Protein kinase C is likely to be involved in zoosporogenesis and maintenance of flagellar motility in the peronosporomycete zoospores

The motility of zoospores is critical in the disease cycles of Peronosporomycetes that cause devastating diseases in plants, fishes, vertebrates, and microbes. In the course of screening for secondary metabolites, we found that ethyl acetate extracts of a marine Streptomyces sp. strain B5136 rapidly impaired the motility of zoospores of the grapevine downy mildew pathogen Plasmopara viticola at 0.1 μg/ml. The active principle in the extracts was identified as staurosporine, a known broad-spectrum inhibitor of protein kinases, including protein kinase C (PKC). In the presence of staurosporine (2 nM), zoospores moved very slowly in their axis or spun in tight circles, instead of displaying straight swimming in a helical fashion. Compounds such as K-252a, K-252b, and K-252c structurally related to staurosporine also impaired the motility of zoospores in a similar manner but at varying doses. Among the 22 known kinase inhibitors tested, the PKC inhibitor chelerythrine was the most potent to arrest the motility of zoospores at concentrations starting from 5 nM. Inhibitors that targeted kinase pathways other than PKC pathways did not practically show any activity in impairing zoospore motility. Interestingly, both staurosporine (5 nM) and chelerythrine (10 nM) also inhibited the release of zoospores from the P. viticola sporangia in a dose-dependent manner. In addition, staurosporine completely suppressed downy mildew disease in grapevine leaves at 2 μM, suggesting the potential of small-molecule PKC inhibitors for the control of peronosporomycete phytopathogens. Taken together, these results suggest that PKC is likely to be a key signaling mediator associated with zoosporogenesis and the maintenance of flagellar motility in peronosporomycete zoospores.     

Quantification of the particle method for chemotactic bioassay using Peronosporomycete zoospores

We estimated the amount of test solution absorbed by each Chromosorb W AW particle (60-80 mesh) using an isotopic technique to quantitate the particle method. 14C-Labeled standard compounds like carbendazim (MBC), 5-O-methylcochliophilin A, sucrose and proline were dissolved in several solvents, and Chromosorb carrier particles were treated with the solution to coat the particle with these test compounds. The ratios of the radioactivity of 5 microl of the test solution to that of 2 mg of carrier particles treated with the solution at some different concentrations were measured. It was found that each carrier particle holds approx. 3.8 nl of the test solution within a range of 2 x 10(-3) to 1 x 10(-7) M concentrations. Accordingly, it is now possible to widely use the particle method as a quantitative procedure to assay chemotaxis of Peronosporomycete zoospores.     

A photoaffinity probe designed for host-specific signal flavonoid receptors in phytopathogenic Peronosporomycete zoospores of Aphanomyces cochlioides

Aphanomyces cochlioides zoospores show chemotaxis to cochliophilin A (5-hydroxy-6,7-methylenedioxyflavone, 1), a host derived attractant, and also respond to 5,7-dihydroxyflavone (2) known as an equivalent chemoattractant. To investigate the chemotactic receptors in the zoospores, we designed photoaffinity probes 4'-azido-5,7-dihydroxyflavone (3) and 4'-azido-7-O-biotinyl-5-hydroxyflavone (4) considering chemical structure of 2. Both 3 and 4 had zoospore attractant activity which was competitive with that of 1. When zoospores were treated with the biotinylated photoaffinity probe followed by UV irradiation and streptavidin-gold or peroxidase-conjugated streptavidin, probe-labeled proteins were detected on the cell membrane. This result indicated that the 1-specific-binding proteins, a candidate for hypothetical cochliophilin A receptor, were localized on the cell membrane of the zoospores. This is the first experimental evidence of flavonoid-binding proteins being present in zoospores, using chemically synthesized azidoflavone as photoaffinity-labeling reagent.     

Morphology of three polycentric rumen fungi and description of a procedure for the induction of zoosporogenesis and release of zoospores in cultures

Three polycentric rumen fungi, LL, LC2 and Ruminomyces elegans (C2), isolated from the rumen of cattle were grown in six culture media. LL and LC2 were morphologically similar. Their characteristics resembled those of Orpinomyces and Neocallimastix joyonii, and they grew well and produced sporangia after 3-4 d growth in all the media. R. elegans differed morphologically from LL and LC2, but although it also grew well in all media, abundant sporangia occurred only after 2-3 d growth in media containing cellulose. Undifferentiated sporangia were produced by all three isolates; differentiation of the sporangia did not occur in the spent growth media. However, if thalli possessing recently-formed sporangia were transferred to, or flooded with, fresh liquid medium or rumen fluid, zoosporogenesis and liberation of zoospores occurred within 17-20 min for isolates LL and LC2 and 30 min for R. elegans. Procedures for inducing zoosporogenesis by polycentric anaerobic fungi are described.     

Isolation of 2,4-Diacetylphloroglucinol from a Fluorescent Pseudomonad and Investigation of Physiological Parameters Influencing Its Production

Pseudomonas sp. strain F113 was isolated from the rhizosphere of sugar beets and shown to inhibit a range of plant pathogenic fungi by producing an antibioticlike compound. An antibiotic-negative mutant strain, F113G22, was generated by transposon mutagenesis. This mutant has lost the ability to inhibit both bacterial and fungal microorganisms on high-iron medium. The antibioticlike compound was subsequently identified as 2,4-diacetylphloroglucinol (DAPG), and a high-pressure liquid chromatographic assay was developed for to detect it quantitatively in growth culture media and soil. The growth temperature had a direct bearing on DAPG production by strain F113, with maximum production at 12°C. The iron concentration, pH, and oxygen had no influence on DAPG production by strain F113 under the assay conditions used. However, a low ratio of culture volume to surface area available to the microbe in the growth container was critical for optimum DAPG production. Different types of carbon sources influenced DAPG production by strain F113 to various degrees. For example, sucrose, fructose, and mannitol promoted high yields of DAPG by strain F113, whereas glucose and sorbose resulted in very poor DAPG production.     

Quantification of 2,4-Diacetylphloroglucinol Produced by Fluorescent Pseudomonas spp. In Vitro and in the Rhizosphere of Wheat

The broad-spectrum antibiotic 2,4-diacetylphloroglucinol (Phl) is a major determinant in the biological control of a wide range of plant diseases by fluorescent Pseudomonas spp. A protocol was developed to readily isolate and quantify Phl from broth and agar cultures and from the rhizosphere environment of plants. Extraction with ethyl acetate at an acidic pH was suitable for both in vitro and in situ sources of Phl. For soil samples, the addition of an initial extraction step with 80% acetone at an acidic pH was highly effective in eliminating polar organic soil components, such as humic and fulvic acids, which can interfere with Phl detection by high-performance liquid chromotography. The efficiency of Phl recovery from soil by a single extraction averaged 54.6%, and a second extraction added another 6.1%. These yields were substantially greater than those achieved by several standard protocols commonly used to extract polar phenolic compounds from soil. For the first time Phl was isolated from the rhizosphere environment in raw soil. Following application of Pseudomonas fluorescens Q2-87 and the Phl-overproducing strain Q2-87(pPHL5122) to the seeds of wheat, 2.1 and 2.4 (mu)g of Phl/g of root plus rhizosphere soil, respectively, were isolated from wheat grown in a Ritzville silt loam; 0.47 and 1.3 (mu)g of Phl/g of root plus rhizosphere soil, respectively, were isolated from wheat grown in a Shano silt loam. However, when the amount of Phl was calculated on the basis of cell density, Q2-87(pPHL5122) produced seven and six times more antibiotic than Q2-87 in Ritzville silt loam, and Shano silt loam, respectively.     

Secondary metabolites from nonhost plants affect the motility and viability of phytopathogenic Aphanomyces cochlioides zoospores

The motile zoospores of the damping-off pathogen Aphanomyces cochlioides aggregate on host plants (e.g., sugar beet, spinach) guided by the host-specific plant signal cochliophilin A before infection. To assess the potential role of secondary metabolites in nonhost resistance, acetone extracts of 200 nonhost traditional medicinal plants from Chinese and Bangladeshi origins were tested for the motility behaviour of A. cochlioides zoospores using a particle bioassay method. Nearly one third of the tested plant extracts exhibited diverse deleterious activities such as repellent, stimulant, motility halting and lysis against A. cochlioides zoospores. Among these active plants, an extract of the Chinese medicinal plant Dalbergia odorifera displayed potent repellent activity toward zoospores. Chromatographic separation of D. odorifera constituents revealed that the repellent activity was regulated by the cumulative effect of three motility-affecting isoflavonoids, viz. (+/-)-medicarpin (repellent at 150 microg/ml), (-)-claussequinone (stimulant at 100 microg/ml) and formononetin (stimulant and attractant at 50 microg/ml). A mixture (1:1:1, w/w/w) of these three compounds exhibited only repellent activity toward zoospores at a concentration lower than 50 microg/ml. These results suggest that nonhost plants might possess potential bioactive secondary metabolites to ward off zoosporic phytopathogens.     

Effect of 2,4-Diacetylphloroglucinol Producing,Overproducing, and Nonproducing Pseudomonas fluorescens F113 in the Rhizosphere of Pea

Pseudomonas fluorescens F113lacZY and modified strains carrying different function modifications were assessed for their impact in the rhizosphere of pea. Strain F113lacZY naturally produces the anti-fungal metabolite 2,4-diacetylphloroglucinol (Phl) useful in plant disease control. The first modified strain of F113 was repressed in production of Phl, creating the Phl negative strain F113G22. The second was a plasmid based overproducer of Phl (F113Rif (pCUGP)). Both the F113lacZY and the F113Rif (pCUGP) strains increased the rhizoplane fungal populations, whereas the same strains reduced the rhizosphere soil fungal populations with respect to the control. Similar results were found with the rhizoplane and rhizosphere soil bacterial populations. The F113G22 treatment resulted in a significantly greater indigenous fluorescent Pseudomonas population than the F113lacZY and F113Rif (pCUGP) treatments and a greater total Pseudomonas population than the control, F113lacZY, and F113Rif (pCUGP) treatments. Overproduction of Phl did not affect the establishment of the introduced Pseudomonas population. None of the inocula displaced the indigenous populations, but the F113G22 inocula had an additive effect on the total Pseudomonas population. P (phosphatase), S (sulphatase), and N (urease) cycle enzyme activities were increased while C (glucosidase, NAGase) cycle activities were decreased by the F113lacZY and F113Rif (pCUGP) treatments, suggesting C leakage from the roots. Overall, most effects of inoculation compared to the wild type were found with the non-Phl-producing strain. Overproduction of Phl had little environmental effect in relation to wild-type inocula.     

Crystal Structure and Computational Analyses Provide Insights into the Catalytic Mechanism of 2,4-Diacetylphloroglucinol Hydrolase PhlG from Pseudomonas fluorescens

2,4-Diacetylphloroglucinol hydrolase PhlG from Pseudomonas fluorescens catalyzes hydrolytic carbon-carbon (C–C) bond cleavage of the antibiotic 2,4-diacetylphloroglucinol to form monoacetylphloroglucinol, a rare class of reactions in chemistry and biochemistry. To investigate the catalytic mechanism of this enzyme, we determined the three-dimensional structure of PhlG at 2.0 Å resolution using x-ray crystallography and MAD methods. The overall structure includes a small N-terminal domain mainly involved in dimerization and a C-terminal domain of Bet v1-like fold, which distinguishes PhlG from the classical α/β-fold hydrolases. A dumbbell-shaped substrate access tunnel was identified to connect a narrow interior amphiphilic pocket to the exterior solvent. The tunnel is likely to undergo a significant conformational change upon substrate binding to the active site. Structural analysis coupled with computational docking studies, site-directed mutagenesis, and enzyme activity analysis revealed that cleavage of the 2,4-diacetylphloroglucinol C–C bond proceeds via nucleophilic attack by a water molecule, which is coordinated by a zinc ion. In addition, residues Tyr¹²¹, Tyr²²⁹, and Asn¹³², which are predicted to be hydrogen-bonded to the hydroxyl groups and unhydrolyzed acetyl group, can finely tune and position the bound substrate in a reactive orientation. Taken together, these results revealed the active sites and zinc-dependent hydrolytic mechanism of PhlG and explained its substrate specificity as well.     

Loss of cyclin D1 impairs cerebellar development and suppresses medulloblastoma formation

Medulloblastoma, the most common malignant brain tumor of childhood, is believed to derive from immature granule neuron precursors (GNPs) that normally proliferate in the external granule layer before exiting the cell cycle and migrating to their mature location in the inner granule layer. In this study, we examined the expression of D type cyclins in GNPs during cerebellar development and showed that GNPs in early development expressed only cyclin D1, whereas later GNPs expressed both cyclins D1 and D2. Coinciding with the period of cyclin D1-only expression, Ccnd1(-/-) mice showed reduced proliferation of GNPs and impaired growth of the cerebellum. Interestingly, removal of cyclin D1 was sufficient to drastically reduce the incidence of medulloblastoma in Ptch1(+/-) mice, despite the fact that these tumors showed upregulation of both cyclins D1 and D2. We showed that cyclin D1 has an earlier role in tumorigenesis: in the absence of cyclin D1, the incidence and overall volume of ;preneoplastic' lesions were significantly decreased. We propose a model that links a role of cyclin D1 in normal GNP proliferation with its early role in tumorigenesis.     

Proton irradiation suppresses angiogenic genes and impairs cell invasion and tumor growth

The energy deposition characteristics of proton radiation have attracted considerable attention in light of its implications for carcinogenesis risk in space travel, as well for application to cancer treatment. In space, it is the principle component of the galactic cosmic radiation to which astronauts will be exposed. For treatment, an increasing number of proton facilities are being established to exploit the physical advantages of this radiation type. However, the possibility that there may also be biologically based advantages to proton exposure has not been considered in either context. We demonstrate here that high-energy proton irradiation can inhibit expression of major pro-angiogenic factors and multiple angiogenesis-associated processes, including invasion and endothelial cell proliferation, which is prominent in cancer progression. Dose-dependent suppression of angiogenic signaling was demonstrated for both cancer and nontransformed cells. Pan-genomic microarray analysis and RT-PCR revealed that post-irradiation (0.5, 1.0 and 2.0 Gy), critical pro-angiogenic signaling factors including: vascular endothelial growth factor (VEGF), interleukin 6 and 8 (IL-6, IL-8) and hypoxia-inducible factor-1 alpha (HIF-1A), were significantly downregulated. Co-culture studies demonstrated that endothelial cell proliferation and invasion were inhibited by culturing with irradiated cancer or fibroblast cells, which suggests that proton irradiation may, in addition to direct action, contribute to angiogenesis suppression through modulation of paracrine signalings from targeted cells. Addition of recombinant IL-8 or VEGF partially restored these functions in vitro, while in vivo, an attenuated tumor growth rate was demonstrated for proton-irradiated human lung cancer cells. Taken together, these findings provide novel pre-clinical evidence that proton irradiation may, in addition to its physical targeting advantages, have important biological ramifications that should be a consideration in the optimization of proton therapy.     

Identification and Characterization of a Gene Cluster for Synthesis of the Polyketide Antibiotic 2,4-Diacetylphloroglucinol from Pseudomonas fluorescens Q2-87

The polyketide metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) is produced by many strains of fluorescent Pseudomonas spp. with biocontrol activity against soilborne fungal plant pathogens. Genes required for 2,4-DAPG synthesis by P. fluorescens Q2-87 are encoded by a 6.5-kb fragment of genomic DNA that can transfer production of 2,4-DAPG to 2,4-DAPG-nonproducing recipient Pseudomonas strains. In this study the nucleotide sequence was determined for the 6.5-kb fragment and flanking regions of genomic DNA from strain Q2-87. Six open reading frames were identified, four of which (phlACBD) comprise an operon that includes a set of three genes (phlACB) conserved between eubacteria and archaebacteria and a gene (phlD) encoding a polyketide synthase with homology to chalcone and stilbene synthases from plants. The biosynthetic operon is flanked on either side by phlE and phlF, which code respectively for putative efflux and regulatory (repressor) proteins. Expression in Escherichia coli of phlA, phlC, phlB, and phlD, individually or in combination, identified a novel polyketide biosynthetic pathway in which PhlD is responsible for the production of monoacetylphloroglucinol (MAPG). PhlA, PhlC, and PhlB are necessary to convert MAPG to 2,4-DAPG, and they also may function in the synthesis of MAPG.     

Role of 2,4-Diacetylphloroglucinol in the Interactions of the Biocontrol Pseudomonad Strain F113 with the Potato Cyst Nematode Globodera rostochiensis

The potato cyst nematode Globodera rostochiensis is an important pest of potato (Solanum tuberosum). Pseudomonas fluorescens F113, which produces 2,4-diacetylphloroglucinol (DAPG), was investigated as a potential biocontrol agent against G. rostochiensis. Exposure of nematode cysts to the pseudomonad, under in vitro conditions or in soil microcosms, almost doubled the ability of the eggs to hatch. The percentage of mobile juveniles was reduced threefold following their incubation in the presence of the pseudomonad, both in vitro and in soil. Results obtained with a transposon-induced DAPG-negative biosynthetic mutant of F113 and its complemented derivative with restored DAPG synthesis showed that the ability of strain F113 to produce DAPG was responsible for the increase in hatch ability and the reduction in juvenile mobility. Similar effects on egg hatch ability and juvenile mobility of G. rostochiensis were obtained in vitro by incubating nematode cysts and juveniles, respectively, in the presence of synthetic DAPG. DAPG-producing P. fluorescens F113 is proposed as a potential biocontrol inoculant for the protection of potato crops against the potato cyst nematode.     

PHD3-mediated prolyl hydroxylation of nonmuscle actin impairs polymerization and cell motility

Actin filaments play an essential role in cell movement, and many posttranslational modifications regulate actin filament assembly. Here we report that prolyl hydroxylase 3 (PHD3) interacts with nonmuscle actin in human cells and catalyzes hydroxylation of actin at proline residues 307 and 322. Blocking PHD3 expression or catalytic activity by short hairpin RNA knockdown or pharmacological inhibition, respectively, decreased actin prolyl hydroxylation. PHD3 knockdown increased filamentous F-actin assembly, which was reversed by PHD3 overexpression. PHD3 knockdown increased cell velocity and migration distance. Inhibition of PHD3 prolyl hydroxylase activity by dimethyloxalylglycine also increased actin polymerization and cell migration. These data reveal a novel role for PHD3 as a negative regulator of cell motility through posttranslational modification of nonmuscle actins.     

Characterization of PhlG, a Hydrolase That Specifically Degrades the Antifungal Compound 2,4-Diacetylphloroglucinol in the Biocontrol Agent Pseudomonas fluorescens CHA0

The potent antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a major determinant of biocontrol activity of plant-beneficial Pseudomonas fluorescens CHA0 against root diseases caused by fungal pathogens. The DAPG biosynthetic locus harbors the phlG gene, the function of which has not been elucidated thus far. The phlG gene is located upstream of the phlACBD biosynthetic operon, between the phlF and phlH genes which encode pathway-specific regulators. In this study, we assigned a function to PhlG as a hydrolase specifically degrades DAPG to equimolar amounts of mildly toxic monoacetylphloroglucinol (MAPG) and acetate. DAPG added to cultures of a DAPG-negative ΔphlA mutant of strain CHA0 was completely degraded, and MAPG was temporarily accumulated. In contrast, DAPG was not degraded in cultures of a ΔphlA ΔphlG double mutant. To confirm the enzymatic nature of PhlG in vitro, the protein was histidine tagged, overexpressed in Escherichia coli, and purified by affinity chromatography. Purified PhlG had a molecular mass of about 40 kDa and catalyzed the degradation of DAPG to MAPG. The enzyme had a k_cat of 33 s⁻¹ and a K_m of 140 μM at 30°C and pH 7. The PhlG enzyme did not degrade other compounds with structures similar to DAPG, such as MAPG and triacetylphloroglucinol, suggesting strict substrate specificity. Interestingly, PhlG activity was strongly reduced by pyoluteorin, a further antifungal compound produced by the bacterium. Expression of phlG was not influenced by the substrate DAPG or the degradation product MAPG but was subject to positive control by the GacS/GacA two-component system and to negative control by the pathway-specific regulators PhlF and PhlH.     

Metastin suppresses the motility and growth of CHO cells transfected with its receptor

We recently reported having identified of the ligand for an orphan G-protein-coupled receptor, hOT7T175, as the gene product (68-121)-amide of the metastasis suppressor gene KiSS-1. We further showed that the ligand, which we named "metastin," inhibits chemotaxis and invasion of Chinese hamster ovary (CHO) cells transfected with hOT7T175 cDNA (CHO/h175) in vitro, and pulmonary metastasis of hOT7T175-transfected B16-BL6 melanomas in vivo. In the present study, we investigated the activity of metastin in CHO/h175 cells in greater detail. Metastin significantly suppressed motility in a chemotaxis assay and wound healing assay at 10-100 nM order concentrations. Two N-terminally truncated peptides, metastin(40-54) and metastin(45-54) inhibited the migration of CHO/h175 cells as potently as metastin itself. Metastin also inhibited the spreading, monolayer growth and colony formation in agar (0.8%) of CHO/h175 cells at 10-100 nM concentrations. These results indicate that metastin is a potent inhibitor of cell motility, leading to suppression of cell growth and antimetastatic activity, and suggest that low molecular chemical compounds could replace its activity as a novel antimetastatic agent.