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At least 577 records · Page 32Linked to original sources
PubMed1998

Evaluation of respiratory artifact correction techniques in multishot spiral functional MRI using receiver operator characteristic analyses.

Navigator corrections and low-spatial frequency (LSF) oversampling are investigated as methods for reducing respiration-related effects in multishot functional MRI. Both techniques take advantage of the smoothly varying or nearly constant phase variations linked to the respiration cycle. These techniques were tested in functional MRI studies with spiral k-space acquisitions. Receiver operator characteristic (ROC) analyses and the temporal variance averaged across the brain were used to evaluate their effectiveness. Both methods were found to increase the area under the ROC curve and to reduce the standard deviation, with the LSF oversampling method being more effective.

Artifacts↗
PubMed2004

Spiral readout gradients for the reduction of motion artifacts in chemical shift imaging.

A motion artifact reduction method for proton chemical shift imaging (CSI) is presented. The method uses spiral-based readout gradients for data acquisition. A characteristic of spiral-based readout gradients is that data are repeatedly sampled at the kxy origin. These data points are used to estimate and correct for motion-induced phase variations. Both phantom and in vivo spectra reconstructed using the new motion artifact reduction algorithm showed significant signal-to-noise ratio (SNR) improvements as compared to uncorrected data.

Algorithms↗
PubMed2005

Bacterial protein microarrays for identification of new potential diagnostic markers for Neisseria meningitidis infections.

Neisseria meningitidis is the most common cause of meningitis and causes epidemic outbreaks. One trait of N. meningitidis, which is associated with most of the currently recognized virulence determinants, is the presence of phase-variable genes that are suspected to enhance its ability to cause an invasive disease. To detect the immune responses to phase-variable expressed proteins, we applied protein microarray technology for the screening of meningitis patient sera. We amplified all 102 known phase-variable genes from N. meningitidis serogroup B strain MC58 by polymerase chain reaction and subcloned them for expression in Escherichia coli. With this approach, we were able to express and purify 67 recombinant proteins representing 66% of the annotated genes. These were spotted robotically onto coated glass slides to generate protein microarrays, which were screened using 20 sera of patients suffering from meningitis, as well as healthy controls. From these screening experiments, 47 proteins emerged as immunogenic, exhibiting a variable degree of seroreactivity with some of the patient sera. Nine proteins elicited an immune response in more than three patients, with one of them, the phase-variable opacity protein OpaV (NMB0442), showing responses in 11 patient sera. This is the first time that protein microarray technology has been applied for the investigation of genetic phase variation in pathogens. The identification of disease-specific proteins is a significant target in biomedical research, as such proteins may have medical, diagnostic, and commercial potential as disease markers.

Bacterial Proteins↗
PubMed1998

Folding intermediates of wild-type and mutants of barnase. I. Use of phi-value analysis and m-values to probe the cooperative nature of the folding pre-equilibrium.

It is difficult to determine whether transient folding intermediates have a cooperative (or first-order) folding transition without measuring their rates of formation directly. An intermediate I could be formed by a second-order transition from a denatured state D that is progressively changed into I as conditions are changed. We have not been able to monitor the rate of formation of the folding intermediate of barnase directly, but have analysed its reactivity and the equilibrium constant for its formation over a combination of wide ranges of temperature, concentration of denaturant and structural variation. Phase diagrams have been constructed for wild-type and 16 mutant proteins to map out the nature of the energy landscape of the denatured state. The free energy of unfolding of I, delta GD-I, changes with [urea] according to a highly cooperative transition. Further, mD-I (= delta delta GD-I/delta [urea]) for wild-type and several mutants is relatively insensitive to temperature, as would be expected for an intermediate that is formed cooperatively, rather than one that melts out according to a second-order transition. The phi-values for the formation of I change abruptly through the folding transitions rather than have the smooth changes expected for a second-order transition. There is a subset of mutants for which both mD-I and phi-value analysis indicate that a second intermediate becomes populated close to the melting temperatures of the native proteins. The folding intermediate of barnase is, thus, a relatively discrete and compact entity which is formed cooperatively.

Amino Acid Sequence↗
PubMedDate not supplied

Three-dimensional structure of Bordetella pertussis fimbriae.

We describe the helical structure of Bordetella pertussis fimbriae of serotype 3/6 as determined to a resolution of approximately 2.5 nm by three-dimensional reconstruction of negatively stained electron micrographs. The fimbria has a distinctly polar structure whose axial repeat of 13 nm contains five copies of the fim3 gene product (22 kDa) in two complete turns. These subunits are connected by interactions along the fimbrial backbone which, unlike other classes of bacterial fimbriae, has no axial channel. Its outer diameter is approximately 5.7 nm, and the most pronounced feature is a radially protruding domain that gives the fimbria its characteristic serrated appearance. Serotype 2 fimbriae, composed of the fim2 subunit which is 60% homologous with fim3, have essentially the same quaternary structure. These observations are discussed in relation to fimbrial phase variation and structure-based classification of fimbriae/pili.

Antigens, Bacterial↗
PubMed1993

Escherichia coli F-18 phase locked 'on' for expression of type 1 fimbriae is a poor colonizer of the streptomycin-treated mouse large intestine.

Escherichia coli F-18, a human fecal isolate, makes type 1 fimbriae in vitro and in the streptomycin-treated mouse large intestine in vivo, and is an excellent colonizer of the cecal mucus layer in the streptomycin-treated mouse large intestine. E. coli F-18(pPKL91) harbors an extra fimB gene on a parB stabilized pPBR322 plasmid and is therefore phase-locked 'on' such that all cells express type 1 fimbriae. E. coli F-18(pPR633) contains essentially the same plasmid minus the fimB gene and in L-broth about 30% of the cells express type 1 fimbriae. When fed alone to streptomycin-treated mice, E. coli F-18(pPKL91) colonized the large intestine at about 10(7) cfu/g of feces. However, when simultaneously fed with E. coli F-18(pPR633) at either high (10(10) cfu), or low doses (10(4) cfu), E. coli F-18(pPKL91) was a poor colonizer dropping to a level of between 10(2) and 10(3) cfu/g of feces. When given enough time to establish the state of colonization (10 days), E. coli F-18(pPKL91) persisted in feces in high numbers despite subsequent challenge by E. coli F-18(pPR633). Moreover, although both E. coli F-18(pPR633) and E. coli F-18(pPKL91) grew equally well in cecal mucus in vitro, E. coli F-18(pPR633) traveled through a layer of cecal mucus in vitro much faster than E. coli F-18(pPKL91). Together, the data suggest that type 1 fimbriated cells are at a disadvantage in initiating the colonization state because they have difficulty entering the mucus layer of the intestine as rapidly as non-fimbriated cells. The data also point to the possible biological significance of type 1 fimbrial phase-variation in the mouse large intestine.

Animals↗
PubMed1997

Localization of the in vivo expression of P and F1 fimbriae in chickens experimentally inoculated with pathogenic Escherichia coli.

Escherichia coli causing septicemia in poultry often possess F1 (type 1) and/or P fimbriae which may be involved in bacterial colonization and infection. To investigate the expression of these fimbriae in vivo, two pathogenic E. coli strains with different fimbrial profiles, TK3 (fim+/pap+) and MT78 (fim+/pap-), were administered to 2-week-old chickens by either the intratracheal or caudal thoracic air sac inoculation route. Antibodies specific for native F1 fimbriae were detected by ELISA and immunodot in the serum of chickens inoculated with either strain MT78 or strain TK3, irrespective of the route of inoculation. Antibodies specific for P fimbriae of serotype F11 were detected by ELISA and immunoblotting in the serum of chickens inoculated by either route with strain TK3. F1, but not P fimbriae, were expressed by bacteria colonizing the trachea of chickens inoculated by the air sac route with strain MT78 or TK3, as demonstrated by examination of frozen tissue sections using immunofluorescence. F1 fimbriae were also expressed by bacteria colonizing the air sacs and lungs, but not by bacteria in the blood or other internal organs, of chickens inoculated with either strain. P fimbriae were expressed by bacteria colonizing the air sacs, lungs, kidney, blood, and pericardial fluid, but not by bacteria colonizing the trachea, of chickens inoculated with strain TK3. Fimbriae-like structures were observed by electron microscopy on bacteria adhering to the epithelial cells of the air sacs of chickens inoculated with strain TK3. These results demonstrate that both strains MT78 and TK3 undergo in vivo phase variation with respect to their fimbrial profiles and site of bacterial colonization in different organs of infected chickens and suggest that F1 fimbriae are important for initial bacterial colonization of the upper respiratory tract whereas P fimbriae are important for later stages of the infection.

Air Sacs↗
PubMed1981

The relationship of two invertible segments in bacteriophage Mu and Salmonella typhimurium DNA.

A Mu gin- mutant which lacks the function required for inversion of the G segment can be complemented by hybrid plasmids and phages carrying the invertible segment that controls flagellar phase variation in Salmonella typhimurium. This suggests that the same kind of site-specific recombination mechanism is responsible for these inversions. Based on the different features of the two invertible segments we propose a model for their evolutionary relationship.

Genetic Complementation Test↗
PubMed2005

Stable transformation of Chromobacterium violaceum with a broad-host-range plasmid.

Stable transformants of Chromobacterium violaceum were obtained by high-voltage electroporation with a 7-kilobase binary plasmid. The technique was reliable, reproducible, and simple, with efficiencies of 10(5) transformants/microg of plasmid DNA. The electrical conditions that resulted in the highest efficiencies were short pulse length (4.4-4.5 ms) and high voltage (12.5 kV/cm). The numbers of transformants were almost the same during the growth exponential phase (variation at optical density) and resulted in the highest efficiencies at DNA concentration of 250 pg/ml. Saturation appeared to begin at 4 microg/ml of DNA. This method of C. violaceum transformation should enhance the genetic and biotechnological research by providing a valuable, widely used procedure of introducing DNA into this bacterium.

Chromobacterium↗
PubMed2003

Gene content and organization of an 85-kb DNA segment from the genome of the phytopathogenic mollicute Spiroplasma kunkelii.

Spiroplasma kunkelii, the causative agent of corn stunt disease in maize (Zea maysL.), is a helical, cell wall-less prokaryote assigned to the class Mollicutes. As part of a project to sequence the entire S. kunkelii genome, we analyzed an 85-kb DNA segment from the pathogenic strain CR2-3x. This genome segment contains 101 ORFs and two tRNA genes. The majority of the ORFs code for predicted proteins that can be assigned to respective clusters of orthologous groups (COGs). These COGs cover diverse functional categories including genetic information storage and processing, cellular processes, and metabolism. The most notable gene cluster in this genome segment is a super-operon capable of encoding 24 ribosomal proteins. The organization of genes in this operon reflects the unique evolutionary position of the spiroplasma. Gene duplications, domain rearrangements, and frameshift mutations in the segment are interpreted as indicators of phase variation in the spiroplasma. To our knowledge, this is the first analysis of a large genome segment from a plant pathogenic spiroplasma.

Base Sequence↗
PubMed1995

Differential codon usage: a safeguard against inappropriate expression of specialized genes?

Recent work has suggested that rare codons are sometimes used for the regulation of specialized gene expression in bacteria. Moreover, the cellular levels of certain tRNAs may fluctuate with growth conditions. Evidence implicating such mechanisms in the control of photosynthesis in Rhodobacter, solventogenesis in Clostridium, sporulation in Streptomyces, and fimbrial phase variation in E. coli is summarized. It is suggested that such mechanisms will prove applicable to the control of numerous additional specialized functions, and that the empirical tools for testing this possibility are currently available.

Bacteria↗
PubMed1984

Involvement of the invertible G segment in bacteriophage mu tail fiber biosynthesis.

The orientation [G(+) or G(-)] of the invertible G segment of bacteriophage Mu DNA determines the host range specificity of the phage particles. In this study the hypothesis that the G segment genes are involved in synthesis of Mu tail fibers has been tested. Serum blocking power (SBP) assays demonstrated that among Mu late gene mutants only those defective in genes S or U encoded by the G segment were defective in G(+) SBP and that they lacked the same antigens. Electron microscopy of lysates produced by inversion-defective gin mutants (isolated by their inability to complement a hin inversion-defective mutant of the Salmonella phase variation segment) showed that G(+) phages with amber mutations in S or U made tail-fiberless particles with contracted tail sheaths. Inversion of G to the G(-) orientation or suppression of the amber mutations restored the normal phage particle morphology. These experiments demonstrate that genes S and U are required for Mu G(+) tail fiber biosynthesis and/or attachment.

Antigens, Viral↗
PubMed1986

Gene conversion involving the pilin structural gene correlates with pilus+ in equilibrium with pilus- changes in Neisseria gonorrhoeae.

Gonococci (Gc) exhibit pilus+----pilus- "phase transitions" at high frequency, but only some of the pilus- Gc can revert to pilus+ phenotype. We examined reversible phase transitions between pilus+ Gc and a particular pilus- variant (P-rp+ phenotype) whose pilin mRNA carries a unique block of nucleotides encoding an "assembly missense" pilin polypeptide. The results show that Gc pilus+ in equilibrium with P-rp+ transitions can result from intragenic recombination in which there is nonreciprocal exchange of partially homologous DNA sequences from a partial pilin gene (in silent, storage form) into the expression locus' complete pilin gene. Hence Gc pilus phase variation, like pilus antigenic variation, can occur by gene conversion of the pilin structural gene expression locus.

Bacterial Adhesion↗
PubMed1986

Host protein requirements for in vitro site-specific DNA inversion.

Flagellar phase variation is mediated by a recombination event that occurs at specific sites leading to inversion of a chromosomal segment of DNA. The presence of a 60 bp recombinational enhancer sequence on the DNA substrate molecule results in a 150-fold stimulation in the initial rate of inversion. The protein components required for inversion have been purified. They include the 21,000 dalton recombinase (Hin), a 12,000 dalton host protein (Factor II), and one of the major histone-like proteins of E. coli HU. The dependence of the initial rate of recombination on HU varies with respect to the location of the recombinational enhancer. The role of HU, Factor II, and the enhancer in facilitating site-specific recombination is discussed.

Bacterial Proteins↗
PubMed1988

A physiological role for DNA supercoiling in the osmotic regulation of gene expression in S. typhimurium and E. coli.

The proU locus encodes an osmotically inducible glycine betaine transport system that is important in the adaptation to osmotic stress. We present evidence that DNA supercoiling plays a key role in the osmotic induction of proU transcription. An increase in extracellular osmolarity increases in vivo DNA supercoiling, and the expression of proU is highly sensitive to these changes. Furthermore, topA mutations can mimic an increase in osmolarity, facilitating proU expression even in media of low osmolarity in which it is not normally expressed. Selection for trans-acting mutations that affect proU expression has yielded only mutations that alter DNA supercoiling, either in topA or a new genetic locus, osmZ, which strongly influences in vivo supercoiling. Mutations in osmZ are highly pleiotropic, affecting expression of a variety of chromosomal genes including ompF, ompC, fimA, and the bgl operon, as well as increasing the frequency of site-specific DNA inversions that mediate fimbrial phase variation.

Betaine↗
PubMed1986

Plasmids related to RSF1010 from Bordetella bronchiseptica.

Six out of 14 Bordetella bronchiseptica isolates from U.K. pigs each contained one plasmid, of 8.7-44 kb. All plasmid-containing isolates were sulfonamide resistant, and this property was shown to be plasmid-encoded. Five of the plasmids were related; two were indistinguishable from the broad-host-range plasmid, RSF1010. The other three, two of which appeared to be identical, were shown to have regions of homology with RSF1010. One of these regions encompassed the sulfonamide resistance determinant while the other contained oriV, which also determines plasmid incompatibility. None of the plasmids could be associated with virulence or phase variation, and it appears likely that they have been acquired in response to antibiotic pressure.

Animals↗
PubMed1989

The assessment of relative surface hydrophobicity as a factor involved in the activation of human polymorphonuclear leukocytes by uropathogenic strains of Escherichia coli.

The initiation of the respiratory burst and the degranulation of human polymorphonuclear leukocytes (PMN) in response to stimulation by uropathogenic strains of Escherichia coli is dependent on the expression of Type 1 fimbriae by those strains. These PMN responses correlate with an increasing tendency of the interacting E. coli strain to be retained on hydrophobic columns. The present work assessed the measurement of relative surface hydrophobicity in relation to PMN activation. Type 1 fimbriate organisms bound most readily to Octyl-Sepharose columns and were strongly agglutinated in the salt aggregation test. In contrast, the same organisms partitioned to the dextran-rich (hydrophilic) phase of aqueous two-polymer phase systems. Electron microscopic observation of the organisms eluted from the Octyl-Sepharose columns and of the organisms recovered from both phases of the aqueous two-phase systems demonstrated, however, that both Type 1 and P-fimbriate organisms were retained on the columns and partitioned into the dextran-rich phase as a consequence of their being fimbriate and failed to identify this as a major factor in the activation of PMN. In addition electron microscopy demonstrated that each P-fimbriate population had fewer organisms expressing fimbriae than did Type 1 fimbriate populations, confirming the importance of phase variation as a factor affecting the physicochemical characteristics of a bacterial population.

Agglutination↗
PubMed1990

Structure of the Bordetella pertussis gene coding for the serotype 3 fimbrial subunit.

Bordetella pertussis strains contain at least three distinct genes coding for fimbrial subunits, designated fim2, fim3, and fimX. The sequences of the fim2 and fimX genes have been published. Here we present the sequence of the fim3 gene. Proximal and distal to the fim3 gene, regions were observed that could function as rho-independent terminators, suggesting that the gene is not part of a larger operon. Comparison of the putative promoter regions of the fim2 and fim3 genes revealed a conserved region containing a stretch of approximately 13 C's. This region may be involved in fimbrial phase variation. A comparison of the deduced amino acid sequences of the three fimbrial subunits revealed conserved, variable, and hypervariable regions. The hypervariable regions coincided with predicted antigenic determinants. Peptides derived from the conserved regions may be incorporated into a future pertussis vaccine to induce antibodies which confer protection against strains producing different fimbrial serotypes.

Amino Acid Sequence↗