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PubMed2005

Characterisation of surface-modified solid lipid nanoparticles (SLN): influence of lecithin and nonionic emulsifier.

Solid lipid nanoparticles (SLN), an alternative colloidal drug delivery system to polymer nanoparticles, emulsions and liposomes, are generally produced by high pressure melt-emulsification. However, the harsh production process is not applicable for formulations containing shear and temperature sensitive compounds. For that reason, subsequent adsorptive SLN loading might be a promising alternative. The aim of the present study was the development and characterisation of surface-modified SLN for adsorptive protein loading by variation of both the lipid matrix and the emulsifier concentration in the continuous phase. Variations in SLN composition resulted in particle sizes between 674 and 61 nm corresponding to specific surfaces of 4.5 m(2)/g and 48.9 m(2)/g and zeta potentials between -23.4 mV and -0.9 mV. In dependence of SLN surface properties, albumin payload ranged from 2.5 to 15%. Thermoanalysis, X-ray diffraction and electron microscopy revealed anisometrical and crystalline particles. In vitro cytotoxicity was low in terms of both haemolysis, which was between 1 and 2%, and neutral red test (NRT) showing a half lethal dose between 1.1 and 4.6%.

Cell Survival↗
PubMed1984

Picogram detection of eicosanoids by ultraviolet absorbance after narrow-bore high-performance liquid chromatography. Comparison with conventional-bore column.

Mobile-phase variations were employed to achieve optimal separation by narrow-bore reversed-phase high-performance liquid chromatography of eleven eicosanoids. Separation and quantitation by ultraviolet absorbance at 190 nm using conventional-bore ODS columns were compared. Using the improved sensitivity obtained by means of the narrow-bore column, i.e. 250-pg detection limits of a standard solution, analysis of eicosanoids in kidney medulla was achieved. Parallel quantitation by radioactivity, using [1-(14)C]arachidonic acid as substrate, was applied.

Animals↗
PubMed2002

Self-perpetuating epigenetic pili switches in bacteria.

Bacteria have developed an epigenetic phase variation mechanism to control cell surface pili-adhesin complexes between heritable expression (phase ON) and nonexpression (phase OFF) states. In the pyelonephritis-associated pili (pap) system, global regulators [catabolite gene activator protein (CAP), leucine-responsive regulatory protein (Lrp), DNA adenine methylase (Dam)] and local regulators (PapI and PapB) control phase switching. Lrp binds cooperatively to three pap DNA binding sites, sites 1-3, proximal to the papBA pilin promoter in phase OFF cells, whereas Lrp is bound to sites 4-6 distal to papBA in phase ON cells. Two Dam methylation targets, GATC(prox) and GATC(dist), are located in Lrp binding sites 2 and 5, respectively. In phase OFF cells, binding of Lrp at sites 1-3 inhibits methylation of GATC(prox), forming the phase OFF DNA methylation pattern (GATC(dist) methylated, GATC(prox) nonmethylated). Binding of Lrp at sites 1-3 blocks pap pili transcription and reduces the affinity of Lrp for sites 4-6. Together with methylation of GATC(dist), which inhibits Lrp binding at sites 4-6, the phase OFF state is maintained. We hypothesize that transition to the phase ON state requires DNA replication to dissociate Lrp and generate a hemimethyated GATC(dist) site. PapI and methylation of GATC(prox) act together to increase the affinity of Lrp for sites 4-6. Binding of Lrp at the distal sites protects GATC(dist) from methylation, forming the phase ON methylation pattern (GATC(dist) nonmethyated, GATC(prox) methylated). Lrp binding at sites 4-6 together with cAMP-CAP binding 215.5 bp upstream of the papBA transcription start, is required for activation of pilin transcription. The first gene product of the papBA transcript, PapB, helps maintain the switch in the ON state by activating papI transcription, which in turn maintains Lrp binding at sites 4-6.

Base Sequence↗
PubMed2005

Genetic determinants of biofilm development of opaque and translucent Vibrio parahaemolyticus.

Vibrio parahaemolyticus isolates display variation in colony morphology, alternating between opaque (OP) and translucent (TR) cell types. Phase variation is the consequence of genetic alterations in the locus encoding the quorum sensing output regulator OpaR. Here, we show that both cell types form stable, but distinguishable biofilms that differ with respect to attachment and detachment profiles to polystyrene, pellicle formation and stability at the air/medium interface, and submerged biofilm architecture and dispersion at a solid/liquid interface. The pellicle, which is a cohesive mat of cells, was exploited to identify mutants having altered or defective biofilm formation. Transposon insertion mutants were obtained with defects in genes affecting multiple cell surface characteristics, including extracellular polysaccharide, mannose-sensitive haemagglutinin type 4 pili and polar (but not lateral) flagella. Other insertions disrupted genes coding for potential secreted proteins or transporters of secreted proteins, specifically haemolysin co-regulated protein and an RTX toxin-like membrane fusion transporter, as well as potential modifiers of cell surface molecules (nagAC operon). The pellicle screen also identified mutants with lesions in regulatory genes encoding H-NS, a CsgD-like repressor and an AraC-like protein. This work initiates the characterization of V. parahaemolyticus biofilm formation in the OP and TR cell types and identifies a diverse repertoire of cell surface elements that participate in determining multicellular architecture.

Bacterial Proteins↗
PubMed1988

Alternative model for Neisseria gonorrhoeae pilin variation.

The pilus of Neisseria gonorrhoeae, a dominant outer membrane organelle, is a major virulence factor. The pilus undergoes phase variation and antigenic variation has also been observed, both in vitro and in vivo. The current model of pilus variation invokes a gene conversion type recombination between a silent pilin locus and the pilin expression site. Experimental results which led to the creation of this hypothesis are reviewed and data are presented which support an alternative model based on DNA transformation.

Antigenic Variation↗
PubMed1988

Steric hindrance of antibody binding to surface proteins of Coxiella burnetti by phase I lipopolysaccharide.

The exposure of surface protein antigens on virulent phase I Coxiella burnetti was compared with that on avirulent phase II cells. Although anti-phase II antibodies did not bind to the surfaces of native intact phase I cells, they bound to phase I proteins if the proteins were solubilized for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by immunoblotting. In addition, removal of the phase I lipopolysaccharide (LPS) by trichloroacetic acid exposed surface proteins for reactivity with anti-phase II antibodies, as shown by immunofluorescence assays, direct antibody binding, and immunoelectron microscopy using protein A-colloidal gold conjugates. Based on these observations, a simple model of phase variation is proposed to explain the apparently conflicting notions of the identity of the phase II antigen(s). The model suggests that the phase I LPS sterically hinders access of anti-phase II antibodies to a multitude of shared protein antigens, any one of which may confer phase II specificity. Exposure of these shared protein antigens through the appearance of a more truncated LPS (phase II) or extraction of the smooth-type phase I LPS allows antibody accessibility and therefore confers apparent phase II serospecificity.

Antibodies, Bacterial↗
PubMed1977

Bacteriological variation among Bordetella bronchiseptica isolates from dogs and other species.

Bacteriological properties of 50 isolates of Bordetella bronchiseptica were compared. Phase variation, which involved colonial morphology and its associated characters of hemagglutination, hemolysis, acriflavine agglutination; crystal violet staining, flagellation, and fimbriation, occurred among these isolates. Organisms representing the three observed morphotypes did not have different growth rates, nor were any differences in their bacteriological characteristics observed after repeated subculture on agar. There were also variations in antimicrobial drug susceptibility, especially to sulfonamide-trimethoprim, and in nitrate reduction. The relationships among these variable parameters were not apparent. None of the observed variations could be attributed to differences in the species of origin.

Animals↗
PubMed2000

Sensory modification of leech swimming: rhythmic activity of ventral stretch receptors can change intersegmental phase relationships.

For segmented animals to generate optimal locomotory movements, appropriate phase relationships between segmental oscillators are crucial. Using swimming leeches, we have investigated the role of sensory input in establishing such relationships. We found that the stretch receptors associated with ventral longitudinal muscles encode the information of muscle contraction during swimming via membrane potential oscillations, with amplitudes of up to 10 mV at our recording site. We subsequently modified the activity of ventral stretch receptors (VSRs) by injecting rhythmic current at different phases of the swim cycle and determined intersegmental phase lags by comparing the delay between the discharges of serially homologous motoneurons in three adjacent segments of isolated nerve cords. When no current was injected, the phase lag between neighboring segments was 8.6 +/- 0.8 degrees (mean +/- SEM; n = 20), with large phase variations from cycle to cycle, between different episodes, and between different preparations. When the phase of stretch receptor activity was set to 90-150 degrees by current injection, the phase of the motoneuron activity in the ganglion was consistently retarded by approximately 5 degrees. It was advanced by approximately 5 degrees when the VSR phase was set to 240-300 degrees. Therefore, the rhythmic activity of the ventral stretch receptor generated during swimming can change intersegmental phase lags of leech ganglia in a phase-dependent manner. These stretch receptors may set the optimal intersegmental phases during swimming movement in intact leeches.

Animals↗
PubMed1999

Manual handling performance: the effects of menstrual cycle phase.

Physiological and subjective responses to physical performance have been shown to interrelate with fluctuations in the female hormonal environment throughout the menstrual cycle. The aim of this study was to examine whether these fluctuations affect the strenuous performance required in manual handling. Seventeen eumenorrheic females performed lifting tasks in five phases of their menstrual cycle. These tasks were maximal isometric lifting strength (MILS) and an endurance lift at 45% MILS (t), at both knee and waist height; and the selection of a maximal acceptable load (MAL) to lift six times per min, for 10 min, in both the sagittal and asymmetric planes. Heart rate response (HR) and rating of perceived exertion (RPE) were recorded throughout each of the lifting tasks. MILS, t and the chosen MAL were unaffected by menstrual phase over both heights and planes of lift (p > 0.05). HR to the isometric endurance lift was greater following ovulation than prior to ovulation by approximately 7 beats.min-1 (p < 0.05). This was true when the data were analysed at 50, 80 and 100% of the time to volitional fatigue, and by an area under the curve procedure. HR to the dynamic lifting tasks was also elevated by approximately 7 beats.min-1 following ovulation. This difference was non-significant due to the low power of the analysis. Re-analysis of the data by re-sampling 1000 matched comparisons produced significant phase variations (p < 0.05). The RPE for all of the lifting tasks was independent of menstrual phase (p > 0.05). The impact of the eumenorrheic menstrual cycle on lifting capability was negligible in the present study. However, the results of this study indicate that all further investigations utilizing HR data to produce recommendations for health and safety in manual handling tasks must control for menstrual cycle phase in female populations.

Adolescent↗
PubMed2005

Statistical behavior of joint least-square estimation in the phase diversity context.

The images recorded by optical telescopes are often degraded by aberrations that induce phase variations in the pupil plane. Several wavefront sensing techniques have been proposed to estimate aberrated phases. One of them is phase diversity, for which the joint least-square approach introduced by Gonsalves et al. is a reference method to estimate phase coefficients from the recorded images. In this paper, we rely on the asymptotic theory of Toeplitz matrices to show that Gonsalves' technique provides a consistent phase estimator as the size of the images grows. No comparable result is yielded by the classical joint maximum likelihood interpretation (e.g., as found in the work by Paxman et al.). Finally, our theoretical analysis is illustrated through simulated problems.

Computer Simulation↗
PubMed1991

Cytometry and time-dependent variations in peripheral blood and bone marrow cells: a literature review and relevance to the chronotherapy of cancer.

By flow cytometry of individual cells, multiple cell properties can be analyzed. Such parameters may be important in relation to cytotoxic treatment of cancer. For example, DNA measurements will answer questions regarding cell kinetics. Myelosuppression is the major dose-limiting toxicity during cancer treatment. Therefore, the study of cell cycle parameters in bone marrow cells is highly relevant. However, inattention to the existence and potential importance of biological rhythms may introduce artifacts and misleading results. The literature of rhythms in hematology is reviewed. Time-dependent variations in hematological variables have been extensively studied and rhythms have been described for all kinds of blood cells. Also the numbers of hemopoietic stem cells in the bone marrow undergo circadian variations. Our group has shown how such variations change with aging in mice. The relevance of time sequence studies in aging research of hemopoiesis was clearly demonstrated. In animal studies using cytometry, our group has demonstrated extensive circadian variations in cell cycle distribution of bone marrow cells, especially the DNA synthesis (S-phase). In humans a few and rather small time sequence studies of the bone marrow have been performed, so far. In this overview the clinical implications of circadian rhythms of S-phase variations measured by flow cytometry of human bone marrow cells are discussed. Male volunteers were examined every 4 h around-the-clock. The data indicated a lower proliferative activity during night, suggesting the possibility of reducing the bone marrow toxicity to cancer treatment when taking these time-dependent variations into consideration.

Animals↗
PubMed2006

Analysis of phase of LUCIFERASE expression reveals novel circadian quantitative trait loci in Arabidopsis.

In response to exogenous rhythms of light and temperature, most organisms exhibit endogenous circadian rhythms (i.e. cycles of behavior and gene expression with a periodicity of approximately 24 h). One of the defining characteristics of the circadian clock is its ability to synchronize (entrain) to an environmental rhythm. Entrainment is arguably the most salient feature of the clock in evolutionary terms. Previous quantitative trait studies of circadian characteristics in Arabidopsis (Arabidopsis thaliana) considered leaf movement under constant (free-running) conditions. This study, however, addressed the important circadian parameter of phase, which reflects the entrained relationship between the clock and the external cycle. Here it is shown that, when exposed to the same photoperiod, Arabidopsis accessions differ dramatically in phase. Variation in the timing of circadian LUCIFERASE expression was used to map loci affecting the entrained phase of the clock in a recombinant population derived from two geographically distant accessions, Landsberg erecta and Cape Verde Islands. Four quantitative trait loci (QTL) were found with major effects on circadian phase. A QTL on chromosome 5 contained SIGNALING IN RED LIGHT REDUCED 1 and PSEUDORESPONSE REGULATOR 3, both genes known to affect the circadian clock. Previously unknown polymorphisms were found in both genes, making them candidates for the effect on phase. Fine mapping of two other QTL highlighted genomic regions not previously identified in any circadian screens, indicating their effects are likely due to genes not hitherto considered part of the circadian system.

Amino Acid Sequence↗
PubMed1998

Insertional inactivation of genes encoding the crystalline inclusion proteins of Photorhabdus luminescens results in mutants with pleiotropic phenotypes.

The entomopathogenic bacterium Photorhabdus luminescens exhibits phase variation when cultured in vitro. The variant forms of P. luminescens are pleiotropic and are designated phase I and phase II variants. One of the characteristic phenotypes of phase I cells is the production of two types of intracellular protein inclusions. The genes encoding the protein monomers that form these inclusions, designated cipA and cipB, were cloned and characterized. cipA and cipB encode hydrophobic proteins of 11,648 and 11,308 Da, respectively. The deduced amino acid sequences of CipA and CipB have no significant amino acid sequence similarity to any other known protein but have 25% identity and 49% similarity to each other. Insertional inactivation of cipA or cipB in phase I cells of P. luminescens produced mutants that differ from phase I cells in bioluminescence, the pattern and activities of extracellular products, biochemical traits, adsorption of dyes, and ability to support nematode growth and reproduction. In general, the cip mutants were phenotypically more similar to each other than to either phase I or phase II variants.

Amino Acid Sequence↗
PubMed1990

Analysis of time trends, individual subject responses and background variation in crossover factorial studies with antihypertensive drugs.

In studying patients with mild hypertension, we used a double-blind, randomly allocated crossover, 2 x 2 factorial design to obtain more precise estimates of the effectiveness of individual hypertensive drugs and of the interactions when used in combination. In the present study, we reanalysed data from our own previously completed crossover factorial studies in order to determine the optimal length of phases and to try to exploit patient heterogeneity by comparing blood pressure responses within each patient to the two different drugs in each study. An analysis of the 'weeks within phases' variation (analysis of variance) applied to all phases, to the placebo phases alone and to the active treatment phases alone, of the individual factorial studies and of pooled data from studies of similar design, only revealed differences in the first week of our 4-week phases. Accordingly, we suggest that 4 weeks is an optimal phase length, since only the values from the third and fourth weeks showed the full expression of a drug's antihypertensive effect, avoiding the problem of a carryover effect and the need for a more complex intervening washout placebo phase. Comparing the blood pressure reductions shown by individual patients in response to different antihypertensive drugs using arbitrary criteria of greater than or equal to 5, 10 and 15 mmHg for diastolic and greater than or equal to 5, 10 and 20 mmHg for systolic pressure, we were unable to distinguish between the two separate drugs in each factorial study, due to the magnitude of the background variation which had a coefficient of variation of 5-7%.(ABSTRACT TRUNCATED AT 250 WORDS)

Antihypertensive Agents↗
PubMed2004

An X-ray BBB Michelson interferometer.

A new X-ray Michelson interferometer based on the BBB interferometer of Bonse and Hart and designed for X-rays of wavelength approximately 1 A was described in a previous paper. Here, a further test carried out at the SPring-8 1 km beamline BL29XUL is reported. One of the BBB's mirrors was displaced by a piezo to introduce the required path-length difference. The resulting variation of intensity with piezo voltage as measured by an avalanche photodiode could be ascribed to the phase variation resulting from the path-length change, with a small additional contribution from the change of the position of the lattice planes of the front mirror relative to the rest of the crystal. This 'Michelson fringe' interpretation is supported by the observed steady movement across the output beam of the interference fringes produced by a refractive wedge when the piezo voltage was ramped. The front-mirror displacement required for one complete fringe at the given wavelength is only 0.675 A; therefore, a quiet environment is vital for operating this device, as previous experiments have shown.

Journal Article↗
PubMed1995

In vivo variation of Mycoplasma gallisepticum antigen expression in experimentally infected chickens.

The antigen expression profiles of Mycoplasma gallisepticum isolates obtained from tracheal swabs of chickens after aerosol-inoculation with M. gallisepticum strain R or clonal variant R/E were examined in western immunoblots. A reference anti-M. gallisepticum chicken antiserum and antisera from individual infected chickens as well as monoclonal antibodies (mAbs) specific for surface proteins were used to monitor in vivo antigenic variation. mAbs 1E5 and 12D8, recognizing PvpA and p67a, recently shown to undergo high-frequency in vitro phase variation, were used for consecutive staining of colony and western immunoblots in order to distinguish between the resultant phenotypes with respect to the corresponding epitopes. Marked differences in the expression of major immunogenic proteins, including p67a, were observed between the two inocula as well as among reisolates recovered at different times of infection. Comparative western immunoblot analysis of the rapidly changing chicken serum antibody response and reisolates recovered during the course of an experimental infection with M. gallisepticum R or clonal variant R/E suggest that immune modulation may have a key role in generating surface diversity. In addition, comparison of colony immunoblots of strain R inoculum and of reisolated colonies from tracheas of birds 8 days post infection indicated an in vivo selection of the PvpA+p67a- phenotype. This study established that surface antigens of M. gallisepticum are subjected in vivo to rapid alteration in their expression. This variability may function as a crucial adaptive mechanism, enabling the organism to escape from the host immune defense and to adapt to the changing host environment at different stages of a natural infection.

Animals↗
PubMed2001

Phase retrieval in TEM using Fresnel images.

As an alternative to sideband holography in an electron microscope, methods for phase recovery by in-line holography utilising Fresnel images of aperiodic objects were tested with computed simulations and experimental data. Phases were recovered by minimising an error functional defined as a measure of the differences between experimental and calculated image intensities. The probability of convergence to local minima of the error function was reduced by increasing the ratio of known to unknown parameters, partly by use of several Fresnel images at different defoci, and also by an incremental relaxation of the phase bandwidth. Iterative methods did not converge reliably to a global minimum, but a conjugate gradient algorithm usually recovered the phases exactly, even for object arrays which included large phase variations. In practice, it was essential to use analytic expressions for the error gradients with respect to the phases, defoci and beam direction. Phase shifts of several radians were measured near the edge of a contaminated aperture. The factors that limit the accuracy and reliability of phase recovery from Fresnel images are discussed.

Journal Article↗
PubMed2003

Salmonella enterica serovar typhimurium rdoA is growth phase regulated and involved in relaying Cpx-induced signals.

The disulfide oxidoreductase, DsbA, mediates disulfide bond formation in proteins as they enter or pass through the periplasm of gram-negative bacteria. Although DsbA function has been well characterized, less is known about the factors that control its expression. Previous studies with Escherichia coli demonstrated that dsbA is part of a two-gene operon that includes an uncharacterized, upstream gene, yihE, that is positively regulated via the Cpx stress response pathway. To clarify the role of the yihE homologue on dsbA expression in Salmonella enterica serovar Typhimurium, the effect of this gene (termed rdoA) on the regulation of dsbA expression was investigated. Transcriptional assays assessing rdoA promoter activity showed growth phase-dependent expression with maximal activity in stationary phase. Significant quantities of rdoA and dsbA transcripts exist in serovar Typhimurium, but only extremely low levels of rdoA-dsbA cotranscript were detected. Activation of the Cpx system in serovar Typhimurium increased synthesis of both rdoA- and dsbA-specific transcripts but did not significantly alter the levels of detectable cotranscript. These results indicate that Cpx-mediated induction of dsbA transcription in serovar Typhimurium does not occur through an rdoA-dsbA cotranscript. A deletion of the rdoA coding region was constructed to definitively test the relevance of the rdoA-dsbA cotranscript to dsbA expression. The absence of RdoA affects DsbA expression levels when the Cpx system is activated, and providing rdoA in trans complements this phenotype, supporting the hypothesis that a bicistronic mechanism is not involved in serovar Typhimurium dsbA regulation. The rdoA null strain was also shown to be altered in flagellar phase variation. First it was found that induction of the Cpx stress response pathway switched flagellar synthesis to primarily phase 2 flagellin, and this effect was then found to be abrogated in the rdoA null strain, suggesting the involvement of RdoA in mediating Cpx-related signaling.

Bacterial Outer Membrane Proteins↗