Quantification of Magnetically Induced Changes in ECM Local Apparent Stiffness

The stiffness of the extracellular matrix (ECM) is known to influence cell behavior. The ability to manipulate the stiffness of ECM has important implications in understanding how cells interact mechanically with their microenvironment. This article describes an approach to manipulating the stiffness ECM, whereby magnetic beads are embedded in the ECM through bioconjugation between the streptavidin-coated beads and the collagen fibers and then manipulated by an external magnetic field. It also reports both analytical results (obtained by formal modeling and numerical simulation) and statistically meaningful experimental results (obtained by atomic force microscopy) that demonstrate the effectiveness of this approach. These results clearly suggest the possibility of creating desired stiffness gradients in ECM in vitro to influence cell behavior.     

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.     

Quantification of Local Morphodynamics and Local GTPase Activity by Edge Evolution Tracking

Advances in time-lapse fluorescence microscopy have enabled us to directly observe dynamic cellular phenomena. Although the techniques themselves have promoted the understanding of dynamic cellular functions, the vast number of images acquired has generated a need for automated processing tools to extract statistical information. A problem underlying the analysis of time-lapse cell images is the lack of rigorous methods to extract morphodynamic properties. Here, we propose an algorithm called edge evolution tracking (EET) to quantify the relationship between local morphological changes and local fluorescence intensities around a cell edge using time-lapse microscopy images. This algorithm enables us to trace the local edge extension and contraction by defining subdivided edges and their corresponding positions in successive frames. Thus, this algorithm enables the investigation of cross-correlations between local morphological changes and local intensity of fluorescent signals by considering the time shifts. By applying EET to fluorescence resonance energy transfer images of the Rho-family GTPases Rac1, Cdc42, and RhoA, we examined the cross-correlation between the local area difference and GTPase activity. The calculated correlations changed with time-shifts as expected, but surprisingly, the peak of the correlation coefficients appeared with a 6–8 min time shift of morphological changes and preceded the Rac1 or Cdc42 activities. Our method enables the quantification of the dynamics of local morphological change and local protein activity and statistical investigation of the relationship between them by considering time shifts in the relationship. Thus, this algorithm extends the value of time-lapse imaging data to better understand dynamics of cellular function.     

Environmentally Induced Heritable Changes in Flax

Some flax varieties respond to nutrient stress by modifying their genome and these modifications can be inherited through many generations. Also associated with these genomic changes are heritable phenotypic variations ^1,2. The flax variety Stormont Cirrus (Pl) when grown under three different nutrient conditions can either remain inducible (under the control conditions), or become stably modified to either the large or small genotroph by growth under high or low nutrient conditions respectively. The lines resulting from the initial growth under each of these conditions appear to grow better when grown under the same conditions in subsequent generations, notably the Pl line grows best under the control treatment indicating that the plants growing under both the high and low nutrients are under stress. One of the genomic changes that are associated with the induction of heritable changes is the appearance of an insertion element (LIS-1) ^{3, 4} while the plants are growing under the nutrient stress. With respect to this insertion event, the flax variety Stormont Cirrus (Pl) when grown under three different nutrient conditions can either remain unchanged (under the control conditions), have the insertion appear in all the plants (under low nutrients) and have this transmitted to the next generation, or have the insertion (or parts of it) appear but not be transmitted through generations (under high nutrients) ⁴. The frequency of the appearance of this insertion indicates that it is under positive selection, which is also consistent with the growth response in subsequent generations. Leaves or meristems harvested at various stages of growth are used for DNA and RNA isolation. The RNA is used to identify variation in expression associated with the various growth environments and/or t he presence/absence of LIS-1. The isolated DNA is used to identify those plants in which the insertion has occurred.     

Angiotensin-Induced Changes in the Apparent Size of Rat Liver Angiotensin Receptors

Abstract

Angiotensin receptors from rat liver were labeled using four different ligands : (Sar¹-(³H)Tyr⁴)-Angiotensin II ((³H)SarAII); (Sar¹-(³H)Tyr⁴-IIe⁸) -Angiotensin II ((³H)SarIIeAII); (Sar¹-(¹²⁵I)Tyr⁴-(4′-N₃)Phe⁸)-Anglotensin II (IN₃AII); (Sar¹-(¹²⁵I)Tyr⁴-(4′N₃D-Phe)⁸)-Angiotensin II (IN₃DPheAII) (³H) SarAII and IN₃AII behaved like agonists and (³H) SarlleAII and IN₃DPheAII like antagonists. All four ligands labeled the same population of sites. The azido derivatives allowed covalent labeling of receptors with a high yield (about 40%). Membranes were solubilized by Triton X-100 under experimental conditions which ensured complete solubilization of the liganded receptors in a stable form (less than 40% dissociation after 20 h). The apparent size of liganded angiotensin receptors was determined by gel filtration on Ultrogel ACA-34 columns and by SDS gel electrophoresis (in the case of covalent labeling). The apparent Stokes radius of solubilized angiotensin receptors was different wether the receptor was labeled with an agonist (Stokes radius = 6.2 ± 0. 1 nm (6) after labeling with (³H) SarAII) or with an antagonist (Stokes radii of 5. 5 ± 0. 1 (7), and 5.6 ± 0.1 nm (4) after labeling with (³H) SarIIeAII and IN₃DPheAII respectively). After covalent labeling with IN₃All anglotensin receptors were eluted as a mixture of light and heavy forms. SDS gel electrophoresis revealed only one molecular entity of M_r 64,000. It is concluded that binding of an agonist to liver angiotensin receptors triggers or stabilizes an interaction with another membrane component Involved in the coupling of the receptor to its primary effector.     

利用竞争型ELISA法鉴定硒诱导的金属硫蛋白

利用竞争型ELISA法鉴定硒诱导的金属硫蛋白 (MT) ,发现对照组小鼠肝脏MT含量为 (2 .47± 0 .90 )μg/g湿重组织 ,硫酸锌组小鼠肝脏MT含量为 (8.15± 2 .2 0 ) μg/ g湿重组织 ,硒麦芽组小鼠肝脏MT含量为(12 .80± 1.44 ) μg/ g湿重组织。锌组和硒组的MT含量与对照组相比有显著性差异 (P <0 .0 5 ,P <0 .0 5 )。硒组MT含量要显著高于锌组的MT含量 (P <0 .0 5 )。     

Changes in Pollen Autofluorescence Induced by Ozone

The changes in the autofluorescence spectra of intact Philadelphus grandiflorus, Epiphyllum hybridum, and Plantago major pollen have been observed with microspectrofluorometric method after 100-h exposure to ozone (3 h per day for 5 d per week in total dose 5.0 cm³ m^-3). The fluorescence maximum at 530 - 550 nm disappeared in carotenoid-containing pollen of Philadelphus grandiflorus and Epiphyllum hybridum and new maximum at 475 - 480 nm arose that correlated with lipofuscin-like substances observed in the extracts from the pollen grains. The carotenoid-less pollen of Plantago major showed only the increase of the maximum at 470 nm, and no lipofuscin in the extracts. It is supposed that main indicator of the pollen damage by ozone in carotenoid-containing microspores are the lipofuscin-like compounds. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cytoskeleton Remodeling Induces Membrane Stiffness and Stability Changes of Maturing Reticulocytes

Reticulocytes, the precursors of erythrocytes, undergo drastic alterations in cell size, shape, and deformability during maturation. Experimental evidence suggests that young reticulocytes are stiffer and less stable than their mature counterparts; however, the underlying mechanism is yet to be fully understood. Here, we develop a coarse-grained molecular-dynamics reticulocyte membrane model to elucidate how the membrane structure of reticulocytes contributes to their particular biomechanical properties and pathogenesis in blood diseases. First, we show that the extended cytoskeleton in the reticulocyte membrane is responsible for its increased shear modulus. Subsequently, we quantify the effect of weakened cytoskeleton on the stiffness and stability of reticulocytes, via which we demonstrate that the extended cytoskeleton along with reduced cytoskeleton connectivity leads to the seeming paradox that reticulocytes are stiffer and less stable than the mature erythrocytes. Our simulation results also suggest that membrane budding and the consequent vesiculation of reticulocytes can occur independently of the endocytosis-exocytosis pathway, and thus, it may serve as an additional means of removing unwanted membrane proteins from reticulocytes. Finally, we find that membrane budding is exacerbated when the cohesion between the lipid bilayer and the cytoskeleton is compromised, which is in accord with the clinical observations that erythrocytes start shedding membrane surface at the reticulocyte stage in hereditary spherocytosis. Taken together, our results quantify the stiffness and stability change of reticulocytes during their maturation and provide, to our knowledge, new insights into the pathogenesis of hereditary spherocytosis and malaria.     

Time-dependent Changes in the Auxin Sensitivity of Coleoptile Segments: Apparent Sensory Adaptation

When segments are excised from corn (Zea mays L.) coleoptiles they exhibit a very low rate of elongation for about 3.5 hours. A strong increase in growth rate (the spontaneous growth response) then occurs and persists for many hours. During the latent period preceding the spontaneous growth response there is an apparent increase with time in the sensitivity of the segments to indoleacetic acid (IAA). This increase in sensitivity is expressed as a 2- to 3-fold increase in the magnitude of the growth response to low levels of IAA and a 3-fold decrease in the latent period of the response during the first 3 hours following excision. A similar increase in sensitivity to low levels of IAA is noted if application of IAA is timed from the point of termination of a previous exposure to the hormone. Since the increase in responsiveness to low levels of IAA is not paralleled by an increase in the rate of uptake of the hormone, the data may be interpreted as evidence for a type of time-dependent sensory adaptation to auxin. The IAA dose-response relationship also changes with time, and there is indirect evidence that an auxin-dependent inhibitor may influence the expression of the apparent sensory adaptation to auxin.     

Spectral Changes in Anacystis nidulans Induced by Chilling

When Anacystis nidulans, strain TX 20 was grown at 39 C, then rapidly chilled to 0 C, a pigment with a carotenoid-like spectrum was bleached. This effect was not seen when cells which had been grown at 25 C were chilled. The effect seen in 39 C-grown cells was not reversible except under extreme conditions such as heating to near boiling for several minutes. Bleaching could be prevented by prior exposure of cells to glutaraldehyde, but could not be reversed by glutaraldehyde treatment following chilling. The effect occurred upon chilling 39 C-grown cells even after extensive heating at 85 C, a treatment which destroys phycocyanin and metabolic activities. 25 C-grown cells were induced to bleach by chilling when suspended in 50% glycerol. The results are interpreted as indicating a chill-induced change in aggregation state of a carotenoid, which changes its specific absorbance.     

Changes Induced in Peroxidase Activity under Simulated Hypo-gravity

A new method of inducing and maintaining a hypo-gravity environment is described. Using this new technique (buoyancy compensation), the effect of reduced gravitational stress on dwarf marigolds (Tagetes patula) was studied. It was found that the specific activity of peroxidase was elevated significantly in gravity compensated seedlings. This effect, discovered with the buoyancy compensators, was confirmed with plants grown on traditional clinostats.     

Light Induced Concentration Changes of Adenosine-Triphosphate in Phycomyces Sporangiophores

The concentrations of extractable adenosine triphosphate (ATP) following the induction of positive light-growth responses in Phycomyces sporangiophores by blue light stimuli have been measured by means of the luciferin-luciferase assay. The ATP concentration in the light-sensitive growing zone increases 30 to 50% within 30 seconds after the start of a light stimulus and returns to the normal adapted level within 1 minute after stimulation. The ATP concentration is constant for any level of light adaptation and is uniform along the length of sporangiophores even though the light sensitivity is confined to a growing zone less than 5 mm long. These results suggest that one of the initial biochemical steps after a light stimulus is the production of extractable ATP.     

Applications of ECM analogs in surgery

The loss of tissue mass in human has been conventionally treated as an irreversible change. Treatments have emaphesized repalcement of the missing function by use of a transparent, an autograft, tissue synthesize in vitro or, mst commanly, by use of engineering deviced based on biomaterials. During the last few years solid progress has been made in the area of tissue and organ regeneratioin. This new approach is based on the discovery that certain simple chemiaal analouges of extraculler matpices synthesized by graft copolymerization of a glycosaminoglycan onto type 1 collagen can induced sylthesis of physiologic tissue in lesion which otherwise heal spontaneousely by synthesis of scar host tissue. However, regration in the adult mammal has been sucessfully demonstrated so far only in skin (human, guinea pig), scitic nerve(rat) and the inee menisaus (dog).     

An Apparent Trade-Off between Direct and Signal-Based Induced Indirect Defence against Herbivores in Willow Trees

Signal-based induced indirect defence refers to herbivore-induced production of plant volatiles that attract carnivorous natural enemies of herbivores. Relationships between direct and indirect defence strategies were studied using tritrophic systems consisting of six sympatric willow species, willow leaf beetles (Plagiodera versicolora), and their natural predators, ladybeetles (Aiolocaria hexaspilota). Relative preferences of ladybeetles for prey-infested willow plant volatiles, indicating levels of signal-based induced indirect defence, were positively correlated with the vulnerability of willow species to leaf beetles, assigned as relative levels of direct defence. This correlation suggested a possible trade-off among the species, in terms of resource limitation between direct defence and signal-based induced indirect defence. However, analyses of volatiles from infested and uninfested plants showed that the specificity of infested volatile blends (an important factor determining the costs of signal-based induced indirect defence) did not affect the attractiveness of infested plant volatiles. Thus, the suggested trade-off in resource limitation was unlikely. Rather, principal coordinates analysis showed that this ‘apparent trade-off’ between direct and signal-based induced indirect defence was partially explained by differential preferences of ladybeetles to infested plant volatiles of the six willow species. We also showed that relative preferences of ladybeetles for prey-infested willow plant volatiles were positively correlated with oviposition preferences of leaf beetles and with the distributions of leaf beetles in the field. These correlations suggest that ladybeetles use the specificity of infested willow plant volatiles to find suitable prey patches.     

Frequency-Dependent Changes of Local Resting Oscillations in Sleep-Deprived Brain

Sleep deprivation (SD) adversely affects brain function and is accompanied by frequency dependent changes in EEG. Recent studies have suggested that BOLD fluctuations pertain to a spatiotemporal organization with different frequencies. The present study aimed to investigate the frequency-dependent SD-related brain oscillatory activity by using the amplitude of low-frequency fluctuation (ALFF) analysis. The ALFF changes were measured across different frequencies (Slow-4: 0.027–0.073 Hz; Slow-5: 0.01–0.027 Hz; and Typical band: 0.01–0.08 Hz) in 24 h SD as compared to rested wakeful during resting-state fMRI. Sixteen volunteers underwent two fMRI sessions, once during rested wakefulness and once after 24 h of SD. SD showed prominently decreased ALFF in the right inferior parietal lobule (IPL), bilateral orbitofrontal cortex (OFC) and dorsolateral prefrontal cortex (DLPFC), while increased ALFF in the visual cortex, left sensorimotor cortex and fusiform gyrus. Across the Slow-4 and Slow-5, results differed significantly in the OFC, DLPFC, thalamus and caudate in comparison to typical frequency band; and Slow-4 showed greater differences. In addition, negative correlations of behavior performance and ALFF patterns were found mainly in the right IPL across the typical frequency band. These observations provided novel insights about the physiological responses of SD, identified how it disturbs the brain rhythms, and linked SD with frequency-dependent alterations in amplitude patterns.     

Gibberellin Induced Changes in Diffusible Auxins from Savoy Cabbage

Diffusates from apices of young plants of savoy cabbage treated with gibberellie acid (GA) and apices of control plants have been examined with respect to their content of Indole auxins. Three indole Compounds were detected and identified on the basis of their chromatographic characteristics in several systems. These compounds were: glucoubrassicin, indole-3-acetic acid (IAA) and indole-3-acetonitrile (IAN). An effect of GA on the total auxin activity of the diffusate was noted 90 hours after treatment, while an increase in stem height occurred 48 hours later. This increase in auxin effect of the entire diffusates was shown bv chromogenic development and bioassay of chromatograms of diffusates to be a result of an increase in level of the IAA content. A concomitant decrease in I the glucobrassicin content was indicated. Since GA was found to have no effect on the enzymatic conversion of tryptophan or tryptamine to IAA, it is proposed that the effect of GA is on the conversion of glucobrassicin to IAA.