Secreted Acid Phosphatase Research Articles

Immunomodulatory properties of Leishmania tarentolae extracellular vesicles containing the Spike protein of SARS-CoV-2

Extracellular vesicles released by the protozoan parasite Leishmania display immunomodulatory properties towards mammalian immune cells. In this study, we have evaluated the potential of extracellular vesicles derived from the non-pathogenic protozoan Leishmania tarentolae towards the development of a vaccine adjuvant. As a proof of concept, we expressed in L. tarentolae a codon-optimized SARS-CoV-2 Spike protein fused to the L. mexicana secreted acid phosphatase signal peptide in the N-terminal and to a 6×-His stretch in the C-terminal. Extracellular vesicles released by the engineered L. tarentolae were isolated by ultracentrifugation and fast protein liquid chromatography and were characterized via nanoparticle tracking analysis and transmission electron microscopy. The recombinant S protein was present in extracellular vesicles released by L. tarentolae, as determined by Western blot analyses and immunoelectron microscopy. Next, we evaluated the immunomodulatory potential of extracellular vesicles containing the S protein towards bone-marrow-derived macrophages and bone-marrow-derived dendritic cells. Our data show that in bone-marrow-derived dendritic cells, extracellular vesicles containing the S protein induced an increased expression of proinflammatory genes compared to plain extracellular vesicles whereas the opposite was observed in bone-marrow-derived macrophages. These findings reveal the immunomodulatory potential of L. tarentolae extracellular vesicles and provide a proof of concept that they can be used as adjuvant in the context of dendritic cell stimulation.

Revealing the Mechanistic Basis of Regulation of Phosphorus Uptake in Soybean (Glycine max) Roots by Molybdenum: An Integrated Omics Approach.

While molybdenum (Mo) application can improve phosphorus (P) availability to plants by changing P speciation in the rhizosphere, the mechanistic basis of this process remains unclear. This work investigated the impact of various combinations of Mo and P treatments on root morphology, P and Mo uptake, and root transcriptome and metabolome. Mo application significantly increased soybean biomass and the number of lateral roots at both low (5 μmol) or normal (500 μmol) P levels and significantly improved P concentration and accumulation in Normal P treatment. Compared with the Normal P treatment, Low P significantly increased the number of roots, root surface area, and root acid phosphatase secretion. A total of 6811 Mo-responsive differentially expressed genes and 135 differential metabolites were identified at two P levels. At Low P, transcriptional changes significantly increased root synthesis and secretion of succinic acid, methylmalonic acid, and other organic acids as well as acid phosphatase, thereby increasing the conversion of soil aluminum-bound P and organic P into available P. At Normal P, Mo application increased P uptake mainly by increasing the number of lateral roots. Thus, Mo helps crops adapt to different P levels by regulating root anatomy and transcriptional and metabolic profiles of their roots.

C-reactive protein binds to short phosphoglycan repeats of Leishmania secreted proteophosphoglycans and activates complement.

Human C-reactive protein (CRP) binds to lipophosphoglycan (LPG), a virulence factor of Leishmania spp., through the repeating phosphodisaccharide region. We report here that both major components of promastigote secretory gel (PSG), the filamentous proteophosphoglycan (fPPG) and the secreted acid phosphatase (ScAP), are also ligands. CRP binding was mainly associated with the flagellar pocket when LPG deficient Leishmania mexicana parasites were examined by fluorescent microscopy, consistent with binding to secreted material. ScAP is a major ligand in purified fPPG from parasite culture as demonstrated by much reduced binding to a ScAP deficient mutant fPPG in plate binding assays and ligand blotting. Nevertheless, in sandfly derived PSG fPPG is a major component and the major CRP binding component. Previously we showed high avidity of CRP for LPG ligand required multiple disaccharide repeats. ScAP and fPPG only have short repeats but they retain high avidity for CRP revealed by surface plasmon resonance because they are found in multiple copies on the phosphoglycan. The fPPG from many species such as L. donovani and L. mexicana bound CRP strongly but L. tropica and L. amazonensis had low amounts of binding. The extent of side chain substitution of [-PO4-6Galβ1-4Manα1-] disaccharides correlates inversely with binding of CRP. The ligand for the CRP on different species all had similar binding avidity as the half maximal binding concentration was similar. Since the PSG is injected with the parasites into host blood pools and phosphoglycans (PG) are known to deplete complement, we showed that CRP makes a significant contribution to the activation of complement by PSG using serum from naive donors.

Surprising Effects of Rocking Motion on Leishmania tarentolae Behavior in Culture and Implications for Cell Stress

Leishmania are an understudied genus of parasitic protozoans causing significant health problems for people, particularly in tropical climates. To better understand the growth of Leishmania and potential drug sensitivity implications, the effects of motion on cells grown in vitro were probed. Using a stock Leishmania tarentolae cell culture, cells were placed in 10 mL of a Brain–Heart Infusion medium in either a non-moving (static) environment or on a flat platform of one of two lab rockers (set at a minimal speed) in a dark environment for 13 days. Also, the addition of 0.5 M of L-Proline was evaluated. Microscopy, cell clumping, cell viability, and secreted acid phosphatase (SAP) activity data were collected. Results show that a constant slow rocking motion changed cell growth, clumping behavior, and detectable SAP activity relative to the no-motion cultures, but this change was dependent on which rocker was used, indicating a complexity in the growth of these cells in culture. Thus, continuous motion affects the stresses placed on the cells during a growth curve under some conditions. The implications of this study lead to questions about the effects of motion on the efficacy of pharmaceutical testing in vitro. Further study of the effects of motion on Leishmania is important.

Investigating the Leishmania donovani sacp Gene and Its Role in Macrophage Infection and Survival in Mice.

The protozoan parasite Leishmania donovani is a causative agent of the neglected tropical disease known as visceral leishmaniasis, which can be lethal when untreated. Studying Leishmania viru-lence factors is crucial in determining how the parasite causes disease and identifying new targets for treatment. One potential virulence factor is L. donovani's abundantly secreted protein: secreted acid phosphatase (SAcP). Whole-genome analysis revealed that the sacp gene was present in three copies in wild type L. donovani. Using CRISPR-Cas9 gene editing; we generated a sacp gene knockout termed LdΔSAcP, which demonstrated a loss of both the SAcP protein and an associated reduction in secreted acid phosphatase activity. Genome sequencing confirmed the precise dele-tion of the sacp gene in LdΔSAcP and identified several changes in the genome. LdΔSAcP demonstrated no significant changes in promastigote proliferation or its ability to infect and survive in macrophages compared to the wildtype strain. LdΔSAcP also demonstrated no change in murine liver infection; however, survival was impaired in the spleen. Taken together these results show that SAcP is not necessary for the survival of promastigotes in culture but may support long-term survival in the spleen. These observations also show that the use of CRISPR gene editing and WGS together are effective to investigate the function and phenotype of complex potential drug targets such as multicopy genes.

Characterization of the Secreted Acid Phosphatase SapS Reveals a Novel Virulence Factor of Staphylococcus aureus That Contributes to Survival and Virulence in Mice.

Staphylococcus aureus possesses a large arsenal of immune-modulating factors, enabling it to bypass the immune system's response. Here, we demonstrate that the acid phosphatase SapS is secreted during macrophage infection and promotes its intracellular survival in this type of immune cell. In animal models, the SA564 sapS mutant demonstrated a significantly lower bacterial burden in liver and renal tissues of mice at four days post infection in comparison to the wild type, along with lower pathogenicity in a zebrafish infection model. The SA564 sapS mutant elicits a lower inflammatory response in mice than the wild-type strain, while S. aureus cells harbouring a functional sapS induce a chemokine response that favours the recruitment of neutrophils to the infection site. Our in vitro and quantitative transcript analysis show that SapS has an effect on S. aureus capacity to adapt to oxidative stress during growth. SapS is also involved in S. aureus biofilm formation. Thus, this study shows for the first time that SapS plays a significant role during infection, most likely through inhibiting a variety of the host's defence mechanisms.

Plant Foraging Strategies Driven by Distinct Genetic Modules: Cross-Ecosystem Transcriptomics Approach.

Plants have evolved diverse strategies for foraging, e.g., mycorrhizae, modification of root system architecture, and secretion of phosphatase. Despite extensive molecular/physiological studies on individual strategies under laboratory/greenhouse conditions, there is little information about how plants orchestrate these strategies in the field. We hypothesized that individual strategies are independently driven by corresponding genetic modules in response to deficiency/unbalance in nutrients. Roots colonized by mycorrhizal fungi, leaves, and root-zone soils were collected from 251 maize plants grown across the United States Corn Belt and Japan, which provided a large gradient of soil characteristics/agricultural practice and thus gene expression for foraging. RNA was extracted from the roots, sequenced, and subjected to gene coexpression network analysis. Nineteen genetic modules were defined and functionally characterized, from which three genetic modules, mycorrhiza formation, phosphate starvation response (PSR), and root development, were selected as those directly involved in foraging. The mycorrhizal module consists of genes responsible for mycorrhiza formation and was upregulated by both phosphorus and nitrogen deficiencies. The PSR module that consists of genes encoding phosphate transporter, secreted acid phosphatase, and enzymes involved in internal-phosphate recycling was regulated independent of the mycorrhizal module and strongly upregulated by phosphorus deficiency relative to nitrogen. The root development module that consists of regulatory genes for root development and cellulose biogenesis was upregulated by phosphorus and nitrogen enrichment. The expression of this module was negatively correlated with that of the mycorrhizal module, suggesting that root development is intrinsically an opposite strategy of mycorrhizae. Our approach provides new insights into understanding plant foraging strategies in complex environments at the molecular level.

Harnessing root-foraging capacity to improve nutrient-use efficiency for sustainable maize production

Producing more food with less input is imperative in feeding the increasing world population. Here, for the first time we characterize the multipartite responses of maize to nutrient-rich patches and demonstrate significant synergistic effects between root morphological and physiological responses on improving nutrient-use efficiency and yield. Our results showed that maize root length, lateral root formation, acid phosphatase (APase) secretion, and expression of ammonium transporter genes increased in the ammonium-containing patches; moreover, ammonium-induced rhizosphere acidification enhanced activity of APase. These root responses were associated with improved maize nutrition and growth even with reduced fertilizer input, suggesting an adaptive benefit of synergistic root foraging strategies. These new findings improve our understanding of root-foraging responses to nutrient-specific cues and are crucial for engineering root and rhizosphere properties to improve nutrient-use efficiency for sustainable crop production. • Producing more food with less input is imperative for sustainable intensification of agriculture. • Rhizosphere dynamics is key to controlling nutrient-use efficiency (NUE). • Localized nutrient-supply maximizes root/rhizosphere efficiency for improving NUE. • Harnessing root foraging provides a new approach for sustainable maize production.

Acaulospora as the Dominant Arbuscular Mycorrhizal Fungi in Organic Lowland Rice Paddies Improves Phosphorus Availability in Soils

Flooding in rainfed lowlands greatly impairs the mutualistic relationship between indigenous arbuscular mycorrhizal fungi (AMF) and rice. In flooded soils, root colonization by AMF is arrested, but some AMF genera, defined as the core AMF, remain present. However, the core AMF in rainfed lowlands and their symbiotic roles remain unknown. Here, we showed that Acaulospora fungi were the core AMF in rice seedling roots of the Sangyod Muang Phatthalung (SMP) landrace rice variety grown in non-flooded and flooded paddy soils. Subsequently, indigenous Acaulospora spores were propagated by trap cultures using maize as the host plants. Therefore, to clarify the roles of cultured Acaulospora spores in a symbiotic partnership, the model japonica rice variety Nipponbare was grown in sterile soil inoculated with Acaulospora spores, and recolonized with a native microbial filtrate from the organic rice paddy soil. Our data demonstrated that the inoculation of Acaulospora spores in well-drained soil under a nutrient-sufficient condition for six weeks enabled 70 percent of the rice roots to be colonized by the fungi, leading to higher phosphate (Pi) accumulation in the mycorrhizal roots. Unexpectedly, the growth of rice seedlings was significantly suppressed by inoculation while photosynthetic parameters such as fractions of incoming light energy and relative chlorophyll content were unaltered. In the soil, the Acaulospora fungi increased soil phosphorus (P) availability by enhancing the secretion of acid phosphatase in the mycorrhizal roots. The findings of this work elucidate the symbiotic roles of the dominant Acaulospora fungi from lowland rice paddies.

The wheat secreted root proteome: Implications for phosphorus mobilisation and biotic interactions

Root secreted acid phosphatases and organic anions are widely perceived as major players of plant phosphorus (P) mobilisation from the rhizosphere under P limiting growth conditions. Previous research indicated that other mechanisms play a role, especially in species with fine roots, such as wheat. In this study we characterised the plant-derived extracellular proteome of wheat roots by profiling root tip mucilage, soluble root secreted and root tip proteomes. Extracellular acid phosphatases and enzymes of the central carbon metabolism were targeted using selected reaction monitoring. More than 140 proteins with extracellular localisation prediction were identified in mucilage. P starvation induced proteins predicted to be localised to the apoplast which are related to cell wall modification and defence in both, root tip and soluble root-secreted proteomes. Glycolytic enzymes were strongly increased in abundance by P limitation in root tips, as were PEPC and plastidial MDH. Soluble acid phosphatases were not identified in extracellular protein samples. Our results indicate that root tip mucilage contains proteins with the functional potential to actively shape their immediate environment by modification of plant structural components and biotic interactions. Wheat acid phosphatases appear to play a minor role in P mobilisation beyond the immediate root surface. SignificancePhosphorus (P) is a plant growth limiting nutrient in many agricultural situations and the development of phosphorus efficient crops is of paramount importance for future agricultural management practices. As P is relatively immobile in soils, processes occurring at the root-soil interface, the rhizosphere, are suspected to play a key role in plant-induced P mobilisation. According to the current view, the secretion of extracellular acid phosphatases and organic anions enhances P mobilisation within several millimetres beyond the root surface, either directly or indirectly through the selection and appropriate soil microbes. However, the mechanisms of P mobilisation in species with fine roots, such as wheat, and the role of other secreted root proteins are poorly understood. Here, we carried out the profiling of wheat root tip mucilage, soluble root secreted and root tip proteomes. We analysed proteome changes in response to P starvation. We found that proteins with a predicted localisation to the apoplast made up a major proportion of stress-responsive proteins. Acid phosphatases were not identified within extracellular protein samples, which were enriched in proteins with predicted extracellular localisation. The absence of extracellular APases was further validated by multiple reaction monitoring. Our data indicates that wheat acid phosphatases play a minor role in P mobilisation beyond the immediate root surface and provides a resource for breeding strategies and further investigations of the functional roles of root tip-released proteins in the rhizosphere under P limitation.

Secreted acid phosphatases maintain replicative lifespan via inositol polyphosphate metabolism in budding yeast.

Secreted acid phosphatases (APases) dephosphorylate extracellular organic phosphate compounds to supply inorganic phosphate (Pi) to maintain cellular functions. Here, we show that APases are necessary to maintain a normal replicative lifespan in Saccharomyces cerevisiae. Deletion of all four APase genes shortened the lifespan in yeast strains on synthetic media (irrespective of the concentrations of Pi in the media), but it did not affect the intracellular ortho- and polyphosphate levels. Deletion of inositol-pentakisphosphate 2-kinase (IPK1), which encodes inositol-pentakisphosphate 2-kinase, restored the lifespan in APase-null mutants, and IPK1 overexpression shortened the lifespan in wild-type strains. Overexpression of inositol hexakisphosphate (IP6 ) and heptakisphosphate kinases, KCS1 and VIP1, recovered the lifespan in APase-null mutants. Thus, yeast APases modulate the replicative lifespan, probably through dephosphorylation of intracellular IP6 .

Leishmania tarentolae novel responses to Bi3+-doped strontium aluminum oxyfluorides

Novel therapeutics for the treatment of leishmaniasis are of interest as the disease not only is becoming more prevalent, but drug resistance is increasing in certain regions of the world. Reported here is the use of Bi3+-doped strontium aluminum oxyfluoride phosphors and protease inhibitors to test in vitro inhibitory activity against cultured promastigote Leishmania tarentolae and effects on L. tarentolae secreted acid phosphatase (SAP) activity. Cell viability did not significantly decrease in the presence of 50 μM anti-perovskite compounds, implying limited cytotoxicity. Yet SAP activity did increase in the cell free preparations with time in the presence of strontium compounds. Of interest was the observation that cell free SAP activity did not increase in the presence of protease inhibitors with or without added strontium compounds. Since secreted proteases may play a role in the maturation of Leishmania SAP and thus be involved with parasite-host infection establishment, this is in further need of evaluation. Nitric oxide production on day 4 post-addition of the strontium compounds was evaluated and showed an approximately 50% decrease in NO production in the presence of two test compounds relative to DMSO control cells. This is the first report of anti-perovskite compound inhibition of NO production by Leishmania.

Nitrogen and Phosphorus Interplay in Lupin Root Nodules and Cluster Roots.

Nitrogen (N) and phosphorus (P) are two major plant nutrients, and their deficiencies often limit plant growth and crop yield. The uptakes of N or P affect each other, and consequently, understanding N–P interactions is fundamental. Their signaling mechanisms have been studied mostly separately, and integrating N–P interactive regulation is becoming the aim of some recent works. Lupins are singular plants, as, under N and P deficiencies, they are capable to develop new organs, the N2-fixing symbiotic nodules, and some species can also transform their root architecture to form cluster roots, hundreds of short rootlets that alter their metabolism to induce a high-affinity P transport system and enhance synthesis and secretion of organic acids, flavonoids, proteases, acid phosphatases, and proton efflux. These modifications lead to mobilization in the soil of, otherwise unavailable, P. White lupin (Lupinus albus) represents a model plant to study cluster roots and for understanding plant acclimation to nutrient deficiency. It tolerates simultaneous P and N deficiencies and also enhances uptake of additional nutrients. Here, we present the structural and functional modifications that occur in conditions of P and N deficiencies and lead to the organogenesis and altered metabolism of nodules and cluster roots. Some known N and P signaling mechanisms include different factors, including phytohormones and miRNAs. The combination of the individual N and P mechanisms uncovers interactive regulation pathways that concur in nodules and cluster roots. L. albus interlinks N and P recycling processes both in the plant itself and in nature.

Structure–Function Relationship Study of a Secretory Amoebic Phosphatase: A Computational-Experimental Approach

Phosphatases are hydrolytic enzymes that cleave the phosphoester bond of numerous substrates containing phosphorylated residues. The typical classification divides them into acid or alkaline depending on the pH at which they have optimal activity. The histidine phosphatase (HP) superfamily is a large group of functionally diverse enzymes characterized by having an active-site His residue that becomes phosphorylated during catalysis. HP enzymes are relevant biomolecules due to their current and potential application in medicine and biotechnology. Entamoeba histolytica, the causative agent of human amoebiasis, contains a gene (EHI_146950) that encodes a putative secretory acid phosphatase (EhHAPp49), exhibiting sequence similarity to histidine acid phosphatase (HAP)/phytase enzymes, i.e., branch-2 of HP superfamily. To assess whether it has the potential as a biocatalyst in removing phosphate groups from natural substrates, we studied the EhHAPp49 structural and functional features using a computational-experimental approach. Although the combined outcome of computational analyses confirmed its structural similarity with HP branch-2 proteins, the experimental results showed that the recombinant enzyme (rEhHAPp49) has negligible HAP/phytase activity. Nonetheless, results from supplementary activity evaluations revealed that rEhHAPp49 exhibits Mg2+-dependent alkaline pyrophosphatase activity. To our knowledge, this study represents the first computational-experimental characterization of EhHAPp49, which offers further insights into the structure–function relationship and the basis for future research.

High level of zinc triggers phosphorus starvation by inhibiting root-to-shoot translocation and preferential distribution of phosphorus in rice plants.

Since the urbanization and industrialization are wildly spread in recent decades, the concentration of Zn in soil has increased in various regions. Although the interactions between P and Zn has long been recognized, the effect of high level of Zn on P uptake, translocation and distribution in rice and its molecular mechanism are not fully understood. In this study, we conducted both hydroponic culture and field trial with different combined applications of P and Zn to analyze the rice growth and yield, the uptake, translocation and distribution of P and Zn, as well as the P- and Zn-related gene expression levels. Our results showed that high level of Zn decreased the rice biomass and yield production, and inhibited the root-to-shoot translocation and distribution of P into new leaves by down-regulating P transporter genes OsPT2 and OsPT8 in shoot, which was controlled by OsPHR2-OsmiR399-OsPHO2 module. High Zn supply triggered P starvation signal in root, thereafter increased the activities of both root-endogenous and -secreted acid phosphatase to release more Pi, and induced the expression OsPT2 and OsPT8 to uptake more P for plant growth. On the other hand, high level of P significantly decreased the Zn concentrations in both root and shoot, and the root uptake ability of Zn through altering the expression levels of OsZIPs, which were further confirmed by the P high-accumulated mutant osnla1-2 and OsPHR2-OE transgenic plant. Taken together, we revealed the physiological and molecular mechanisms of P-Zn interactions, and proposed a working model of the cross-talk between P and Zn in rice plants. Our results also indicated that appropriate application of P fertilizer is an effective strategy to reduce rice uptake of excessive Zn when grown in Zn-contaminated soil.

Proteome adaptations under contrasting soil phosphate regimes of Rhizophagus irregularis engaged in a common mycorrhizal network

For many plants, their symbiosis with arbuscular mycorrhizal fungi plays a key role in the acquisition of mineral nutrients such as inorganic phosphate (Pi), in exchange for assimilated carbon. To study gene regulation and function in the symbiotic partners, we and others have used compartmented microcosms in which the extra-radical mycelium (ERM), responsible for mineral nutrient supply for the plants, was separated by fine nylon nets from the associated host roots and could be harvested and analysed in isolation. Here, we used such a model system to perform a quantitative comparative protein profiling of the ERM of Rhizophagus irregularis BEG75, forming a common mycorrhizal network (CMN) between poplar and sorghum roots under a long-term high- or low-Pi fertilization regime. Proteins were extracted from the ERM and analysed by liquid chromatography-tandem mass spectrometry.This workflow identified a total of 1301 proteins, among which 162 displayed a differential amount during Pi limitation, as monitored by spectral counting. Higher abundances were recorded for proteins involved in the mobilization of external Pi, such as secreted acid phosphatase, 3′,5′-bisphosphate nucleotidase, and calcium-dependent phosphotriesterase. This was also the case for intracellular phospholipase and lysophospholipases that are involved in the initial degradation of phospholipids from membrane lipids to mobilize internal Pi. In Pi-deficient conditions. The CMN proteome was especially enriched in proteins assigned to beta-oxidation, glyoxylate shunt and gluconeogenesis, indicating that storage lipids rather than carbohydrates are fuelled in ERM as the carbon source to support hyphal growth and energy requirements. The contrasting pattern of expression of AM-specific fatty acid biosynthetic genes between the two plants suggests that in low Pi conditions, fatty acid provision to the fungal network is mediated by sorghum roots but not by poplar. Loss of enzymes involved in arginine synthesis coupled to the mobilization of proteins involved in the breakdown of nitrogen sources such as intercellular purines and amino acids, support the view that ammonium acquisition by host plants through the mycorrhizal pathway may be reduced under low-Pi conditions. This proteomic study highlights the functioning of a CMN in Pi limiting conditions, and provides new perspectives to study plant nutrient acquisition as mediated by arbuscular mycorrhizal fungi.

Synthesis of Polycyclic Ether-Benzopyrans and In Vitro Inhibitory Activity against Leishmania tarentolae.

Construction of a focused library of polycyclic ether-benzopyrans was undertaken in order to discover new therapeutic compounds that affect Leishmania growth and infectivity. This is especially of interest since there are few drug therapies for leishmaniasis that do not have serious drawbacks such high cost, side effects, and emerging drug resistance. The construction of these polycyclic ether-benzopyrans utilized an acetoxypyranone-alkene [5+2] cycloaddition and the Suzuki-Miyaura cross-coupling. The multi-gram quantity of the requisite aryl bromide was obtained followed by effective Pd-catalyzed coupling with boronic acid derivatives. Compounds were tested in vitro using the parasitic protozoan, Leishmania tarentolae. Effects of concentration, time, and exposure to light were evaluated. In addition, the effects on secreted acid phosphatase activity and nitric oxide production were investigated, since both have been implicated in parasite infectivity. The data presented herein are indicative of disruption of the Leishmania tarentolae and thus provide impetus for the development and testing of a more extensive library.

Chemical genetic analysis of FTY720- and Ca2+ -sensitive mutants reveals a functional connection between FTY720 and membrane trafficking.

FTY720, a sphingosine-1-phosphate (S1P) analog, is used as an immune modulator to treat multiple sclerosis. Accumulating evidence has suggested the mode of action of FTY720 independent of an S1P modulator. In fission yeast, FTY720 induces an increase in intracellular Ca2+ and ROS levels. We have previously identified 49 genes of which deletion causes FTY720 sensitivity. Here, we characterized the FTY720-sensitive mutants in terms of their relevance to the Ca2+ homeostasis and identified the 16 FTY720- and Ca2+ -sensitive mutants (fcs mutants). Most of the FTY720-sensitive mutants showed elevated Ca2+ levels and exhibited Ca2+ dysregulation by FTY720 treatment. One of the functional categories among the genes whose deletion renders cells susceptible to FTY720 and Ca2+ include the Golgi/endosomal membrane trafficking. Notably, FTY720, but not phosphorylated FTY720 incapable of inducing Ca2+ increase, inhibited the secretion of acid phosphatase in the wild-type cells. Importantly, secretory defects of the Golgi/endosomal trafficking mutants, Vps45, or Ryh1 deletion, were further exacerbated by FTY720. Our fcs mutant screen also identified the adenylyl cyclase-associated protein Cap1 and a Rictor homolog Ste20, whose deletion markedly exacerbated FTY720-sensitive secretory impairment. Collectively, our data may suggest a synergistic impact of FTY720 combined with secretion perturbation on proliferation and Ca2+ homeostasis.

Immunological Activities of Crude Polysaccharides Extracts of Climacodon pulcherrimus (Phanerochaetaceae) in Lipopolysaccharide-Induced Rat Macrophages and Neutrophils' Responses.

Mushroom contains a wide variety of components with high immune-modulating properties. Mushroom polysaccharides are known as immunocorrectors with wide areas of usage, which balance the immune system without resulting in the entire immune suppression or overreaction. To explore the potentially immunomodulatory role of Climacodon pulcherrimus, the present study was intended to evaluate the in vitro immunomodulatory activity of C. pulcherrimus polysaccharides (CPP) on rat neutrophils and macrophages. CPP was studied on lipopolysaccharide-induced neutrophils and macrophages' responses. The effect on phagocytosis, release of reactive oxygen intermediates (ROI), nitric oxide (NO), lysosomal enzyme release and neutrophil myeloperoxidase, and neutrophil adherence was assessed. The cytotoxicity of CPP against macrophages using 3-(4,5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide assay and neutral red inclusion assay was also assessed. ENPC (endopolysaccharides) markedly enhanced phagocytosis of macrophages and neutrophils, whereas the EXPC (exopolysaccharides) increased only that of neutrophils at 0.34 and 0.68 mg/mL. The production of NO evidently increased in ENPC- and EXPC-treated neutrophil and macrophage cultures. The production of ROI in a concentration-dependent manner increased in ENPC-treated (0.08-5.55 mg/mL) and EXPC-treated (0.17 and 0.34 mg/mL) macrophage cultures and ENPC-treated neutrophils, whereas it decreased in EXPC-treated neutrophils. The secretion of acid phosphatase increased in ENPC- and EXPC-treated macrophage and neutrophil cultures. Neutrophil myeloperoxidase release increased in EXPC-treated (5.55 mg/mL) cultures, whereas it decreased in ENPC-treated cultures. ENPC and EXPC increased the adhesion of neutrophils to microplate. These findings demonstrated that CPP displayed a dual mode of action, inhibiting or enhancing numerous phagocytic activities of macrophages and neutrophils.

Fine structure of Aldrovanda vesiculosa L: the peculiar lifestyle of an aquatic carnivorous plant elucidated by electron microscopy using cryo-techniques.

The aquatic carnivorous plant Aldrovanda vesiculosa L. is critically endangered worldwide; its peculiar lifestyle raises many questions and poses problems both intriguing on their own and relevant to conservation. While establishing a culture system for its propagation and restoring its natural habitat in Hozoji pond in Saitama, Japan, we conducted ultrastructural observations to examine the various aspects of Aldrovanda's way of life. Electron microscopic observation in combination with cryo-techniques produced novel information which could not be obtained by other methods. Some of the results are: phosphorous is stored in petiole cells of turions during winter; mucilaginous guides are provided for pollen tubes in parietal placental ovaries; storage of potassium in the vicinity of the midrib of carnivorous leaves may contribute to the rapid closing of the carnivorous leaves; dynamic sequential changes of the ultrastructure of digestive glands are involved in the synthesis and secretion of digestive enzymes, including protease and acid phosphatase. These results should contribute significantly to our understanding of Aldrovanda and the detailed mechanisms of its life.