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.     

Circadian rhythms in mouse epidermal basal cell proliferation. Variations in compartment size, flux and phase duration.

Several kinetic parameters of basal cell proliferation in hairless mouse epidermis were studied, and all parameters clearly showed circadian fluctuations during two successive 24 hr periods. Mitotic indices and the mitotic rate were studied in histological sections; the proportions of cells with S and G2 phase DNA content were measured by flow cytometry of isolated basal cells, and the [3H]TdR labelling indices and grain densities were determined by autoradiography in smears from basal cell suspensions. The influx and efflux of cells from each cell cycle phase were calculated from sinusoidal curves adapted to the cell kinetic findings and the phase durations were determined. A peak of cells in S phase was observed around midnight, and a cohort of partially synchronized cells passed from the S phase to the G2 phase and traversed the G2 phase and mitosis in the early morning. The fluctuations in the influx of cells into the S phase were small compared with the variations in efflux from the S phase and the flux through the subsequent cell cycle phases. The resulting delay in cell cycle traverse through S phase before midnight could well account for the accumulation of cells in S phase and, therefore, also the subsequent partial synchrony of cell cycle traverse through the G2 phase and mitosis. Circadian variations in the duration of the S phase, the G2 phase and mitosis were clearly demonstrated.     

Leishmania braziliensis: protein, carbohydrate, and antigen differences between log phase and stationary phase promastigotes in vitro

When Leishmania species are grown in vitro, parasites from the stationary phase differ from those in log phase growth in being more infective and more resistant to complement and macrophage mediated killing. In the present study, log phase and stationary phase promastigotes of Leishmania braziliensis panamensis were compared at the molecular level. Differences in polypeptide and glycoprotein composition and antigenicity between log and stationary phase promastigotes of L. b. panamensis were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting; the former showed that two polypeptides were unique to log phase promastigotes and one was unique to stationary phase promastigotes. There were also differences in surface lectin binding characteristics of log and stationary phase promastigotes. Live stationary phase promastigotes bound more concanavalin and lentil lectin than log phase promastigotes, indicating a greater number of mannose residues on their surfaces.     

CIRCADIAN RHYTHMS IN MOUSE EPIDERMAL BASAL CELL PROLIFERATION

Several kinetic parameters of basal cell proliferation in hairless mouse epidermis were studied, and all parameters clearly showed circadian fluctuations during two successive 24 hr periods. Mitotic indices and the mitotic rate were studied in histological sections; the proportions of cells with S and G₂ phase DNA content were measured by flow cytometry of isolated basal cells, and the [³H]TdR labelling indices and grain densities were determined by autoradiography in smears from basal cell suspensions. The influx and efflux of cells from each cell cycle phase were calculated from sinusoidal curves adapted to the cell kinetic findings and the phase durations were determined. A peak of cells in S phase was observed around midnight, and a cohort of partially synchronized cells passed from the S phase to the G₂ phase and traversed the G₂ phase and mitosis in the early morning. The fluctuations in the influx of cells into the S phase were small compared with the variations in efflux from the S phase and the flux through the subsequent cell cycle phases. The resulting delay in cell cycle traverse through S phase before midnight could well account for the accumulation of cells in S phase and, therefore, also the subsequent partial synchrony of cell cycle traverse through the G₂ phase and mitosis. Circadian variations in the duration of the S phase, the G₂ phase and mitosis were clearly demonstrated.     

Fungal melanin inhibitor and related compounds from Penicillium decumbens

In Silene vulgaris (M.) G. cell culture three growth phases were distinguished, namely, a lag phase, an exponential phase and a stationary phase. Pectin termed silenan and an acidic arabinogalactan were isolated as cell wall polysaccharides of S. vulgaris callus at the different growth phases during culture. Production of silenan as the galacturonan (or rhamnogalacturonan) core was observed at the beginning of the exponential phase and at the stationary phase of the callus growth. Arabinogalactan, containing the galacturonic acid residues, is formed at the exponential phase followed by attachment to the core of silenan in the middle of the exponential phase. The arabinogalactan constituent of silenan appeared to be destroyed gradually at the stationary growth phase. The monosaccharide compositions of silenan and arabinogalactan were determined at various phases of the callus growth. Silenan was found to be formed in maximum amounts at the exponential phase of the cell growth. Insignificant alterations of the yields of acidic arabinogalactan were found during culture while total productivity per litre of medium and rate of production per day of arabinogalactan were found to be maximal at the exponential phase of growth.     

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.     

Annual reproductive cycle of a bitterling, Tanakia tanago, reared in an outdoor tank

From June 2000 to September 2001, we investigated the presence of eggs spawned in Margaritifera laevis and the seasonal changes in the gonads of Tanakia tanago. Eggs were observed from mid-March to mid-September. In females with a shrunken ovipositor, as the GSI gradually increased, most ovaries were in the prespawning phase (Oct-Mar). As the GSI increased further, most ovaries were in the early spawning phase (Mar-Jun). As the GSI gradually deceased, ovaries in the late spawning phase appeared (Jun-Sep). When the GSI was very low, most ovaries were in the postspawning phase (Sep-Oct). In males, when the GSI was low, most testes were in the early prespawning phase from Oct-Dec. As the GSI gradually increased, most testes were in the late prespawning phase (Dec-Jan). As the GSI increased further, testes were in the early spawning phase (Jan-Jun). As the GSI gradually decreased, amost testes were in the late spawning phase (Jun-Sep). When the GSI was very low, most testes were in the postspawning phase (Sep-Oct). These results indicate that T. tanago has a distinct annual reproductive cycle and is a spring-autumn spawner. Based on the relationship between reproductive activity and environmental factors, the spawning season of T. tanago appears to be initiated by increasing temperature and / or longer days in spring and to be terminated by shorter days in autumn.     

Siberian hamsters that fail to reentrain to the photocycle have suppressed melatonin levels

Siberian hamsters readily reentrain to a 3-h phase delay of the photocycle (16 h light/day) but fail to reentrain to a 5-h phase delay. This study tested whether melatonin production was suppressed in animals that failed to reentrain. Melatonin was measured on the day before, day of, or several days after each phase shift. Melatonin levels measured 4 h after dark onset were approximately 83 microg/ml on the day before each phase delay and undetectable (<6 microg/ml) during the light phase on the day of the phase shift. Activity onsets regained their prior phase relationship to the photocycle 4 (3 h) or 5 (5 h) days after the phase shift; on that day, melatonin levels were measured 4 h after dark onset. Melatonin levels were unaffected by the 3-h phase delay (>57.6 microg/ml) but were undetectable after a 5-h phase delay (<8 microg/ml). Thus melatonin remained suppressed only after the phase delay to which hamsters also fail to reentrain. This relationship suggests that the propensity for reentrainment may be influenced by changes in melatonin production following a phase shift of the photocycle.     

Androgen receptor regulates Cdc6 in synchronized LNCaP cells progressing from G1 to S phase

We have shown previously that androgen receptor (AR) activity is required for the progression of cells from G(1) to S phase. In an attempt to elucidate the mechanism of androgen- and androgen-receptor-mediated proliferation of prostate cancer cells, we studied the effect of anti-androgen bicalutamide (Casodex) on the expression of cell-cycle regulatory genes in synchronized LNCaP cells progressing from G(1) to S phase. LNCaP cells were synchronized by isoleucine-deprivation. Expression of cell-cycle regulatory genes in S phase control cells versus Casodex-treated cells that fail to enter S phase was studied using a microarray containing cDNA probes for 111 cell-cycle specific genes. RT-PCR and Western-blots were used to validate microarray data. Casodex blocked synchronized LNCaP cells from entering S phase. Microarrays revealed downregulation of eight genes in cells prevented from entering into S phase by Casodex. Of these eight genes, only Cdc6, cyclin A, and cyclin B were downregulated at both the mRNA and protein level in Casodex treated cells as compared to control cells. The mRNA and protein levels of Cdc6 increased as synchronized LNCaP cells progressed from G(1) to S phase, and were attenuated in Casodex-treated cells failed to enter S phase. Cyclins A and B were detected when cells entered S phase, but not when they were in G(1) phase. Like Cdc6, the levels of both cyclins A and B were attenuated in Casodex-treated cells. AR may play an important role in the onset of DNA synthesis in prostate cancer cells by regulating the expression and stability of Cdc6, which is critically required for the assembly of the pre-replication complex(pre-RC).     

The relation between membrane lipid phase separation and frost tolerance of cereals and other cool climate plant species

Abstract. The temperatures at which liposomes prepared from membrane phospholipids begin to phase separate were compared to the temperatures at which intact plants were damaged. Woody perennials tolerated temperatures below which their membrane phospholipids began to phase separate. By contrast, rye and wheat seedlings were damaged about 25°C above their phase separation temperature. Differences in tolerance among cultivars pre-hardened to frost were reflected by changes of the phase separation temperature. The results support the notion that alterations in membrane lipid composition are associated with frost hardening. A correlation between the temperature of phase separation and frost tolerance suggests that lipid properties may influence freezing tolerance of cereals; however, the lethal event is apparently not phase separation of the membrane phospholipids.     

Immune response induced by airway sensitization after influenza A virus infection depends on timing of antigen exposure in mice

To study which phase of viral infection promotes antigen sensitization via the airway and which type of antigen-presenting cells contributes to antigen sensitization, BALB/c mice were sensitized by inhalation of ovalbumin (OA) during the acute phase or the recovery phase of influenza A virus infection, and then 3 weeks later animals were challenged with OA. The numbers of eosinophils and lymphocytes, the amounts of interleukin-4 (IL-4) and IL-5 in the bronchoalveolar lavage fluid, and the serum levels of OA-specific immunoglobulin G1 (IgG1) and IgE increased in mice sensitized during the acute phase (acute phase group), while a high level of gamma interferon production was detected in those sensitized during the recovery phase (recovery phase group). In the acute phase group, both major histocompatibility complex class II molecules and CD11c were strongly stained on the bronchial epithelium; in the recovery phase group, however, neither molecule was detected. OA-capturing dendritic cells (DCs) migrated to the regional lymph nodes, and a small number of OA-capturing macrophages were also observed in the lymph nodes of the acute phase group. In the recovery group, however, no OA-capturing DCs were detected in either the lungs or the lymph nodes, while OA-capturing macrophages were observed in the lymph nodes. These results indicate that the timing of antigen sensitization after viral infection determines the type of immune response.     

Nucleoid structure and partition in Methanococcus jannaschii: an archaeon with multiple copies of the chromosome.

We measured different cellular parameters in the methanogenic archaeon Methanococcus jannaschii. In exponential growth phase, the cells contained multiple chromosomes and displayed a broad variation in size and DNA content. In most cells, the nucleoids were organized into a thread-like network, although less complex structures also were observed. During entry into stationary phase, chromosome replication continued to termination while no new rounds were initiated: the cells ended up with one to five chromosomes per cell with no apparent preference for any given DNA content. Most cells in stationary phase contained more than one genome equivalent. Asymmetric divisions were detected in stationary phase, and the nucleoids were found to be significantly more compact than in exponential phase.     

Effect of counterions on the influence of dodecyltrimethylammonium halides on thermotropic phase behaviour of phosphatidylcholine bilayers

Effects of dodecyltrimethylammonium chloride (DTAC), dodecyltrimethylammonium bromide (DTAB) and dodecyltrimethylammonium iodide (DTAI) on thermotropic phase behaviour of phosphatidylcholine bilayers as well as on 1H NMR spectra were studied. In order to enhance the effect of counterions on water structure two series of experiments were performed. In the first one the surfactants were added to the water phase and in the other one directly to lipid phase (a mixed film was formed). The effects of particular surfactants on the main phase-transition temperature were more pronounced when they were added to the water phase (1st method) instead of the lipid phase (2nd method). Furthermore, in the case of the first method the transitions were found asymmetrical while in the second method nearly symmetrical. It is suggested that surfactant-poor and surfactant-rich domains are formed when surfactants are added to the water phase.     

Changes in phospholipids, cholesterol and protein content of oviduct fluid of cows during the oestrous cycle

The oviducts of 4 cows were cannulated and oviduct fluid was collected daily from the exteriorized cannulas for a total of 5 oestrous cycles. Daily serum samples were assayed for oestradiol-17 beta and progesterone to monitor the oestrous cycle. Data for each cycle were compared for oviduct fluid collected during the non-luteal phase (serum progesterone less than or equal to 1.5 ng/ml) and the luteal phase (serum progesterone greater than 1.5 ng/ml). During the non-luteal phase oviduct fluid volume was higher and the osmolality was lower than during the luteal phase. Total protein, cholesterol and phospholipid secreted daily was greater during the non-luteal phase. Cholesterol and protein concentrations were generally lower during the non-luteal phase, but phospholipid concentrations were generally higher. About 40% of the phospholipid in oviduct fluid was phosphatidylcholine and lysophosphatidylcholine, while phosphatidylinositol and lysophosphatidylinositol accounted for 20%. The ratio of 1-acyl-phospholipid to diacylphospholipid increased during the non-luteal phase. An increased cholesterol to phospholipid ratio, and a decreased cholesterol to protein ratio in oviduct fluid also were associated with the non-luteal phase. Changes in the lipid composition of oviduct fluid during the oestrous cycle may play a role in the preparation of gametes for fertilization.     

Influence of dodecyltrimethylammonium halides on thermotropic phase behaviour of phosphatidylcholine/cholesterol bilayers

Effects of dodecyltrimethylammonium chloride (DTAC), dodecyltrimethylammonium bromide (DTAB) and dodecyltrimethylammonium iodide (DTAI) on thermotropic phase behaviour of phosphatidylcholine bilayers containing cholesterol as well as on 1H NMR spectra were studied. Two series of experiments were performed. In the first one the surfactants were added to the water phase while in the other directly to the lipid phase (a mixed film from cholesterol, surfactant and phosphatidylcholine was formed). The effects of particular surfactants on the main phase transition temperature, Tm, were more pronounced when added to the lipid phase (2nd method) than to the water phase (1st method); the opposite happened when cholesterol was absent (Rózycka-Roszak and Pruchnik 2000, Z. Naturforsch. 55c, 240-244). Furthermore, in the case of the first method the transitions were asymmetrical while in the second method nearly symmetrical. It is suggested that surfactant poor and surfactant rich domains are formed when surfactants are added to the water phase.     

Relative participation of the gas phase and total particulate matter in the imbalance in prostacyclin and thromboxane formation seen following chronic cigarette smoke exposure

Chronic exposure to cigarette smoke causes an imbalance in the ratio of PGI2 and TXA2 production and is believed to favor the development of atherosclerosis. Components of the particulate phase of smoke (especially nicotine) as well as the gas phase of smoke have been shown to adversely alter arachidonic acid metabolism. To determine the relative participation of nicotine, particulate and gas phases in eliciting an imbalance in TXA2 formation, male Sprague-Dawley rats were chronically exposed (7 days/wk/mo.) to freshly generated whole smoke or gas phase from University of Kentucky Reference cigarettes and allowed access to regular drinking water or to water supplemented with nicotine (10 micrograms/ml). COHb levels were monitored to confirm smoke or gas phase inhalation. All treatment groups had lower body weights than shams. No differences in body weights were observed between smoke (+/- oral nicotine) and gas phase (+/- oral nicotine) treatment groups but all were significantly lower than oral nicotine treated animals. Platelet TXA2 production was elevated in all treatment groups compared to shams. No differences in TXA2 production were observed between smoke (+/- oral nicotine), gas phase and oral nicotine treated animals. Animals receiving gas phase/oral nicotine exhibited significantly higher platelet TXA2 production compared to the other treatments. Constituents of the gas phase as well as the particulate phase of whole smoke were both shown to elevate platelet TXA2 formation. Components of the particulate matter appear to modulate the effects of nicotine and the gas phase in the perturbation of TXA2 production in the rat smoking model.     

Phase equilibria in four lysophosphatidylcholine/water systems. Exceptional behaviour of 1-palmitoyl-glycerophosphocholine

The phase equilibria in four lysophosphatidylcholine/water systems were investigated at different temperatures. Each of the 1-palmitoyl-, 1-stearoyl-, 1-oleoyl- and 1-linoleoyl-sn-glycero-3-phosphocholines was dispersed in heavy water at different concentrations. The phase structures were determined by 2H-, 14N- and 31P-NMR, polarization microscopy and low-angle X-ray diffraction. The phase diagrams of the oleoyl and linoleoyl systems were quite similar. At room temperature and with decreasing water content the isotropic micellar solution was followed by a hexagonal phase and then a cubic phase. Finally the lamellar phase appeared before the region of hydrated crystals. The same sequence of phases was observed in the stearoyl system at elevated temperatures. The palmitoyl system differed from the others: here a cubic phase followed after the micellar solution, then came a hexagonal phase and after this a lamellar phase. In general the lysophosphatidylcholines seem to behave similarly to the many soaps and detergents as they show the same sequence of isotropic micellar solution, hexagonal phase, lamellar phase with interspersed cubic phases. The presently established phase diagrams demonstrate that the major lysophosphatidylcholines which may be generated by phospholipase A2 in mammalian cell membranes, viz. 1-palmitoyl- and 1-stearoyl-glycerophosphocholines differ greatly in their packing properties. The extraordinary ability of 1-palmitoyl-glycerophosphocholine to form a cubic phase in equilibrium with a micellar solution is of particular interest with regard to the possible occurrence of cubic structures in biomembranes during the process of fusion.     

Phase relationships between sleep-wake cycle and underlying circadian rhythms in Morningness-Eveningness

A shorter phase angle between habitual wake time and underlying circadian rhythms has been reported in evening types (E types) compared to morning-types (M types). In this study, phase angles were compared between 12 E types and 12 M types to verify if this difference was observed when the sleep schedule was relatively free from external social constraints. Subjects were selected according to their Morningness-Eveningness Questionnaire score (MEQ score). There were 6 men and 6 women in each group (ages 19-34 years), and all had a habitual sleep duration between 7 and 9 h. Sleep schedule was recorded by actigraphy and averaged over 7 days. Circadian phase was estimated by the hour of temperature minimum (T(min)) in a 26-h recording and by the timing of the onset of melatonin secretion (dim-light melatonin onset [DLMO]) measured in saliva samples. Phase angles were defined as the interval between phase markers and averaged wake time. Results showed that, in the present experimental conditions, phase angles were very similar in the 2 groups of subjects. However, results confirmed the previously reported correlation between phase and phase angle, showing that a later circadian phase was associated with a shorter phase angle. Gender comparisons showed that for a same MEQ score, women had an earlier DLMO and a longer phase angle between DLMO and wake time. Despite a significant difference in the averaged circadian phases between E-type and M-type groups, there was an overlap in the circadian phases of the subjects of the 2 groups. Further comparisons were made between the 2 circadian types, separately for the subgroups with overlapping or nonoverlapping circadian phases. In both subgroups, the significant difference between MEQ scores, bedtimes, and wake times were maintained in the expected direction. In the subgroup with nonoverlapping circadian phases, phase angles were shorter in E-type subjects, in accordance with previous studies. However, in the overlapping subgroup, phase angles were significantly longer in E types compared to M types. Results suggest that the morningness-eveningness preference identified by the MEQ score refers to 2 distinct mechanisms, 1 associated with a difference in circadian period and phase of entrainment and the other associated with chronobiological aspects of sleep regulation.