Promastigote secretory gel from natural and unnatural sand fly vectors exacerbate Leishmania major and Leishmania tropica cutaneous leishmaniasis in mice.

Leishmania rely heavily on glycans to complete their digenetic life cycle in both mammalian and phlebotomine sand fly hosts. Leishmania promastigotes secrete a proteophosphoglycan-rich gel (Promastigote Secretory Gel, PSG) that is regurgitated during transmission and can exacerbate infection in the skin. Here we explored the role of PSG from natural Leishmania-sand fly vector combinations by obtaining PSG from Leishmania (L.) major-infected Phlebotomus (P.) papatasi and P. duboscqi and L. tropica-infected P. arabicus. We found that, in addition to the vector's saliva, the PSG from L. major and L. tropica potently exacerbated cutaneous infection in BALB/c mice, improved the probability of developing a patent cutaneous lesion, parasite growth and the evolution of the lesion. Of note, the presence of PSG in the inoculum more than halved the prepatent period of cutaneous L. tropica infection from an average of 32 weeks to 13 weeks. In addition, L. major and L. tropica PSG extracted from the permissive experimental vector, Lutzomyia (Lu.) longipalpis, also exacerbated infections in mice. These results reinforce and extend the hypothesis that PSG is an important and evolutionarily conserved component of Leishmania infection that can be used to facilitate experimental infection for drug and vaccine screening.

Effects of soil-applied imidacloprid on Asian citrus psyllid (Hemiptera: Psyllidae) feeding behavior.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is one of the most important pests of citrus (Citrus spp.) because of its status as a vector of Candidatus Liberibacter asiaticus (Las), the bacterium associated with citrus greening disease. The use of insecticides for vector control is the primary method of managing the spread of this pathogen. Imidacloprid is an insecticide commonly applied to the root zone of young citrus trees to provide systemic protection from pests. The effects of imidacloprid on feeding behavior of D. citri have not been studied in much detail. The purpose of this study was to examine the effects of imidacloprid application on feeding behavior of D. citri and to determine whether use of this systemic insecticide could have any effect on pathogen transmission by D. citri. A direct current electrical penetration graph monitor was used to record D. citri feeding behaviors for 12-h periods on mature and young leaves of imidacloprid-treated and -untreated citrus seedlings. Overall, compared with untreated plants, the feeding behavior of D. citri was disrupted on imidacloprid-treated plants via reduction in the number of probes, as well as durations of average probes, initial stylet contact with phloem, phloem salivation, and phloem ingestion. The results of this study demonstrate that soil applications of imidacloprid can reduce the probability of citrus plants becoming inoculated with Las through a reduction in the number and duration of phloem salivation events by D. citri. Furthermore, Las acquisition from infected citrus is greatly reduced as a result of decreased phloem ingestion by D. citri on imidacloprid-treated plants.

Transmission parameters for Candidatus liberibacter asiaticus by Asian citrus psyllid (Hemiptera: Psyllidae).

The purpose of this investigation was to evaluate acquisition and inoculation (together, transmission) efficiency of Candidatus Liberibacter asiaticus (Las), the pathogen associated with citrus huanglongbing (HLB) by the Asian citrus psyllid, Diaphorina citri (Kuwayama) (Hemiptera: Psyllidae). In laboratory studies, nymphs reared on Las infected plants were more likely to acquire the bacterium than adults. Acquisition by nymphs ranged from 60 to 100%, whereas acquisition by adults only reached 40% after 5 wk of feeding on Las-infected plants. Similar rates of pathogen acquisition by psyllids after nymphal and adult feeding were observed in the field. Transmission of Las from parent to offspring (transovarial) occurred at a rate of 2-6%. One year after psyllid inoculations, successful transmission by individual D. citri ranged from 4 to 10%, whereas groups of 100 or more D. citri transmitted the pathogen at a rate of approximately 88%. In addition, the proportion of Las-positive adult psyllids, determined using quantitative real-time polymerase chain reaction, decreased over time when held on healthy plants. Due to the low rate of pathogen acquisition and long time period required for successful inoculation by adult D. citri, experiments designed to determine the latent period required for replication and successful inoculation of Las by D. citri did not result in Las-infected plants after >1 yr of incubation after inoculation. Collectively, these results indicate that adult D. citri which acquire the HLB pathogen as adults are poor vectors of the pathogen compared with adults that acquired the pathogen as nymphs.

Ribonucleoprotein particles in the amphibian oocyte nucleus. Possible intermediates in ribosome synthesis.

Studies of the sedimentation properties of RNP(1) material from the nucleus of the amphibian oocyte have indicated (1) that there are few, if any, 78S ribosomes in the nucleus, (2) that there are smaller particles sedimenting at 50-55S and 30S, and (3) that the larger of these is the precursor of the 60S subunit of the cytoplasmic ribosomes. Although the nature of the 30S material is not completely clear, it probably includes precursor particles to the 40S ribosomal subunit. Heavy (50-55S) particles are predominant in immature oocytes of Triturus viridescens, whereas in immature oocytes of Triturus and Amblystoma mexicanum they are reduced greatly in amount, but are still detectable. Double-labeling studies of RNA and protein reveal that both types of particle incorporate uridine-(3)H, but that the 50-55S material of immature oocytes does not incorporate (14)C-labeled amino acids. However, other evidence exists that favors the RNP nature of this material. Sedimentation analyses after SDS extraction show that 50-55S particles contain 40 and 30S RNA, whereas 30S particles contain 20S RNA. These types of RNA represent at least 80% of all the extractable nuclear RNA. The 50-55S particles are probably heterogeneous, including both particles containing mostly 40S RNA and particles containing only 30S RNA.

Evaluating the Effect of Imidacloprid Administered in Artificial Diet on Feeding Behavior of Diaphorina citri (Hemiptera: Liviidae) Using Electropenetrography.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae) is the vector of Candidatus Liberibacter asiaticus (CLas), the presumed cause of Huanglongbing (HLB) in citrus. Management strategies were developed in Florida that used soil-applied neonicotinoids to protect young trees. Despite the implementation of intense management programs, infection spread among the most intensively managed groves. We used electopenetrography to test five imidacloprid doses (0.55, 5.5, 55, 550, and 5,500 ppm) administered in artificial diet to approximate the dosage required to reduce feeding activity and prevent salivation/ingestion activity. We failed to detect a significant effect of 0.55 ppm imidacloprid on probing behavior, pathway, or salivation/ingestion activity when compared with the untreated control. We observed a significant reduction in the number of probes and the number of pathway with both 5.5 and 55 ppm imidacloprid. We detected a significant reduction in the number of salivation/ingestion events at both 5.5 ppm and 55 ppm imidacloprid (57 and 54 percent, respectively) compared with the untreated control, and a reduction in number of sustained (>600 s) salivation/ingestion at 55 ppm. While reductions in feeding activity were apparent at dosages of at least 5.5 ppm, we were unable to prevent salivation/ingestion with dosages as high as 5,500 ppm, which is greater than what is known to occur following application in the field. While soil-applied imidacloprid may slow the spread of CLas, our findings suggest that prevention of CLas inoculation in the field is unlikely. Management strategies must be refined to prevent the spread of HLB in Florida.

Influence of Tree Size and Application Rate on Expression of Thiamethoxam in Citrus and Its Efficacy Against Diaphorina citri (Hemiptera: Liviidae).

Neonicotinoids are a key group of insecticides used to manage Diaphorina citri Kuwayama (Hemiptera: Liviidae), in Florida citrus. Diaphorina citri is the vector of Candidatus Liberibacter asiaticus, the presumed causal agent of huanglongbing, a worldwide disease of citrus. A two-season field study was conducted to evaluate the effect of tree size and application rate on the expression of thiamethoxam in young citrus following application to the soil. D. citri adult and nymph abundance was also correlated with thiamethoxam titer in leaves. Tree size and application rate each significantly affected thiamethoxam titer in leaf tissue. The highest mean thiamethoxam titer observed (33.39 ppm) in small trees (mean canopy volume = 0.08 m3) occurred after application of the high rate (0.74 g Platinum 75SG per tree) tested. There was a negative correlation between both nymph and adult abundance with increasing thiamethoxam titer in leaves. A concentration of 64.63 ppm thiamethoxam was required to reach a 1% probability of encountering a flush shoot with at least one adult D. citri, while 19.05 ppm was required for the same probability of encountering nymphs. The LC90 for the field population was 7.62 ppm thiamethoxam when administered through ingestion. Exposure to dosages as low as 7.62 ppm would likely result in sublethal exposure of some proportion of the population, which could exacerbate resistance development. Based on our results, subsequent work should investigate the use of neonicotinoids by foliar rather than soil application to maintain the chemical class in future insecticide management programs in Florida citrus.

Opportunistic Aspergillus pathogens measured in home and hospital tap water by quantitative PCR (QPCR).

Opportunistic fungal pathogens are a concern because of the increasing number of immunocompromised patients. The goal of this research was to test a simple extraction method and rapid quantitative PCR (QPCR) measurement of the occurrence of potential pathogens, Aspergillus fumigatus, A. flavus, A. terreus and A. niger, in home tap water and a hospital water supply. Water samples were taken from the kitchen tap in the homes of 60 patients who were diagnosed with legionellosis. Water samples were also taken from three locations in a hospital that generated all of its hot water by flash heating. Opportunistic infectious agents Aspergillus fumigatus, A. flavus, A. terreus and A. niger were measured using QPCR. Aspergillus terreus DNA was found in 16.7% and A. fumigatus DNA in 1.7% of the samples taken from the kitchen tap. None of the Aspergillus species were found in any of the hospital water samples.The development of a simple DNA extraction method along with QPCR analysis is suitable for rapid screening of tap water for opportunistic fungal pathogens. This simple method can be used to obtain pathogen occurrence results in about 3 h, instead of waiting days to weeks for culture data. Obtaining pathogen occurrence data in a timely manner could promote the elimination of the pathogens from the water supply of immunocompromised patients.

A monoclonal antibody to a multiphosphorylated, conformational epitope at the carboxy-terminus of p53.

Mutations of the gene encoding the tumor suppressor protein p53 are the most common molecular alterations of cancer cells found in about half of all human tumors. Mutations which cluster in well-defined hot spots change the structure of the protein thus affecting its ability to bind to DNA. Post-translational modifications, primarily phosphorylation, might also influence how p53 binds to DNA or folds to its active tetrameric form. However, the lack of appropriate biochemical markers to characterize the status of phosphorylation in different cell types and in cells at different stages of tumor progression has prohibited such investigations. To generate a sensitive and phosphorylation-specific monoclonal antibody (mAb), we chemically synthesized the C-terminal 23 amino acid stretch of human p53 in a double-phosphorylated form. The peptide 371-393, carrying phosphate groups on Ser378 and Ser392, was co-synthesized with a turn-inducing spacer and peptide 31D, an immunodominant T-helper cell epitope in mice of the H-2k haplotype. After immunization and fusion of splenocytes with myeloma cells, a number of mAbs were obtained, from which mAb p53-18 emerged as a highly sensitive reagent. By enzyme-linked immunosorbent assay, p53-18, a mAb of the IgM isotype, recognized phosphorylated p53, expressed in insect cells infected with a recombinant baculovirus but not p53 expressed in Escherichia coli. Moreover, murine p53 from insect cells could be immune purified with mAb p53-18. Mass spectrometry following tryptic digestion of the purified protein and liquid chromatography of the fragments verified the presence of phosphate groups at both Ser375 and Ser389. From the corresponding human protein fragments, mAb p53-18 bound to the immunizing peptide phosphorylated on Ser378 and on Ser392, but failed to cross-react with the unphosphorylated peptide, or peptides phosphorylated individually on either Ser378 or Ser392. The binding to the unphosphorylated peptide could be restored, however, if the peptide conformation was stabilized to that of an alpha-helix. The immunogenic nature of the multiphosphorylated C-terminus of p53 is indicated by the finding that human sera, mostly from cancer patients, preferentially recognized the double-phosphorylated peptide over the monophosphorylated or unphosphorylated analogs. Antibody p53-18 appears to be a highly useful biochemical marker to detect low levels of p53 protein in different tissues, and to be a key tool to characterize the phosphorylation status of the C-terminus of p53 protein originated from various sources.

New insights into the developmental biology and transmission mechanisms of Leishmania.

Leishmania alternates between two main morphological forms in its life cycle: intracellular amastigotes in the mammalian host and motile promastigotes in the sandfly vector. Several different forms of promastigote can be recognised in sandfly infections. The first promastigote forms, which are found in the sandfly in the bloodmeal phase, are multiplicative procyclic promastigotes. These differentiate into nectomonad promastigotes, which are a non-dividing migratory stage moving from the posterior to the anterior midgut. When nectomonad promastigotes arrive at the anterior midgut they differentiate into leptomonad forms, a newly named life cycle stage, which resume replication. Leptomonad promastigotes, which are found in the anterior midgut, are the developmental precursors of the metacyclic promastigotes, the mammal-infective stages. Leptomonad forms also produce promastigote secretory gel, a substance that plays a key role in transmission by forming a physical obstruction in the gut, forcing the sandfly to regurgitate metacyclic promastigotes during bloodfeeding.

Filamentous proteophosphoglycan secreted by Leishmania promastigotes forms gel-like three-dimensional networks that obstruct the digestive tract of infected sandfly vectors.

Development of Leishmania parasites in the digestive tract of their sandfly vectors involves several morphological transformations from the intracellular mammalian amastigote via a succession of free and gut wall-attached promastigote stages to the infective metacyclic promastigotes. At the foregut midgut transition of Leishmania-infected sandflies a gel-like plug of unknown origin and composition is formed, which contains high numbers of parasites, that occludes the gut lumen and which may be responsible for the often observed inability of infected sandflies to draw blood. This "blocked fly" phenotype has been linked to efficient transmission of infectious metacyclic promastigotes from the vector to the mammalian host. We show by immunofluorescence and immunoelectron microscopy on two Leishmania/sandfly vector combinations (Leishmania mexicana/Lutzomyia longipalpis and L. major/Phlebotomus papatasi) that the gel-like mass is formed mainly by a parasite-derived mucin-like filamentous proteophosphoglycan (fPPG) whereas the Leishmania polymeric secreted acid phosphatase (SAP) is not a major component of this plug. fPPG forms a dense three-dimensional network of filaments which engulf the promastigote cell bodies in a gel-like mass. We propose that the continuous secretion of fPPG by promastigotes in the sandfly gut, that causes plug formation, is an important factor for the efficient transmission to the mammalian host.

Novel fast atom bombardment mass spectrometric procedures for glycoprotein analysis.

We describe procedures, based upon fast atom bombardment mass spectrometry, for characterising the carbohydrate chains in glycoproteins and for determining sites of glycosylation. Strategies for rapidly screening glycoproteins to ascertain the types of sugar chains present and the degree of heterogeneity are presented. Protocols are given for sequencing O- and N-linked glycans. These strategies exploit simple purification steps and afford data at high sensitivity.

In situ stimulation of a T helper cell hybridoma with a cellulose-bound peptide antigen.

Many enzyme-linked immunosorbent assays take advantage of immobilized antigens for the identification of antibody binding sites. Generally, the analysis of cellulose membrane-bound B-cell epitopes is currently considered of high utility. We adapted this methodology for the stimulation of a T helper cell hybridoma with known specificity. Forty overlapping peptides corresponding to the entire rabies virus nucleoprotein were synthesized in duplicates on a single sheet of 90x130 mm size amino-modified paper. The efficacy of the peptide assembly was monitored by color staining of the unreacted amino groups. After completion of the synthesis, the side-chain protecting groups were removed, and the membrane was thoroughly cleaned of all organic and inorganic contaminants. The membrane was cut into pieces, and a standard lymphokine release assay was performed directly from the paper-bound antigens. From all the 40 peptide spots only peptide 31D stimulated the proliferation of the 9C5.D8-H T-cell hybridoma, known to react to this peptide. By using this protocol, as little as 0.4 microgram (approximately 200 pmole) of peptide could be detected. According to mass spectrometry the T-cell stimulation proceeded as a true solid-phase assay. The peptide neither leached from the membrane nor was cleaved by the medium-splenocyte mixture. Additionally, tryptic digestion of the cellulose membrane released the expected peptide fragments.

Detection of IgM to hepatitis B core antigen in a reductant containing, chemiluminescence assay.

The Abbott PRISM(R) hepatitis B core (HBc) antigen assay is an automatic in vitro competitive chemiluminescence immunoassay for the detection of total antibody to HBc (anti-HBc) antigen in human serum or plasma. The assay utilizes cysteine solution as a reducing reagent in order to maximize specificity. To help understand the effect of cysteine on detection of anti-HBc antigen, we separated and purified anti-HBc IgM and IgG from human plasma using size exclusion, protein A/G, and affinity chromatography techniques. We showed that cysteine affected the reactivity of anti-HBc IgM with recombinant HBc (rHBc) antigen but not the reactivity of anti-HBc IgG. Anti-HBc IgM treated with cysteine yielded byproducts which were reactive in the PRISM HBcore assay. Reduction-sensitive factor (RSF) - IgM fraction from serum known to be non-specific for anti-HBc activity, similarly treated with cysteine, was no longer reactive in the PRISM HBcore assay. We showed that cysteine treatment is effective against non-specific IgM in human blood. Also, the inclusion of cysteine in the PRISM HBcore assay does not compromise the detection of HBc specific antibodies.

3-Arylquinolizidines, potential antidepressant agents.

The synthesis, structure elucidation, and pharmacological evaluation of some 3-arylquinolizidines as semirigid phenethylamines are described. Many of the derivatives posses antidepressant activity. Some anticonvulsant effects are noted.

N-(2-Cyanoethyl) derivatives of meperidine, ketobemidone, and a potent 6,7-benzomorphan.

The N-(2-cyanoethyl)-9alpha-ethyl-5-methyl-6,7-benzomorphan (1c) is a more potent antinociceptive and has stronger receptor binding affinity than its N-methyl analogue 1b. The N-(2-cyanoethyl)-4-phenylpiperidine compounds 2b and 3b were almost inactive compared to their N-methyl congeners 2a and 3a, respectively. It appears that the pharmacological effect of the N-(2-cyanoethyl) moiety is dependent on the opioid on which it is substituted.

Racemic and optically active 2,9-dimethyl-5-(m-hydroxyphenyl)morphans and pharmacological comparison with the 9-demethyl homologues.

2,9 alpha-Dimethyl-5-(m-hydroxyphenyl)morphan (3a) has been synthesized from 5-(m-methoxyphenyl)-2-methyl-9-oxomorphan (4) and resolved into its enantiomers (+)-3a and (-)-3a. The assigned alpha-orientation of the 9-methyl group was derived from studies of induced NMR shifts using Eu(fod)3-d27. Compound (+)-3a has inappreciable agonist (antinociceptive) activity in mice, and (-)-3a shows codeine-like potency in the hot-plate and writhing tests only. The 9-demethyl homologues, (+)-1 and (-)-1, are strong agonists, about as potent as morphine in these tests as well as in the tail-flick assay. The racemic compound 3a and (+)-3a, but not (-)-3a, exhibit low-potency, narcotic-antagonist activity in mice (tail-flick test, vs. morphine). All three, however, precipitate abstinence in nonwithdrawn, morphine-dependent rhesus monkeys. Monkey studies with the 9-demethyl homologues confirmed earlier results showing that (+)-1, suppressing abstinence in withdrawn animals, has high physical dependence capacity, while (-)-1 has none. Instead, (-)-1 precipitates abstinence in nonwithdrawn animals. Studies in rats and isolated organs (guinea pig ileum and mouse vas deferens) and receptor-binding assays confirm the quite different opioid-action profiles of (+)-1 and (-)-1, which thus might interact with different opioid receptors. Catalytic hydrogenation of the methiodide (7) of 5 gave, instead of the expected epimer of 3a, ring-opened compound 8.

Mesoionic xanthine analogues: phosphodiesterase inhibitory and hypotensive activity.

Several mesoionic thiazolo[3,2-alphapyrimidines and mesoionic 1,3,4-thiadiazol[3,2-alpha-pyrimidines were evaluated as inhibitors of cyclic-AMP phosphodiesterase. While small alkyl substituents at the 6 position have no significant effect on activity, phenyl and benzyl substituents enhance activity. Mesoionic structures such as 1 (R2 = H; R8 = Et) possess 20 to 40 times the activity of theophylline when the R6 substituent is phenyl or 4-chlorobenzyl. methyl and ethyl substitution at the 2 position essentially abolishes activity. Although plagued by solubility problems, several of the mesoionic derivatives were found to display weak hypotensive effects in vivo.

Lipophilic 4-isoxazolyl-1,4-dihydropyridines: synthesis and structure-activity relationships.

A series of 4-isoxazolyl-1,4-dihydropyridines bearing lipophilic side chains at the C-5 position of the isoxazole ring have been prepared. The calcium channel antagonistic activity of these compounds has been evaluated. A hypothetical model for binding of these compounds in the calcium channel is proposed, and the validity of this model is evaluated based on the SAR of this series of calcium binding, especially for the two most active derivatives, 1a, g. The solid-state structure for the most active compound, 1a, has also been determined, and its important features are reported.

Some spiro analogues of the potent analgesic ketobemidone.

A series of spiro analogues of the potent narcotic ketobemidone have been prepared and found to be devoid of opiate activity. Additional pharmacology and possible implications for the mode of binding of ketobemidone to the analgesic receptor are discussed.

1-(4-Aminobenzyl)-and 1-(4-aminophenyl)isoquinoline derivatives: synthesis and evaluation as potential irreversible cyclic nucleotide phosphodiesterase inhibitors.

In an effort to increase the specificity of the potent phosphodiesterase inhibitor papaverine, we synthesized two series of novel 1-(4-aminobenzyl)- and 1-(4-aminophenyl)isoquinoline derivatives, incorporating alkylating moieties on the amine substituents. These compounds were evaluated for their inhibitory action on phosphodiesterase preparations from bovine heart and rat cerebral cortex. Studies were also conducted to determine whether these compounds were reacting with the enzymes in an irreversible manner. The compounds were potent inhibitors of the phosphodiesterases; however, no evidence was found for an irreversible inhibition.