Effect of normalization and phase angle calculations on continuous relative phase

The purpose of this investigation was to determine if phase plot normalization and phase angle definitions would have an affect on continuous relative phase calculations. A subject ran on a treadmill while sagittal plane kinematic data were collected with a high-speed (180 Hz) camera. Segmental angular displacements and velocities were used to create phase plots, and examine the coordination between the leg and thigh. Continuous relative phase was calculated with a combination of two different amplitude normalization techniques, and two different phase angle definitions. Differences between the techniques were noted with a root mean square (RMS) calculation. RMS values indicated that there were differences in the configuration of the non-normalized and normalized continuous relative phase curves. Graphically and numerically, it was noted that normalization tended to modify the continuous relative phase curve configuration. Differences in continuous relative phase curves were due to a loss in the aspect ratio of the phase plot during normalization. Normalization tended to neglect the nonlinear forces acting on the system since it did not maintain the aspect ratio of the phase plot. Normalization is not necessary because the arc tangent function accounts for differences in amplitudes between the segments. RMS values indicated that there were profound differences in the continuous relative phase curve when the phase angle was normalized and a phase angle was calculated relative to the right horizontal axis.     

Glucocorticoids and acute phase proteins

Glucocorticoids as well as acute phase proteins participate in non-specific host defence as well as in restoring host integrity after injury. Plasma levels of both compounds augment during the inflammatory reaction. However, glucocorticoids also have physiological effects that share similar molecular mechanisms with the family of steroids. During the inflammatory reaction, and for participating in host defense, glucocorticoids, together with augmented cytokines, use new signalling pathways. In doing so, they participate in the positive or negative control of inflammatory mediator synthesis. For example, they induce the synthesis of acute phase proteins in synergy with interleukin 6, interleukin 1 and TNF alpha.     

Protein crystallization and phase diagrams

The phase diagram is a map which represents the state of a material (e.g., solid and liquid) as a function of the ambient conditions (e.g., temperature and concentration). It is therefore a useful tool in processing many different classes of materials. In this article, methods to determine the phase diagram of an aqueous solution of a globular protein are described, focusing on the solid (crystal) and condensed liquid states. The use of the information contained in the phase diagram for protein crystallization is also discussed.     

Electron microscopic studies of lipopolysaccharides from phase I and phase II Coxiella burnetii

Lipopolysaccharides from phase I (LPSI) Coxiella burnetii Ohio and Nine Mile strains and from phase II (LPSII) Nine Mile stain were negatively and positively and examined with the electron microscope. The ultrastructure of LPSI and LPSII positively stained with uranyl formate or uranyl acetate was ribbon-like. When negatively stained with uranyl acetate, LPSI was ribbon-like but LPSII exhibited hexagonal lattice structures. However, LPSII stained negatively with sodium phosphotungstate and ammonium molybdate exhibited hexagonal lattice ultrastructures which were not identical to those observed when negatively stained with uranyl acetate. The hexagonal lattice structures formed in vitro were due to the interactions of LPSII and the staining reagents rather than to protein-LPS interactions. The differences in the ultrastructures of LPSI and LPSII are undoubtedly based on variations in their chemical composition.     

Defective S phase chromatin assembly causes DNA damage,activation of the S phase checkpoint,and S phase arrest

The S phase checkpoint protects the genome from spontaneous damage during DNA replication, although the cause of damage has been unknown. We used a dominant-negative mutant of a subunit of CAF-I, a complex that assembles newly synthesized DNA into nucleosomes, to inhibit S phase chromatin assembly and found that this induced S phase arrest. Arrest was accompanied by DNA damage and S phase checkpoint activation and required ATR or ATM kinase activity. These results show that in human cells CAF-I activity is required for completion of S phase and that a defect in chromatin assembly can itself induce DNA damage. We propose that errors in chromatin assembly, occurring spontaneously or caused by genetic mutations or environmental agents, contribute to genome instability.     

Nucleic acid actions on abnormal protein aggregation,phase transitions and phase separation

Liquid–liquid phase separation (LLPS) and phase transitions (PT) of proteins, which include the formation of gel- and solid-like species, have been characterized as physical processes related to the pathology of conformational diseases. Nucleic acid (NA)-binding proteins related to neurodegenerative disorders and cancer were shown by us and others to experience PT modulated by different NAs. Herein, we discuss recent work on phase separation and phase transitions of two amyloidogenic proteins, i.e. the prion protein (PrP) and p53, which undergo conformational changes and aggregate upon NA interaction. The role of different NAs in these processes is discussed to shed light on the relevance of PSs and PTs for both the functional and pathological roles of these mammalian proteins.     

Chemical and immunological characterization of lipopolysaccharides from phase I and phase II Coxiella burnetii.

Lipopolysaccharides (LPSs) isolated from phase I and phase II Coxiella burnetii (LPS I and LPS II, respectively) were analyzed for chemical compositions, molecular heterogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological properties. The yields of crude phenol-water extracts from phase I cells were roughly three to six times higher than those from phase II cells. Purification of LPSs by ultracentrifugation gave similar yields for both LPS I and LPS II. Purified LPS I and LPS II contained roughly 0.8 and 0.6% protein, respectively. The fatty acid constituents of the LPSs were different in composition and content, with branched-chain fatty acids representing about 15% of the total. beta-Hydroxymyristic acid was not detected in either LPS I or LPS II. A thiobarbituric acid-periodate-positive compound was evident in the LPSs; however, this component was not identified as 3-deoxy-D-mannooctulosonic acid by gas and paper chromatographies. LPS II contained D-mannose, D-glucose, D-glyceromannoheptose, glucosamine, ethanolamine, 3-deoxy-D-mannooctulosonic acid-like material, phosphate, and fatty acids. LPS I contained the unique disaccharide galactosaminuronyl glucosamine and nine unidentified components in addition to the components of LPS II. The hydrophobic, putative lipid A fraction of LPS I and LPS II contained the above constituents, but the hydrophilic fraction was devoid of ethanolamine. The LPS I disaccharide galactosaminuronyl glucosamine was found in both fractions of the acetic acid hydrolysates. Analysis of LPSs by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining indicated that LPS II was composed of only one band, whereas LPS I consisted of six or more bands with irregular spacing. Ouchterlony immunodiffusion tests demonstrated that LPS I reacted with phase I but not with phase II whole-cell hyperimmune antibody, and LPS II reacted neither with phase I nor phase II hyperimmune antibody. From these results, it was concluded that the chemical structures of LPSs from C. burnetii were different from those of the LPSs of gram-negative bacteria; however, the LPS structural variation in C. burnetii may be similar to the smooth-to-rough mutational variation of saccharide chain length in gram-negative bacteria.     

The ternary phase diagram of lecithin, cholesteryl linolenate and water: phase behavior and structure

The ternary phase diagram of cholesteryl linolenate-egg lecithin-water has been determined by polarizing light microscopy, calorimetry and X-ray diffraction at 23 °C. Hydrated lecithin forms a lamellar liquid-crystalline structure into which small amounts of cholesteryl linolenate are incorporated. The maximum incorporation of cholesterol ester into this lamellar structure varies with the degree of hydration. Increasing the water concentration from 10 to 15% (w/w) increased the limiting molar ratio of cholesteryl linolenate to lecithin in the lamellar phase from 1:50 to 1:22. At intermediate concentrations (15 to 30% water) the cholesteryl linolenate:lecithin ratio remains constant at 1:22. When water is increased to 42.5%, the maximum water content in the lamellar phase, the molar ratio decreased to 1:32. At low water concentrations the cholesterol ester appears to be entirely in the apolar region of the lecithin bilayer, while at higher water concentrations the ester groups of cholesteryl linolenate may be located at the lipid-water interface. At high water concentrations the ester appears to disorder the alkyl chains of the lecithin, giving rise to a thinner lipid layer and an increased surface area per lipid molecule when compared to the lecithin-water system in the absence of cholesteryl linolenate.The lamellar phase is the only phase (except at water concentrations less than 5%) in which all three components mutually interact. All mixtures of the three components having compositions outside the one-phase (lamellar) zone produce additional phases of cholesteryl linolenate or water, or both. Between 23 °C and 60 °C only minor changes in the phase diagram are observed.     

Physiological and morphological characteristics of stationary phase vibrio cells able to support phase growth

Growth of phase alpha 3a on stationary phase Vibrio cultures requires micro-aerophilic conditions and is inhibited by aeration. Since pre-conditioning of the bacteria by allowing them to stand for 24 h after shaking for 3 d is an important aspect of the stationary phase phage growth system, various physiological and morphological characteristics of the stationary phase cells during the transition from shaking to standing were investigated. Shaken stationary phase cells were less viable and more sensitive to ultraviolet irradiation and heat than standing stationary phase cells. During pre-conditioning the small, non-flagellated cells present in shaken stationary phase cultures underwent morphological changes and became large, flagellated rods which resembled exponential phase cells. The transition of stationary phase cells from shaking to standing was associated with a marked increase in total RNA synthesis but a rapid and large decrease in total protein synthesis. Intracellular concentrations of ATP in shaken stationary phase cells were 53% lower than those in standing stationary phase cells. Studies on leucine uptake indicated that its transport was inhibited by isoleucine and that the major part (90%) of the total leucine uptake was due to a shared system for uptake of both amino acids. Shaken stationary phase cells transported less leucine than standing stationary phase cells. Inhibition of phage growth in aerated stationary phase cultures was not due to the prevention of phase absorption by shaking. It is suggested that the observed differences between shaken and standing stationary phase cells could be due to aeration affecting the template specificity of the Vibrio RNA polymerase.     

Modeling and estimating bacterial lag phase

A branching process model of a bacterial population with initial lag phase is developed. Approximations are established in order to facilitate parameter estimation. The validity of approximations and estimation procedures is tested with simulated data.     

Exercise elicits phase shifts and acute alterations of melatonin that vary with circadian phase

To examine the immediate phase-shifting effects of high-intensity exercise of a practical duration (1 h) on human circadian phase, five groups of healthy men 20-30 yr of age participated in studies involving no exercise or exposure to morning, afternoon, evening, or nocturnal exercise. Except during scheduled sleep/dark and exercise periods, subjects remained under modified constant routine conditions allowing a sleep period and including constant posture, knowledge of clock time, and exposure to dim light intensities averaging (+/-SD) 42 +/- 19 lx. The nocturnal onset of plasma melatonin secretion was used as a marker of circadian phase. A phase response curve was used to summarize the phase-shifting effects of exercise as a function of the timing of exercise. A significant effect of time of day on circadian phase shifts was observed (P < 0.004). Over the interval from the melatonin onset before exercise to the first onset after exercise, circadian phase was significantly advanced in the evening exercise group by 30 +/- 15 min (SE) compared with the phase delays observed in the no-exercise group (-25 +/- 14 min, P < 0.05). Phase shifts in response to evening exercise exposure were attenuated on the second day after exercise exposure and no longer significantly different from phase shifts observed in the absence of exercise. Unanticipated transient elevations of melatonin levels were observed in response to nocturnal exercise and in some evening exercise subjects. Taken together with the results from previous studies in humans and diurnal rodents, the current results suggest that 1) a longer duration of exercise exposure and/or repeated daily exposure to exercise may be necessary for reliable phase-shifting of the human circadian system and that 2) early evening exercise of high intensity may induce phase advances relevant for nonphotic entrainment of the human circadian system.     

A theoretical model for lipid mixtures, phase transitions, and phase diagrams.

We present a new model for the thermodynamic properties of lipid bilayers. The model consists of a system of hard cylinders of varying radii that correspond to the different molecular radii of lipids having different numbers of gauche rotations in their chains. Scaled particle theory is used to provide an accurate estimate of the entropy of packing of the cylinders. To apply the model to bilayers we introduce a semiempirical attractive potential energy. Once the form of this potential is chosen, we adjust one parameter, the interaction strength, so that the model fits the transition temperatures and entropies for various phospholipids. The model then agrees quite well with other published data for these systems. We also directly generalize our model to lipid mixtures, and we obtain phase diagrams that we compare to existing data for these systems. We use the model to describe lipid protein interactions in bilayers as well.     

Comparison of definitions of the lag phase and the exponential phase in bacterial growth

M.H. ZWIETERING, F.M. ROMBOUTS AND K. VAN 'T RIET. 1992. Different definitions of the lag time and of the duration of the exponential phase can be used to calculate these quantities from growth models. The conventional definitions were compared with newly proposed definitions. It appeared to be possible to derive values for the lag time and the duration of the exponential phase from the growth models, and differences between the various definitions could be quantified. All the different values can be calculated from the growth parameters μ _m , and a. Therefore, it appeared to be unnecessary to use complicated mathematical equations: simple equations were adequate. For the Gompertz model the conventional definition of the lag time did not differ appreciably from the newly proposed definition. The end-point of the exponential phase and thus the duration of the exponential phase differed considerably for the two definitions. For the logistic model the two definitions lead to considerable differences for all quantities. It is recommended that the conventional definition is used for calculating the lag time. For the duration of the exponential phase it is recommended that the new definition is used. The value can be calculated, however, directly from the conventional growth parameters.     

Three phase liquid phase microextraction of phenylacetic acid and phenylpropionic acid from biological fluids

Three phase liquid phase microextraction (three phase LPME) technique coupled with HPLC-UV has been applied as a sensitive and efficient sample preparation method to determine phenylacetic acid (PAA) as a biomarker of depressive disorders and phenylpropionic acid (PPA) in biological fluids. The compounds were extracted from 3.0 ml aqueous solution with the adjustment of pH at a fixed value in the range of 2.0-3.5 (donor solution) into an organic phase (1-hexanol) layered on the surface of the donor solution and finally back-extracted into 4.0 microl of the acceptor microdrop (pH 11.1) located at the end of the microsyringe needle. After a prescribed back-extraction time, the acceptor microdrop was withdrawn into the microsyringe and then directly injected into the HPLC system. In order to achieve maximum extraction efficiency, different parameters affecting the extraction conditions were optimized. At the optimum conditions (donor solution: 2.3M Na(2)SO(4), pH 2.0-3.5; organic membrane: 95 microl of 1-hexanol; acceptor solution: 4.0 microl of 0.1M NH(3)/NH(4)(+) with pH 11.1; donor solution temperature: 45-50 degrees C; extraction time: 20 min and back-extraction time: 12 min), up to 110-fold enrichment factor was obtained. The calibration curve for these analytes was linear in the range of 1-5000 microg/l with r(2)>0.998. The intraday and interday RSD% were below 6.5% and the limits of detection (LODs) for both analytes were 0.2 microg/l (based on S/N=3). The proposed technique is a low cost, simple and sensitive method with highly clean-up effect. Finally, this technique was successfully utilized for the detection of target analytes in human urine, serum and plasma.     

Acute phase proteins and pesticide exposure

Gamma mobility C-reactive protein (CRP) level was determined in the sera of persons occupationally exposed to pesticides and controls in conjunction with serum protein analysis and other biochemical and enzymologic tests. Workers chronically exposed to dieldrin and pentachlorophenol showed significantly higher prevalence of CRP than the unexposed persons. In addition, the pentachlorophenol-exposed subjects revealed significantly elevated levels of total bilirubin and creatine phosphokinase, although the levels were within normal limits. The results suggest that chronic exposure to pentachlorophenol may have been responsible for the difference in the prevalence of CRP between the pentachlorophenol and control groups.