Biphasic kinetic behavior of rat cytochrome P-4501A1-dependent monooxygenation in recombinant yeast microsomes

Rat cytochrome P-4501A1-dependent monooxygenase activities were examined in detail using recombinant yeast microsomes containing rat cytochrome P-4501A1 and yeast NADPH-P-450 reductase. On 7-ethoxycoumarin, which is one of the most popular substrates of P-4501A1, the relationship between the initial velocity (v) and the substrate concentration ([S]) exhibited non-linear Michaelis-Menten kinetics. Hanes-Woolf plots ([S]/v vs. [S]) clearly showed a biphasic kinetic behavior. Aminopyrine N-demethylation also showed a biphasic kinetics. The regression analyses on the basis of the two-substrate binding model proposed by Korzekwa et al. (Biochemistry 37 (1998) 4137-4147) strongly suggest the presence of the two substrate-binding sites in P-4501A1 molecules for those substrates. An Arrhenius plot with high 7-ethoxycoumarin concentration showed a breakpoint at around 28 degrees C probably due to the change of the rate-limiting step of P-4501A1-dependent 7-ethoxycoumarin O-deethylation. However, the addition of 30% glycerol to the reaction mixture prevented observation of the breakpoint. The methanol used as a solvent of 7-ethoxycoumarin was found to be a non-competitive inhibitor. Based on the inhibition kinetics, the real V(max) value in the absence of methanol was calculated. These results strongly suggest that the recombinant yeast microsomal membrane containing a single P-450 isoform and yeast NADPH-P-450 reductase is quite useful for kinetic studies on P-450-dependent monooxygenation including an exact evaluation of inhibitory effects of organic solvents.     

Regulation of the H+ pump activity in the plasma membrane of internally perfused Chara corallina

The role of cytoplasm for the maintenance of the H+ pump activity in Chara corallina internodal cells was examined by the intracellular perfusion technique. Cytoplasm-rich and -poor states were obtained by changing the perfusion time, short-term (less than 2 min) and long-term (more than 5 min), respectively. A large portion of cytoplasm was left by short-term perfusion but most of the cytoplasm was removed by long-term perfusion. The activities of the H+ pump of these two different conditions were examined by measuring current-voltage relation (I-V curve) and conductance-voltage relation (G-V curve) under voltage clamp conditions. The H+ pump conductance decreased to 37%, 9% and zero by short-term, long-term and hexokinase perfusion, respectively, whereas the passive channel conductance decreased to 71%, 39% and 73% by short-term, long-term and hexokinase perfusion, respectively. On the other hand, the electromotive-force of the H+ pump (approximately -260 mV) and the passive channel (approximately -130 mV) were not affected by either short- or long-term perfusion. It is indicated that the cytoplasm plays an essential role to regulate the activity of both the H+ pump and the passive channel together with ATP.     

Plasma lipoproteins behave as carriers of extracellular sphingosine 1-phosphate: is this an atherogenic mediator or an anti-atherogenic mediator?

Sphingosine 1-phosphate (S1P) concentration in plasma and serum has been estimated to be within 200-900 nM. Among plasma and serum components, S1P is concentrated in lipoprotein fractions with a rank order of high-density lipoprotein (HDL)>low-density lipoprotein (LDL)>very low-density lipoprotein (VLDL)>lipoprotein-deficient plasma (LPDP) when expressed as the per unit amount of protein. It is well known that LDL, especially oxidized LDL, is closely correlated and HDL is inversely correlated, with the risk of cardiovascular disease, such as atherosclerosis. Evidence was presented that a part of HDL-induced actions previously reported are mediated by the lipoprotein-associated S1P. Furthermore, S1P content in LDL was markedly decreased during its oxidation. This paper will discuss whether S1P is an atherogenic mediator or an anti-atherogenic mediator.     

Forced symbiosis between synechocystis spp. PCC 6803 and apo-symbiotic Paramecium bursaria as an experimental model for evolutionary emergence of primitive photosynthetic eukaryotes

Single-cell green paramecia (Paramecium bursaria) is a swimming vehicle that carries several hundred cells of endosymbiotic green algae. Here, a novel model for endo-symbiosis, prepared by introducing and maintaining the cells of cyanobacterium (Synechocystis spp. PCC 6803) in the apo-symbiotic cells of P. bursaria is described.Key words: green paramecia, cyanobacteria, evolution, diagostic PCR, Escherichia coli, food vacuoleSingle-cell Paramecium bursaria, or green paramecia, is often described as a swimming vehicle that carries several hundred cells of endo-symbiotic green algae that are morphologically and genetically almost identical to Chlorella species.¹ Recent bioengineering studies have demonstrated that the high capacity for symbiotic algae inside the host cells can be replaced with various natural and artificial particles such as fluorescent and magnetic microspheres.^2,3This organism has attracted the attention of cell biologists, biochemists and ecologists since P. bursaria serves as an excellent experimental model for studying the nature of endo-symbiosis in which one species propagates inside the cells of other species under the precise control through the chemical communications between the host and symbiont cells, and knowledge on the recognition of the symbiotic partners, exchange of chemicals and regulation of metabolic processes have been documented.^4–6 It is well known that synchronization on the algal cell division is imposed by the hosting paramecia possibly through chemical communication between the partners.⁷ In our recent study focusing on the impacts of the host''s cell cycle and growth status on the life cycle in endo-symbiotic algae, flow-cytometric analysis has revealed that the life cycle of symbiotic algae is largely affected by the growth status of the hosting cells.⁸Occasionally, apo-symbiotic cells of P. bursaria (thus lacking algae) can be found in natural water environments⁹ and also in dark-grown culture of P. bursaria.¹⁰ Interestingly, alga-free cell strains of P. bursaria can be artificially prepared by treating the stocks of green paramecia with cycloheximide¹¹ or some herbicides.^12,13 Some groups have shown that independently cultured apo-symbiotic host cells and ex-symbiotic algae can re-associate and re-establish the symbiotic relationship.^14,15 Due to this experimentally reproducible symbiotic nature, P. bursaria can be the best model for studying the mechanism (and possibly the origins) of endo-symbiosis. In our effort to elucidate the mechanism required for successful symbiosis, our recent report has described a case of symbiosis distortion leading to unregulated growth of symbiotic algae, and the significance and advantage of such material for studying the nature and origin of endo-symbiosis were discussed in reference ³.Above studies suggest that in the evolutional time scale required for emergence of a novel photosynthetic organism, the history of symbiosis in P. bursaria is likely recent; thus, both the symbiont and host organisms are still on the process for developing the mechanism in which partners become highly dependent on each other. From such evolutional points of view, the symbiosis between algae and ciliate in P. bursaria is most likely a fruit of co-evolution between two organisms in which host species developed its tolerance to the presence of photosynthetic symbionts which behave as the source of both the sugars and photosynthesis-associated oxidative stresses.¹⁶Our aim in this study was for experimentally reproducing the conditions mimicking the first contact and development of symbiosis between unicellular ciliate protozoa and photosynthetic bacteria as a novel model for studying the very early evolutional processes for the emergence of photosynthetic eukaryotes, the hypothetical ancestorial organisms of plants.¹⁷ In fact, in our system, the preparation of apo-sympiotic P. bursaria (white cells) after forced algal removal from symbiotic P. bursaria (green cells) allows us loading of any particles of interests both biological and non-biological into the ciliate cells;^2,3 therefore the fate of experimentally loaded particles or organism is of great interest. Here, we describe a novel model endo-symbiotic complex formed between the cells of cyanobacterium (Synechocystis spp. PCC 6803) and the hosting cells derived from alga-removed P. bursaria.P. bursaria strain INA-1 (Fig. 1A; syngen 1, shown to be mating type I as recently tested),¹⁷ which has endosymbiotic green algae (Fig. 1B) was originally collected from the Ongagawa River (Kama-city, Fukuoka Pref., Japan) as described in reference ².Open in a separate windowFigure 1Light microscopic images of P. bursaria and its ex-endosymbiotic algae. (A) Matured cell of P. bursaria harboring the symbiotic green algae. (B) Ex-symbiotic algae isolated from P. bursaria. (C) Alga-free apo-symbiotic host cell of P. bursaria. (D) Cyanobacteria (Synechocystis spp. PCC 6803).Since the cell line was established after single cell isolation, all the cells in the culture were clones sharing identical genetic background. Apo-symbiotic white strain of P. bursaria was prepared from natural green strain (INA-1) as previously described in reference ². These strains were maintained in the lettuce infusion inoculated with the food bacterium Klebsiella pneumoniae 24 h prior to the subculturing of ciliate cells, as described before in reference ⁸. The ciliate culture was initiated with ca. 10–20 cells/ml and propagated to the confluent level (over 1,000 cells/ml) under a light cycle of 12 h light and 12 h dark with ca. 3,500 lux (30 cm from the light source) of fluorescent natural-white light at 23°C.The protocol of Tanaka et al.¹⁸ was employed for preparation of apo-symbiotic white cells. Briefly, the green cells were incubated in the presence of 0.1 µM paraquat for over 24 h under light condition (with a fluorescent white lamp, 3,000 lux at least). Then, a single ciliate lacking algae was separated under a microscope and the cell line of apo-symbiotic paramecia (Fig. 1C) derived from this single cell was propagated in the lettuce infusion inoculated with food bacteria as described above. We found that this herbicide treatment merely enhances the excretion of algae from the ciliate but many portions of resultant ex-symbiotic algae excreted from the ciliates are still alive and capable of growing in vitro.¹⁷As a model organism in order to develop an experimental model for studying the origin of photosynthetic organisms leading to evolution of plants, cyanobacteria would be the best organism since the photosynthetic apparatus in this organism is very similar to the one found in plants.¹⁹ Among cyanobacteria as the material to be loaded to apo-symbiotic P. bursaria cells, Synechocystis sp. PCC 6803 (Fig. 1D) was chosen since it is one of the most highly studied cyanobacteria capable of growth in both autotrophical and heterotrophical manners.²⁰ PCC 6803 cells were propagated in BG-11 medium under a continuous light at ca. 3,000 lux with fluorescent natural-white light at 23°C. Then alga-free apo-symbiotic cells isolated and propagated after forced algal excretion, were used for the introduction of PCC 6803 cells based on the modified procedure used for re-introduction of green algae (re-greening process). Apo-symbiotic P. bursaria cells were incubated with suspension of PCC 6803 cells (10 µl of confluent culture of PCC 6803 into 1 ml of ciliate culture, i.e., 2,000 cells) in lettuce infusion for 30 min in an Eppendorf tube at room temperature. The resultant Synechocystis-fed re-greened cells were collected on a nylon mesh (pore size, 10 µm), and washed out with 10 ml of fresh medium three times.Intact live cells were used for routine observations under a stereomicroscope (SMZ645; Nikon, Tokyo, Japan; Fig. 2A). For obtaining the digital microscopic images with higher resolutions, the P. bursaria cells with and without symbionts were fixed in 3% (w/v) formaldehyde added to the culture medium and fixation was allowed at room temperature for 5 min. Confocal laser scanning microscopic red fluorescent images and differential interference contrast (DIC) images of P. bursaria cells were acquired using a Radiance 2100 microscope (Bio-Rad Laboratories, Hercules, CA; Fig. 2B). The obtained images were processed using Adobe Photoshop software.Open in a separate windowFigure 2Microscopic images of cyanobacteria-loaded cell of P. bursaria. (A) Light microscopic image. (B) Laser scanning confocal microscopic images (Left: nomarski differential interference image, Right: chlorophyll fluorescence image)Diagnostic polymerase chain reactions (PCR) were performed for confirming the presence of PCC 6803 cells in the one-year-old symbiotic culture; following primers and reaction condition were employed. For cyanobacteria specific detection, a set of primers was designed from the Cyanobase.²¹ The PCR products were obtained from positions 1,600,000 to 1,601,783 of the chromosome. The specific primers were Forward primer 5′-GTG GTT TGG GTC AAT GTT-3′ and Reverse primer 5′-CAG GGC CTT GTA AAC TTT-3′. The other standard methods for culture and DNA manipulations of cyanobacteria were described in reference ²².Sysmex flow particle image analyzer FPIA-2100 (Sysmex Co., Kobe, Japan) was used for statistical analysis of algal cell size as previously described in reference ³. PCC 6803 cells were suspended in the sheath medium and used for direct measurement by FPIA-2100.After addition of PCC 6803 suspension to apo-symbiotic cells of P. bursaria, bacterial particles were rapidly taken up by the ciliate through the oral groove. Based on microscopic observation with video, the moment that one bacterium located at the deepest end of oral groove is incorporated in cytosolic space (i.e., constriction and isolation of bacterium-containing membrane-like structure resembling the digestive vacuoles) was recorded, indicating that the ciliate takes up the PCC 6803 cells in a manner similar to take up the food bacterium. However, the fate of food bacteria and cyanobacteria drastically differed. As previously reported, the emerald green fluorescent protein (EmGFP)-labeled cells of E. coli can be used as a model food bacterium and its fate can be visualized by following the changes in fluorescence level and localization.² Based on observation of EmGFP E. coli-loaded apo-symbiotic ciliate, localization of bacteria in several compartments was observed; thus instead of being dispersed throughout the hosting cells, presence of food bacteria are restricted and localized within the food vacuoles (Fig. 3B). The time required for digestion of EmGFP-labeled cells of E. coli was estimated to be 30–60 min (fluorescence could not be observed after 1 h).²Open in a separate windowFigure 3Continuous culturing of Synechocystis spp. PCC 6803 in the host cell. (A) Light microscopic images of wild-type P. bursaria. (B) Laser scanning confocal microscopic image of EmGFP-labeled E. coli-loaded cell of apo-symbiotic P. bursaria.² (C) Light microscopic images of Synechocystis spp. PCC 6803-loaded cell of P. bursaria in the one-year-old symbiotic culture. (D) Statistical analysis of Synechocystis spp. PCC 6803 cells in 1 year-old symbiotic culture using a Symex flow particle image analyzer FPIA -2100.On the other hand, the presence of PCC 6803 cells inside the host ciliate could be observed even after continuous propagation of the ciliate for over 1 year (Fig. 3C), thus indicative of the bacterial growth in the host cells. Initially, number of PCC 6803 cells in the host cells were limited within few particles and restricted within the localized compartments in the ciliate cells, but in the samples after continuous culturing for 1, 3, 6 and 12 months, PCC 6803 cells increased and maintained its population inside the hosting cells and their presence were shown to be dispersed throughout the cytosol of hosting cells.Irie et al.³ tested the loading of various fluorescence-labeled microspheres, green algae and bacteria into the apo-symbiotic P. bursaria. According to their work, there is a tight relationship between the size of particles loaded and the mode of localization within the ciliate. Small nano-particles (sized 50–250 nm in diameter) and polystyrene micro-beads sized around 2–3 µm in diameter are likely packed as groups in localized compartments, thus some tens of particles were gathered in spherical structures (resembling the digestive vacuoles) within the ciliate cells, thus their presence was restricted. Larger micro-beads sized around 4–10 µm in diameter did not gather around each other but likely packed individually, thus showing dispersed localization throughout the cytosol of the ciliate cells.³ While E. coli (sized ca. 2 µm) obeys this size-based localization pattern, PCC 6803 showed dispersed distribution similar to the localization of natural green algae (symbiotic Chlorella species), despite its small size (1.61 µm in diameter, mean size; Fig. 3D).According to Tanaka et al.¹⁸ for discussing the presence or absence of symbiotic microorganisms inside the ciliate, microscopic morphological observation is not sufficient, but instead diagnostic PCR is required. Therefore, the presence and stability of PCC 6803 cells likely propagated within the one-year-old symbiotic complex was examined by diagnostic PCR (Fig. 4). Presence of the genome of PCC 6803 was confirmed in the one-year-old culture of PCC 6803-loaded P. bursaria, supporting our microscopic observation at molecular level.Open in a separate windowFigure 4Diagostic polymerase chain reaction for confirming the presence of Synechocystis spp. PCC 6803 cells in host cell from one-year-old symbiotic culture.We previously proposed two possible biochemical models (models 1 and 2) explaining the co-evolution between Paramecium species and algal symbionts by focusing on the dual roles of algal photosynthesis providing the energy source and the risk of oxidative damage to the hosting cells.¹⁶ The first model is based on the correlation between the (re)greening ability and the tolerance to oxidative stress among the paramecium species, whereas the second model is deduced through discussion on the possible evolutionary selection leading to emergence of Paramecium species tolerant against alga-derived reactive oxygen species through alga-paramecium contacts. These views could be applicable to the events required for the emergence of photosynthetic eukaryotes after incorporation of photosynthetic bacteria as primitive chloroplasts. These views should be critically tested in the future researches using our experimental model system.     

Functional activation of Src family kinase yes protein is essential for the enhanced malignant properties of human melanoma cells expressing ganglioside GD3

The possible roles of Src family kinases in the enhanced malignant properties of melanomas related to GD3 expression were analyzed. Among Src family kinases only Yes, not Fyn or Src, was functionally involved in the increased cell proliferation and invasion of GD3-expressing transfectant cells (GD3+). Yes was located upstream of p130Cas and paxillin and at an equivalent level to focal adhesion kinase. Yes underwent autophosphorylation even before serum treatment and showed stronger kinase activity in GD3+ cells than in GD3- cells following serum treatment. Coimmunoprecipitation experiments revealed that Yes bound to focal adhesion kinase or p130Cas more strongly in GD3+ cells than in GD3- cells. As a possible mechanism for the enhancing effects of GD3 on cellular phenotypes, it was shown that majority of Yes was localized in glycolipid-enriched microdomain/rafts in GD3+ cells even before serum treatment, whereas it was scarcely detected in glycolipid-enriched microdomain/rafts in GD3- cells. An in vitro kinase assay of Yes revealed that coexistence of GD3 with Yes in membranous environments enhances the kinase activity of GD3- cell-derived Yes toward enolase, p125, and Yes itself. Knockdown of GD3 synthase resulted in the alleviation of tumor phenotypes and reduced activation levels of Yes. Taken together, these results suggest a role of GD3 in the regulation of Src family kinases.     

Tetrahydropyridine derivatives with inhibitory activity on the production of proinflammatory cytokines: Part 3

In order to develop a new class of anti-rheumatic drug which inhibits production of proinflammatory cytokines such as TNFα, IL-1β, IL-6, and IL-8, a series of 3-pyridylpyrrole derivatives possessing a bicyclic tetrahydropyridine moiety at the 4-position of the pyrrole ring were synthesized and their pharmacological activities were evaluated. The derivatives were found to have potent inhibitory activities on the production of the cytokines both in vitro and in vivo. Among them, compound 4a, (S)-2-(4-fluorophenyl)-4-(1,2,3,5,6,8a-hexahydroindolizin-7-yl)-3-(pyridin-4-yl)-1H-pyrrole (R-132811), achieved the most promising results in various in vitro and in vivo tests including several rheumatoid arthritis models ((i) inhibition of p38α, p38β, p38γ, and p38δ MAP kinases: IC₅₀ = 0.034, 0.572, >10, and >10 μM, respectively; (ii) inhibition of TNFα, IL-1β, IL-6, and IL-8 production in human whole blood: IC₅₀ = 0.026, 0.020, 0.88, and 0.016 μM, respectively; (iii) inhibition of LPS induced TNFα, IL-1β and IL-6 production in mice: ID₅₀ = 0.93, 8.63, and 0.11 mg/kg, po, respectively; (iv) inhibition of anti-collagen antibody-induced arthritis in mice: ID₅₀ = 2.22 mg/kg, po; (v) inhibition of collagen-induced arthritis in mice: ID₅₀ = 2.38 mg/kg, po; (vi) prophylactic effect on adjuvant-induced arthritis in rats: ID₅₀ = 3.1 mg/kg, po; (vii) therapeutic effect on adjuvant-induced arthritis in rats: ID₅₀ = 4.9 mg/kg, po; (viii) analgesic effect on adjuvant-induced arthritic pain in rats: ID₅₀ = 2.9 mg/kg, po). As a result, compound 4a was chosen as a candidate for further pre-clinical studies.     

Mutation of the SNF2 family member Chd2 affects mouse development and survival

The chromodomain helicase DNA-binding domain (Chd) proteins belong to the SNF2-like family of ATPases that function in chromatin remodeling and assembly. These proteins are characterized by the presence of tandem chromodomains and are further subdivided based on the presence or absence of additional structural motifs. The Chd1-Chd2 subfamily is distinguished by the presence of a DNA-binding domain that recognizes AT-rich sequence. Currently, there are no reports addressing the function of the Chd2 family member. Embryonic stem cells containing a retroviral gene-trap inserted at the Chd2 locus were utilized to generate mice expressing a Chd2 protein lacking the DNA-binding domain. This mutation in Chd2 resulted in a general growth delay in homozygous mutants late in embryogenesis and in perinatal lethality. Animals heterozygous for the mutation showed decreased neonatal viability and increased susceptibility to non-neoplastic lesions affecting most primary organs. In particular, approximately 85% of the heterozygotes showed gross kidney abnormalities. Our results demonstrate that mutation of Chd2 dramatically affects mammalian development and long-term survival.