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PubMed2003

Cell cycle-dependent protein fingerprint from a single cancer cell: image cytometry coupled with single-cell capillary sieving electrophoresis.

Study of cell cycle-dependent protein expression is important in oncology, stem cell research, and developmental biology. In this paper, we report the first protein fingerprint from a single cell with known phase in the cell cycle. To determine that phase, we treated HT-29 colon cancer cells with Hoescht 33342, a vital nuclear stain. A microscope was used to measure the fluorescence intensity from one treated cell; in this form of image cytometry, the fluorescence intensity is proportional to the cell's DNA content, which varies in a predictable fashion during the cell cycle. To generate the protein fingerprint, the cell was aspirated into the separation capillary and lysed. Proteins were fluorescently labeled with 3-(2-furoylquinoline-2-carboxaldehyde, separated by capillary sieving electrophoresis, and detected by laser-induced fluorescence. This form of electrophoresis is the capillary version of SDS-PAGE. The single-cell electropherogram partially resolved approximately 25 components in a 30-min separation, and the dynamic range of the detector exceeded 5000. There was a large cell-to-cell variation in protein expression, averaging 40% relative standard deviation across the electropherogram. The dominant source of variation was the phase of the cell in the cell cycle; on average, approximately 60% of the cell-to-cell variance in protein expression was associated with the cell cycle. Cells in the G1 and G2/M phases of the cell cycle had 27 and 21% relative standard deviations in protein expression, respectively. Cells in the G2/M phase generated signals that were twice the amplitude of the signals generated by G1 phase cells, as expected for cells that are soon to divide into two daughter cells. When electropherograms were normalized to total protein content, the expression of only one component was dependent on cell cycle at the 99% confidence limit. That protein is tentatively identified as cytokeratin 18 in a companion paper.

Algorithms↗
PubMed1977

Study of the role of puring phosphoribosyltransferases in the uptake of adenine and guanine by Schizosaccharomyces pombe cells.

1. In the yeast Schizosaccharomyces pombe 972h-, the uptake rate of both adenine and guanine is related to variations in the specific activity of the corresponding phosphoribosyltransferases during the growth of the culture. Furthermore, the mutant strains dap 1, devoid of adenine phosphoribosyltransferase activity, and pur 1, devoid of guanine phosphoribosyltransferase activity have a lowered uptake rate of adenine and guanine respectively, along with an increased apparent Km value for these purines in comparison to the wild-type 972h. 2. The uptake rate of the purines is strongly dependent on the pH of the uptake medium in 972h- as well as in the strains dap 1 and pur 1, the optimum being between pH4 and pH5. 3. A new method of extraction of 5-phosphoribosyl-1-pyrophosphate from the yeast has been devised. Important fluctuations of the P-Rib-P2 pool were measured in S. pombe at different stages of growth, the maximum taking place at the start of the exponential phase, whereas no variations in the specific activity of the P-Rib-P2 synthetase could be observed during the growth. The P-Rib-P2 intracellular content in the mutants devoid of purine phosphoribosyltransferases, namely pur 1, dap 1 and pur 1, dap 1, was increased up to 5-fold as compared to the wild-type strain. 4. The effect of intracellular concentrations of P-Rib-P2, a substrate for phosphoribosyltransferases, on the uptake rate of purines has been studied: addition of formycin to the growth medium lowered simultaneously the P-Rib-P2 intracellular content and the uptake of adenine and guanine. 5. Although our results demonstrate the activating effect of phosphoribosyltransferase activities on the uptake of adenine and guanine, they do not support the hypothesis of a 'group translocation' mechanism.

Adenine↗
PubMed1991

[Simultaneous effects and aftereffects of the intensity and success aspect in coping with stresses].

Intensity and success of coping processes in achievement-related situations are very often tightly connected. Therefore, the specific empirical status of each of the two aspects is unclear. In the current study a technique is introduced which allows orthogonal variation of both components. 64 subjects were confronted with a simple perceptual-speed task in which objective success rate was held constant by a computer-based adaptive process. Graphic feedback was provided about the objective success rate in relation to one of two different success criteria. For half of the subjects the criterion was above (failure condition) and for the other half it was below (success condition) the adaptively controlled success rate. Intensity, the second between-subjects factor, was manipulated by presenting either a high or a low number of items per time unit. As an additional quasi-experimental factor, the degree of action- vs. state-orientation (Kuhl, 1983) was included. The results showed highly specific effects for both the intensity and the failure/success dimension: High intensity resulted in an increase of electrodermal activity which could be observed throughout a one hour post-stressor phase. The failure/success variation had effects mainly on subjective variables representing appraisal of controllability and produced no after-effects. Especially strong effects on subjective variables could be observed when state-oriented subjects were confronted with both failure and high intensity.

Achievement↗
PubMed2006

[Aging and biological rhythms in primates].

All living organisms exhibit rhythmic activities in a wide variety of endocrine and behavioural parameters. These biological rhythms are endogenously generated by a circadian clock, and they are entrained by cyclic variations of environmental factors called synchronizers. Aging is associated with changes in amplitude and temporal organization of many daily and seasonal rhythms. In humans, daily rhythms of sleep, thermoregulation and hormonal secretion are severely altered with aging. Except in humans, studies on primates are scarce. However, age-related effects on biological rhythms are relatively consistent among primate species studied to date, including humans. Therefore, non human primates are of valuable use for such investigations. Most studies have been performed on the Rhesus macaque (longevity 35-40 years) and on the gray mouse lemur (longevity 10-12 years). Like in humans, the rest-activity rhythm becomes fragmented in aged primates, and shows an increased activity during the resting period. Aging induces a decrease in amplitude of the body temperature rhythm and an increase in energy consumption. Various hormonal secretions exhibit a decrease with aging, but the rhythmic components of these declines have not always been depicted. Moreover, changes (amplitude or phase) in daily variations depended of the hormonal secretion tested. Taken together, these results suggest that the biological clock in the brain would be a primary target of aging. The main central clock is located in the suprachiasmatic nucleus of the hypothalamus whose endogenous oscillations are entrained by light. In this brain structure, cellular function and sensitivity to light show drastic changes with age in the mouse lemur. The precise knowledge of age-related alterations of biological rhythms in primates can have important consequences on the development of new treatments to maintain or restore biological rhythmicity in the elderly.

Aging↗
PubMed1987

Phase separation in lens cytoplasm is genetically linked to cataract formation in the Philly mouse.

The variation of the phase-separation temperature, Tc, in lenses was studied during the postnatal development of three genetically different mouse strains: Swiss-Webster, Philly, and the (Swiss-Webster x Philly)F1 hybrid. The general behavior of Tc during early postnatal development has two stages: in stage I, Tc increased to a maximum and then, in stage II, Tc decreased. Philly mice are a strain that develops hereditary cataracts about 36 days following birth. In F1 hybrids of Philly and Swiss-Webster mice, cataracts appeared about 49 days following birth, approximately equal to 13 days later in development than in the Philly mice. In the Philly and hybrid mice, stage I and stage II were followed by stage III in which Tc reached a minimum value and then increased toward body temperature. The values of Tc at birth, the slope of the increase during stage I, and the maximum Tc were characteristic for each mouse strain. These results establish that the behavior of the temperature of the phase separation Tc in mouse lens is linked to the genetic strain of the mice and that the value of Tc at birth is an early indicator of lenses that will develop cataracts and lenses that will develop normally.

Animals↗
PubMed2005

The development of a mouse model to explore resistance and susceptibility to early Ascaris suum infection.

Ascaris suum and Ascaris lumbricoides exhibit an over-dispersed frequency distribution in their host populations in both the adult and larval stages. The impact of host factors on this observed distribution is still poorly understood and difficult to investigate in the natural host populations. The use of a mouse model has been supported by the observations that the larval migratory pattern, in this host, mimics the pattern observed in the pig. We explored the extrinsic factors that might affect the quantitative recovery of larvae during this migration in order to standardize a model system facilitating accurate future assessment of host genetic variation on this phase of the infection. In Exp. 1 larvae accumulated in the livers of both C57BL/6j and BALB/c mice up to and including days 4-5 p.i. and then declined in both strains until day 9. Loss of larvae from the livers corresponded to arrival in the lungs and maximum accumulation on day 7 p.i. but recovery was considerably higher in C57BL/6j mice. It was concluded that day 7 recoveries gave the best indication of relative resistance/susceptibility to this parasite. In Exp. 2 A/J, BALB/c, CBA/Ca, C57BL/6j, C3H/HeN, DBA/2, NIH, SJL, and SWR mice were compared. C57BL/6j mice were identified as the most susceptible strain and CBA/Ca mice as having the most contrasting phenotype, but with a similar kinetic pattern of migration. Finally, in Exp. 3, a strong positive correlation between the size of the inoculum and the mean worm recovery from the lungs was found in CBA/Ca and C57BL/6j mice, but the difference between these strains was highly consistent, 66.6-80%, regardless of the initial dose. These results demonstrate that, using our protocols for infection and recovery, between-experiment variation in A. suum worm burdens is minimal, and that C57BL/6j mice are highly susceptible to infection compared to other strains. The mechanistic basis of this susceptibility in relation to the resistance of other strains is unknown, but the possibilities are reviewed.

Analysis of Variance↗
PubMed2006

Timing of follicular phase events and the postovulatory progesterone rise following synchronisation of oestrus in cows.

In cows the timing of both ovulation and the subsequent postovulatory progesterone rise are critical to successful fertilisation and early embryo development. The aim of this study was to determine the degree of variability in the timing of ovulation relative to other follicular phase events and to determine how variations in the timing of follicular phase events contribute to the timing of the postovulatory progesterone rise. Plasma concentrations of progesterone, oestradiol and luteinising hormone (LH) and the timing of oestrus and ovulation were determined following induction of luteolysis were determined in 18 mature, non-lactating Holstein-Friesian cows. Four cows were excluded on the basis of abnormal reproductive function. In the remaining 14 cows oestrus occurred at 57.4+/-4.3h and the LH surge at 54.6+/-4.0h following luteolysis (progesterone <1ngmL(-1)) followed by a fall in circulating oestradiol concentration at 64.6+/-4.4h. Cows ovulated at 88.0+/-4.7h with the postovulatory progesterone rise (to >1ngmL(-1)) occurring 159+/-7.2h after luteolysis. There was considerable variation in the timing of ovulation following luteolysis (range 64-136h) onset of oestrus (range 24-40h) and onset of the LH surge (range 24-44h). Cows were then split on the basis of interval from progesterone fall to progesterone rise giving groups (n=7 per group) with intervals of 180.6+/-6.7 and 138.3+/-5.7h (P<0.001). Between groups, both the intervals from luteolysis to ovulation (98.3+/-6.9 vs 77.7+/-3.4h; P<0.05) and ovulation to progesterone rise (82.3+/-4.2 vs. 60.6+/-5.5h; P<0.01) were longer in late rise cows. There was no difference between groups in the interval from oestrus or LH surge to ovulation. In conclusion the results of this study further highlight the high variability that exists in the timing and interrelationships of follicular phase events in the modern dairy cow, reemphasising the challenges that exist in optimising mating strategies. However, the data do suggest that in cows with poor post ovulatory progesterone secretion, the key problem appears to be poor post ovulatory development rather than a delay in ovulation.

Animals↗
PubMed1991

Variations in performance in simple muscle tests at different phases of the menstrual cycle.

The effect of the menstrual cycle on the performance of women in sporting activities is a very confused subject. In this study, performances in simple muscle tests--the handgrip and standing long jump, were studied at three phases of the menstrual cycle--menstrual (day 1-4), follicular (day 12-14) and luteal (day 19-21). Within subject paired "t" testing showed that in the handgrip test, performance was significantly superior during the menstrual phase than those during both the follicular and luteal phases. In the standing long jump test, performance was again superior during the menstrual phase, although not significantly with respect to the luteal phase. This finding is discussed in terms of the reported effects of the menstrual cycle on sporting performance, the variation in the types of exercise, and the possible role of the female sex hormones.

Adolescent↗
PubMed1990

Double labelling of cells with tritiated thymidine and bromodeoxyuridine reveals a circadian rhythm-dependent variation in duration of DNA synthesis and S phase flux rates in rodent oral epithelium.

We describe a double labelling method for estimating the duration of DNA synthesis (Ts) and the flux of cells into and from the S phase of the cell cycle, based on labelling with tritiated thymidine [( 3H]TdR) followed by bromodeoxyuridine (BrdU) and combining immunohistological detection of BrdU with conventional autoradiography. In practice, the change in size of a window of double labelled cells occurs as the time interval between the two labels increases. In mouse tongue epithelium there is a marked circadian variation in the number of cells in DNA synthesis. From 0900 to 1500 h this labelling index (LI) falls, but from 2100 to 0300 h it increases. Our results show that the circadian decrease in LI is associated with a short Ts (5.8 +/- 0.3 h), a high S phase efflux and an initially low influx of cells from G1 into S. Conversely, the rising circadian LI is associated with a longer Ts (9.4 +/- 0.1 h), an initially low efflux and a moderate to high influx. Two time-points exist on the circadian LI curve when influx and efflux rates change abruptly. At 0100 h the efflux rate rises from low (5 cells %/h) to high (15-16 cells %/h) and simultaneously the influx rate changes from high to low. Similarly at 1300-1400 h, efflux rate falls from high (19-20 cells %/h) to low (4-8 cells %/h) values and influx rates change from low to high. This double labelling method has revealed that the duration of DNA synthesis varies across the circadian cycle, as do influx and efflux values which generally fall within a discrete range of high or low values. The timing of the changes in flux suggests the presence of two 'control' points on the circadian LI cycle that were previously unrecognized.

Animals↗
PubMed1997

The Phase Co(1)(-)(x)()Ni(x)()Sn(2): Structural Variations Based on the Stacking of Two Different Planar Nets.

The investigation of the tin-rich part of the ternary system Co/Ni/Sn yielded the phase Co(1)(-)(x)()Ni(x)()Sn(2) with the range of composition 0.23(3) < x < 0.59(3). When using a large excess of tin, Co(1)(-)(x)()Ni(x)()Sn(2) crystallizes at 500 degrees C in a structure isotypic to that of tetragonal PdSn(2) whereas a modification with the orthorhombic CoGe(2) structure type always forms from a stoichiometric mixture of the elemental components or from a tin melt at temperatures above 550 degrees C. The structures of Co(0.625)Ni(0.375)Sn(2) were determined by single-crystal X-ray diffraction methods (PdSn(2)-type space group I4(1)/acd, a = 6.2360(5) Å, c = 23.588(2) Å, Z = 16; CoGe(2)-type space group Aba2, a = 6.2439(4) Å, b = 6.2493(4) Å, c = 11.778(1) Å, Z = 8). Both structures have the same building unit consisting of three consecutive planar nets 4(4), 3(2)434, 4(4) formed by tin atoms. The condensation of the building blocks in the c direction gives rise to different stacking sequences. In the PdSn(2)-type, a ABCD sequence is realized and the c axis is doubled compared to the ideal CoGe(2)-type with AB stacking. Electron diffraction and high-resolution electron microscopy studies revealed the existence of other, complex, stacking variants as well as stacking faults in the Co(1)(-)(x)()Ni(x)()Sn(2) system.

Journal Article↗
PubMed2003

Solid phase micro-extraction in a miniature ion trap mass spectrometer.

Fiber introduction mass spectrometry (FIMS), a variation of solid-phase microextraction (SPME) and membrane introduction mass spectrometry (MIMS), is employed with a miniature mass spectrometer. The inlet system, constructed of commercially available vacuum parts, allows the direct introduction of the SPME needle vacuum chamber into the mass spectrometer. Thermal desorption of the analyte from the poly(dimethylsiloxane) (PDMS) coated fiber was achieved with a built in nichrome heater, followed by electron ionization of the analytes internal to the cylindrical ion trap (CIT). The system has been tested with several volatile organic compounds (VOC) in air and to analyze the headspace over aqueous solutions, with limits of detection in the low ppb range. The signal rise (10-90%) and fall (90-10%) times for the system ranged from 0.1 to 1 s (rise) and 1.2 to 6 s (fall) using heated desorption. In addition, this method has been applied to quantitation of toluene in benzene, toluene, xylene (BTX) mixtures in water and gasoline. This simple and rapid analysis method, coupled to a portable mass spectrometer, has been shown to provide a robust, simple, rapid, reproducible, accurate and sensitive (low ppb range) fieldable approach to the effective in situ analysis of VOC in various matrices.

Ions↗
PubMed2004

Bi-phasic growth patterns in rice.

BACKGROUND AND AIMS: When examining the growth patterns of rice crops for a 5-year period, it was found that the time course of accumulation of above-ground dry matter did not follow a simple sigmoid curve as expected for a monocarpic plant. Instead, there was a decrease in growth around flowering, followed by an increase and then a final decrease of growth at crop maturity. There are two nearly equal phases of growth in rice, with about half of the first phase of vegetative growth preceding reproductive growth. METHODS: Logistic curves were fitted separately to the vegetative parts of the crop and to the reproductive parts (the panicle). When the curves were summed, the combined curve gave a good description of the time course of above-ground dry matter, capturing the pause in growth and its resumption. The overall pattern of growth can be seen to be the result of this bi-phasic nature of the crop. KEY RESULTS: Variations in the panicle phase of growth were shown to be largely a consequence of year-to-year variations in the weather, whereas the vegetative phase seemed largely independent of those variations. CONCLUSIONS: Analysing rice growth as two components, each with a logistic curve, provides insight into the growth processes of the plant and the pattern of yield formation.

Logistic Models↗
PubMed1999

The effect of residual Nyquist ghost in quantitative echo-planar diffusion imaging.

Single-shot diffusion-weighted echo-planar imaging (EPI) is typically used for most clinical diffusion studies due to its low sensitivity to patient motion. Although the Nyquist ghost artifact in EPI can be substantially reduced, there is frequently a residual ghost with low signal intensity. As reported in this study, this residual ghost can produce severe artifacts when maps of the apparent diffusion coefficient (ADC) are calculated from single-shot echo-planar images. The artifacts presented in this paper appear as regions of apparently low ADC which simulate regions of reduced diffusion, but are in fact generated by b-value dependent Nyquist ghosts of the orbits. Data acquired in vivo were used to demonstrate that these artifacts can be avoided by including standard methods of spatial presaturation or fluid-suppression in the diffusion-weighted EPI protocol. In addition, phantom studies were used to illustrate how phase and amplitude variations in the ghost generate the artifacts and theoretical expressions, derived elsewhere, were used to provide a detailed understanding of the artifacts observed in vivo. The level of Nyquist ghost reported for the current generation of commercial scanners suggests that this is a general phenomenon which should be a consideration in all EPI-based diffusion studies. Magn Reson Med 42:385-392, 1999.

Artifacts↗
PubMed1995

Dreaming in the late morning: summation of REM and diurnal cortical activation.

Since the discovery that the characteristics of dreaming sleep are far stronger in Stage 1 rapid eye movement (REM) sleep than in any other biological state, investigators have attempted to determine the relative responsibility of the tonic versus the phasic properties of REM sleep for the different characteristics of dreaming--features such as the amount of information in the dream report, the brightness and clarity of the visual images, shifts in thematic continuity, and incongruities of image and meaning. The present experiment is designed to identify dream characteristics that are specifically associated with tonic changes in level of cortical activation within sleep. It samples reports of imagery and thought during spontaneous variations within one phase of the 24-h diurnal rhythm and across the REM-NREM (non-REM) sleep cycle in order to identify the independent and joint contributions of the two cycles to imagery and thought. The rising phase of the diurnal cycle in the late night and morning was estimated from clock time during the late night and early morning and was varied by delaying the sleep onset and waking time of the subjects. Considered together with other studies, the results suggest that the major determinant of vivid visual imagery and enhanced cognitive activity during sleep is a pattern of subcortical and cortical activation that is common to both the REM phase of the REM-NREM cycle and the activated phase of the 24-h diurnal wake-sleep cycle.

Adult↗
PubMed1998

Five-quantum coherence of I = 52 nuclei: 27Al in an Al2O3 single crystal

Optimal conditions were calculated for excitation and detection of the five-quantum coherence of quadrupolar nuclei with I = 52 in single crystals, observed by the two-pulse sequence (theta1)x - tau1 - (theta2)alpha - tau2, where alpha is the phase cycling angle. Variations in the pulse lengths, the relative values of the nutation frequency omega1 = gammaB1, and the quadrupolar frequency omegaQ as well as in the resonance offset were taken into account. In addition, the effect of the pulse length on the intensity of spectral lines was considered. Theoretical results were compared with experiments on 27Al nuclei in an Al2O3 single crystal. Copyright 1998 Academic Press.

Journal Article↗
PubMed1996

Protein-detergent interactions in single crystals of membrane proteins studied by neutron crystallography.

The detergent micelles surrounding membrane protein molecules in single crystals can be investigated using neutron crystallography combined with H2O/D2O contrast variation. If the protein structure is known then the contrast variation method allows phases to be determined at a contrast where the detergent dominates the scattering. The application of various constraints allows the resulting scattering length density map to be realistically modeled. The method has been applied to two different forms of the membrane protein porin. In one case both hydrogenated and partially deuterated protein were used, allowing the head group and tail to be distinguished.

Crystallography↗
PubMed1999

Mechanisms of Helicobacter pylori infection: bacterial factors.

Since the discovery of H. pylori in 1982 (MARSHALL 1983; WARREN 1983), research on the mechanisms of virulence of H. pylori has advanced substantially. It is now well established that urease and flagella are virulence factors of H. pylori. Although known for some time to be toxic to epithelial cells in vitro, VacA has only recently been established as a virulence factor. The cag pathogenicity island has also emerged as another virulence contender, although the specific genes involved in virulence are still being determined. Other possible virulence factors, not yet confirmed by gene disruptions, are hapA, katA, sodA, cagA, and iron-regulated genes. As of yet, no adhesins have been confirmed as being important for in vivo survival of H. pylori. With the sequence of the H. pylori genome in hand, it should be possible to more easily determine the role of specific genes in virulence. Genes of immediate interest are the OMPs, which may under go phase and antigenic variation and may represent adhesins. Additionally, virulence-related orthologs and vacA-related genes may provide some interesting findings. Once we define the genes that contribute to H. pylori virulence, we may be able to more easily develop novel therapeutic drugs or vaccines to treat and prevent H. pylori infection.

Adhesins, Bacterial↗