely identical to ITS-L. The two spacer regions of NOSTOC: PCC 7120 were aligned to published ITS sequences of cyanobacteria, the cyanelle of Cyanophora paradoxa and Escherichia coli. Although the ITS regions of cyanobacteria vary in length from 283 to 545 nucleotides and contain either both tRNA(Ile) and tRNA(Ala) genes, only the tRNA(Ile) gene, or neither, there is no correlation between ITS size and coding capacity for tRNAs. Putative secondary structures were determined for the deduced transcripts of the rrn operons of several cyanobacteria and were compared to that of E. coli. Highly conserved motifs important for folding and for maturation of the rRNA transcripts were identified, and regions homologous to bacterial antiterminators (box B-box A) were located. The conserved and variable regions of the cyanobacterial ITS are potential targets of PCR primers and oligonucleotide probes for detection and identification of cyanobacteria at different taxonomic levels. (+info)
Cytochrome c6 from Cyanophora paradoxa. Characterization of the protein and the cDNA of the precursor and import into isolated cyanelles.
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In the eukaryotic alga Cyanophora paradoxa, which does not contain plastocyanin, photosynthetic electron transport from the cytochrome b6/f complex to photosystem I is mediated by cytochrome c6. Cytochrome c6 was purified to homogeneity by column chromatography and FPLC. The relative molecular mass of the holoprotein was determined by two different mass spectrometric methods (californium-252 plasma desorption and UV matrix-assisted laser desorption ionization) giving 9251 +/- 3.3 Da. N-terminal Edman microsequencing yielded information on approx. 30 amino acid residues. Based on these data and on highly conserved regions of cytochromes c6, degenerate oligonucleotides were designed and used for PCR to amplify the genomic DNA of C. paradoxa. Screening of a C. paradoxa cDNA library yielded several clones coding for preapo-cytochrome c6. The deduced sequence of the mature protein was verified by plasma desorption mass spectrometric peptide mapping and shows high similarity to those of cytochromes c6 from cyanobacteria and algae. Cytochrome c6 appears to be encoded by a single nuclear gene (petJ) in C. paradoxa. As the mature protein is located in the lumen of the thylakoid membrane, it has to traverse three biological membranes as well as the unique peptidoglycan layer of the cyanelles before it reaches its final subcellular locale. Thus the transit sequence is composed of two different targeting signals: a stroma targeting peptide resembling those of higher plants with respect to hydropathy plots and amino acid composition and a hydrophobic signal peptide functioning as a thylakoid-traversing domain. There are indications for alternative sorting of part of the cyanelle cytochrome c6 pool to the periplasmic space. This is the first known bipartite transit sequence of a cyanelle precursor protein from C. paradoxa, a model organism concerning the endosymbiotic origin of plastids. Labeled precursor is efficiently imported into isolated cyanelles, then routed into thylakoids and processed to the mature protein. Hitherto, in vitro protein translocation was not reported for cyanobacterial-type thylakoids. (+info)
The mitochondrial genome of the stramenopile alga Chrysodidymus synuroideus. Complete sequence, gene content and genome organization.
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This is the first report of a complete mitochondrial genome sequence from a photosynthetic member of the stramenopiles, the chrysophyte alga Chrysodidymus synuroideus. The circular-mapping mitochondrial DNA (mtDNA) of 34 119 bp contains 58 densely packed genes (all without introns) and five unique open reading frames (ORFs). Protein genes code for components of respiratory chain complexes, ATP synthase and the mitoribosome, as well as one product of unknown function, encoded in many other protist mtDNAs (YMF16). In addition to small and large subunit ribosomal RNAs, 23 tRNAs are mtDNA-encoded, permitting translation of all codons present in protein-coding genes except ACN (Thr) and CGN (Arg). The missing tRNAs are assumed to be imported from the cytosol. Comparison of the C.SYNUROIDEUS: mtDNA with that of other stramenopiles allowed us to draw conclusions about mitochondrial genome organization, expression and evolution. First, we provide evidence that mitochondrial ORFs code for highly derived, unrecognizable versions of ribosomal or respiratory genes otherwise 'missing' in a particular mtDNA. Secondly, the observed constraints in mitochondrial genome rearrangements suggest operon-based, co-ordinated expression of genes functioning in common biological processes. Finally, stramenopile mtDNAs reveal an unexpectedly low variability in genome size and gene complement, testifying to substantial differences in the tempo of mtDNA evolution between major eukaryotic lineages. (+info)
Intracellular targets of cyclin-dependent kinase inhibitors: identification by affinity chromatography using immobilised inhibitors.
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BACKGROUND: Chemical inhibitors of cyclin-dependent kinases (CDKs) have great therapeutic potential against various proliferative and neurodegenerative disorders. Olomoucine, a 2,6,9-trisubstituted purine, has been optimized for activity against CDK1/cyclin B by combinatorial and medicinal chemistry efforts to yield the purvalanol inhibitors. Although many studies support the action of purvalanols against CDKs, the actual intracellular targets of 2,6, 9-trisubstituted purines remain unverified. RESULTS: To address this issue, purvalanol B (95. ) and an N6-methylated, CDK-inactive derivative (95M. ) were immobilized on an agarose matrix. Extracts from a diverse collection of cell types and organisms were screened for proteins binding purvalanol B. In addition to validating CDKs as intracellular targets, a variety of unexpected protein kinases were recovered from the 95. matrix. Casein kinase 1 (CK1) was identified as a principal 95. matrix binding protein in Plasmodium falciparum, Leishmania mexicana, Toxoplasma gondii and Trypanosoma cruzi. Purvalanol compounds also inhibit the proliferation of these parasites, suggesting that CK1 is a valuable target for further screening with 2,6,9-trisubstituted purine libraries. CONCLUSIONS: That a simple batchwise affinity chromatography approach using two purine derivatives facilitated isolation of a small set of highly purified kinases suggests that this could be a general method for identifying intracellular targets relevant to a particular class of ligands. This method allows a close correlation to be established between the pattern of proteins bound to a small family of related compounds and the pattern of cellular responses to these compounds. (+info)
Eukaryotically encoded and chloroplast-located rubredoxin is associated with photosystem II.
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We analyzed a eukaryotically encoded rubredoxin from the cryptomonad Guillardia theta and identified additional domains at the N- and C-termini in comparison to known prokaryotic paralogous molecules. The cryptophytic N-terminal extension was shown to be a transit peptide for intracellular targeting of the protein to the plastid, whereas a C-terminal domain represents a membrane anchor. Rubredoxin was identified in all tested phototrophic eukaryotes. Presumably facilitated by its C-terminal extension, nucleomorph-encoded rubredoxin (nmRub) is associated with the thylakoid membrane. Association with photosystem II (PSII) was demonstrated by co-localization of nmRub and PSII membrane particles and PSII core complexes and confirmed by comparative electron paramagnetic resonance measurements. The midpoint potential of nmRub was determined as +125 mV, which is the highest redox potential of all known rubredoxins. Therefore, nmRub provides a striking example of the ability of the protein environment to tune the redox potentials of metal sites, allowing for evolutionary adaption in specific electron transport systems, as for example that coupled to the PSII pathway. (+info)