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Biomedical subjects

G Christiansen

Publications and source records attributed to G Christiansen.

At least 19 recordsLinked to original sources
PubMed2001

Nonribosomal peptide synthetase genes occur in most cyanobacterial genera as evidenced by their distribution in axenic strains of the PCC.

Previous studies largely carried out with environmental samples or axenic and non-axenic cultures suggested that cyanobacteria may be a rich source of hitherto unexplored bioactive compounds. This has been confirmed in the present study by a screening of 146 axenic strains from the Pasteur Culture Collection (PCC) of cyanobacteria. Use of degenerate PCR primers, designed on the basis of conserved sequence motifs in the aminoacyl-adenylation domain of peptide synthetases, revealed the presence of the corresponding genes in the majority (75.3%) of the strains examined. Among unicellular cyanobacteria, only Chamaesiphon sp. strain PCC 6605, two strains of Gloeocapsa and most Microcystis isolates (22 out of 24) contained these genes; no amplicons were detected for any members of the genera Cyanothece, Gloeobacter and Gloeothece and the genetically diverse representatives of Synechococcus and Synechocystis. By contrast, eight out of ten pleurocapsalean members, 16 out of 25 oscillatorian strains, and all but two of the 63 filamentous heterocystous cyanobacteria tested gave positive amplification results. This information will be highly valuable for further exploring the corresponding cyanobacterial peptides and for elucidating the bioactivity of such non-ribosomally synthesized molecules.

Cyanobacteria↗
PubMed2001

Differential expression of Pmp10 in cell culture infected with Chlamydia pneumoniae CWL029.

The complete genome of Chlamydia pneumoniae contains a total of 21 genes encoding polymorphic membrane proteins (Pmp). From this large Pmp family three genes, pmp8, pmp10 and pmp11, were cloned and antibodies against recombinant full-length Pmp proteins were produced. Indirect immunofluorescence microscopy of HEp-2 cells infected with C. pneumoniae CWL029 was performed with the Pmp antibodies in combination with a Chlamydia-specific anti-lipopolysaccharide (LPS) antibody. This double staining technique clearly showed that expression of Pmp10 was differential. Additional double staining with monoclonal antibodies to the surface of C. pneumoniae elementary bodies and the anti-LPS antibody resulted in identification of seven monoclonal antibodies that reacted identically to the Pmp10 antibody indicating that Pmp10 is an immunodominant protein. Finally, the molecular mechanism responsible for differential expression is suggested to be variation in the guanine residues in the polyG tract of pmp10.

Antibodies, Monoclonal↗
PubMed2001

Characterization of a hypervariable region in the genome of Chlamydophila pneumoniae.

Chlamydophila pneumoniae displays surprisingly little genomic variation, as seen by comparisons of the published genomes from three different isolates and sequencing of four different genes from different isolates. We have in the present study, however, demonstrated genomic variation between 10 C. pneumoniae isolates in the 11690-bp region between the two outer membrane protein genes pmp1 and pmp2. This region of the C. pneumoniae CWL-029 isolate contains seven C. pneumoniae-specific open reading frames (hb1-7, encoding hydrophobic beta-sheet-containing proteins). We identified additionally 12 open reading frames in the C. pneumoniae CWL-029 genome encoding hypothetical proteins with similarity to the seven hypothetical Hb-proteins. Compared to other isolates, genomic variation is seen to cause frame-shifting of three of the 19 hb-open reading frames, which are proposed to be three full-length genes and eight frame-shifted pseudogenes. The hypothetical proteins encoded by these proposed genes contain an N-terminally located highly hydrophobic stretch of 50-60 residues. A similar motif is found in all identified Chlamydia inclusion membrane proteins and therefore the Hb-proteins are candidate inclusion proteins.

Bacterial Outer Membrane Proteins↗
PubMed2001

Proteome analysis of the Chlamydia pneumoniae elementary body.

Chlamydia pneumoniae is an obligate intracellular human pathogen that causes acute and chronic respiratory tract diseases and that has been implicated as a possible risk factor in the development of atherosclerotic heart disease. C. pneumoniae cultivated in Hep-2 cells were 35S-labeled and infectious elementary bodies (EB) were purified. The EB proteins were separated by two-dimensional gel electrophoresis. Excised protein spots were in-gel digested with trypsin and peptides were concentrated on reverse-phase chromatographic beads for identification analysis by matrix-assisted laser desorption/ionization-mass spectrometry. In the pH range from 3-11, 263 C. pneumoniae protein spots encoded from 167 genes were identified. These genes constitute 15% of the genome. The identified proteins include 31 hypothetical proteins. It has recently been suggested that EB should be able to synthesize ATP. This view may be strengthened by the identification of several proteins involved in energy metabolism. Furthermore, proteins have been found which are involved in the type III secretion apparatus important for pathogenesis of intracellular bacteria. Proteome maps and a table of all identified proteins have been made available on the world wide web at www.gram.au.dk.

Amino Acids↗
PubMed2001

Time-dependent expression and processing of a hypothetical protein of possible importance for regulation of the Chlamydia pneumoniae developmental cycle.

Chlamydia pneumoniae is an obligate intracellular human pathogen infecting epithelial cells of the upper respiratory tract. It is a Gram-negative bacteria and has a unique biphasic developmental cycle. In this study, we use two-dimensional gel electrophoresis in combination with radioactive labeling to investigate time-dependent expression and processing of C. pneumoniae proteins. We report on (i) the identification of a hypothetical protein which is expressed late in the developmental cycle and subsequently processed; we speculate that this protein may be of importance for the developmental cycle of Chlamydia; (ii) the identification of the major outer membrane protein in three different variants, which may all be present in vivo.

Bacterial Proteins↗
PubMed2001

Evaluation of real-time quantitative PCR for identification and quantification of Chlamydia pneumoniae by comparison with immunohistochemistry.

Chlamydia pneumoniae is a common cause of community-acquired pneumonia and it has been associated with atherosclerosis. C. pneumoniae has usually been diagnosed by serology using a microimmunofluorescence test, but more recently polymerase chain reaction (PCR) has been viewed as an advantageous alternative. We developed a quantitative real-time PCR for detection of C. pneumoniae. Primers were targeted for the pmp4 gene, and the PCR fragment was detected real-time with a fluorescence resonance energy transfer probe set using a LightCycler instrument. The PCR was used on DNA released from 50 microm sections of paraffin-embedded formalin-fixed lung tissue from experimentally infected mice. Thereby, the number of C. pneumoniae genomes was determined. To our knowledge this is the first time quantification of C. pneumoniae DNA has been attempted on paraffin-embedded formalin-fixed tissue. C. pneumoniae-specific immunohistochemistry (IHC) was done on 5 microm sections adjacent to the sections used in PCR, and the number of inclusions were counted in each section. Good correlation was found when comparing results from PCR and IHC, which is in contrast to many previous studies.

Animals↗
PubMed2001

Glistenings in the AcrySof intraocular lens: pilot study.

PURPOSE: To evaluate the prevalence, severity, and visual significance of glistenings seen in the AcrySof intraocular lens (IOL) and determine whether a large in-depth study of this lens is warranted. SETTING: John Moran Eye Center, University of Utah, Salt Lake City, Utah, USA. METHODS: Patients who had the AcrySof IOL implanted at 1 institution over the past 4 years were serially selected. Forty-two eyes that had phacoemulsification with implantation of the AcrySof IOL were evaluated. The examination consisted of visual acuity, glare, contrast sensitivity, and dilated fundus and dilated slitlamp evaluations. Glistenings in the IOL were graded at the slitlamp from trace to 4+. RESULTS: All 42 IOLs had some degree of lenticular glistenings. The glistenings were graded as trace in 27 IOLs (64%), 1+ in 5 (12%), 2+ in 5 (12%), 3+ in 3 (7%), and 4+ in 2 (5%). The Snellen acuity in eyes with severe glistenings (> or = 2+) was one-half line lower than in eyes with mild glistenings (+) (P =.01). In all eyes, the Brightness Acuity Tester score was a little more than one-half line lower than the Snellen acuity, and the difference was 1 full line in eyes with glistenings graded above 2+. Fourteen of 15 IOLs (93%) with glistenings graded greater than trace had been in the eye for more than 1 year. There was no evidence that contrast sensitivity was affected by the glistenings. CONCLUSIONS: Glistenings occurred frequently in AcrySof IOLs, with most cases mild. A larger study of this lens is needed to determine whether severe presentations affect visual function and to understand how glistenings change over time.

Acrylates↗
PubMed2001

FimH-mediated autoaggregation of Escherichia coli.

Autoaggregation is a phenomenon thought to contribute to colonization of mammalian hosts by pathogenic bacteria. Type 1 fimbriae are surface organelles of Escherichia coli that mediate d-mannose-sensitive binding to various host surfaces. This binding is conferred by the minor fimbrial component FimH. In this study, we have used random mutagenesis to identify variants of the FimH adhesin that confer the ability of E. coli to autoaggregate and settle from liquid cultures. Three separate autoaggregating clones were identified, all of which contained multiple amino acid changes located within the N-terminal receptor-binding domain of FimH. Autoaggregation could not be inhibited by mannose, but was inhibited by growth at temperatures at or below 30 degrees C. Using green fluorescent protein (GFP) as a reporter, we show that the autoaggregating clones do not mix with wild-type fimbriated cells. Electron microscopy shows that autoaggregating cells produce fimbriae with a twisted and entangled appearance. We present evidence that autoaggregating versions of FimH also occur in nature. Our results stress the highly adaptive nature of the ubiquitous FimH adhesin.

Adhesins, Bacterial↗
PubMed2001

Serological investigation of Mycoplasma genitalium in infertile women.

BACKGROUND: The role of Mycoplasma genitalium in the pathogenesis of pelvic inflammatory disease has not been characterized. METHODS: Sera from 308 infertile women were investigated for antibodies to M. genitalium by immunoblotting. Women with tubal factor infertility (TFI) made up 132 of the patients, 67 of the women had an infertile male partner and 109 were infertile for unknown reasons. RESULTS: Of the TFI patients 29 (22.0%) were seropositive to the major adhesin, MgPa, of M. genitalium versus 11 (6.3%) in the group of women with normal tubes. No cross-reactions between MgPa and P1 of the related Mycoplasma pneumoniae were found. Besides, MgPa positive sera were confirmed by immunoblotting using a cloned fragment of the C-terminal part of MgPa specific to M. genitalium. Chlamydia trachomatis is known to be able to cause infertility as a result of salpingitis. Therefore, the sera were tested against C. trachomatis using a commercial ELISA test. Seventy-five (56.8%) of the TFI patients were seropositive to C. trachomatis. Eight (27.6%) TFI patients seropositive to MgPa were negative to C. trachomatis. CONCLUSIONS: This study indicates that M. genitalium may be an independent risk factor in the development of an inflammatory process leading to scarring of the uterine tubes in women and thereby causing infertility.

Adhesins, Bacterial↗
PubMed2001

Molecular design of Mycoplasma hominis Vaa adhesin.

The variable adherence-associated (Vaa) adhesin of the opportunistic human pathogen Mycoplasma hominis is a surface-exposed, membrane-associated protein involved in the attachment of the bacterium to host cells. The molecular masses of recombinant 1 and 2 cassette forms of the protein determined by a light-scattering (LS) method were 23.9 kD and 36.5 kD, respectively, and corresponded to their monomeric forms. Circular dichroism (CD) spectroscopy of the full-length forms indicated that the Vaa protein has an alpha-helical content of approximately 80%. Sequence analysis indicates the presence of coiled-coil domains in both the conserved N-terminal and antigenic variable C-terminal part of the Vaa adhesin. Experimental results obtained with recombinant proteins corresponding to the N- or C-terminal parts of the shortest one-cassette form of the protein were consistent with the hypothesis of two distinct coiled-coil regions. The one-cassette Vaa monomer appears to be an elongated protein with a axial shape ratio of 1:10. Analysis of a two-cassette Vaa type reveals a similar axial shape ratio. The results are interpreted in terms of the topological organization of the Vaa protein indicating the localization of the adherence-mediating structure.

Adhesins, Bacterial↗
PubMed2000

Characterization of the variability of a 75-kDa membrane protein in Mycoplasma hominis.

The gene p75 encoding a 75-kDa surface-exposed membrane protein P75 was cloned and sequenced from Mycoplasma hominis type strain PG21T. To investigate the intraspecies variability, sequences were obtained from an additional two isolates 7488 and 183, and the three sequences were compared. The nucleotide and amino acid differences were not confined to specific regions of the gene/protein, but when comparing the three sequences, differences were present as single site substitutions or small insertions or deletions of nucleotides/amino acids. The intraspecies variability was further investigated by restriction enzyme analysis with two restriction enzymes (Alul and MboII) of PCR products amplified from p75 from 28 M. hominis isolates. On the basis of band patterns produced by the two restriction enzymes, the isolates could be divided into five and six groups. These groups neither matched categories of the M. hominis vaa gene nor the M. hominis p120 gene classes, indicating that the three genes vary by different mechanisms and possibly indicating horizontal gene transfer. Federation of European Microbiological Societies.

Amino Acid Sequence↗
PubMed2000

Membrane proteins PmpG and PmpH are major constituents of Chlamydia trachomatis L2 outer membrane complex.

The outer membrane complex of Chlamydia is involved in the initial adherence and ingestion of Chlamydia by the host cell. In order to identify novel proteins in the outer membrane of Chlamydia trachomatis L2, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. By silver staining of the protein profile, a major protein doublet of 100-110 kDa was detected. In-gel tryptic digestion and matrix-assisted laser desorption/ionization mass spectrometry identified these proteins as the putative outer membrane proteins PmpG and PmpH.

Amino Acid Sequence↗
PubMed2000

Cloning, sequencing and variability analysis of the gap gene from Mycoplasma hominis.

The gap gene encodes the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The gene was cloned and sequenced from the Mycoplasma hominis type strain PG21(T). The intraspecies variability was investigated by inspection of restriction fragment length polymorphism (RFLP) patterns after polymerase chain reaction (PCR) amplification of the gap gene from 15 strains and furthermore by sequencing of part of the gene in eight strains. The M. hominis gap gene was found to vary more than the Escherichia coli counterpart, but the variation at nucleotide level gave rise to only a few amino acid substitutions. To verify that the gene was expressed in M. hominis, a polyclonal antibody was produced and tested against whole cell protein from 15 strains. The enzyme was expressed in all strains investigated as a 36-kDa protein. All strains except type strain PG21(T) showed reaction to a 104-kDa band in addition to the expected 36-kDa band. The protein reacting at 104 kDa is a M. hominis protein with either an epitope similar to one on GAPDH, or it is an immunoglobulin binding protein.

Amino Acid Sequence↗
PubMed2000

Genetic differences in the Chlamydia trachomatis tryptophan synthase alpha-subunit can explain variations in serovar pathogenesis.

The human pathogen Chlamydia trachomatis is an obligate intracellular bacterium, characterized by a developmental cycle that alternates between the infectious, extracellular elementary bodies and intracellular, metabolically active reticulate bodies. The cellular immune effector interferon gamma (IFN-gamma) inhibits chlamydial multiplication in human epithelial cells by induction of the tryptophan degrading enzyme indoleamine 2,3 dioxygenase. IFN-gamma causes persistent C. trachomatis serovar A infections with atypical reticulate bodies that are unable to redifferentiate into elementary bodies and show diminished expression of important immunogens, but not of GroEL. However, the sensitivity to IFN-gamma varies among serovars of C. trachomatis. In our previous study significant IFN-gamma-specific, but tryptophan reversible, induction of proteins in C. trachomatis A and L2 with molecular masses of approximately 30 and 40 kDa was observed on 2D-gels. The 30-kDa protein from C. trachomatis L2 migrated with a significantly lower molecular weight in C. trachomatis A. In this paper we include C. trachomatis B, C and D in our investigations and identify the proteins as alpha- and beta-subunits of the chlamydial tryptophan synthase using matrix-assisted laser desorption/ionization mass spectrometry. DNA sequencing of the trpA genes from C. trachomatis A and C shows that the TrpA in these serovars is a 7.7-kDa truncated version of C. trachomatis D and L2 TrpA. The truncation probably impairs the TrpA activity, thus elucidating a possible molecular mechanism behind variations in the pathogenesis of C. trachomatis serovars.

Amino Acid Sequence↗
PubMed2000

Endotracheal tube introducer for failed intubations: a variant of the gum elastic bougie.

There is no universally accepted nonsurgical adjunct for management of the difficult airway in the emergency department. The gum elastic bougie is widely accepted in the British anesthesia literature. One model of endotracheal tube introducer, the Flex-Guide ET Tube Introducer (GreenField Medical Sourcing, Inc, Northborough, MA), is a less expensive plastic version of the gum elastic bougie with the same properties, available in the United States. We present 3 cases of its use in obtaining airway control in difficult airways in the ED. The bougie facilitates intubation where the cords are not visualized or neck movement is contraindicated, allows verification of correct placement before placing the endotracheal tube, is simple to use, and inexpensive to obtain. Reports of 100% first-attempt intubation success in difficult airways are published in the anesthesia literature. We advocate use of this device in the emergency department as a nonsurgical adjunct for difficult airway management.

Adult↗
PubMed2000

Potential relevance of Chlamydia pneumoniae surface proteins to an effective vaccine.

The surface of Chlamydia pneumoniae is covered with proteins but their exact identification is not known probably because of the presence of conformational epitopes. A family of 21 pmp genes has been found by DNA sequencing. In common, these genes have the capacity to encode the amino acid motif GGAI. Several of the genes have the capacity to encode outer membrane proteins of about 100 kDa. Thus, they are candidate genes to encode the protein(s) present in the 98-kDa protein band of the C. pneumoniae outer membrane complex. The production of recombinant GGAI proteins is described as is the use of polyclonal antibodies raised against the recombinant GGAI proteins to determine their expression in C. pneumoniae elementary bodies. At least three of the proteins, Omp4, 5, and 11, are expressed.

Amino Acid Motifs↗