Mechanism of Photoregulated Carotenogenesis in Rhodotorula minuta VII. Action Spectrum for Photoinduced Carotenogenesis

An action spectrum for photoinduced carotenogenesis in the yeast,Rhodotorula minuta, was determined over the wavelength rangefrom 250 nm to 770 nm. The action spectrum had a prominent peak at about 280 nm, withshoulders at 340, 370 and 400 nm. In addition, at wavelengthsfrom 260 nm to 400 nm, all slopes of fluence-response curveswere approximately equal, and reciprocity was obtained at eachwavelength tested. The action spectrum obtained was different from any action spectrumso far reported for photoinduced carotenogenesis and suggeststhat a new type of chromoprotein plays a major role as a photoreceptor. ³Present address: Koshi Agricultural Extension Office of FukuiPrefecture, Matsumoto, Fukui, 910 Japan (Received March 31, 1989; Accepted December 8, 1989)     

Latent Enhancers Activated by Stimulation in Differentiated Cells

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Highlights? Latent enhancers are regulatory elements unmarked and unbound in differentiated cells ? Stimulus-dependent TFs co-opt lineage-determining TFs to activate latent enhancers ? Latent enhancers may confer short-term memory of environmental exposure ? Environmental stimulation qualitatively alters the pre-existing enhancer repertoire     

Mechanism of Photoregulated Carotenogenesis in Rhodotorula minuta VIII. Effect of Mevinolin on Photoinduced Carotenogenesis

Mevinolin, which is a highly specific competitive inhibitorof 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase,was used in a search for photoinducible enzyme(s) other thanHMG-CoA reductase in the pathway of carotenoid biosynthesisin Rhodotorula minuta. The photoinduced production of carotenoids was competitivelyinhibited by mevinolin. The concentration of mevinolin thatis required to inhibit completely the production of carotenoidsdepends on the light dose given to the cells. However, the relationshipbetween the inhibition ratio and the concentration of mevinolinwas almost identical regardless of the light dose. These resultssuggest that the activity of enzymes involved in the formationof HMG-CoA may not be affected by light. When an adequate amount of mevalonate was added to the growthmedium that contained sufficient mevinolin for the completeinhibition of the photoinduction of the production of carotenoids,the same quantity of carotenoids was produced as in the absenceof mevinolin. Moreover, the production of carotenoids in thepresence of both mevinolin and mevalonate was inhibited by cycloheximide. It appears from these results that one or more photoinducibleenzymes, such as HMG-CoA reductase, may be present in the carotenogenicpathway beyond mevalonate. (Received April 12, 1989; Accepted January 16, 1990)     

Closing of Venus Flytrap by Electrical Stimulation of Motor Cells

Electrical signaling and rapid closure of the carnivorous plant Dionaea muscipula Ellis (Venus flytrap) have been attracting the attention of researchers since XIX century, but the exact mechanism of Venus flytrap closure is still unknown. We found that the electrical stimulus between a midrib and a lobe closes the Venus flytrap leaf by activating motor cells without mechanical stimulation of trigger hairs. The closing time of Venus flytrap by electrical stimulation of motor cells is 0.3 s, the same as mechanically induced closing. The mean electrical charge required for the closure of the Venus flytrap leaf is 13.6 µC. Ion channel blockers such as Ba²⁺, TEACl as well as uncouplers such as FCCP, 2,4-dinitrophenol and pentachlorophenol dramatically decrease the speed of the trap closing. Using an ultra-fast data acquisition system with measurements in real time, we found that the action potential in the Venus flytrap has a duration time of about 1.5 ms. Our results demonstrate that electrical stimulation can be used to study mechanisms of fast activity in motor cells of the plant kingdom.Key Words: action potential, electrophysiology, electrical signaling, Venus flytrap, motor cells     

Stimulation of Tubulin Synthesis by Lactate in Isolated Spermatogenic Cells

The effect of lactate on synthesis of new proteins in isolated spermatids and spermatocytes of rats was examined. Lactate stimulated[³⁵S]methionine ([³⁵S]met) incorporation into both spermatids and spermatocytes. The rate of protein synthesis was positively correlated with the intracellular level of ATP. The [³⁵S]met-labeled proteins in the two types of cells were compared by one and two dimensional polyacrylamide gel electrophoresis (1D and 2D-PAGE) and autoradiography. The syntheses of several stagespecific and non-specific proteins were observed. When spermatids and spermatocytes were cultured in medium without lactate, two major proteins of molecular weight (Mr) 43 kD and 55 kD were detected in the water-soluble fraction (105,000 g supernatant), and one major protein of Mr 24 kD was observed in the membrane-rich fraction. Addition of lactate to the incubation medium dramatically increased the synthesis of six proteins (Mr 14 kD, 16 kD, 43 kD, 55 kD, 84 kD and 135 kD) in the water-soluble fractions of spermatids and spermatocytes, but did not stimulate the synthesis of the Mr 24 kD protein in the membrane-rich fraction. In addition, after 1D and 2D-PAGE and electrophoretic transfer to nitrocellulose, two proteins of Mr 43 kD and 55 kD were identified as actin and tubulin, respectively, on the basis of their reactivities with specific antisera. Tubulin was also produced by in vitro translation using a spermatid lysate. These results suggest that lactate may play an important role in changing the cell structure and shape during spermatogenesis by regulating the syntheses of actin and tubulin.     

Differential Stimulation of PEP-carboxylation in Guard Cells and Mesophyll Cells by Ammonium or Fusicoccin

Dark CO₂-fixation in guard cells of Vicia faba was much moresensitive to ammonium than in mesophyll cells. Addition of ammonium(5.0 mol m^–3; pH₀ 7.6) caused up to a 7-fold increasein dark CO₂-fixation rates in guard cell protoplasts (GCP),whereas in leaf slices, mesophyll cells, and mesophyll protoplaststhe increase was only about 1.4-fold. In both cell or tissuetypes, total CO₂-fixation rates were higher in the light (2–12-foldhigher in GCP and 28-fold in mesophyll); these rates were onlyslightly changed by ammonium treatment. However, separationof ¹⁴C-labelled products after fixation of CO₂ in the lightby GCP revealed a large ammonium-induced shift in carbon flowfrom starch and sugars to typical products of C₄-metabolism(mainly malate and aspartate). In contrast, in mesophyll cellsamino acid and malate labelling was only moderately increasedby ammonium at the expense of sucrose. The data suggest thatin vivo ammonium might facilitate stomatal opening and/or delaystomatal closing through an increased production of organicacids. Key words: PEP-carboxylation, guard cell protoplasts, ammonium, fusicoccin     

Biodesulfurization of Dibenzothiophene by Microbial Cells Coated with Magnetite Nanoparticles

Microbial cells of Pseudomonas delafieldii were coated with magnetic Fe₃O₄ nanoparticles and then immobilized by external application of a magnetic field. Magnetic Fe₃O₄ nanoparticles were synthesized by a coprecipitation method followed by modification with ammonium oleate. The surface-modified Fe₃O₄ nanoparticles were monodispersed in an aqueous solution and did not precipitate in over 18 months. Using transmission electron microscopy (TEM), the average size of the magnetic particles was found to be in the range from 10 to 15 nm. TEM cross section analysis of the cells showed further that the Fe₃O₄ nanoparticles were for the most part strongly absorbed by the surfaces of the cells and coated the cells. The coated cells had distinct superparamagnetic properties. The magnetization (δ_s) was 8.39 emu · g⁻¹. The coated cells not only had the same desulfurizing activity as free cells but could also be reused more than five times. Compared to cells immobilized on Celite, the cells coated with Fe₃O₄ nanoparticles had greater desulfurizing activity and operational stability.     

Reliable Detection of Dead Microbial Cells by Using Fluorescent Hydrazides

We have developed a new method for accurate quantification of dead microbial cells. This technique employs the simultaneous use of fluorescent hydrazides and nucleic acid dyes. Fluorescent hydrazides allow detection of cells that cannot be detected with currently used high-affinity nucleic acid dyes. This is particularly important for nongrowing bacterial populations and for multicellular communities containing physiologically heterogeneous cell populations, such as colonies and biofilms.Many different approaches are used to assess the viability of bacterial cells. Currently, the most-used method is determination of the number of CFU, which reflects the ability of cells to reproduce. However, this method is not reliable because a fraction of live bacterial cells cannot divide under standard growth conditions (2) and because some bacteria are killed due to oxidative stress that occurs upon plating (3). Another approach detects dead cells through staining with fluorescent dyes that have high affinities for nucleic acids (RNA and DNA). Examples of this class of dye include Sytox Green (SG) (7). Cells incorporating these stains are then identified using flow cytometry or fluorescence microscopy. Discrimination between live and dead cells is based on the selective entry of dyes such as SG into dead cells, whose membrane integrity is compromised. However, the accessibility of DNA to SG staining also depends on the bacterial cell cycle stage. Cells that die during stationary phase are poorly stained by SG, probably because the DNA topology is altered (7). In addition, it is not possible to detect dead cells whose nucleic acids are degraded.In order to detect dead cells that escape detection by the methods described above, we used an Alexa Fluor hydrazide (AFH) dye. AFH dyes are low-molecular-weight, bright, photostable fluorescent molecules that are generally used as cell tracers by microinjection into eukaryotic cells. Because hydrazine components interact with carbonyl groups (aldehydes and ketones), AFH can be used for the detection of carbonylated proteins (1, 10). The quantity of carbonylated proteins, which are irreversibly damaged, increases after various lethal stresses such as oxidative stress, heat shock, and acidic stress (4). In stationary phase, bacteria also accumulate carbonylated proteins (5). As AFH cannot pass freely across the functional membranes of living cells, we hypothesized that it can be used for the detection of dead cells by tagging carbonylated proteins even when cells are devoid of nucleic acids. For the development of this method, we used Escherichia coli as a model organism.We first compared the results of staining E. coli cells killed by heat treatment with SG and Alexa Fluor 633 hydrazide (AF633H; both purchased from Invitrogen, Carlsbad, CA). Cells from 1- and 15-day-old liquid cultures were killed by incubation at 95°C for 10 min, which reduced the CFU counts in the cultures from approximately 2 × 10⁸/ml to undetectable levels (below 10 CFU/ml). Aliquots of cells taken before and after heat treatment were stained with the two dyes and analyzed with a FACSAria cell sorter and flow cytometer (Becton Dickinson Biosciences, San Jose, CA) (Fig. ​(Fig.1).1). Among cells from the 1-day-old cultures, less than 1% collected before treatment and 99.5% collected after treatment were stained with the two dyes. The 15-day cultures contained large proportions of dead cells even before treatment, as indicated by a decrease in the CFU count from approximately 2 × 10⁹/ml on day 1 to 1 × 10⁸/ml on day 15 (a reduction of 95%). In 15-day cultures, 26 and 89% of cells before heat treatment were stained with SG and AF633H dyes, respectively. After heat treatment, CFU levels were again undetectable, and 99.9% of cells from 15-day cultures were stained with AF633H, while only 36% were stained with SG. This difference in staining of dead cells by SG and AF633H indicates that the ability of dead cells to be stained by SG decreases dramatically with cell age but that the staining of dead cells by AF633H increases with cell age. This may be due at least partly to the fact that AF633H can stain cells devoid of nucleic acids while SG cannot. Reconstruction experiments in which heat-treated and untreated cells from 1- and 15-day-old cultures were mixed in fixed ratios and stained by the two dyes gave expected values (Table ​(Table1),1), showing that AF633H can be used for precise quantification of dead cells. In addition, we observed that AF633H staining remained stable after cell fixation with paraformaldehyde (see Fig. S1 in the supplemental material).Open in a separate windowFIG. 1.Levels of fluorescence of heat-killed microbial cells stained by AF633H and SG are indicated by fluorescence intensity histograms for untreated microbial cells (white histograms) and heat-treated cells (brown histograms). Cells were stained with AF633H (left panels) and SG (right panels). E. coli (MG1665) cells were from 1- and 15-day-old cultures. D. radiodurans, B. subtilis, and S. cerevisiae cells were from 1-day-old liquid cultures. The presence of two peaks of fluorescence for cells stained with SG probably indicates the existence of cells with different numbers of copies of the chromosome.

TABLE 1.

Detection of dead E. coli cells in liquid cultures by AF633H and SG staining^a

Carotenogenesis in Phycomyces

Time course studies of carotenoid production and of mycelial growth in liquid cultures of Phycomyces blakesleeanus wild type [NRRL 1555 (?)], red mutants C9, C10 and C13 and the heterokaryon C2 * C9 are reported. The ratios of the concentrations of lycopene, γ-carotene and β-carotene in the red mutant C13 and in the heterokaryon C2 * C9 during the growth periods were measured. In these strains the concentration of lycopene is close to its final value after 2 days of growth, at a time at which β-carotene is just beginning to be produced. It is suggested that the β-carotene produced late is possibly synthesized via β-zeacarotene.     

Stimulation of Autochthonous Lymphocytes by Cells from Normal and Leukaemic Lines

THE mixed lymphocyte reaction (MLR) can possibly be regarded as an in vitro form of an in vivo phenomenon reflecting the recognition of “non-self” tumour specific or neo-antigens on the surface of lymphoid cells. A reaction similar to the normal MLR but of greater magnitude occurs when irradiated lymphoid cells from lymphoblastoid cell lines (LCL) are added to freshly isolated peripheral lymphocytes from allogeneic individuals^1,2. The intense stimulation which occurred in every case when irradiated cells from various LCL were added to lymphocytes from a large number of individuals³ suggested the presence of extra surface determinants on the cells, which are not present on normal freshly isolated cells. We have investigated whether freshly isolated lymphoid cells could detect and respond to extra antigenic determinants on the surface of cell lines derived more than 3 months earlier from their own lymphoid cells.     

Stimulation of Phosphoinositide Hydrolysis by Serotonin in C6 Glioma Cells

5-Hydroxytryptamine (serotonin or 5-HT) stimulated the incorporation of 32Pi into phosphatidylinositol (PI) but not into polyphosphoinositides in C6 glioma cells with an EC50 of 1.2 X 10(-7) M. The phosphoinositide response was blocked by the 5-HT2 antagonists ketanserin and spiperone but inhibited only partly by methysergide and mianserin. Atropine, prazosin, and yohimbine did not block the response, whereas fluphenazine and haloperidol did so partially but also inhibited basal incorporation by approximately 30%. The 5-HT1A agonist 8-hydroxy-2(di-n-propylamino)tetralin did not cause stimulation. Incubation with 5-HT (1 microM) for 1 h increased the incorporation of [2-3H]myoinositol into all phosphoinositides but not into inositol phosphates (IPs). Li+ alone at 10 mM increased labeling in inositol bisphosphate (IP2) and trisphosphate (IP3), whereas labeling in IP and phosphoinositides remained unaltered. Addition of 5-HT had no effect on this increase. Mn2+ at 1 mM enhanced labeling in PI, PI-4-phosphate, lyso-PI, glycerophosphoinositol, and IP, but the presence of 5-HT again did not cause further stimulation. 5-HT also stimulated the release of IPs in cells prelabeled with [2-3H]myo-inositol, incubated with LiCl (10 mM) and inositol (10 mM), and then exposed to 5-HT (1 microM). Radioactivity in IP2 and IP3 was very low, was stimulated approximately 50% as early as 30 s, and remained elevated for at least 20 min. Radioactivity in IP was at least 10 times as high as in IP3 but was increased only from 3 min on with a peak at 20 min, when the elevation was approximately 40 times that in IP3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photoinduced Carotenogenesis in Chlorotic Euglena gracilis

Light induces β-carotene synthesis in streptomycin-bleached Euglena gracilis Z. Light-adapted, chemostat-grown cells have up to 10-fold as much β-carotene and 25% more protein than similarly grown, dark-adapted cells. Carotenogenesis does not occur under anaerobic conditions or in the presence of diphenylamine, cyanide, or cycloheximide. The blue portion of the spectrum (360-560 nm) is most active in initiating carotenogenesis. The level of cellular carotenoids is influenced by the type of carbon source and to some degree by pH. Phytofluence and ζ-carotene are present in dark-grown cells but not in cells grown aerobically in white light (360-1120 nm). These pigments, however, were present in cells grown in yellow or green light (above 486 nm) or in cells exposed to white light anaerobically. The carotenoids are localized in two types of structures at the light microscope level. A protoporphyrin was isolated from Euglena, and its role as a possible photoreceptor during carotenogenesis is suggested.     

Production of Microbial Cells from Hydrocarbons

Hydrocarbon-assimilating yeasts and bacteria were isolated from soil and sewage. The optimal conditions of cell yield from liquid paraffine by a Torulopsis yeast and a Pseudomonas strain were studied. A Torulopsis yeast gave, in optimal condition, 70 percent cell yield on a weight conversion basis from light oil fraction. In a strain of Pseudomonas the additions of amino acids, Fe^{+ +}, Mg^{+ +} and Ca^{+ +} ions were effective for cell production. This strain showed, in optimal condition, 80 percent cell yield (wt%) from kerosene.     

Carotenogenesis diversification in phylogenetic lineages of Rhodophyta

Carotenoid composition is very diverse in Rhodophyta. In this study, we investigated whether this variation is related to the phylogeny of this group. Rhodophyta consists of seven classes, and they can be divided into two groups on the basis of their morphology. The unicellular group (Cyanidiophyceae, Porphyridiophyceae, Rhodellophyceae, and Stylonematophyceae) contained only β‐carotene and zeaxanthin, “ZEA‐type carotenoids.” In contrast, within the macrophytic group (Bangiophyceae, Compsopogonophyceae, and Florideophyceae), Compsopogonophyceae contained antheraxanthin in addition to ZEA‐type carotenoids, “ANT‐type carotenoids,” whereas Bangiophyceae contained α‐carotene and lutein along with ZEA‐type carotenoids, “LUT‐type carotenoids.” Florideophyceae is divided into five subclasses. Ahnfeltiophycidae, Hildenbrandiophycidae, and Nemaliophycidae contained LUT‐type carotenoids. In Corallinophycidae, Hapalidiales and Lithophylloideae in Corallinales contained LUT‐type carotenoids, whereas Corallinoideae in Corallinales contained ANT‐type carotenoids. In Rhodymeniophycidae, most orders contained LUT‐type carotenoids; however, only Gracilariales contained ANT‐type carotenoids. There is a clear relationship between carotenoid composition and phylogenetics in Rhodophyta. Furthermore, we searched open genome databases of several red algae for references to the synthetic enzymes of the carotenoid types detected in this study. β‐Carotene and zeaxanthin might be synthesized from lycopene, as in land plants. Antheraxanthin might require zeaxanthin epoxydase, whereas α‐carotene and lutein might require two additional enzymes, as in land plants. Furthermore, Glaucophyta contained ZEA‐type carotenoids, and Cryptophyta contained β‐carotene, α‐carotene, and alloxanthin, whose acetylenic group might be synthesized from zeaxanthin by an unknown enzyme. Therefore, we conclude that the presence or absence of the four enzymes is related to diversification of carotenoid composition in these three phyla.     

利用异化金属还原菌构建含糖微生物燃料电池

环境中的一些微生物通过还原金属氧化物进行无氧呼吸,而石墨电极与金属氧化物相似,也可以作为这类微生物呼吸作用的最终电子受体,利用这类微生物构建微生物燃料电池,以糖类物质为燃料,对电池产电情况、产电原理进行研究。实验结果表明,以Rhodoferaxferrireducens为产电微生物,在外接电阻510Ω条件下,以葡萄糖为燃料,常温下产生的电流密度达158mAm2(平台电压为0.46V,电极有效接触表面积为57cm2),且循环性能良好。更换燃料为其它糖,发现微生物可以利用多种糖进行产电;通过SEM观察发现大量微生物吸附在石墨电极上,用Bradford法对运行20d后电池的细胞量进行定量,测得悬浮细胞蛋白浓度为140mgL,吸附在电极上的生物量为1180mgm2。通过数据采集分析和细菌还原实验,发现吸附在电极上的微生物对电压的产生贡献最大,具有电化学和生物学活性;悬浮细胞对产电贡献很小,不具有电化学和生物学活性。     

Microbial Community Analysis of Anodes from Sediment Microbial Fuel Cells Powered by Rhizodeposits of Living Rice Plants

By placing the anode of a sediment microbial fuel cell (SMFC) in the rhizosphere of a rice plant, root-excreted rhizodeposits can be microbially oxidized with concomitant current generation. Here, various molecular techniques were used to characterize the composition of bacterial and archaeal communities on such anodes, as influenced by electrical circuitry, sediment matrix, and the presence of plants. Closed-circuit anodes in potting soil were enriched with Desulfobulbus-like species, members of the family Geobacteraceae, and as yet uncultured representatives of the domain Archaea.Living plants release substantial amounts of carbon in the soil as rhizodeposits, which are to a large extent transformed into the greenhouse gas methane in wetlands (21). It was recently demonstrated (8, 33) that the rhizodeposits can be harvested by plant microbial fuel cells (plant MFCs) and transformed into electricity. In its most straightforward form, a plant MFC is an adaptation of a sediment MFC (SMFC), which has an anode buried in (planted) sediment, allowing (microbial) oxidation of reduced compounds, and a cathode in the overlying water.The roots and surrounding rhizosphere in a plant SMFC add an extra parameter to the as yet multifaceted SMFC system. In the present study, two molecular profiling techniques (denaturing gradient gel electrophoresis [DGGE] and terminal restriction fragment length polymorphism [T-RFLP]) will be applied to evaluate the effect of plant presence, support material, operation of the electrical circuit, and anode depth on the bacterial and archaeal communities associated with rice SMFC anodes. Phylogenetic analysis will give further insight in their composition.     

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared light by water in neuronal tissue. This technique holds great potential for replacing or complementing standard stimulation techniques, due to the potential for increased localization of the stimulus and minimization of mechanical contact with the tissue. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Moreover, thermal modelling suggests that cumulative heating effects might be potentially hazardous when multiple stimulus sites or high laser repetition rates are used. The protocol outlined below describes an enhanced approach to the infrared stimulation of neuronal cells. The underlying mechanism is based on the transient heating associated with the optical absorption of gold nanorods, which can cause triggering of neuronal cell differentiation and increased levels of intracellular calcium activity. These results demonstrate that nanoparticle absorbers can enhance and/or replace the process of infrared neural stimulation based on water absorption, with potential for future applications in neural prostheses and cell therapies.     

High-Frequency Stimulation of Excitable Cells and Networks

High-frequency (HF) stimulation has been shown to block conduction in excitable cells including neurons and cardiac myocytes. However, the precise mechanisms underlying conduction block are unclear. Using a multi-scale method, the influence of HF stimulation is investigated in the simplified FitzhHugh-Nagumo and biophysically-detailed Hodgkin-Huxley models. In both models, HF stimulation alters the amplitude and frequency of repetitive firing in response to a constant applied current and increases the threshold to evoke a single action potential in response to a brief applied current pulse. Further, the excitable cells cannot evoke a single action potential or fire repetitively above critical values for the HF stimulation amplitude. Analytical expressions for the critical values and thresholds are determined in the FitzHugh-Nagumo model. In the Hodgkin-Huxley model, it is shown that HF stimulation alters the dynamics of ionic current gating, shifting the steady-state activation, inactivation, and time constant curves, suggesting several possible mechanisms for conduction block. Finally, we demonstrate that HF stimulation of a network of neurons reduces the electrical activity firing rate, increases network synchronization, and for a sufficiently large HF stimulation, leads to complete electrical quiescence. In this study, we demonstrate a novel approach to investigate HF stimulation in biophysically-detailed ionic models of excitable cells, demonstrate possible mechanisms for HF stimulation conduction block in neurons, and provide insight into the influence of HF stimulation on neural networks.     

Alleviation of Stress-Induced Damage to Rat Brain Cells by Transcranial Electromagnetic Stimulation

Biophysics - The effect of transcranial electromagnetic stimulation on immobilization stress-induced damage to rat brain cells was studied. Electromagnetic stimulation was performed by microwave...