Diesel exhaust particles modulate the tight junction protein occludin in lung cells in vitro

ABSTRACT: BACKGROUND: Using an in vitro triple cell co-culture model consisting of human epithelial cells (16HBE14o-), monocyte-derived macrophages and dendritic cells, it was recently demonstrated that macrophages and dendritic cells create a transepithelial network between the epithelial cells to capture antigens without disrupting the epithelial tightness. The expression of the different tight junction proteins in macrophages and dendritic cells, and the formation of tight junction-like structures with epithelial cells has been demonstrated. Immunofluorescent methods combined with laser scanning microscopy and quantitative real-time polymerase chain reaction were used to investigate if exposure to diesel exhaust particles (DEP) (0.5, 5, 50, 125 mug/ml), for 24 h, can modulate the expression of the tight junction mRNA/protein of occludin, in all three cell types. RESULTS: Only the highest dose of DEP (125 mug/ml) seemed to reduce the occludin mRNA in the cells of the defence system however not in epithelial cells, although the occludin arrangement in the latter cell type was disrupted. The transepithelial electrical resistance was reduced in epithelial cell mono-cultures but not in the triple cell co-cultures, following exposure to high DEP concentration. Cytotoxicity was not found, in either epithelial mono-cultures nor in triple cell co-cultures, after exposure to the different DEP concentrations. CONCLUSION: We concluded that high concentrations of DEP (125 mug/ml) can modulate the tight junction occludin mRNA in the cells of the defence system and that those cells play an important role maintaining the epithelial integrity following exposure to particulate antigens in lung cells.

Virosomes can enter cells by non-phagocytic mechanisms

Phagocytosis of fine particles (1 mum) by macrophages is a ligand-receptor-mediated, actin-based process, whereas the entering of smaller particles (cells, in particular penetration into monocyte-derived macrophages. The microscopic analysis of phagocytic cells incubated with virosomes and polystyrene particles showed that virosomes and particles penetrated cells even in the presence of cytochalasin D, a drug inhibiting actin-based phagocytosis. The charge of the virosomes and particles did not influence their penetration. Also, different inhibitors of endocytotic pathways did not prevent the particles and virosomes from penetrating into the cells. Additionally, to study the ability of virosomes to overcome the epithelial airway barrier, a triple cell co-culture model composed of epithelial cells, monocyte-derived macrophages and dendritic cells of the respiratory tract was used. We found virosomes and polystyrene particles in both populations of antigen-presenting cells, monocyte-derived macrophages, and dendritic cells, in the latter even if they were not directly exposed. In conclusion, virosomes are readily taken up by monocyte-derived macrophages, both by conventional phagocytosis and by actin-independent mechanisms. Further, they can penetrate the airway barrier and reach resident dendritic cells. Therefore, virosomes are promising vaccine candidates.

Isolation of myeloid dendritic cells and epithelial cells from human thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c(+)), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45(hi)) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

The immune response of bovine mammary epithelial cells to live or heat-inactivated Mycoplasma bovis

Mycoplasma bovis is an emerging bacterial agent causing bovine mastitis. Although these cell wall-free bacteria lack classical virulence factors, they are able to activate the immune system of the host. However, effects on the bovine mammary immune system are not yet well characterized and detailed knowledge would improve the prevention and therapy of mycoplasmal mastitis. The aim of this study was to investigate the immunogenic effects of M. bovis on the mammary gland in an established primary bovine mammary epithelial cell (bMEC) culture system. Primary bMEC of four different cows were challenged with live and heat-inactivated M. bovis strain JF4278 isolated from acute bovine mastitis, as well as with the type strain PG45. The immune response was evaluated 6 and 24h after mycoplasmal challenge by measuring the relative mRNA expression of selected immune factors by quantitative PCR. M. bovis triggered an immune response in bMEC, reflected by the upregulation of tumor necrosis factor-α, interleukin(IL)-1β, IL-6, IL-8, lactoferrin, Toll-like receptor-2, RANTES, and serum amyloid A mRNA. Interestingly, this cellular reaction was only observed in response to live, but not to heat-inactivated M. bovis, in contrast to other bacterial pathogens of mastitis such as Staphylococcus aureus. This study provides evidence that bMEC exhibit a strong inflammatory reaction in response to live M. bovis. The lack of a cellular response to heat-inactivated M. bovis supports the current hypothesis that mycoplasmas activate the immune system through secreted secondary metabolites.

XYLITOL METABOLISM IN RAT LIVER AND HEPATOCELLULAR CARCINOMAS

The significance of nutritional factors in cancer research has been strongly emphasized. Such research is concerned not only with epidemiological effects relative to dietary factors on the causation of cancer, but with nutritional effects as an energy source on the prevention of cancer. Many studies speculate that the energy flow between tumor and host can be regulated by dietary intake. However, little knowledge on the comparison of the specific nutritional and energy requirements of different cells and tissues is available. Most popular and essential energy sources for the body are the carbohydrates. Among them, xylitol is known as efficient an energy source as glucose. In carbohydrate metabolism, glycolysis is one of the major energy producing pathways. However, recently the existence of an alternate catabolic pathway in mammals for carbohydrate besides glycolysis, i.e. bypass through triosephosphates to lactate via methylglyoxal has been suggested. This bypass was implicated to regulate glycolysis and also be responsible for the fluctuation in the levels of a regulator of cell growth. Methylglyoxal itself is known as a cancerostatic agent. The alterations of biochemical parameters in xylitol metabolism in animals indicated that xylitol may be metabolized through a methylglyoxal pathway.^ To elucidate the biological effect of xylitol as an energy source and the biological effect of its metabolites as a cancerostatis agent, the mode and extent of metabolism must be understood in tumor-bearing animals. Differential utilization of xylitol and glucose, if any, between tumor and host in such animals may exert tissue selective effects on both in terms of methylglyoxal formation and energy provision. The aim of this work was to assess the extent to which the differential utilization of xylitol might be used to generate different metabolic pathways in tumor and host, and to consider a role of nutrition in cancer.^ The results disclose that the existence of a pathway for biological methylglyoxal formation in normal rat liver has been confirmed in single cell suspension; the metabolic significance of the methylglyoxal pathway in the metabolism of glucose and xylitol has been evaluated quantitatively in normal rat liver and the differential metabolism of glucose and xylitol through overall catabolic pathways of carbohydrates has been studied in normal hepatic cells, AS-30D hepatoma and other several hepatoma lines. ^

MEASUREMENT OF TUMOR BLOOD FLOW FOLLOWING NEUTRON IRRADIATION (ACTIVATION)

Clinical oncologists and cancer researchers benefit from information on the vascularization or non-vascularization of solid tumors because of blood flow's influence on three popular treatment types: hyperthermia therapy, radiotherapy, and chemotherapy. The objective of this research is the development of a clinically useful tumor blood flow measurement technique. The designed technique is sensitive, has good spatial resolution, in non-invasive and presents no risk to the patient beyond his usual treatment (measurements will be subsequent only to normal patient treatment).^ Tumor blood flow was determined by measuring the washout of positron emitting isotopes created through neutron therapy treatment. In order to do this, several technical and scientific questions were addressed first. These questions were: (1) What isotopes are created in tumor tissue when it is irradiated in a neutron therapy beam and how much of each isotope is expected? (2) What are the chemical states of the isotopes that are potentially useful for blood flow measurements and will those chemical states allow these or other isotopes to be washed out of the tumor? (3) How should isotope washout by blood flow be modeled in order to most effectively use the data? These questions have been answered through both theoretical calculation and measurement.^ The first question was answered through the measurement of macroscopic cross sections for the predominant nuclear reactions in the body. These results correlate well with an independent mathematical prediction of tissue activation and measurements of mouse spleen neutron activation. The second question was addressed by performing cell suspension and protein precipitation techniques on neutron activated mouse spleens. The third and final question was answered by using first physical principles to develop a model mimicking the blood flow system and measurement technique.^ In a final set of experiments, the above were applied to flow models and animals. The ultimate aim of this project is to apply its methodology to neutron therapy patients. ^

The effects of proteasome inhibition in chronic lymphocytic leukemia and prostate cancer

The use of proteasome inhibitors in cancer has received much attention with the recent FDA approval of bortezomib (Velcade/PS-341). However, in the chronic lymphocytic leukemia (CLL) clinical trial, bortezomib was not as effective as it was in vitro. Accordingly, results in prostate cancer were not remarkable, although regression of lymphadenopathy was observed. This response was also seen in CLL. ^ The proteasome degrades ∼80% of intracellular proteins. Although specific pathways affected by proteasome inhibitors are known, there are still unidentified mechanisms by which they induce apoptosis. The efficacy and mechanism of action of the reversible proteasome inhibitor bortezomib were compared to the novel irreversible inhibitor NPI-0052 in this study, and their mechanisms of action in CLL and prostate cancer were examined. ^ NPI-0052 inhibited proteasome activity and induced apoptosis with more rapid kinetics than bortezomib in CLL. Inhibition of proteasome activity with NPI-0052 was also more durable. Interestingly, bortezomib is cleared from the serum within 15min, which is insufficient time for bortezomib to effectively inhibit the proteasome. However, only 5min exposure was needed for NPI-0052 to produce maximal proteasome inhibition. The data suggest that bortezomib's slow kinetics and reversible nature limit its potential in vivo and the use of NPI-0052 should be considered. ^ In examining the mechanism(s) by which bortezomib and NPI-0052 induce apoptosis in CLL, both were found to elicit the ER stress pathway. A stromal cell co-culture system prevented apoptosis induced by both proteasome inhibitors, suggesting that if such factors in vivo were responsible for reducing bortezomib's efficacy, NPI-0052 would not prove useful either. Finally, Lyn, a Src family kinase (SFK), was decreased in response to bortezomib and NPI-0052 and correlated with apoptosis induction in CLL and prostate cancer. Both proteasome inhibitors specifically targeted Lyn rather than SFKs in general. ^ SFKs are overexpressed in cancer and involved in cell signaling, survival, and metastasis. In prostate cancer cells, both proteasome inhibition and Lyn-silencing significantly inhibited migration. Preliminary evidence also suggested that Lyn downregulation decreases invasion potential. Together, these data suggest that proteasome inhibitors are potential candidates for anti-metastasic therapy and further investigation is warranted for the use of Lyn-targeted therapy to treat metastases. ^

The role of plasmacytoid dendritic cells in the regulation of adaptive immunity

Plasmacytoid dendritic cells (pDCs) selectively express TLR7 which allows them to respond to RNA viruses and TLR9 which allows them to respond to DNA viruses and CpG oligonucleotides. Upon exposure to virus pDCs produce vast amounts of type I interferon (IFN) directly inhibiting viral replication and contributing to the activation of other immune cells. The ability of pDCs to promote B and T cell differentiation through type I IFN has been well documented although the role of additional factors including tumor necrosis factor (TNF) family members has not been thoroughly addressed. Here the expression of selected TNF family members in pDCs was examined and the role of TNF receptor-ligand interactions in the regulation of B and T lymphocyte growth and differentiation by pDCs was investigated. Upon stimulation with CpG-B, pDCs exhibit strong and stable expression of CD70, a TNF family ligand that binds to its receptor CD27 on memory B cells and promotes plasma cell differentiation and Ig secretion. Using an in vitro pDC/B cell co-culture system, it was determined that CpG-B-stimulated pDCs induce the proliferation of CD40L-activated human peripheral B cells and Ig secretion. This occurs independently of IFN and residual CpG, and requires physical contact between pDCs and B cells. CpG-stimulated pDCs induce the proliferation of both naive and memory B cells although Ig secretion is restricted to the memory subset. Blocking the interaction of CD70 with CD27 using an antagonist anti-CD70 antibody reduces the induction of B cell proliferation and IgG secretion by CpG-B-stimulated pDCs. Published studies have also indicated an important role for CD70 in promoting the expansion of CD4+ and CD8+ T cells and the development of effector function. CpG-B-stimulated pDCs induce naïve CD4+ T cell proliferation and production of multiple cytokines including IFN-γ, TNF-α, IL-10, IL-4, IL-5 and IL-13. Blocking the function of CD70 with an antagonist anti-CD70 antibody significantly reduced the induction of naïve CD4+ T cell proliferation by CpG-B-stimulated pDCs and the production of IL-4 and IL-13. Collectively these data indicate an important role for CD70 in the regulation of B and T lymphocyte growth and differentiation by pDCs. ^

Proceedings of the Twenty-First Annual Biochemical Engineering Symposium

The 21st Annual Biochemical Engineering Symposium was held at Colorado State University on April 20, 1991. The primary goals of this symposium series are to provide an opportunity for students to present and publish their research work and to promote informal discussions on biochemical engineering research. Contents High Density Fed-Batch Cultivation and Energy Metabolism of Bacillus thuringtensis; W.-M. Liu, V. Bihari, M. Starzak, and R.K. Bajpai Influences of Medium Composition and Cultivation Conditions on Recombinant Protein Production by Bacillus subtilis; K. Park, P.M. Linzmaier, and K.F. Reardon Characterization of a Foreign Gene Expression in a Recombinant T7 Expression System Infected with λ Phages; F. Miao and D.S. Kompala Simulation of an Enzymatic Membrane System with Forced Periodic Supply of Substrate; N. Nakaiwa, M. Yashima, L.T. Fan, and T. Ohmori Batch Extraction of Dilut Acids in a Hollow Fiber Module; D.G. O'Brien and C.E. Glatz Evaluation of a New Electrophoretic Device for Protein Purification; M.-J. Juang and R.G. Harrison Crossflow Microfiltration and Membrane Fouling for Yeast Cell Suspension; S. Redkar and R. Davis Interaction of MBP-β-Galactosidase Fusion Protein with Starch; L. Taladriz and Z. Nikolov Predicting the Solubility of Recombinant Proteins in Escherichia coli; D.L. Wilkinson and R.G. Harrison Evolution of a Phase-Separated, Gravity-Independent Bioractor; P.E. Villeneuve and E.H. Dunlop A Strategy for the Decontamination of Soils Containing Elevated Levels of PCP; S. Ghoshal, S. K. Banelji, and RK. Bajpai Practical Considerations for Implementation of a Field Scale In-Situ Bioremediation Project; J.P. McDonald, CA Baldwin, and L.E. Erickson Parametric Sensitivity Studies of Rhizopus oligosporus Solid Substrate Fermentation; J. Sargantanis, M.N. Karim, and V.G. Murphy, and RP. Tengerdy Production of Acetyl-Xylan Esterase from Aspergillus niger; M.R Samara and J.C. Linden Biological and Latex Particle Partitioning in Aqueous Two-Phase Systems; D.T.L. Hawker, RH. Davis, P.W. Todd, and R Lawson Novel Bioreactor /Separator for Microbial Desulfurization of Coal; H. Gecol, RH. Davis, and J .R Mattoon Effect of Plants and Trees on the Fate, Transport and Biodegradation of Contaminants in the Soil and Ground Water; W. Huang, E. Lee, J.F. Shimp, L.C. Davis, L.E. Erickson, and J.C. Tracy Sound Production by Interfacial Effects in Airlift Reactors; J. Hua, T.-Y. Yiin, LA Glasgow, and L.E. Erickson Soy Yogurt Fermentation of Rapid Hydration Hydrothermal Cooked Soy Milk; P. Tuitemwong, L.E. Erickson, and D.Y.C. Fung Influence of Carbon Source on Pentachlorophenol Degradation by Phanerochaete chrysosportum in Soil; C.-Y.M. Hsieh, RK. Bajpai, and S.K. Banerji Cellular Responses of Insect Cells Spodopiera frugiperda -9 to Hydrodynamic Stresses; P.L.-H. Yeh and RK. Bajpa1 A Mathematical Model for Ripening of Cheddar Cheese; J. Kim, M. Starzak, G.W. Preckshoi, and R.K. Bajpai

Proceedings of the 24th Annual Biochemical Engineering Symposium

The 24th Biochemical Engineering Symposium was held 9-10 September 1994 at the YMCA of the Rockies conference center in Estes Park, Colorado, under the sponsorship of the Department of Chemical Engineering at the University of Colorado. Previous symposia in this series have been hosted by Kansas State University (1st, 3rd, 5th, 9th, 12th, 16th, 20th), University of Nebraska-Lincoln (2nd, 4th), Iowa State University (6th, 7th, 10th, 13th, 17th, 22nd), University of Missouri-Columbia (8th, 14th, 19th), Colorado State University (11th, 15th, 21st), University of Colorado (18th), and the University of Oklahoma (23rd). The next symposium is scheduled to be held at the University of Missouri-Columbia. The symposia are devoted to talks by students about their ongoing research. Because final publication usually takes place elsewhere, the papers included in the proceedings are brief, and often cover work in progress. ContentsIn-Well Aeration: An Innovative Subsurface Remediation TechnologyPrashant Gandhi, X. Yang, L.E. Erickson, and L. T. Fan; Kansas State University Expression of an Antimicrobial Peptide Analog in Eacherlchill coliChris Haught and Roger G. Harrison; University of Oklahoma Using High-frequency Backpulaing to Maximize Croasflow Filtration PerformanceSanjeev G. Redkar and Robert H. Davis; University of Colorado Low Molecular Weight Organic Compositions of Acid Waters from Vegetable Oil SoapstocksSteven L. Johansen, Arunthathi Sivasothy, Peter J. Reilly, and Earl G. Hammond; Iowa State University; Michael K. Dowd; U.S. Department of Agriculture Gas Phase Composition Effects on Suspension Cultures of Taxus cuspidata Noushin Mirjalili and James C. Linden; Colorado State University Cybernetic Modeling of Spontaneous Oscillations in Continuous Cultures of Ssccharomyces cerevisiaeKenneth D. Jones and Dhinakar S. Kompala; University of Colorado The Effect of Turbulent Shear on Calcium Mobilization in Mammalian CellsChristopher M. Cannizzaro, Pradyumna K. Namdev, and Eric H. Dunlop; Colorado State University Experimental Studies of Droplet Ejection at the Free Surface In Sparged ReactorsT. Y. Yiin, L A. Glasgow, and L. E. Erickson; Kansas State University The Role of Domain E (Starch-Binding Region) on the Activity of a Bacillus macersns Cyclodextrln GlucanotransferaseHai-yin Chang, Trang Le, and Zivko L. Nikolov; Iowa State University Use of the Rotating Wall Vessel for Study of Plant Cell Suspension CulturesXinzhi Sun and James C. Linden; Colorado State University A Novel Counter-Current Distribution Apparatus for the Study of Multi-Stage Aqueous Two-Phase Extraction of Biomolecules and Cell ParticlesMartin R. Guinn and Paul Todd; University of Colorado The Dynamics of Unhooking and Contraction of a Polyelectrolyte Chain Around an Isolated PostLin Zhang and Edith M. Sevick; University of Colorado A Laboratory Study of the Fate of Trichloroathylene and 1,1,1-Trlchloroathane In the Presence of Alfalfa PlantsMuralidharan Narayanan, Ryan M. Green, Lawrence C. Davis, and Larry E. Erickson; Kansas State University Modeling the Fate of Pyrene In the RhIzosphereS.K. Santharam, LE. Erickson, and L. T. Fan; Kansas State University Derivatization of MaltooligosaccharidesDaniela Prinz, Peter J. Reilly, and Zivko L. Nikolov; Iowa State University Probing Surfactant-Protein Binding by EPA SpectroscopyNarendra B. Bam, Yale University; Theodore W. Randolph; University of Colorado Optimization of a Stir-Cell Bioreactor for In Vitro Production of RNANeal T. Williams, Kim A. Wicklund, and Robert H. Davis; University of Colorado

The Pto kinase mediates a signaling pathway leading to the oxidative burst in tomato

The Pto gene encodes a serine/threonine kinase that confers resistance in tomato to Pseudomonas syringae pv. tomato strains that express the avirulence gene avrPto. Partial characterization of the Pto signal transduction pathway and the availability of transgenic tomato lines (± Pto) make this an ideal system for exploring the molecular basis of disease resistance. In this paper, we test two transgenic tomato cell suspension cultures (±Pto) for production of H2O2 following independent challenge with two strains of P. syringae pv. tomato (±avrPto). Only when Pto and avrPto are present in the corresponding organisms are two distinct phases of the oxidative burst seen, a rapid first burst followed by a slower and more prolonged second burst. In the remaining three plant–pathogen interactions, we observe either no burst or only a first burst, indicating that the second burst is correlated with disease resistance. Further support for this observation comes from the finding that both resistant and susceptible tomato lines produce the critical second oxidative burst when challenged with P. syringae pv. tabaci, a nonhost pathogen that elicits a hypersensitive response on both tomato lines. The Pto kinase is not required, however, for the oxidative burst initiated by non-specific elicitors such as oligogalacturonides or osmotic stress. A model describing a possible role for the Pto kinase in the overall scheme of oxidative burst signaling is proposed.

Immune surveillance against a solid tumor fails because of immunological ignorance

Many peripheral solid tumors such as sarcomas and carcinomas express tumor-specific antigens that can serve as targets for immune effector T cells. Nevertheless, overall immune surveillance against such tumors seems relatively inefficient. We studied immune surveillance against a s.c. sarcoma expressing a characterized viral tumor antigen. Surprisingly, the tumor cells were capable of inducing a protective cytotoxic T cell response if transferred as a single-cell suspension. However, if they were transplanted as small tumor pieces, tumors readily grew. Tumor growth correlated strictly with (i) failure of tumor cells to reach the draining lymph nodes and (ii) absence of primed cytotoxic T cells. Cytotoxic T cells were not tolerant or deleted because a tumor antigen-specific cytotoxic T cell response was readily induced in lymphoid tissue by immunization with virus or with tumor cells even in the presence of large tumors. Established tumors were rejected by vaccine-induced effector T cells if effector T cells were maintained by prolonged or repetitive vaccination, but not by single-dose vaccination. Thus, in addition to several other tumor-promoting parameters, some antigenic peripheral sarcomas—and probably carcinomas—may grow not because they anergize or tolerize tumor-specific T cells, but because such tumors are immunologically dealt with as if they were in a so-called immunologically privileged site and are ignored for too long.

Nucleotide excision repair in rat male germ cells: low level of repair in intact cells contrasts with high dual incision activity in vitro

The acquisition of genotoxin-induced mutations in the mammalian germline is detrimental to the stable transfer of genomic information. In somatic cells, nucleotide excision repair (NER) is a major pathway to counteract the mutagenic effects of DNA damage. Two NER subpathways have been identified, global genome repair (GGR) and transcription-coupled repair (TCR). In contrast to somatic cells, little is known regarding the expression of these pathways in germ cells. To address this basic question, we have studied NER in rat spermatogenic cells in crude cell suspension, in enriched cell stages and within seminiferous tubules after exposure to UV or N-acetoxy-2-acetylaminofluorene. Surprisingly, repair in spermatogenic cells was inefficient in the genome overall and in transcriptionally active genes indicating non-functional GGR and TCR. In contrast, extracts from early/mid pachytene cells displayed dual incision activity in vitro as high as extracts from somatic cells, demonstrating that the proteins involved in incision are present and functional in premeiotic cells. However, incision activities of extracts from diplotene cells and round spermatids were low, indicating a stage-dependent expression of incision activity. We hypothesize that sequestering of NER proteins by mispaired regions in DNA involved in synapsis and recombination may underlie the lack of NER activity in premeiotic cells.

Potentiation of the Oxidative Burst and Isoflavonoid Phytoalexin Accumulation by Serine Protease Inhibitors1

Treatment of soybean (Glycine max L. cv Williams 82) cell-suspension cultures with Pseudomonas syringae pv glycinea (Psg) harboring an avirulence gene (avrA) or with yeast elicitor resulted in an oxidative burst characterized by the accumulation of H2O2. This burst, and the resultant induction of glutathione S-transferase transcripts, occurred more rapidly and was more prolonged if cells were simultaneously treated with serine protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) or diisopropylfluorophosphate. PMSF and diisopropylfluorophosphate potentiate a large oxidative burst in cells exposed to Psg harboring the avrC avirulence gene, which is not recognized by the soybean cultivar used in this study. The potentiated burst was inhibited by diphenylene iodonium, an inhibitor of NADPH oxidase, and by the protein kinase inhibitor K252a. PMSF treatment of elicited cells or cells exposed to Psg:avrA caused a large increase in the accumulation of the isoflavonoid phytoalexin glyceollin; however, this was not associated with increased levels of transcripts encoding key phytoalexin biosynthetic enzymes. Glyceollin accumulation was inhibited by diphenylene iodonium; however, the oxidative burst in cells treated with Psg:avrC and PMSF was not followed by phytoalexin accumulation. We conclude that active oxygen species from the oxidative burst are necessary but not sufficient for inducing isoflavonoid phytoalexin accumulation in soybean cells.

Piperonylic Acid, a Selective, Mechanism-Based Inactivator of the trans-Cinnamate 4-Hydroxylase: A New Tool to Control the Flux of Metabolites in the Phenylpropanoid Pathway1

Piperonylic acid (PA) is a natural molecule bearing a methylenedioxy function that closely mimics the structure of trans-cinnamic acid. The CYP73A subfamily of plant P450s catalyzes trans-cinnamic acid 4-hydroxylation, the second step of the general phenylpropanoid pathway. We show that when incubated in vitro with yeast-expressed CYP73A1, PA behaves as a potent mechanism-based and quasi-irreversible inactivator of trans-cinnamate 4-hydroxylase. Inactivation requires NADPH, is time dependent and saturable (KI = 17 μm, kinact = 0.064 min−1), and results from the formation of a stable metabolite-P450 complex absorbing at 427 nm. The formation of this complex is reversible with substrate or other strong ligands of the enzyme. In plant microsomes PA seems to selectively inactivate the CYP73A P450 subpopulation. It does not form detectable complexes with other recombinant plant P450 enzymes. In vivo PA induces a sharp decrease in 4-coumaric acid concomitant to cinnamic acid accumulation in an elicited tobacco (Nicotiana tabacum) cell suspension. It also strongly decreases the formation of scopoletin in tobacco leaves infected with tobacco mosaic virus.