Catalytic olefin hydroamination with aminium radical cations: a photoredox method for direct C-N bond formation.

While olefin amination with aminium radical cations is a classical method for C-N bond formation, catalytic variants that utilize simple 2° amine precursors remain largely undeveloped. Herein we report a new visible-light photoredox protocol for the intramolecular anti-Markovnikov hydroamination of aryl olefins that proceeds through catalytically generated aminium radical intermediates. Mechanistic studies are consistent with a process involving amine oxidation via electron transfer, turnover-limiting C-N bond formation, and a second electron transfer step to reduce a carbon-centered radical, rendering the overall process redox-neutral. A range of structurally diverse N-aryl heterocycles can be prepared in good to excellent yields under conditions significantly milder than those required by conventional aminium-based protocols.

Analysis of factor VIII mediated suppression of lentiviral vector titres.

Effective gene therapy for haemophilia A necessitates a vector system that is not subject to a pre-existing immune response, has adequate coding capacity, gives long-term expression and preferably can target non-dividing cells. Vector systems based on lentiviruses such as equine infectious anaemia virus (EIAV) fulfil these criteria for the delivery of factor VIII (FVIII). We have found that B domain-deleted (BDD) FVIII protein inhibits functional viral particle production when co-expressed with the EIAV vector system. Although particle numbers (as measured by reverse transcriptase activity) are near normal, RNA genome levels are reduced and measurement of integrated copies revealed the virus is severely defective in its ability to transduce target cells. This is due to the absence of sufficient vesicular stomatitis virus glycoprotein (VSV-G) envelope on viral particles derived from cells expressing FVIII. By using an internal tissue-specific promoter, that has low activity in the producer cells, to drive expression of FVIII we have overcome this inhibitory effect allowing us to generate titres approaching those obtained with vector genomes encoding reporter genes. Furthermore, we report that codon optimization of the full-length FVIII gene increased vector titres approximately 10-fold in addition to substantially improving expression per integrated vector copy.

A replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based lentiviral vectors.

Lentiviral vectors are being developed to satisfy a wide range of currently unmet medical needs. Vectors destined for clinical evaluation have been rendered multiply defective by deletion of all viral coding sequences and nonessential cis-acting sequences from the transfer genome. The viral envelope and accessory proteins are excluded from the production system. The vectors are produced from separate expression plasmids that are designed to minimize the potential for homologous recombination. These features ensure that the regeneration of the starting virus is impossible. It is a regulatory requirement to confirm the absence of any replication competent virus, so we describe here the development and validation of a replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based vectors. The assay is based on the guidelines developed for testing retroviral vectors, and uses the F-PERT (fluorescent-product enhanced reverse transcriptase) assay to test for the presence of a transmissible reverse transcriptase. We have empirically modelled the replication kinetics of an EIAV-like entity in human cells and devised an amplification protocol by comparison with a replication competent MLV. The RCL assay has been validated at the 20 litre manufacturing scale, during which no RCL was detected. The assay is theoretically applicable to any lentiviral vector and pseudotype combination.

First Demonstration of Koch's Postulates for Lasiodiplodia theobromae Fruit Spot on Eggplant (Solanum melongena).

During October 2004, diseased eggplant fruit from a commercial farm in Colquitt County, Georgia, developed circular, tan, water-soaked lesions. Gray, septate mycelia quickly covered the fruit. Diseased fruit became shriveled, spongy, and mummified. Disease incidence in the field was approximately 1%. Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (synonym Botryodiplodia theobromae Pat.) (2) was isolated from the margins of lesions and cultured on acidified potato dextrose agar. The fungus produced grayish colonies with aerial hyphae and black ostiolate pycnidia massed into stroma. Mature elliptical conidia (25.8 × 15.6 µm) were brown, had a single septation, and longitudinal striations. Isolates obtained from peanut and pecan were included in the pathogenicity tests. Mature fruit cv. Nightshade were surface disinfested for 30 s in 70% ethanol, followed by 60 s in 0.5% sodium hypochlorite, rinsed twice in sterile distilled water, and allowed to dry. Inoculations were made by placing an agar plug containing L. theobromae mycelial side down on the surface of the fruit or wounding with a sterile toothpick containing mycelium of the fungus. Fruit similarly inoculated with agar plugs or sterile toothpicks served as controls. There were a total of three replicates. Fruit were placed in plastic containers lined with moistened paper towels. Containers were placed in a dew chamber and incubated (28°C, relative humidity >95%) for 3 days, and then evaluated. Symptoms identical to those observed on naturally infected fruit developed on inoculated fruit. Controls remained disease free. L. theobromae was reisolated from all symptomatic tissue, satisfying Koch's postulates. Disease damage on wounded fruit was twice that of nonwounded fruit. However, seven of nine inoculations with agar plugs containing L. theobromae resulted in infection. Lesion lengths from wound inoculations were 9.8, 7.3, and 5.2 cm for isolates from peanut, pecan, and eggplant, respectively. Generally, L. theobromae is considered a facultative wound pathogen or a secondary invader (3). However, this study suggests that direct infection can occur. Although fruit spot has been reported previously on eggplant (1), to our knowledge, this is the first report verifying L. theobromae as the causal agent. References: (1) S. A. Alfieri et al. Index of Plant Diseases in Florida. Fla. Dep. Agric. Consum. Serv. Bull. 11, 1984. (2) H. L. Barnett and B. B. Hunter. Illustrated Guide of Imperfect Fungi. 4th ed. The American Phytopathological Society St. Paul, MN, 1998. (3) P. M. Phipps and D. M. Porter. Plant Dis. 82:1205, 1998.

Identification of potential stroke targets by lentiviral vector mediated overexpression of HIF-1 alpha and HIF-2 alpha in a primary neuronal model of hypoxia.

The identification of genes differentially regulated by ischemia will lead to an improved understanding of cell death pathways such as those involved in the neuronal loss observed following a stroke. Furthermore, the characterization of such pathways could facilitate the identification of novel targets for stroke therapy. We have used a novel approach to amplify differential gene expression patterns in a primary neuronal model of stroke by employing a lentiviral vector system to specifically bias the transcriptional activation of hypoxically regulated genes. Overexpression of the hypoxia-induced transcription factor subunits HIF-1 alpha and HIF-2 alpha elevated hypoxia-mediated transcription of many known HIF-regulated genes well above control levels. Furthermore, many potentially novel HIF-regulated genes were discovered that were not previously identified as hypoxically regulated. Most of the novel genes identified were activated by a combination of HIF-2 alpha overexpression and hypoxic insult. These included several genes with particular importance in cell survival pathways and of potential therapeutic value. Hypoxic induction of HIF-2 alpha may therefore be a critical factor in mediating protective responses against ischemic injury. Further investigation of the genes identified in this study may provide increased understanding of the neuronal response to hypoxia and may uncover novel therapeutic targets for the treatment of cerebral ischemia.

First Report of a Field Outbreak of a Bacterial Leaf Spot of Cantaloupe and Squash Caused by Pseudomonas syringae pv. syringae in Georgia.

In March 2000, a leaf spot was reported affecting yellow summer squash (Cucurbita pepo) and cantaloupe (Cucumis melo) in commercial fields in Colquitt, Echols, and Grady counties in Georgia. All of the crops affected were reported within a 10-day period, and average temperatures during that time were 8 to 22.5°C, which is very close to the 50-year normal temperatures for these areas located in southwest Georgia. Incidence in affected fields was 100%. Lesions on squash leaves appeared irregularly shaped, dark, water soaked, somewhat vein restricted, and were 5 to 10 mm in diameter. Lesions on cantaloupe were angular, light tan, and necrotic with a lesion diameter of 2 to 5 mm. A general chlorosis was observed around lesions of both crops. Leaf distortion was observed on squash. Four isolates collected were used in biochemical, pathogenicity, and physiological tests. Gram-negative, rod-shaped bacteria were isolated from diseased tissue from squash and cantaloupes. Bacteria were aerobic, catalase-positive, fluorescent on King's medium B (1), oxidase-negative, nonpectolytic on potato, arginine dihydrolase-negative, utilized sucrose as a carbon source, produced levan, and gave a hypersensitivity response on tobacco (HR). Analysis of fatty acid methyl ester (FAME) profiles using the Microbial identification system (Sherlock version 3.1, Microbial identification system, Newark, DE) characterized representative strains as Pseudomonas syringae (similarity indices 0.65 to 0.80). Upon further characterization, the strains were negative for l (+)-tartarate utilization but utilized l-lactate and betaine and also exhibited ice nucleation activity. These characteristics are consistent with those of Pseudomonas syringae pv. syringae. Squash and cantaloupes were grown in a greenhouse for 4 weeks. Bacteria were grown in nutrient broth, resuspended in sterile tap water, and standardized using a spectrophotometer. Plants were inoculated by infiltrating leaves with 1 ml of bacterial suspensions (1 × 107 CFU/ml) using sterile syringes. Sterile water was used as a negative control, and 1 ml was infiltrated into leaves of squash and cantaloupes. Water-soaked lesions developed in 4 to 6 days on squash and cantaloupes inoculated with bacterial suspensions, and Pseudomonas syringae pv. syringae was isolated from diseased tissue. No symptoms developed on squash and cantaloupes used as negative controls. This outbreak of Pseudomonas syringae pv. syringae did not cause significant economic damage to either crop as symptoms subsided once daily high temperatures reached 28 to 32°C. This disease has been isolated from several cucurbit transplants reared in greenhouses, but to our knowledge, this is the first report of this disease occurring in the field. Reference: (1) E. O. King et al. J. Lab. Clin. Med. 44:301, 1954.

Isolation of antibody to human placental alkaline phosphatase (PLAP) from extracts of human placentae.

PROBLEM: Human placental alkaline phosphatase (PLAP) is a unique placental antigen bound to the syncytiotrophoblast, which may be able to elicit a specific immune response in pregnancy. METHOD OF STUDY: Antibody to PLAP was purified from placental extracts by: a) acid elution of membrane vesicles; b) purifying complexes of PLAP with human antibody on monoclonal antibodies to PLAP followed by denaturation of the enzyme; and c) by denaturation of PLAP in placental extracts and purification of antibody to PLAP on PLAP columns. RESULTS AND CONCLUSIONS: Specific antibody to PLAP is present in placental extracts, and is mostly bound to placental membrane preparations. PLAP is therefore immunogenic in pregnancy and could serve as a useful monitor of pregnancy-specific immunological responses. Since a similar enzyme appears in some cancers, it is possible that the immunization against PLAP in pregnancy will help to protect against the development of ovarian and endometrial cancer (the 'fetal antigen' hypothesis).

First Report of Cabbage leaf curl virus (Family Geminiviridae) in Georgia.

Cabbage and collard greens were inflicted with a previously undescribed virus-like disease during the fall 2000. Symptoms on leaves were yellow spots, vein clearing, mosaic, curling, and puckering. Symptomatic plants were widespread in Brooks, Colquitt, Grady, and Pierce counties in Georgia. Disease incidence ranged from 10 to 20% in the majority of the fields surveyed but some fields had 100% incidence. Fields were heavily infested by Bemisia argentifolii and the symptoms were suggestive of a whitefly-transmitted geminivirus infection. A polymerase chain reaction (PCR)-based diagnostic test for geminivirus was conducted. Total DNA was extracted from symptomatic cabbage and collard green plants collected from commercial fields. The two primers, 5'-GCCCACATYGTCTTYCCNGT-3' and 5'- GGCTTYCTRTACATRGG-3' (2,3), are "universal" for genus Begomovirus of family Geminiviridae. The primer pair could amplify a part of the replicase-associated protein and coat protein and the complete common region of DNA-A. The PCR gave a DNA band of expected size (1.1 kb) from both symptomatic cabbage and collard green samples, whereas no such product was obtained from healthy samples, suggesting that the causal agent could be a geminivirus. To establish the identity of the virus, the 1.1 kb PCR product was cloned into pGEM-T Easy (Promega) and sequenced. GenBank search showed that the geminivirus isolated in Georgia was most closely related (98% sequence identity) to Cabbage leaf curl virus (accession number U65529) reported from Florida (1). The virus was mechanically transmitted to healthy cabbage and collard green plants under experimental conditions. To our knowledge, this is the first report of Cabbage leaf curl virus from Georgia. References: (1) A. M. Abouzid et al. Phytopathology 82:1070, 1992. (2) S. S. Pappu et al. Plant Dis. 84:370, 2000. (3) M. R. Rojas et al. Plant Dis. 77:340-347, 1993.

Investigations into the causes of residue variability on carrots in the UK.

A pilot study was established to determine whether the preferential flow of water and thus insecticide in carrot beds might be responsible for the high residues of organophosphorus insecticides detected in individual carrot roots grown in the UK. A field site typical of UK carrot growing conditions was selected on a sandy soil with a low organic carbon content. Brilliant blue dye was applied in water to a small number of field plots located in the carrot beds to trace water movement through the bed and not to simulate insecticide application or irrigation. The plots were excavated following sufficient time for infiltration and drainage. Horizontal and vertical cross-sections through the soil profile were cut and descriptions of the dye presence in relation to soil features and the position of the carrot roots were made. Dye tracing and soil analyses showed there was a clear mechanical cultivation effect which generated a preferential movement of dye and water within the bed. The subsequent growth of carrots also created additional pathways of preferential movement due to stem-flow or canopy drip. A second study which increased replication of samples and allowed analysis of triazophos and chlorfenvinphos residues in the carrots could not identify any single factor which was conclusively responsible for initiating high residues in individual roots.

Amplified Assay for Specific Dual-Labeled DNA Using the Coagulation Cascade (EDNA-ELCA).

Detection of the products of the PCR reaction using nonisotopically labeled DNA molecules containing biotin, fluorescein, or digoxigenin has become a popular method for identification of specific products of polymerase chain reaction (PCR) (1,3). These labeled molecules are prepared either by PCR synthesis in the presence of labeled deoxyuridine triphosphate (1,3) or by hybridization of labeled probes to the unlabeled PCR product (1,2). Detection is then in a format very similar to enzyme-linked immunosorbent assays (ELISA) using similarly labeled antigens and antibodies, i.e., by capture on the receptor for one ligand (streptavidin or antibody) and using the other ligand for detection.

Detection of colorectal cancer K-ras mutations using a simplified oligonucleotide ligation assay.

It has been suggested that some mutations in codons 12 and 13 of the K-ras gene are associated with the progression of colorectal adenomas to carcinomas. The aim of this study was to develop a rapid, colorimetric assay for K-ras point mutations commonly associated with colorectal cancer. K-ras exon 1 was amplified from colorectal tumor DNA and K-ras activating mutations detected using an oligonucleotide ligation assay (OLA) in combination with immunological and colorimetric detection. Using the OLA with oligonucleotides specific to individual K-ras mutations, 6 (of 17 total colorectal adenomas/carcinomas) were found to have K-ras mutations. The assay could detect as little as 10% mutant allele. A simplified OLA designed to test for either the presence (+) or absence (-) of any of the K-ras activating mutations was developed. The assay was further streamlined by use of a dipstick methodology for colorimetric development. If required, assay sensitivity can be increased by the use of the recently described EDNA-ELCA detection system. The simplified (+/-) mutation OLA in combination with a dipstick or EDNA-ELCA detection system provides a rapid, sensitive assay for K-ras point mutations suitable for use as part of the clinical assessment of colorectal cancer.

Enzyme-linked immunosorbent assay-enzyme-linked coagulation assay for detection of antibodies to Clostridium botulinum neurotoxins A, B, and E and solution-phase complexes.

The solution-phase complex assay for toxins A, B, and E from Clostridium botulinum (Elcatech, Inc., Winston-Salem, N.C.) was modified to measure antibody. The addition of unlabeled polyclonal antibodies to a mixture consisting of toxin with chicken antibody and RVV-XA-labeled horse antibody reduces the sensitivity of detection of neurotoxin. This reduction in sensitivity can be used as a measure of the specific antibody titer.

Enzyme-linked immunosorbent assay and enzyme-linked coagulation assay for detection of Clostridium botulinum neurotoxins A, B, and E and solution-phase complexes with dual-label antibodies.

The measurement of toxins A, B, and E from Clostridium botulinum was accomplished by use of a modified sandwich enzyme-linked immunosorbent assay (ELISA) employing labeled horse antibody and either chicken antibody or biotinylated horse antibody. The complexes formed in solution phase were captured onto solid phases coated with rabbit anti-chicken immunoglobulin G (chicken antibody) or avidin (biotinylated antibody). The assay was brought to the sensitivity of the mouse bioassay (5 to 10 pg/ml, or 0.03 to 0.07 pM) by employing as labeling enzyme the factor X activator of Russell's viper venom (RVV-XA) and a sensitive coagulation-based assay amplification system known as enzyme-linked coagulation assay. Complex formation was found to be a slower reaction than binding to the capture plate, and so the assay used a preincubation step to produce the solution-phase complexes before they were bound to the solid phase. Keeping the concentrations of Russell's viper venom factor X activator antibody and capture antibody constant for diluted samples and diluting complexes into buffer without keeping labeled antibody concentrations constant were equivalent in allowing the detection of low neurotoxin concentrations. This ELISA-enzyme-linked coagulation assay procedure is a convenient alternative to the mouse bioassay, which shows complete resolution of the neurotoxins in addition to the requisite sensitivity.

Amplified Immunoassay ELISA-ELCA for Measuring Clostridium botulinum Type E Neurotoxin in Fish Fillets.

The measurement of Clostridium botulinum type E toxin in fish was accomplished using an amplified immunoassay (enzyme-linked immunosorbent assay-enzyme-linked coagulation assay [ELISA-ELCA]) based on the coagulation cascade. Fresh catfish fillets inoculated with a mixture of spores from five strains of C. botulinum type E were packaged in high barrier film with air, vacuum and modified atmosphere and stored at 4, 8 or 16°C for up to 75 days. Toxin production was monitored during storage by both mouse bioassay (trypsin and non-trypsin treated) and ELISA-ELCA on the non-trypsinized samples. All 26 inoculated products that were positive by the mouse bioassay were also positive by ELISA-ELCA. Of 35 uninoculated samples which were not toxic in mouse bioassay, none were positive by ELISA-ELCA; of 73 inoculated samples which were not toxic by mouse bioassay, 14 had toxin measurable by the ELISA-ELCA. The position of these immunoassay-positives in the sampling sequence indicated that the toxin was identified by the immunoassay before it was found in the mouse bioassay. These results suggest that the ELISA-ELCA technique is a usable alternative to the mouse bioassay for monitoring C. botulinum type E toxin production in fish challenge studies.

Sensitive enzyme-linked immunosorbent assay for detection of Clostridium botulinum neurotoxins A, B, and E using signal amplification via enzyme-linked coagulation assay.

A new immunoassay amplification method has been applied to the measurement of toxins A, B, and E from Clostridium botulinum. The technique is a modified enzyme-linked immunosorbent assay (ELISA) which relies on the detection of sandwich complexes on microtiter plates by a solid-phase coagulation assay known as ELCA, or enzyme-linked coagulation assay. In the method, a coagulation activating enzyme (RVV-XA) isolated from the venom of Russell's viper is conjugated to affinity-purified horse antibodies specific for toxin type A, B, or E. Plates are coated with affinity-purified antibodies, and standard captag (capture-tag) protocols using labeled antibody are employed to bind the toxin from solution. Complexes are detected by adding a modified plasma substrate which contains all the coagulation factors mixed with alkaline phosphatase-labeled fibrinogen and solid-phase fibrinogen; deposition of solid-phase, enzyme-labeled fibrin on the solid phase is then a reflection of formation of toxin-RVV-XA-antibody complexes on the solid phase. Because of the ability to detect RVV-XA by this coagulation assay at concentrations < 0.1 pg/ml, it was possible to measure C. botulinum toxins A, B, and E at mouse bioassay levels (< 10 pg/ml, or < 0.07 pM) for both purified neurotoxin and crude culture filtrates obtained from strains known to produce appropriate single toxins. ELISA-ELCA should be applicable to measurement of toxins in most of the materials (contaminated food, blood, and excreta) for which the comparably sensitive mouse bioassay is currently employed. This method has the potential of broad application to the measurement of low concentrations of any antigen for which appropriate immunochemical reagents are available, in a color test format.

The effects of a carbonated carbohydrate drink on gastric emptying, gastrointestinal distress, and exercise performance.

To determine the effect of a carbonated carbohydrate (CHO) drink on gastric function and exercise performance, eight male cyclists completed four 120-min bouts of cycling. Each bout consisted of a 105-min ride at 70% VO2max followed by a 15-min self-paced performance ride. During each trial, one of four test solutions was ingested: carbonated CHO (C-10%), noncarbonated CHO (NC-10%), carbonated non-CHO (C), and noncarbonated non-CHO (NC). Following the performance ride, the subjects had their stomach contents removed by aspiration. There were no significant differences in gastric emptying (GE) except for Trial C-10%, which averaged 13.3% less than NC. However, there was no difference in the perception of gastrointestinal comfort between this trial and any other. Average power output during the performance ride was not significantly different between carbonated and noncarbonated trials, or between CHO-fed and no-CHO trials; however, the subjects worked at a greater intensity when fed CHO. Finally, acid base status did not change when a carbonated drink was ingested. This indicates that adding carbonation to a sport drink does not significantly alter gastric function, the perception of GI comfort, or exercise performance.

Lingually displaced mandibular canine teeth: orthodontic treatment alternatives in the dog.

Lingual displacements of mandibular canine teeth often occur following retention of deciduous canine teeth. This condition often results in trauma of occlusion to the lingual aspect of the maxillary canine tooth and the further development of a periodontal pocket or an oronasal fistula. This condition can be corrected using orthodontic appliances. The purpose of this paper is to discuss and illustrate the various alternatives available for correction of this common malocclusion in dogs.

Root factors stimulate 32 P uptake and plasmalemma ATPase activity in vesicular-arbuscular mycorrhizal fungus, Gigaspora margarita.

Root-inducing (Ri) T-DNA transformed roots of carrot were used as the plant partner in a study of 32 P absorption and plasmalemma ATPase activity in the hyphae of the vesicular-arbuscular mycorrhizal fungus Gigaspora margarita Becker & Hall. Hyphae from germinating spores were grown in the presence or absence of root exudates and volatiles. In the presence of these root factors, 32 P isotope labelling occurred in hyphae, auxiliary cells and spores, while in the absence of these factors, the labelling only occurred in fungal spores. Similarly, ATPase activity appeared on the fungal plasmalemma only in the presence of root factors. The use of the ATPase inhibitor, diethylstilbestrol, demonstrated the importance of plasmalemma ATPase in the stimulation of hyphal growth of this obligately biotrophic fungus.