Uniform cAMP stimulation of Dictyostelium cells induces localized patches of signal transduction and pseudopodia

The chemoattractant cAMP induces the translocation of cytosolic PHCrac-GFP to the plasma membrane. PHCrac-GFP is a green fluorescent protein fused to a PH domain that presumably binds to phosphatydylinositol polyphosphates in the membrane. We determined the relative concentration of PHCrac-GFP in the cytosol and at different places along the cell boundary. In cells stimulated homogeneously with 1microM cAMP we observed two distinct phases of PHCrac-GFP translocation. The first translocation is transient and occurs to nearly the entire boundary of the cell; the response is maximal at 6-8 s after stimulation and disappears after approximately 20 s. A second translocation of PHCrac-GFP starts after approximately 30 s and persists as long as cAMP remains present. Translocation during this second response occurs to small patches with radius of approximately 4-5 microm, each covering approximately 10% of the cell surface. Membrane patches of PHCrac-GFP are both temporally and spatially closely associated with pseudopodia, which are extended at approximately 10 s from the area with a PHCrac-GFP patch. These signaling patches in pseudopodia of homogeneously stimulated cells resemble the single patch of PHCrac-GFP at the leading edge of a cell in a gradient of cAMP, suggesting that PHCrac-GFP is a spatial cue for pseudopod formation also in uniform cAMP.     

Purification and characterization of beta-actin-rich tumor cell pseudopodia: role of glycolysis

The MSV-MDCK-INV invasive variant of Moloney sarcoma virus (mos) transformed MDCK cells express multiple beta-actin-rich pseudopodia (P. U. Le et al., Cancer Res. 58, 1631-1635, 1998). We show here that the tips of these actively protruding cellular domains are morphologically distinct presenting numerous blebs and selectively pass through 1-microm-pore filters. The pseudopodia were purified from the underside of the filters and a major protein component was identified as the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). By confocal microscopy, GAPDH colocalized with actin in MSV-MDCK-INV pseudopodia localizing this glycolytic enzyme to this site of active actin polymerization. Inhibition of glycolysis with 2-deoxyglucose or oxamate induced a rapid transformation of beta-actin-rich pseudopodia into extended lamellipodia and prevented cell motility. A localized glycolytic supply of energy therefore regulates the formation of beta-actin-rich pseudopodial protrusions and thereby the motility of invasive tumor cells.     

Species-specific effects on the optical signals of voltage-sensitive dyes

Summary The absorption changes of two merocyanine dyes in response to membrane potential changes were measured on several nueronal preparations to see whether the dyes would be useful in recording from these cells.We were able to record large signals without averaging from barnacle and leech neurons. The greatest signal with WW375 was seen at 750 nm. Much smaller increases in transmitted light intensity were seen at all other wavelengths between 500 and 780 nm. In contrast, vertebrate neuronal preparations produced much smaller signals with an entirely different action spectrum. Essentially the same spectrum was seen in cells of the sympathetic ganglion of the bullfrog,Rana catesbiana, dissociated chick spinal cord neurons, or dissociated rat superior cervical ganglion neurons. In each case an action potential was accompanied by increases in transmitted light intensity between 500 and 600 nm and 730 and 780 nm, and decreases in intensity between 600 and 730 nm with the dye WW375, the best dye tested. Similar results were obtained with dye NK2367 on both vertebrate and invertebrate preparations, except that the spectral properties were shifted 30 nm towards the blue. Both dyes caused some photodynamic damage to the cultured neurons after a few minute's exposure to the illuminating light. Several analogues of these dyes were also tried, but did not produce larger signals.     

High resolution dual laser flow cytometry.

Flow cytometers based on optical sensing utilize external light sources and fluorescent dyes to measure one or more specific components or properties of individual cells or subcellular particles in liquid suspension. To provide for independent excitation of two dyes used in double staining experiments we have constructed a high resolution flow cytometer that uses two laser beams to provide two wavelengths of excitation. These beams are separated spatially so that cells flow through them sequentially, with a time separation of about 20 musec. Since the dyes are excited sequentially their emission occurs at different times and their emission spectra may overlap without causing any difficulty in analysis. We have developed new light collection optics that permit up to four measurements to be made on each cell. This approach greatly increases the number of dye combinations that can be used in flow cytometry, thus removing a significant limitation of single illumination instruments.     

涎腺腺样囊性癌细胞蛋白激酶C的活性阻抑对其侵袭能力的影响

用蛋白激酶Ｃ（ＰＫＣ） 抑制剂Ｈ７ 作用于人涎腺腺样囊性癌ＳＡＣＣ８３ 系, 利用扫描电镜观察对人羊膜基底膜侵袭能力的影响。癌细胞接种于羊膜基底膜表面４８－ ７２ 小时后, 在扫描电镜下可以观察到对照组癌细胞侵袭现象, 侵袭的细胞伸出丝状伪足且侵入基底膜内, 伪足周围的基底膜断裂和被溶解, 而实验组癌细胞侵袭现象很难发现。细胞附于基底膜表面, 细胞表面较光滑且无明显伪足伸出, 基底膜无明显受损改变。实验结果表明, 降低涎腺腺样囊性癌细胞的ＰＫＣ活性可明显降低其侵袭能力, ＰＫＣ与涎腺腺样囊性癌的侵袭能力有密切的关系     

Transient induction of photolyase activity in arrested frog cells in response to a short-wave ultraviolet segment of simulated "sunlight"

Induction of photolyase activity was studied in cultured frog cells using clonogenic assays. Exposure of arrested cells to a pre-irradiation (90% survival) of 254 nm ultraviolet light resulted in a transient enhancement of photolyase activity. Cells expressed a decreased level of photolyase activity in response to an equitoxic fluence of simulated "sunlight" wavelengths 280-310 nm. However, no significant increase of enzyme activity was detected in cells following treatment with "sunlight" wavelengths 310-330 nm. In addition, this process depends on newly biosynthesized protein(s).     

Navigation of Lutzomyia longipalpis (Diptera: Psychodidae) under dusk or starlight conditions

The responses of male and female Lutzomyia longipalpis (Lutz & Neiva) to different wavelengths of light was tested by presenting the sandflies with two light sources simultaneously, a series of test wavelengths between 350-670 nm and a 400 nm control. To test whether L. longipalpis could discriminate between the test and control, three sets of experiments were carried out in which the test wavelengths were presented at higher, equivalent or lower intensity than the control. In all three experiments, ultra-violet (350 nm) and blue-green-yellow (490-546 nm) light was more attractive to L. longipalpis than the control wavelength. However, at low intensity, UV was less attractive, than equivalent or higher intensity UV light. At intensities equivalent to or higher than the control wavelength, ultra-violet light was more attractive than blue-green. Furthermore, at low intensity, green-yellow (546 nm) light was more attractive to males whereas blue-green (490 nm) was more attractive to females. Blue-violet (400 nm) and orange-red (600-670 nm) light were least attractive in all three sets of experiments. Response function experiments indicated that the responses were dependent on both intensity and wavelength and that therefore more than one photoreceptor must be involved in the response. The results indicated that L. longipalpis can discriminate between different wavelengths at different intensities and thus have true colour vision. It also suggests that L. longipalpis may be able to navigate at dusk or under moonlight or starlight conditions using light in the blue-green-yellow part of the spectrum. The difference in response of males and females to light in this region is interesting and may indicate the different ecology of the sexes at night. Overall, these results may have important implications for sandfly trap design.     

High light-induced switch from C(3)-photosynthesis to Crassulacean acid metabolism is mediated by UV-A/blue light

The high light-induced switch in Clusia minor from C(3)-photosynthesis to Crassulacean acid metabolism (CAM) is fast (within a few days) and reversible. Although this C(3)/CAM transition has been studied intensively, the nature of the photoreceptor at the beginning of the CAM-induction signal chain is still unknown. Using optical filters that only transmit selected wavelengths, the CAM light induction of single leaves was tested. As controls the opposite leaf of the same leaf pair was studied in which CAM was induced by high unfiltered radiation (c. 2100 micromol m(-2) s(-1)). To evaluate the C(3)-photosynthesis/CAM transition, nocturnal CO(2) uptake, daytime stomatal closure and organic acid levels were monitored. Light at wavelengths longer than 530 nm was not effective for the induction of the C(3)/CAM switch in C. minor. In this case CAM was present in the control leaf while the opposite leaf continued performing C(3)-photosynthesis, indicating that CAM induction triggered by high light conditions is wavelength-dependent and a leaf internal process. Leaves subjected to wavelengths in the range of 345-530 nm performed nocturnal CO(2) uptake, (partial) stomatal closure during the day (CAM-phase III), and decarboxylation of citric acid within the first 2 d after the switch to high light conditions. Based on these experiments and evidence from the literature, it is suggested that a UV-A/blue light receptor mediates the light-induced C(3)-photosynthesis/CAM switch in C. minor.     

Distribution of multiple centrospheres determines migration of BHK syncitia

After fusion of BHK cells with polyethylene glycol, the resulting syncitia contained in 77% of the cases multiple microtubule organizing centers (MTOCs), which were aggregated into a common centrosphere. Based on the observation of phagokinetic tracks, we found that the syncitia were able to locomote if the MTOCs aggregated into a common centrosphere cluster, and the clustered centrospheres were excluded from the cluster of nuclei of the syncitium. The results suggest that each individual pair of one nucleus and one centrosphere contributes, in a process of vectorial addition, its individual polarity to the polarity of the syncitium. Thus the widely accepted idea that the centrosphere is involved in the determination of cell polarity can be generalized beyond the case of single cells.     

CONTACT-INHIBITED REVERTANT CELL LINES ISOLATED FROM SV40-TRANSFORMED CELLS : IV. Microfilament Distribution and Cell Shape in Untransformed, Transformed, and Revertant Balb/c 3T3 Cells

A comparison is made of the ultrastructure of the cell periphery in three cloned cell lines: untransformed Balb/c 3T3 cells, SV40-transformed Balb/c 3T3 cells, and revertant cells obtained from the transformed cell line by a selection technique utilizing concanavalin A. Both thin-section and surface replication techniques are used for in situ examination of the cell lines. Microfilaments, 70 Å in diameter (called alpha filaments), are abundant in untransformed and revertant cell lines, particularly in the anterior expansions of the cells, which tend to have many microvilli and small pseudopodia. Alpha filaments are diminished in the anterior expansions of transformed cells, which contain large blunt pseudopodia and relatively few microvilli. Surface replicas confirm the impression gained from thin sections that transformed cells have a greater proportion of their cell surface involved in bulging pseudopodia than either untransformed or revertant cells. Since alpha filaments are shown to bind heavy meromyosin and are similar to F-actin, these filaments are thought to be important in cell motility. These observations suggest that a close relationship exists between decreased alpha filaments, bulging pseudopodia, and loss of contact inhibition of movement in transformed cells.     

感染环形泰勒焦虫(Theileria annulata)的牛外周血白细胞的形态观察

用光镜及扫描电镜观察了体外高代培养的含牛焦虫颗粒的牛外周血白细胞的形态及在细胞周期中细胞表面的特征性变化。这种经多年传代的含虫的牛外周血白细胞恢复了分裂和繁殖的能力,目前已成为较稳定的细胞系。细胞表面具多种伪足突起,如叶状、丝状及绒毛状。细胞周期中备期细胞表面的主要特征是:S期:细胞平扁,边缘具薄的时状伪足及丝状伪足;G_2期:细胞中部隆起,表面具少量绒毛状伪足;G_1期:绒毛状结构少或无,而出现丝状及小的叶状伪足,细胞仍保持球形;M期:细胞球形,表面密被以绒毛。作者根据扫描电镜的观察认为光镜下所观察的两类细胞,实际上是反映了一种细胞处于不同发育阶段时的特征。     

Platelet-endothelial cell interactions in vitro following freeze-thaw injury or detergent treatment

Summary Platelet interactions with cultured bovine endothelial cells were studied following freeze-thaw damage or detergent tratment. Platelets from whole blood, platelet-rich plasma, or gel-filtered plasma did not interact directly with freeze-thaw-damaged endothelial cells. Freezing and thawing did result in the exposure of an extracellular matrix located beneath the cells, which proved very thrombogenic. Platelets from all sources attached to both microfilament and amorphous components of the extracellular matrix, although only platelets from whole blood demonstrated aggregation and extensive pseudopodia formation. Treatment of cells with Triton-X detergent resulted in exposure of an intracellular cytoskeleton. Most platelets attached to the cytoskeleton were located near the cell border and had one or more pseudopodia either in contact with extracellular or intracellular material. Adhesion of platelets to the extra-cellular matrix may represent platelet-collagen or plateletfibronectin interactions since both are produced by an incorporated into the extracellular matrix. Platelet interaction with endothelial cytoskeletons may represent contact of pseudopodia with the now exposed matrix located beneath the cells. The possibility that platelets also adhered to intra-cellular components could not be eliminated. These findings are in agreement with data from a freeze-thaw injury model of perfused aorta. In addition, they tend to indicate that physical insult is not sufficient to induce platelet interaction with the endothelial surface, but that chemical modification enhances platelet deposition. Disclaimer. The views of the author do not purport to reflect the positions of the Department of the Army or the Department of Defense. (Para. 4-3, AR 360-5).     

Utilization of dimethyl sulfide as a sulfur source with the aid of light by Marinobacterium sp. strain DMS-S1

Strain DMS-S1 isolated from seawater was able to utilize dimethyl sulfide (DMS) as a sulfur source only in the presence of light in a sulfur-lacking medium. Phylogenetic analysis based on 16S ribosomal DNA genes indicated that the strain was closely related to Marinobacterium georgiense. The strain produced dimethyl sulfoxide (DMSO), which was a main metabolite, and small amounts of formate and formaldehyde when grown on DMS as the sole sulfur source. The cells of the strain grown with succinate as a carbon source were able to use methyl mercaptan or methanesulfonate besides DMS but not DMSO or dimethyl sulfone as a sole sulfur source. DMS was transformed to DMSO primarily at wavelengths between 380 and 480 nm by heat-stable photosensitizers released by the strain. DMS was also degraded to formaldehyde in the presence of light by unidentified heat-stable factors released by the strain, and it appeared that strain DMS-S1 used the degradation products, which should be sulfite, sulfate, or methanesulfonate, as sulfur sources.     

Quantitative cytochemistry of 3 beta-hydroxysteroid dehydrogenase activity in avian granulosa cells during follicular maturation

Previous studies have shown that biosynthesis of progesterone, the major steroid product of hen granulosa cells, increases during follicular maturation. However, the contribution of individual granulosa cells to the total progesterone production of each follicle is not known. The objective of the present study was to determine the presence and relative activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in individual granulosa cells isolated from each of the five largest yolk-filled preovulatory follicles of laying hens. 3 beta-HSD cytochemistry in the presence or absence of pregnenolone substrate was performed on digitonin-permeabilized granulosa cells in suspension. The stained cells were fixed in a 70% ethanol solution until 1) the percentage of cells from each follicle that stained dark blue-indicating the presence of 3 beta-HSD activity-was determined by counting under light microscopy, and 2) the intensity of staining-indicating the relative amount of enzyme activity-was quantified using video image analysis. There were three findings. First, 100% of granulosa cells from each of the five largest preovulatory follicles stained positively for the presence of 3 beta-HSD activity. Second, the amount of 3 beta-HSD activity was normally distributed among granulosa cells in the population from each follicle. Third, as follicles matured from the fifth largest to the largest follicle, 3 beta-HSD activity increased steadily in individual cells, as indicated by increased staining intensities. The results indicate uniformity in the steroidogenic capacity of cells in the granulosa layer of hen preovulatory follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

EGF-like module pair 3-4 in vitamin K-dependent protein S: modulation of calcium affinity of module 4 by module 3, and interaction with factor X.

Calcium-binding epidermal growth factor (EGF)-like modules are found in numerous extracellular and membrane proteins involved in such diverse processes as blood coagulation, lipoprotein metabolism, determination of cell fate, and cell adhesion. Vitamin K-dependent protein S, a cofactor of the anticoagulant enzyme activated protein C, has four EGF-like modules in tandem with the three C-terminal modules each harbouring a Ca(2+)-binding consensus sequence. Recombinant fragments containing EGF modules 1-4 and 2-4 have two Ca(2+)-binding sites with dissociation constants ranging from 10(-8) to 10(-5) M. Module-module interactions that greatly influence the Ca(2+) affinity of individual modules have been identified. As a step towards an analysis of the structural basis of the high Ca(2+) affinity, we expressed the Ca(2+)-binding EGF pair 3-4 from human protein S. Correct folding was shown by (1)H NMR spectroscopy. Calcium-binding properties of the C-terminal module were determined by titration with chromophoric chelators; binding to the low-affinity N-terminal site was monitored by (1)H-(15)N NMR spectroscopy. At physiological pH and ionic strength, the dissociation constants for Ca(2+) binding were 1.0x10(-6) M and 4. 8x10(-3) M for modules 4 and 3, respectively, i.e. the calcium affinity of the C-terminal site was about 5000-fold higher than that of the N-terminal site. Moreover, the Ca(2+) affinity of EGF 4, in the pair 3-4, was about 9000-fold higher than that of synthetic EGF 4. The EGF modules in protein S are known to mediate the interaction with factor Xa. We have now found modules 3-4 to be involved in this interaction. However, the individual modules 3 and 4 manifested no measurable activity.     

Application of DNA markers to identify the individual-specific hosts of tsetse feeding on cattle

Primer sets for five different ungulate loci were used to obtain individual microsatellite DNA profiles for 29 Mashona cattle from a herd in Zimbabwe. There were 3-13 alleles for each locus and, using the entire suite of five loci, each animal within the herd, including closely related individuals, could be unequivocally distinguished. Wild-caught Glossina pallidipes Austen (Diptera: Glossinidae) were fed on specific cattle and the bloodmeal was profiled 0.5-72 h after feeding. The individual specific sources of the bloodmeals, including mixe meals produced by allowing tsetse to feed on two different cattle, were reliabl identified up to 24 h after feeding. The technique was used in field studies of hos selection by G. pallidipes and G. morsitans morsitans Westwood (Diptera Glossinidae) attracted to pairs of cattle. When the pair comprised an adult and a calf, 100% of meals were from the adult. For some pairs of adult cattle, tsetse were biased significantly towards feeding on one animal, whereas for other pairs there was no such bias. In general, feeding was greater on the animal known to have lower rate of host defensive behaviour. Results suggest that relatively slight differences in the inherent defensive behaviour of cattle produce large difference in host-specific feeding rates when the hosts are adjacent. For flies attracted to pair of cattle, < 2% contained blood from both hosts. The DNA profiling technique will be useful in studying the epidemiology of vector-borne diseases of livestock.     

Cyanidin-3-O-beta-glucopyranoside protects myocardium and erythrocytes from oxygen radical-mediated damages

The cyanidin-3- O - β-glucopyranoside (C-3-G) antioxidant capacity towards reactive oxygen species (ROS)-mediated damages was assessed in tissue and cells submitted to increased oxidative stress. In the isolated ischemic and reperfused rat heart, 10 or 30 μM C-3-G protected from both lipid peroxidation (66.7 and 94% inhibition of malondialdehyde (MDA) generation in 10 and 30 μM C-3-G-reperfused hearts, respectively, in comparison with control reperfused hearts) and energy metabolism impairment (higher ATP concentration in 10 and 30 μM C-3-G-reperfused hearts than in control reperfused hearts). These effects were associated to C-3-G permeation within myocardial cells, as indicated by results obtained in the isolated rat heart perfused for 30 min in the recirculating Langendorff mode under normoxia with 10 and 30 μM C-3-G. Protective effects were exerted, in a dose-dependent manner, by C-3-G also in 2 mM hydrogen peroxide-treated human erythrocytes. With respect to MDA formation, an apparent IC 50 of 5.12 μM was calculated for C-3-G (the polyphenol resveratrol used for comparison showed an apparent IC 50 of 38.43 μM). The general indications are that C-3-G (largely diffused in dietary plants and fruits, such as pigmented oranges very common in the Mediterranean diet) represents a powerful natural antioxidant with beneficial effects in case of increased oxidative stress, and at pharmacological concentrations it is able to decrease tissue damages occurring in myocardial ischemia and reperfusion.     

Action spectrum of foraging behavior of the Japanese yellow swallowtail butterfly, Papilio xuthus

This paper describes the action spectrum of foraging behavior of a butterfly, Papilio xuthus. We first established an experimental protocol to evaluate learning and discrimination of monochromatic light by the butterflies. We trained butterflies to feed on sucrose solution at the window illuminated with certain monochromatic light produced through a monochromator. After confirming that they learned the monochromatic light, after 10 days of training, we tested the butterflies one by one. We presented training wavelengths for each individual at different intensities, and recorded whether they perform foraging behavior under freely-flying as well as tethered conditions. Freely-flying butterflies responded to light by visiting the window and searching for nectar around it, whereas tethered butterflies responded by extending their proboscides towards the window. The light intensity required to elicit 50% response for each tested monochromatic light was plotted. The resulting action spectrum for the visit was rather flat with the maximum sensitivity a 420 nm, whereas the spectrum for the proboscis extension had prominent peaks at 380, 500 and 600 nm. The difference in action spectra indicates that the visit and the proboscis extension are controlled by two independent mechanisms at least in part.     

Surface-active novel glycolipid and linked 3-hydroxy fatty acids produced by Serratia rubidaea.

A Serratia rubidaea isolate with wetting activity when grown at 30 but not 37 degrees C was examined for the production of specific lipids. Two novel lipids (rubiwettins R1 and RG1) were isolated and shown to be able to lower the surface tension of saline to 26 mN/m. These lipids were located in extracellular vesicles found in a 30 degrees C culture of S. rubidaea. Chemical structures of these biosurfactants were determined by degradation product analyses, infrared spectroscopy, mass spectrometry, and proton nuclear magnetic resonance spectroscopy. Rubiwettin R1 was proposed to be a mixture of 3-(3'-hydroxytetradecanoyloxy)decanoate, 3-(3'-hydroxyhexadecenoyloxy)decanoate, and minor molecular isomers. The structure of rubiwettin RG1 was proposed to be beta-D-glucopyranosyl 3-(3'-hydroxytetradecanoyloxy)decanoate. The importance of such surface-active exolipids in bacterial occupancy on surfaces was suggested.     

Novel Na+ doped Alq3 hybrid materials for organic light‐emitting diode (OLED) devices and flat panel displays

Pure and Na⁺‐doped Alq₃ complexes were synthesized by a simple precipitation method at room temperature, maintaining a stoichiometric ratio. These complexes were characterized by X‐ray diffraction, Fourier transform infrared (FTIR), UV/Vis absorption and photoluminescence (PL) spectra. The X‐ray diffractogram exhibits well‐resolved peaks, revealing the crystalline nature of the synthesized complexes, FTIR confirms the molecular structure and the completion of quinoline ring formation in the metal complex. UV/Vis absorption and PL spectra of sodium‐doped Alq₃ complexes exhibit high emission intensity in comparison with Alq₃ phosphor, proving that when doped in Alq₃, Na⁺ enhances PL emission intensity. The excitation spectra of the synthesized complexes lie in the range 242–457 nm when weak shoulders are also considered. Because the sharp excitation peak falls in the blue region of visible radiation, the complexes can be employed for blue chip excitation. The emission wavelength of all the synthesized complexes lies in the bluish green/green region ranging between 485 and 531 nm. The intensity of the emission wavelength was found to be elevated when Na⁺ is doped into Alq₃. Because both the excitation and emission wavelengths fall in the visible region of electromagnetic radiation, these phosphors can also be employed to improve the power conversion efficiency of photovoltaic cells by using the solar spectral conversion principle. Thus, the synthesized phosphors can be used as bluish green/green light‐emitting phosphors for organic light‐emitting diodes, flat panel displays, solid‐state lighting technology – a step towards the desire to reduce energy consumption and generate pollution free light. Copyright © 2014 John Wiley & Sons, Ltd.