Virus-mediated reprogramming of gene expression in plants.

Plant viruses have made many significant contributions to plant biology over the years: they have provided plant researchers with functional promoters, transient expression systems and, most recently, with critical insights into the phenomenon of posttranscriptional gene silencing. Plant virus expression vectors have the ability to either overexpress genes or suppress gene expression in plants. Whereas the 'rules' for gene expression are generally understood conceptually, the mechanisms for the induction of gene silencing are less well understood. Recent advances in the understanding of both the biological role and the mode of action of posttranscriptional gene silencing will affect both the design and the use of plant viral vectors and transgenic plants for either gene-overexpression or gene-silencing applications.

Rapid, high-level expression of glycosylated rice alpha-amylase in transfected plants by an RNA viral vector.

Tobamoviral vectors have been developed for the heterologous expression of glycoproteins in plants. The rice alpha-amylase gene (OS103) was placed under the transcriptional control of a tobamovirus subgenomic promoter in a RNA viral vector. One to two weeks after inoculation, transfected Nicotiana benthamiana plants accumulated glycosylated alpha-amylase to levels of at least 5% total soluble protein. The 46kDa recombinant enzyme was purified, and its structural and biological properties were analyzed. Post-translational modifications of the secreted protein were compared to rice alpha-amylase expressed in amylolytic strains of Pichia pastoris and Saccharomyces cerevisiae. Endo-H analysis revealed that the alpha-amylase was moderately glycosylated in transfected plants and hyperglycosylated in yeast.

Preparation and characterization of submitochondrial fractions from adrenal cells.

A method for preparing submitochondrial fractions from adrenocortical cells was developed by adapting a procedure that has been successful with yeast mitochondria. The method is based upon osmotic swelling, sonication and centrifugation in sucrose. The preparation yields highly purified fractions of outer membrane, intermembrane space, inner membrane and a less purified fraction of matrix. Recoveries are good so that 10(7) cells yield approximately 170 micrograms of inner membrane protein and 12 micrograms of outer membrane protein. Electron microscopy shows that the outer membrane fraction consists of vesicles (0.2-0.6 micron diameter) while inner membrane appears as densely packed sheets of membranous material. Two-dimensional polyacrylamide gels (isoelectric focusing followed by electrophoresis) of all the fractions give highly reproducible patterns of protein spots with Coomassie staining. Steroidogenic proteins were found only in inner membrane fractions which were shown to contain cytochrome P-450 C27 side-chain cleavage and P-450 11 beta-hydroxylase together with adrenodoxin and adrenodoxin reductase. Inner membrane catalyzes side-chain cleavage of cholesterol (conversions to pregnenolone) and 11 beta-hydroxylation (DOC----corticosterone) when substrate and NADPH are added. The preparation yields highly purified submitochondrial fractions from Y-1 mouse adrenal tumor cells and from porcine and bovine adrenocortical mitochondria. The method has the virtue of yielding highly purified intermembrane fluid which is not true of other methods for fractionation of adrenal mitochondria. The procedure also yields cleaner preparations of the two membranes than two other published methods currently used to prepare submitochondrial fractions from adrenocortical cells.

A 6-month comparison between formoterol and salmeterol in patients with reversible obstructive airways disease.

The aim of this randomized, open, parallel group study was to compare the clinical efficacy of formoterol dry powder capsule 12 micrograms b.i.d. and salmeterol dry powder 50 micrograms b.i.d. in the treatment of patients with reversible obstructive airways disease. The 6-month treatment was preceded by a 2 week run-in period. Morning pre-dose peak expiratory flow (PEF) during the last 7 days of treatment was the primary variable. Throughout the study, patients recorded morning and evening pre-dose PEF, use of rescue medication, respiratory symptoms and adverse events. Clinic visits were scheduled at monthly intervals. Of the 482 patients randomized (equal numbers in the two treatment groups), 428 completed the study. Four hundred and twenty-five patients were included in the efficacy analysis for the primary variable. For mean morning pre-dose PEF during the last 7 days of treatment, the 95% confidence interval (CI) for the treatment contrast formoterol minus salmeterol was included entirely in the pre-defined range of equivalence (CI limits = -8.69, +9.841 min-1). This was also the case for the morning PEF during the last week before each clinic visit. For mean evening pre-dose PEF, the estimated treatment contrasts showed a trend towards superiority of formoterol over salmeterol, which became statistically significant at 2, 3 and 4 months (P < 0.05; estimated contrasts 7.27, 10.45 and 10.511 min-1, respectively). No treatment group differences were found in use of rescue medication and respiratory symptom scores. The incidence of adverse events was similar in the two groups. These findings demonstrate that formoterol 12 micrograms b.i.d. and salmeterol 50 micrograms b.i.d., both formulated as dry powders, have similar long-term efficacy and safety profiles in patients with reversible obstructive airways disease.

Lack of microbial proliferation and phototoxic potential of a new matrix patch for estradiol delivery.

OBJECTIVES: To examine the potential of a new matrix system developed for estradiol delivery to cause microbial proliferation under the occluded site or to cause acute phototoxicity reactions. METHODS: Twenty healthy post-menopausal women participated in a microbial proliferation study and 11 in a phototoxicity study. Both studies were single centre, single blind and placebo controlled. Microbial proliferation was assessed by quantitative counts of the total aerobic bacterial population and of eight individual species before patch application and after removal following a 4 day application period on the abdomen. Acute phototoxicity potential was assessed following an 8 h application period on the abdomen by irradiating the application site after patch removal with ultra violet A radiation and visible light and evaluating the sites for up to 48 h post irradiation. Non-irradiated active and placebo patches on the other side of the abdomen served as controls. RESULTS: Total aerobic bacterial populations both before and after the matrix patch application period were low as expected for dry skin. Separate counts of microbial species were also low and did not change in any meaningful or consistent manner after patch application. In the phototoxicity study, mild erythema was observed in some patients at 0, 0.5 and 24 h post patch removal with no differences between irradiated and non-irradiated sites. CONCLUSIONS: These two studies demonstrate that a new matrix patch developed for estradiol delivery does not promote microbial proliferation under the occluded patch site or cause acute phototoxicity following removal.

[Neurological manifestations of systemic lupus erythematosus. Study of 53 cases]. / Manifestazioni neurologiche nel lupus eritematoso sistemico. Studio di 53 casi.

The Authors submitted 53 randomly selected patients affected by systemic lupus erythematosus (SLE) to neurologic evaluation to investigate the prevalence of neurologic manifestations, establish relationships to clinical and epidemiological findings and antinuclear antibodies and/or lupus anticoagulant (LAC), as well as to assess the usefulness of electroencephalogram (EEG), saccadic eye movements (SEM) analysis, brain computerized tomography (CT). Twenty-two patients (41.5%) had nervous system involvement on anamnestic and/or clinical examination: there were seizures in 5 patients, headache in 3, involuntary movements in 3, psychosis in 2 and cerebrovascular disorders in 9. The patients were subdivided into 2 groups, with neuro-SLE and without neuro-SLE, according to clinical and/or anamnestic evidence of nervous system involvement. There were no differences between the two groups of patients regarding disease duration, disease activity, presence of antinuclear antibodies and/or LAC. EEG and/or SEM and/or brain CT abnormalities were found in 38 cases, 18 of which had no clinical evidence of neuro-SLE. Instrumental evaluation can thus document subtle nervous dysfunction and offers the possibility of classification into: a) non-neuro-SLE; b) subclinical neuro-SLE; c) overt neuro-SLE.

Melanin production in Escherichia coli from a cloned tyrosinase gene.

We have cloned and functionally expressed a tyrosinase gene from Streptomyces antibioticus in Escherichia coli under the control of an inducible bacteriophage T7 promoter. Recombinant E. coli cells containing the induced tyrosinase gene produced melanin pigments on agar plates and in liquid culture when supplemented with copper and tyrosine. The expression of an additional open reading frame from the mel gene locus of S. antibioticus was required for high-level melanin production in E. coli. Our results also show that it is possible to screen other classes of precursor compounds for incorporation into melanin pigments with unique colors and other biochemical features. In addition, it may be possible to screen for enhanced melanin production in the absence of added precursors to identify overproducing mutants in the amino acid biosynthetic pathways of E. coli. The ability to screen for a melanin phenotype in recombinant E. coli provides new opportunities for production of novel melanins and for protein engineering of tyrosinases with altered catalytic properties.

Conversion of starch to ethanol in a recombinant Saccharomyces cerevisiae strain expressing rice alpha-amylase from a novel Pichia pastoris alcohol oxidase promoter.

A recombinant Saccharomyces cerevisiae, expressing and secreting rice alpha-amylase, converts starch to ethanol. The rice alpha-amylase gene (OS103) was placed under the transcriptional control of the promoter from a newly described Pichia pastoris alcohol oxidase genomic clone. The nucleotide sequences of ZZA1 and other methanol-regulated promoters were analyzed. A highly conserved sequence (TTG-N3-GCTTCCAA-N5-TGGT) was found in the 5' flanking regions of alcohol oxidase, methanol oxidase, and dihydroxyacetone synthase genes in Pichia pastoris, Hansenula polymorpha, and Candida boidinii S2. The yeast strain containing the ZZA1-OS103 fusion secreted biologically active enzyme into the culture media while fermenting soluble starch.

Import of a precursor protein into chloroplasts is inhibited by the herbicide glyphosate.

Import of the precursor to 5-enolpyruvylshikimate-3-phosphate synthase (pEPSPS) into chloroplasts is inhibited by the herbicide glyphosate. Inhibition of import is maximal at glyphosate concentrations of >/=10 mum and occurs only when pEPSPS is present as a ternary complex of enzyme-shikimate-3-phosphate-glyphosate. Glyphosate alone had no effect on the import of pEPSPS since it is not known to interact with the enzyme in the absence of shikimate-3-phosphate. Experiments with wild-type and glyphosate-resistant mutant forms of pEPSPS show that inhibition of import is directly proportional to the binding constants for glyphosate. Inhibition of import is thus a direct consequence of glyphosate binding to the enzyme-shikimate-3-phosphate complex. The potential for non-specific effects of glyphosate on the chloroplast transport mechanism has been discounted by showing that import of another chloroplast-designated protein was unaffected by high concentrations of glyphosate and shikimate-3-phosphate. The mechanism of import inhibition by glyphosate is consistent with a precursor unfolding/refolding model.

A developmental perspective of gambling behavior in children and adolescents.

Although it has been determined that gambling is a popular activity amongst the young, there seems to be a lack of studies examining developmental differences in children's gambling behavior. This study examines developmental differences in children's blackjack gambling behavior. One hundred and four students (51 males; 53 females) from grades 4, 6, and 8 completed a questionnaire examining their gambling behavior in general and individually played a computerized blackjack game with the following data being recorded: percentage of accuracy, amounts of money bet, gross winnings, percentage of wins, number of hands played, and end balance. Findings revealed few developmental differences in prevalence and frequency of gambling behavior and performance on a blackjack task. Males were found to wager greater amounts of money and have larger gross winnings than females on the blackjack task. Furthermore, males were more likely to view gambling as involving both large amounts of skill and luck, thus suggesting an illusion of control for gambling activities. The results are discussed from a cognitive developmental perspective.

Formoterol delivered via the dry powder Aerolizer inhaler versus albuterol MDI and placebo in mild-to-moderate asthma: a randomized, double-blind, double-dummy trial.

The objectives of this study were to compare the efficacy and tolerability of twice-daily formoterol dry powder 12 microg and 24 microg (Foradil) delivered via Aerolizer inhaler with four times daily albuterol (salbutamol) 180 microg delivered via metered dose inhaler (MDI) and placebo. A total of 554 adolescents and adults (ages 12-75 years) with mild-to-moderate asthma were randomized to this 12-week, multicenter, double-blind, double-dummy, placebo-controlled, parallel-group study. Twelve-hour spirometry measurements were taken at weeks 0, 4, 8, and 12. A total of 484 patients completed the study (122, 116, 127, and 119 given formoterol 12 microg, formoterol 24 microg, albuterol, and placebo, respectively). For the primary efficacy variable, the forced expiratory volume in 1 second (FEV1), both formoterol 12 microg and 24 microg were statistically superior to placebo at all time points on all test days (p < or = 0.017) and to albuterol at most time points on all test days (p < or = 0.001). The onset of improvement in FEV1 was rapid, with 15% increase within 5 min in 57%, 71%, and 65% of formoterol 12 microg, formoterol 24 microg, and albuterol patients, respectively. Formoterol was also superior to placebo and albuterol in terms of secondary efficacy variables: FEV1 area under the curve, percentage of predicted FEV1, forced vital capacity and forced expiratory flow, asthma symptom scores, and peak expiratory flows. In conclusion, both formoterol doses were superior to placebo in all lung function measurements. Overall, compared with albuterol, both formoterol doses produced superior bronchodilation. Formoterol and albuterol were safe and well-tolerated.

Clinical efficacy with formoterol in the absence of a response to salmeterol: a review.

Salmeterol and formoterol are both beta 2-agonist bronchodilators with a long duration of action and are often classified together, yet they are distinctly different in their pharmacology. Recent evidence suggests there is a subpopulation of asthmatic patients who do not respond to salmeterol, yet can attain clinical benefit with formoterol. Following a literature search, three published case reports are reviewed as well as results from two published clinical trials designed specifically to document response to formoterol in 'non-responders to salmeterol' asthmatics. Possible mechanisms underlying this observation are discussed, including pharmacological differences of the two drugs relating to agonism at the beta 2-receptor and to effect on nuclear transcription factors.

Effects of budesonide and formoterol on NF-kappaB, adhesion molecules, and cytokines in asthma.

The asthmatic inflammatory response can be attenuated by corticosteroids and in part by beta(2)-agonists. We investigated if these effects are accompanied by a downregulation in nuclear factor kappa B (NF-kappaB), a transcription factor regulating many of the cytokine and adhesion molecule genes expressed in allergic inflammation. Bronchial biopsies were taken before and after 8 wk treatment with formoterol, budesonide, or placebo from atopic asthmatics. Biopsies were processed into glycol methacrylate and stained immunohistochemically for eosinophils (as an index of inflammation), activated and total NF-kappaB, adhesion molecules, and cytokines. After budesonide treatment there was a significant decrease in the number of submucosal cells staining for total NF-kappaB, granulocyte macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha), accompanied by a significant decrease in mucosal eosinophils and expression of vascular cell adhesion molecule-1 (VCAM-1) in the endothelium and interleukin-8 (IL-8) in the epithelium. After formoterol treatment there was a significant decrease in eosinophils and the epithelial expression of activated NF-kappaB, but these changes were not accompanied by reduced immunoreactivity for adhesion molecules or cytokines. We conclude that at least some of the therapeutic efficacy of inhaled corticosteroids is mediated through inhibition of NF-kappaB-regulated gene expression, whereas the reduction in airway eosinophilia by long-acting beta(2)-agonists probably operates through alternative pathways.

The extrapulmonary effects of increasing doses of formoterol in patients with asthma.

OBJECTIVE: To assess the cardiovascular and metabolic responses to increasing doses of formoterol administered from a dry powder inhaler. METHODS: Twenty patients with mild to moderate asthma were given 12, 24, 48 and 96 microg of formoterol or a matched placebo on separate days. The doses were administered using a randomised, cross-over, double-blind design. The effects on heart rate, blood pressure, electromechanical systole (QS2I), the electrocardiographic QTc interval, plasma potassium (K); blood glucose and FEV1 were assessed prior to, and for 9 h following each dose. RESULTS: There was no difference between the maximum effects of formoterol 12 microg and placebo; the 24 microg dose significantly decreased plasma K (-0.2 mmol x l(-1)) and increased blood glucose (1.8 mmol x l(-1)) compared to placebo. The two highest doses affected most of the variables with the 96 microg dose being significantly different from placebo for all indices, heart rate (9 beats x min(-1)), systol BP (4 mmHg), diastolic BP (-3 mmHg), QS2I (-11 ms), QTc (17 ms), plasma K (-0.5 mmol x l(-1)) and blood glucose (2.6 mmol x l(-1)). All doses of formoterol increased FEV1. CONCLUSION: Although there were dose-dependent effects on the extrapulmonary measurements, only the effects at the highest dose may be of clinical significance.

Pharmacokinetics and tolerability of formoterol in healthy volunteers after a single high dose of Foradil dry powder inhalation via Aerolizer.

OBJECTIVE: The pharmacokinetics of the long-acting beta2-agonist formoterol fumarate, which is a racemate of the (S,S)- and (R,R)-enantiomers were evaluated in 12 healthy (eight male, four female) volunteers after a single inhaled high dose of 120 microg of formoterol fumarate. The tolerability and safety were also assessed. METHODS: Each volunteer inhaled the single 120-microg dose through the Aerolizer device within 2-5 min, using ten 12-microg dry powder capsules for inhalation. Formoterol, i.e., the sum of both enantiomers, was determined in plasma over 24 h, whereas the separate enantiomers were determined in urine over 48 h. Incidence, seriousness and severity of adverse experiences, electrocardiogram (ECG), including the corrected QT interval (QTc) calculation, systolic blood pressure, heart rate, and plasma potassium levels were recorded. RESULTS: In nine of the 12 volunteers, the peak plasma concentration of formoterol was observed already at 5 min after inhalation. The absorption kinetics were complex, as depicted by multiple peaks or shoulders within 0.5-6 h after inhalation. Mean with (SD; n = 12) of maximum concentration (Cmax) and area under the curve (AUC) of formoterol in plasma were 266 (108) pmol x l(-1) and 1330 (398) pmol x l(-1), respectively. The moderate inter-individual variability in systemic exposure of formoterol reflects the homogeneous pharmacokinetics of the drug. A predominant slow elimination of formoterol from plasma with a mean half-life (t1/2) of 10 h was demonstrated. Assuming linear kinetics in plasma suggested by urinary data, the steady-state trough plasma levels of formoterol for a b.i.d. dosing regimen are predicted to amount to 20% of Cmax. In urine, mean with (SD; n = 10) of the amount excreted over 48 h was 3.61 (0.89)% of dose for the pharmacologically active (R,R)-enantiomer and 4.80 (1.33)% of dose for the (S,S)-enantiomer. The terminal half-lives calculated from the excretion rate-time curves, i.e., 13.9 h and 12.3 h for the (R,R)- and (S,S)-enantiomer, respectively, confirm the slow elimination of formoterol from plasma. The dose inhaled was 10 times the most frequently recommended dose (12 microg) and 5 times the highest recommended dose (24 microg). Ten of 12 subjects experienced mild and transient nervousness. Pulse readings demonstrated the maximum mean increase of 25.8 beats x min(-1) at 6 h. The mean maximum QTc increase was 25 msec at 6 h. Pulse and QTc values returned to baseline or close to baseline values at 24 h or before. Potassium levels in plasma decreased in eight out of 12 subjects; the lowest mean value was 3.53 mmol x l(-1) at 2 h post-dose. The lowest individual potassium measurement was 2.95 mmol x l(-1) between 15 min and 6 h. By 8 h post-dose all values had returned to within the normal ranges. CONCLUSIONS: The extremely fast appearance of formoterol in plasma shows the predominance of airways absorption shortly after inhalation. Due to a terminal elimination half-life of about 10 h, sustained systemic concentrations of formoterol are predicted for a twice daily treatment regimen without noteworthy accumulation. The excreted amounts in percent of dose of the enantiomers in urine and the enantiomer ratio are similar to data reported previously after lower doses and suggest linear kinetics for doses between 12 microg and 120 microg of formoterol fumarate. The expected side effects on heart rate, QTc interval, and plasma potassium were small and had no clinical consequences in spite of the very high dose of 120 microg (5 to 10 times the recommended therapeutic dose of Foradil). It should be noted that the impact of high doses may be greater in patients. Nevertheless these findings provide reassurance on the safety margin of formoterol after accidental and intentional overdosing.

Protein trafficking in plant cells.

The cells of higher plants contain distinct subcellular compartments (organelles) that perform specialized functions such as photosynthesis, carbohydrate and lipid metabolism, and so forth. The majority of the protein constituents of plant organelles are formed as cytosolic precursors with N-terminal extensions that direct transport across one or more membrane bilayers in a post- or co-translational fashion. Since the majority of proteins in plant cells are products of nuclear gene expression, there must be precise sorting mechanisms in the cytoplasm that direct proteins to their correct cellular locations. Based on recent studies of protein targeting to chloroplasts and vacuoles, the details of these intracellular sorting mechanisms are becoming clear. The ability to direct proteins to specific compartments within cells provides new opportunities for improvement of plants by genetic manipulation.

Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA.

The carotenoid biosynthetic pathway in higher plants was manipulated by using an RNA viral vector. A cDNA encoding phytoene synthase and a partial cDNA encoding phytoene desaturase (PDS) were placed under the transcriptional control of a tobamovirus subgenomic promoter. One to two weeks after inoculation, systemically infected Nicotiana benthamiana plants were analyzed for phytoene. Leaves from transfected plants expressing phytoene synthase developed a bright orange phenotype and accumulated high levels of phytoene. Cytoplasmic inhibition of plant gene expression by viral RNA was demonstrated with an antisense RNA transcript to a partial PDS cDNA derived from tomato. The leaves of the plants transfected with the antisense PDS sequence developed a white phenotype and also accumulated high levels of phytoene. A partial cDNA to the corresponding N. benthamiana PDS gene was isolated and found to share significant homology with the tomato antisense PDS transcript. This work demonstrates that an episomal RNA viral vector can be used to deliberately manipulate a major, eukaryotic biosynthetic pathway. In addition, our results indicate that an antisense transcript generated in the cytoplasm of a plant cell can turn off endogenous gene expression.

Translocation of the precursor of 5-enolpyruvylshikimate-3-phosphate synthase into chloroplasts of higher plants in vitro.

5-enolPyruvylshikimate-3-phosphate synthase (EPSP synthase; 3-phosphoshikimate 1-carboxyvinyl-transferase; EC 2.5.1.19) is a chloroplast-localized enzyme of the shikimate pathway in plants. This enzyme is the target for the nonselective herbicide glyphosate (N-phosphonomethylglycine). We have previously isolated a full-length cDNA clone of EPSP synthase from Petunia hybrida. DNA sequence analysis suggested that the enzyme is synthesized as a cytosolic precursor (pre-EPSP synthase) with an amino-terminal transit peptide. Based on the known amino terminus of the mature enzyme, and the 5' open reading frame of the cDNA, the transit peptide of pre-EPSP synthase would be maximally 72 amino acids long. To confirm this prediction and to assay directly for translocation of pre-EPSP synthase into chloroplasts in vitro, we cloned the full-length cDNA into an SP6 transcription system to produce large amounts of mRNA for in vitro translation. The translation products, when analyzed by NaDodSO(4)/PAGE autoradiography, indicate a relative molecular mass for pre-EPSP synthase of approximately 55 kDa. Uptake studies with intact chloroplasts, in vitro, indicate that pre-EPSP synthase was rapidly taken up into chloroplasts and proteolytically cleaved to the mature approximately 48-kDa enzyme. The transit peptide was shown to be essential for import of the precursor enzyme into the chloroplast. To our knowledge, post-translational import into chloroplasts of a precursor enzyme involved in amino acid biosynthesis has not been reported previously. Furthermore, enzymatic analysis of translation products indicates that pre-EPSP synthase is catalytically active and has a similar sensitivity to the herbicide glyphosate as the mature enzyme. To our knowledge, pre-EPSP synthase represents the only example of a catalytically competent chloroplast-precursor enzyme.