Iturin levels on wheat spikes linked to biological control of Fusarium head blight by Bacillus amyloliquefaciens.

The TrigoCor strain of Bacillus amyloliquefaciens provides consistent control against Fusarium head blight of wheat in controlled settings but there is a lack of disease and deoxynivalenol suppression in field settings. Since production of antifungal compounds is thought to be the main mode of action of TrigoCor control, we quantified levels of a key family of antifungal metabolites, iturins, as well as monitored Bacillus populations on wheat spikes over 14 days post-application in both the greenhouse and the field. We found that initial iturin levels on spikes in the greenhouse were three times greater than on spikes in the field, but that by 3 days post-application, iturin levels were equivalent and very low in both settings. We also determined that iturins declined rapidly over a 3-day post-application period on wheat spikes in both environments, despite the presence of significant Bacillus populations. Greenhouse trials and antibiosis tests indicated that the lower iturin levels on wheat spikes in the field could be a major factor limiting disease control in field settings. Future efforts to improve Bacillus disease control on wheat spikes and in the phyllosphere of various plants should focus on maintaining higher levels of iturins over critical infection periods.

First Report of Blight Caused by Sclerotium rolfsii on the Invasive Exotic Weed, Vincetoxicum rossicum (Pale Swallow-Wort), in Western New York.

Pale (Vincetoxicum rossicum) and black swallow-wort (V. nigrum) are perennial, twining vines that are increasingly invasive in natural and managed ecosystems in the northeastern United States and southeastern Canada. Both species, introduced from Europe in the 1800s, are listed as noxious weeds or banned invasive species by the USDA-Natural Resource Conservation Service. Observations by C. Southby, a local naturalist, over several years at a meadow populated by pale swallow-wort in Powder Mill Park, Monroe County, NY, revealed a gradual disappearance of pale swallow-wort with restoration of native grasses and some dicotyledonous plant species, in a 6.7-m-diameter area. Diseased swallow-wort plants had extensive yellowing and wilting of foliage, likely due to splitting of the basal stem, with white mycelium throughout the stem and crown; small, reddish brown sclerotia were evident, but roots were not affected. Stem tissue sections from 20 symptomatic plants were vacuum infiltrated with 2% NaOCl for 20 min, then plated onto malt yeast agar and potato dextrose agar amended with 60 mg/liter of penicillin and 80 mg/liter of streptomycin, resulting in development of fast-growing, white mycelium which then formed numerous, irregularly shaped (2 to 4 mm diameter), reddish brown sclerotia at the plate edges. Two individual cultures were identified as S. rolfsii (1) based on size, shape, and color of the sclerotia and presence of characteristic clamp connections in the mycelium. The isolate was suspected to be S. rolfsii var. delphinii due to the reported inability of S. rolfsii to persist in regions with extremely low winter temperatures (4), but molecular data showed otherwise. Sequences of the 18S gene (GenBank JN543690), internal transcribed spacer region (JN543691), and 28S gene (JN543692) of the ribosomal DNA identified the isolate, VrNY, as S. rolfsii (2,3). Pathogenicity tests were conducted with individual 2-month-old seedlings of V. rossicum and V. nigrum grown in steam-sterilized Metromix 360 in SC10 polypropylene conetainers in a growth chamber with a diurnal cycle of 25/20°C, a photoperiod of 14-h light/10-h dark, and fertilized at 3 week intervals. Two independent replications of 12 plants of each species were each inoculated at the stem base with a 4-mm-diameter mycelial agar plug from the growing edge of a colonized plate. The agar plug was held in place with 5 g of sterile sand. Control plants (12 of each species per replication) were treated with sterile agar plugs. Plants for each treatment were placed within a clear plastic bag to maintain 90% relative humidity for 72 h, and then removed from the bags. Disease symptoms developed over 21 days, with >90% of inoculated plants showing symptoms within 2 weeks. Control plants were symptomless. Incidence of mortality was 66 and 60% for V. rossicum and V. nigrum, respectively, by 3 weeks. The fungus reisolated from diseased stem and crown tissue produced characteristic mycelium with irregular sclerotia, consistent with those of S. rolfsii. Since spread of this fungus is based on movement of soilborne sclerotia, this isolate may offer potential as a bio-herbicide for control of swallow-wort in natural ecosystems if the isolate can be demonstrated to have a host range restricted to this invasive weed. References: (1) B. A. Edmunds and M. L. Gleason. Plant Dis. 87:313, 2003. (2) C. E. Harlton et al. Phytopathology 85:1269, 1995. (3) I. Okabe and N. Matsumoto. Mycol. Res. 107:164, 2003. (4) Z. Xu et al. Plant Dis. 92:719, 2008.

A simple method for enhancing paclitaxel release from Taxus canadensis cell suspension cultures utilizing cell wall digesting enzymes.

Paclitaxel storage in Taxus suspension cell cultures was studied through the simple use of cell wall digesting enzymes. The application of cellulase (1%) and pectolyase (0.1%) to Taxus canadensis suspension cultures induced a significant increase in the paclitaxel present in the extracellular medium while maintaining membrane integrity, suggesting that paclitaxel is stored in the cell wall. The addition of cell wall digesting enzymes to a cell culture bioprocess may be an effective way of enhancing paclitaxel release to the extracellular medium and hence simplify product recovery.

Involvement of a cytochrome P450 monooxygenase in thaxtomin A biosynthesis by Streptomyces acidiscabies.

The biosynthesis of the thaxtomin cyclic dipeptide phytotoxins proceeds nonribosomally via the thiotemplate mechanism. Acyladenylation, thioesterification, N-methylation, and cyclization of two amino acid substrates are catalyzed by the txtAB-encoded thaxtomin synthetase. Nucleotide sequence analysis of the region 3' of txtAB in Streptomyces acidiscabies 84.104 identified an open reading frame (ORF) encoding a homolog of the P450 monooxygenase gene family. It was proposed that thaxtomin A phenylalanyl hydroxylation was catalyzed by the monooxygenase homolog. The ORF was mutated in S. acidiscabies 84.104 by using an integrative gene disruption construct, and culture filtrate extracts of the mutant were assayed for the presence of dehydroxy derivatives of thaxtomin A. Reversed-phase high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry indicated that the major component in culture filtrate extracts of the mutant was less polar and smaller than thaxtomin A. Comparisons of electrospray mass spectra as well as (1)H- and (13)C-nuclear magnetic resonance spectra of the purified compound with those previously reported for thaxtomins confirmed the structure of the compound as 12,15-N-dimethylcyclo-(L-4-nitrotryptophyl-L-phenylalanyl), the didehydroxy analog of thaxtomin A. The ORF, designated txtC, was cloned and the recombinant six-His-tagged fusion protein produced in Escherichia coli and purified from cell extracts. TxtC produced in E. coli exhibited spectral properties similar to those of cytochrome P450-type hemoproteins that have undergone conversion to the catalytically inactive P420 form. Based on these properties and the high similarity of TxtC to other well-characterized P450 enzymes, we conclude that txtC encodes a cytochrome P450-type monooxygenase required for postcyclization hydroxylation of the cyclic dipeptide.

Polyketide synthase genes in insect- and nematode-associated fungi.

Production of polyketides is accomplished through complex enzymes known as polyketide synthases (PKS); these enzymes have highly conserved domains that might be useful in screens for PKSs in diverse groups of organisms. A degenerate PCR-based approach was used to amplify PKS fragments of the ketosynthase domain from genomic DNA of a group of insect- and nematode-associated fungi. Of 157 isolates (representing 73 genera and 144 species) screened, 92 isolates generated PCR products of predicted size (approximately 300 bp). The ability to detect PKS domains was a function of the number of different primer pairs employed in the screen. Cloning and sequencing revealed that 66 isolates had at least one unique PKS sequence; ten members of this set contained multiple PKS fragments, for a total of 76 unique PKS fragments. Since PKS genes appear to be widespread among fungi, a PCR-based screening system appears to be an efficient, directed means to identify organisms having the potential to produce polyketides.

A new coding region polymorphism of human IgLC2.

A new allelic form of the human IgLC2 gene is described. The marker involves a T to C substitution in the C lambda 2 constant region gene, a silent substitution at amino acid coding position 178 (YAASSYLSL) and two substitutions in the 3'-flanking region. Analysis of IgLC2 alleles in a total of 60 individuals has indicated a frequency of 0.32 for the new allele, which has been designated IgLC2*B2. The *B1 and *B2 alleles encode T and C, respectively, at nucleotide position 212 in the IgLC2 coding region. Both the *B1 and *B2 alleles are found in individuals homozygous for the single-copy RFLP allele of IgLC2/IgLC3 (8 kb EcoRI). Knowledge of alleles of this marker will be important for studies on the expression of the IgLC2 and IgLC3 isotypes in normal and autoimmune lymphocyte populations, as the coding regions of the two isotypes differ only at this position. The marker will also be useful in further studies of linkage with other IgLV and IgLC markers and to establish possible correlations with susceptibility to autoimmune disorders.

Survival and differential development of Entomophaga maimaiga and Entomophaga aulicae (Zygomycetes: Entomophthorales) in Lymantria dispar hemolymph.

The closely related entomophthoralean fungi Entomophaga aulicae and E. maimaiga are both host-specific pathogens of lepidopteran larvae. However, these fungi do not have the same host range. The first objective of this study was to compare the fate of E. aulicae in the nonpermissive host Lymantria dispar with the fate of the successful pathogen E. maimaiga over the same time period. In the hemolymph of L. dispar injected with E. maimaiga protoplasts, the number of hemocytes demonstrated a decreasing trend after the first day postinjection and hemocytes completely disappeared by day 5, with the majority of larvae dying in 5.6 +/- 0.1 days. In L. dispar larvae, E. maimaiga infections developed successfully, evidenced by increasing numbers of protoplasts and hyphal bodies prior to host mortality. In contrast, at day 5 hemocytes were readily visible in hemolymph of E. aulicae-injected larvae, but E. aulicae cells did not increase in numbers, although persisting in the hemolymph for at least 16 days postinjection. For both fungal species, when hemolymph samples from injected insects were introduced to culture media viable fungal cultures were always produced. Both E. aulicae and E. maimaiga occurred in hemolymph initially after injection as protoplasts. For E. maimaiga, after day 3, <50% of fungal cells were hyphal bodies until insect death when most cells regenerated cell walls. For E. aulicae, from day 2 equal numbers of fungal cells in the hemolymph occurred as protoplasts and hyphal bodies. To investigate the cause of fungistasis in E. aulicae-injected larvae, E. aulicae cell cultures exposed to partially purified protein fractions from hemolymph of larvae infected with either fungus displayed increased lysis and decreased viability at lower concentrations of protein fractions compared with E. maimaiga cell cultures. These studies demonstrate that E. aulicae does not increase in L. dispar hemolymph, although it persists and results suggest that proteinaceous factors induced within the hemolymph may limit the capacity of E. aulicae to develop successful infections.

The txtAB genes of the plant pathogen Streptomyces acidiscabies encode a peptide synthetase required for phytotoxin thaxtomin A production and pathogenicity.

Four Streptomyces species have been described as the causal agents of scab disease, which affects economically important root and tuber crops worldwide. These species produce a family of cyclic dipeptides, the thaxtomins, which alone mimic disease symptomatology. Structural considerations suggest that thaxtomins are synthesized non-ribosomally. Degenerate oligonucleotide primers were used to amplify conserved portions of the acyladenylation module of peptide synthetase genes from genomic DNA of representatives of the four species. Pairwise Southern hybridizations identified a peptide synthetase acyladenylation module conserved among three species. The complete nucleotide sequences of two peptide synthetase genes (txtAB) were determined from S. acidiscabies 84.104 cosmid library clones. The organization of the deduced TxtA and TxtB peptide synthetase catalytic domains is consistent with the formation of N-methylated cyclic dipeptides such as thaxtomins. Based on high-performance liquid chromatography (HPLC) analysis, thaxtomin A production was abolished in txtA gene disruption mutants. Although the growth and morphological characteristics of the mutants were identical to those of the parent strain, txtA mutants were avirulent on potato tubers. Moreover, introduction of the thaxtomin synthetase cosmid into a txtA mutant restored both pathogenicity and thaxtomin A production, demonstrating a critical role for thaxtomins in pathogenesis.

Effect of food on the pharmacodynamics and pharmacokinetics of atorvastatin, an inhibitor of HMG-CoA reductase.

The pharmacodynamics and pharmacokinetics of atorvastatin, an HMG-CoA reductase inhibitor, were characterized in 16 healthy subjects following administration of 10 mg atorvastatin tablets with, or 3 h after, evening meals for 15 days in an open-label, randomized, 2-way crossover study. Atorvastatin was well tolerated. Atorvastatin administration with evening meals resulted in 25.2% lower mean Cmax and 29.8% longer mean tmax values relative to administration after meals. The mean AUC(0-24) value was 8.6% lower for atorvastatin administration with meals compared to after meals. In contrast to the effect of food on pharmacokinetics, LDL-C reductions were similar after atorvastatin administration with or after evening meals. Average reductions from baseline were 24.4% for total cholesterol, 39.6% for LDL-C and 10% for triglycerides. Therefore, atorvastatin may be administered with or without food.

Reduced rehospitalizations and reintegration of persons with mental illness into community living: a holistic approach.

The goal of inpatient rehabilitation services should be directed towards returning persons with mental illness to the community by producing measurable gains in functioning that promote increased personal independence, self-direction, and care to prepare the patient to live in the least restrictive environment. Aftercare compliance was increased and the recidivism rate was cut in half when the aftercare nurse saw the person with mental illness before discharge and a specific appointment was scheduled with the aftercare nurse. The scope of community outpatient services that can be authorized is determined by the outcome of negotiating and communicating between the clinician and case manager.

Isolation of labeled 9-dihydrobaccatin III and related taxoids from cell cultures of taxuscell cultures of taxus canadensis elicited with m.

Cell suspension cultures of Taxus canadensis rapidly produced paclitaxel (1) and other taxoids in response to elicitation with methyl jasmonate. Three of these taxoids, of potential value in the synthesis of taxoid analogues, have been isolated from cell cultures of Taxus canadensis and identified as 13-acetyl-9-dihydrobaccatin III (2), baccatin VI (3), and 9-dihydrobaccatin III (4). Of these metabolites, 9-dihydrobaccatin III (4) has not been isolated from any Taxus species, whereas 13-acetyl-9-dihydrobaccatin III (2) and baccatin VI (3) have been isolated from a number of natural sources. 2D NMR techniques, mass spectrometry, and partial synthesis were used to rigorously elucidate the structure and stereochemistry of these natural products.

Characterization of 6-epi-3-anhydroophiobolin B from Cochliobolus heterostrophus.

The new sesterterpenoid 6-epi-3-anhydroophiobolin B (1) and six known ophiobolins were isolated from the extracts of the fungus Cochliobolus heterostrophus race O. The structure of 6-epi-3-anhydroophiobolin B was deduced from analysis of spectral data and the structural characterization of dehydration and dimerization products. Ophiobolin A (2) showed potent activity in cytotoxicity assays and marginal activity in antimalarial assays.

The kinetics of taxoid accumulation in cell suspension cultures of Taxus following elicitation with methyl jasmonate.

Cell suspension cultures of Taxus canadensis and Taxus cuspidata rapidly produced paclitaxel (Taxol) and other taxoids in response to elicitation with methyl jasmonate. By optimizing the concentration of the elicitor, and the timing of elicitation, we have achieved the most rapid accumulation of paclitaxel in a plant cell culture, yet reported. The greatest accumulation of paclitaxel occurred when methyl jasmonate was added to cultures at a final concentration of 200 microM on day 7 of the culture cycle. The concentration of paclitaxel increased in the extracellular (cell-free) medium to 117 mg/day within 5 days following elicitation, equivalent to a rate of 23.4 mg/L per day. Paclitaxel was only one of many taxoids whose concentrations increased significantly in response to elicitation. Despite the rapid accumulation and high concentration of paclitaxel, its concentration never exceeded 20% of the total taxoids produced in the elicited culture. Two other taxoids, 13-acetyl-9-dihydrobaccatin III and baccatin VI, accounted for 39% to 62% of the total taxoids in elicited cultures. The accumulation of baccatin III did not parallel the pattern of accumulation for paclitaxel. Baccatin III continued to accumulate until the end of the culture cycle, at which point most of the cells in the culture were dead, implying a possible role as a degradation product of taxoid biosynthesis, rather than as a precursor.

Information technology education for health professionals: opportunities and challenges.

Computer applications are on the increase in virtually every aspect of health care, from the routine management of information to clinical diagnoses or clinical decision making. Indeed, the use of computers extends from patient monitoring and point-of-care testing to basic and applied research. Given the ubiquity of computers in health care, it is crucial that health professions education programs provide students with the computer proficiencies they need to apply to their roles as health professionals. This article highlights the importance of such training. It also provides results of two separate needs assessment surveys, and presents curriculum design strategies and competencies to meet health care's present and emerging information technology needs.

Cimetidine does not alter atorvastatin pharmacokinetics or LDL-cholesterol reduction.

OBJECTIVE: To determine the effects of cimetidine on the steady-state pharmacokinetics and pharmacodynamics of atorvastatin, a 3-hydroxymethyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitor. METHODS: Twelve healthy subjects participated in a randomized two-way crossover study. Each subject received atorvastatin 10 mg every morning for 2 weeks and atorvastatin 10 mg every morning with cimetidine 300 mg four times a day for 2 weeks, separated by a 4-week washout period. Steady-state pharmacokinetic parameters (based on an enzyme inhibition assay) and lipid responses were compared. RESULTS: Pharmacokinetic parameters and lipid responses were similar following administration of atorvastatin alone and atorvastatin with cimetidine. Mean values for Cmax (the maximum concentration) were 5.11 ng eq.ml(-1) and 4.54 ng eq.ml(-1), for tmax (the time to reach maximum concentration) 2.2 h and 1.3 h, for AUC0-24 (area under the concentration-time curve from time 0 h to 24 h) 58.6 ng eq.h.ml(-1) and 58.5 ng eq.h.ml(-1), and for t1/2 (terminal half-life) 10.1 h and 17.0 h, respectively, following administration of atorvastatin alone and atorvastatin with cimetidine. Following treatment with atorvastatin alone and atorvastatin with cimetidine, mean values for the percentage change from baseline for total cholesterol were -29.5% and -29.9%, for low-density lipoprotein (LDL) cholesterol -41.0% and -42.6%, for high-density lipoprotein (HDL) cholesterol 6.3% and 5.8%, and for triglycerides -33.8% and -25.8%, respectively. CONCLUSIONS: The rate and extent of atorvastatin absorption and the effects of atorvastatin on LDL-cholesterol responses are not influenced by coadministration of cimetidine.

The Potential of Thiarubrine C as a Nematicidal Agent against Plant- parasitic Nematodes.

Thiarubrine C, a polyacetylenic 1,2-dithiin isolated from the roots of Rudbeckia hirta (Asteraceae), exhibited strong nematicidal activity in in vitro and growth chamber assays. Thiarubrine C was toxic, in the absence of light, to the plant-parasitic nematodes Meloidogyne incognita and Pratylenchus penetrans at LCs of 12.4 ppm and 23.5 ppm, respectively. A minimum exposure time between 12 and 24 hours was the critical period for nematode mortality due to thiarubrine C. Although thiarubrine C was not totally dependent on light for toxicity, activity was enhanced in the presence of light, especially with the microbivorous nematode, Teratorhabditis dentifera. Upon exposure of M. incognita juveniles to 20 ppm thiarubrine C for 1 hour, infection of tomato plants was greatly reduced compared to untreated checks. Thiarubrine C was also effective in reducing plant infection when mixed with soil 24 hours prior to or at planting, unlike other related compounds such as delta-terthienyl.

Protein phosphatase 2A subunit assembly: the catalytic subunit carboxy terminus is important for binding cellular B subunit but not polyomavirus middle tumor antigen.

The carboxy terminus of protein phosphatase 2A (PP2A) catalytic subunit is highly conserved. Seven out of the last nine residues, including two potential in vivo phosphorylation sites, threonine 304 and tyrosine 307, are completely invariant in all known PP2As. Mutational analysis of the carboxy terminus in vivo was facilitated by efficient immunoprecipitation of trimeric PP2A holoenzyme via an epitope-tagged catalytic subunit. The results indicate that the catalytic subunit carboxy terminus is important for complex formation with the PP2A 55 kDa regulatory B subunit, but not with polyomavirus oncogene, middle tumor antigen (MT), a viral B-type regulatory subunit. Replacing catalytic subunit threonine 304 or tyrosine 307 with a negatively charged amino acid abolished binding of the B subunit to the dimeric enzyme core and altered substrate specificity. Certain other amino acid substitutions of different size and/or charge also abolished or greatly reduced B subunit binding. Substitution of alanine at position 304 or phenylalanine at position 307 did not dramatically reduce B subunit binding or phosphatase activity in vitro, yet the latter substitutions are not found in naturally occurring PP2As. Thus, the wild-type residues are important for a yet unknown function in vivo. Additionally, deleting the carboxy terminal nine amino acids inhibited binding of the B subunit to the dimeric enzyme core, indicating a requirement for one or more of these amino acids for complex formation. MT interaction with the dimeric PP2A enzyme core was not inhibited by any of these mutations. Finally, unlike B subunit, MT does not activate the phosphatase activity of the PP2A heterodimer towards cdc2-phosphorylated histone H1.

Taxol production and taxadiene synthase activity in Taxus canadensis cell suspension cultures.

The cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene represents the first committed, and a slow, step in the complex biosynthetic pathway leading to the anticancer drug Taxol. The cyclization enzyme, taxadiene synthase, has been previously purified from Pacific yew (Taxus brevifolia) stem and characterized, and the corresponding cDNA has been isolated. To better assess the role of taxadiene synthase in the control of pathway flux in Canadian yew (T. canadensis) cells, a reliable system for production of Taxol in suspension culture, the enzyme from this source was isolated and shown to be chromatographically, electrophoretically, and kinetically identical to that of T. brevifolia stem. Results from the analysis of enzyme activity levels during the time course of Taxol accumulation in developing cell cultures of T. canadensis indicate that rate-limiting transformations lay farther down the pathway than the cyclization step in this system.