Trypanosoma rhodesiense: semiautomated microtesting for quantitation of antitrypanosomal activity in vitro

A rapid, semiautomated microdilution method was developed to measure the antitrypanosomal activity of large numbers of compounds against blood form Trypanosoma rhodesiense at 37 C in vitro. Parasites harvested from infected rats were incubated for 3 hr in microtitration plates with serial dilutions of test compounds. Inhibition of uptake of radiolabeled thymidine and l-leucine by the parasites served as indicators of antitrypanosomal activity. Repeated measurements of the activity of ethidium bromide demonstrated the sensitivity and precision of the method. Several additional antitrypanosomal drugs were tested in vitro and all except tryparsamide (a pentavalent arsenical) and suramin (a complex polyanion) were consistent with in vivo activity. Antimicrobial agents which are not effective antitrypanosomal drugs failed to show activity in vitro. Other compounds active in vitro included allopurinol and a number of aromatic diamidines, carbamodithioic acid derivatives, and 2-acetylpyridine thiosemicarbazones. This in vitro microtest system was developed as a primary screen for the selection of active compounds in a new Antitrypanosomal Drug Development Program.     

Antitrypanosomal activity of some pregnane glycosides isolated from Caralluma species

Pregnane glycosides previously isolated from genus Caralluma (C. Penicillata, C. tuberculata and C. russelliana) were tested for their antitrypanosomal activity. Penicilloside E showed the highest antitrypanosomal activity (IC₅₀ 1.01 μg/ml) followed by caratuberside C (IC₅₀ 1.85 μg/ml), which exhibited the highest selectivity index (SI 12.04). It was noticed that acylation is required for the antitrypanosomal activity while glycosylation at C-20 has no significant effect on the activity.     

Synthesis,in vitro antitrypanosomal and antibacterial activity of phenoxy,phenylthio or benzyloxy substituted quinolones

Chagas’ disease, caused by Trypanosoma cruzi (T. cruzi), is one of the most serious parasitic diseases in Latin America. The currently available chemotherapy, based on nifurtimox or benznidazole, is unsatisfactory due to the limited efficacy in the prevalent chronic stage of the disease and toxic side effects. In order to address these deficiencies, a series of quinolones based novel molecules have been synthesized and evaluated as potential antitrypanosomal agents. The most active analogue 10 inhibited T. cruzi with an IC₅₀ of 1.3 μg/mL. The results of this study have implications in the development of novel quinolone’s antitrypanosomal agents.     

Trypanosoma rhodesiense: evaluation of the antitrypanosomal action of 2,5-bis(4-guanylphenyl)furan dihydrochloride

The antitrypanosomal activity of 2,5-bis(4-guanylphenyl)furan dihydrochloride was evaluated with Trypanosoma rhodesiense in a primate model and its toxicological profile determined. The new antitrypanosomal agent failed to show chemotherapeutic advantages over pentamidine and investigation of the compound was halted.     

Bioassay-guided isolation of a sesquiterpene lactone of deoxyelephantopin from Elephantopus scaber Linn. active on Trypanosome brucei rhodesience

Methanolic extracts of 70 Malaysia plants were screened for their in vitro antitrypanosomal activity using Trypanosome brucei rhodesience, strain STIB 900 and mouse skeletal cell (L-6) in cytotoxicity activity assay. Results indicated that methanol extract from Elephantopus scaber Linn. (E. scaber) possessed the highest value of antitrypanosomal activity with good selectivity index (antitrypanosomal IC₅₀ of 0.22 ± 0.02 μg/ml, SI value of 204.55). Based on these results, E. scaber was chosen for further study by applying bioassay guided fractionation to isolate its antiprotozoal principle. The antiprotozoal principle was isolated from the ethyl acetate partition through solvent fractionation and crystallization process. The isolated active compound 1 was identified as deoxyelephantopin on the basis of its spectral analysis (FTIR, MS, 1D and 2D NMR).     

Effect of vertebrate hypoglycemic and β-cell cytotoxic agents on insects

1. The effect of several vertebrate hypoglycemic and β-cell cytotoxic chemicals on insects was examined.2. The dietary LC₅₀'s for phenformin fed to Manduca sexta, Plodia interpunctella, and Tribolium confusum were 1.4 × 10³, 3.3 × 10³ and 13.0 × 10³ ppm.3. Chlorpropamide and tolbutamide were similarly effective against M. sexta at 8.5 × 10³ and ∼17 × 10³ppm.4. Chlorpropamide produced a 23% hypotrehalosemic response in M. sexta fed 10⁴ ppm supplemented diet.5. The β-cell cytotoxic agents, alloxan and streptozotocin, produced 50% mortality when injected into the hemocoel of fourth-instar M. sexta at doses of 150 and 85 μg/g body weight.     

Persistence of spores of Pleistophora schubergi (Cnidospora: Microsporida) in the field,and their application in microbial control

Spores of Pleistophora schubergi, when applied to oak trees in the field at 2 × 10⁸ spores/ml with a uv protectant, “Shade,” infected 88% of Anisota senatoria larvae at 4 days after spray application. Spores without the uv protectant infected only 10% of the larvae at 4 days after application. When the spores were applied at the rate of 2 × 10⁸ and 2 × 10⁷ spores/ml in the field, 96 and 72% of the A. senatoria larvae and 100 and 100% of the Symmerista canicosta larvae were infected 14 days after spray application.     

Identity and Behavior of Xylem-Residing Bacteria in Rough Lemon Roots of Florida Citrus Trees

An aseptic vacuum extraction technique was used to obtain xylem fluid from the roots of rough lemon (Citrus jambhiri Lush.) rootstock of Florida citrus trees. Bacteria were consistently isolated from vascular fluid of both healthy and young tree decline-affected trees. Thirteen genera of bacteria were found, the most frequently occurring genera being Pseudomonas (40%), Enterobacter (18%), Bacillus, Corynebacterium, and other gram-positive bacteria (16%), and Serratia (6%). Xylem bacterial counts fluctuated seasonally. Bacterial populations ranged from 0.1 to 22 per mm³ of root tissue (about 10² to 2 × 10⁴ bacteria per g of xylem) when bacterial counts were made on vascular fluid, but these numbers were 10- to 1,000-fold greater when aseptically homogenized xylem tissue was examined similarly. Some of the resident bacteria (4%) are potentially phytopathogenic. It is proposed that xylem bacteria have an important role in the physiology of citrus.     

Protoplast isolation for species in the Chamelaucium group and the effect of antioxidant enzymes (superoxide dismutase and catalase) on protoplast viability

An effective protocol for protoplast isolation from young leaves and somatic embryogenic cells of species in the Chamelaucium group and the use of superoxide dismutase (SOD) and catalase (CAT) to enhance protoplast viability are described. Mesophyll protoplasts were isolated from young leaves of a white Geraldton waxflower (Chamelaucium uncinatum) line 583, using a mixture of 1% (w/v) cellulase R10, 0.5% (w/v) macerozyme R10, and 0.1% (w/v) pectolyase. Viability of isolated mesophyll protoplasts increased dramatically when SOD and CAT were added. The highest increase of 7.61-fold in viability and 4.34-fold of viable protoplast yield were achieved when a combination of SOD at 500 units mL^?1 and CAT at 2,000 units mL^?1 was added to the enzyme mixture. Somatic embryogenic cell-derived protoplasts were isolated from embryogenic suspension cells of C. uncinatum line 583 when 1% (w/v) hemicellulase was added to a combination of 2% (w/v) cellulase R10, and 1% (w/v) macerozyme R10. Addition of SOD at 500 units mL^?1 and CAT at 2,000 units mL^?1 to the enzyme mixture improved viability only slightly, to above 90%, but improved yield significantly (6.6-fold). This combination of enzymes was also used to isolate protoplasts from embryogenic suspension cells of Chamelaucium repens and from young leaves of C. uncinatum, Actinodium calocephalum, Verticordia etheliana, Verticordia grandis, Verticordia hughanii, and Verticordia mitchelliana successfully with viability >80% and viable yield >7?×?10⁵ cells g^?1 fresh weight (or per milliliter packed cell volume in the case of suspension cells).     

Conidiation of Hirsutella thompsonii var. synnematosa in submerged culture

Fourteen monosporal isolates of Hirsutella thompsonii grew vegetatively in various liquid media producing typical phialidic-like conidiophores. Hirsutella thompsonii var. synnematosa from Ivory Coast (HtIC) was the only pathotype which produced true conidia in submerged culture. HtIC began producing conidia after 3 days incubation reaching a peak ranging from 6.8 × 10⁵ to 9.7 × 10⁷ conidia/ml between 6 and 11 days. A concentration of 10 g/liter of corn steep liquor and 0.2% Tween 80 were essential for maximum conidiation. Submerged conidia had a smooth but somewhat rugose conidial walls, whereas aerially formed conidia were distinctly verrucose. Germination of submerged conidia ranged from 5.2 to 12.9% and were virulent causing 32.5% infection to adult citrus rust mites when sprayed on citrus foliage at 1.2 × 10⁹ conidia/ml.     

Absorption and mobility of foliar-applied boron in soybean as affected by plant boron status and application as a polyol complex

In the present study (i) the impact of plant Boron (B) status on foliar B absorption and (ii) the effect of B complexation with polyols (sorbitol or mannitol) on B absorption and translocation was investigated. Soybean (Glycine max (L.) Meer.) plants grown in nutrient solution containing 0 ??M, 10 ??M, 30 ??M or 100 ??M ¹¹B labelled boric acid (BA) were treated with 50 mM ¹⁰B labelled BA applied to the basal parts of two leaflets of one leaf, either pure or in combination with 500 mM sorbitol or mannitol. After one week, ¹⁰B concentrations in different plant parts were determined. In B deficient leaves (0 ??M ¹¹B), ¹⁰B absorption was significantly lower than in all other treatments (9.7% of the applied dose vs. 26%?C32%). The application of BA in combination with polyols increased absorption by 18?C25% as compared to pure BA. The absolute amount of applied ¹⁰B moving out of the application zone was lowest in plants with 0 ??M ¹¹B supply (1.1% of the applied dose) and highest in those grown in 100 ??M ¹¹B (2.8%). The presence of sorbitol significantly decreased the share of mobile ¹⁰B in relation to the amount absorbed. The results suggest that ¹¹B deficiency reduces the permeability of the leaf surface for BA. The addition of polyols may increase ¹⁰B absorption, but did not improve ¹⁰B distribution within the plant, which was even hindered when applied a sorbitol complex.     

Pseudomonas aeruginosa UCBPP-PA14 a useful bacterium capable of lysing Microcystis aeruginosa cells and degrading microcystins

To identify a useful bacterium capable of controlling both Microcystis aeruginosa and microcystins (MCs), 30 strains of Pseudomonas were screened. Two of them (Pseudomonas aeruginosa UCBPP-PA14 and Pseudomonas putida KCCM 10464) could cause significant lysis of M. aeruginosa. PA14 exhibited higher degradation activity against microcystins than KCCM 10464, and hence, it was selected as the bacterium for further analysis. Following its introduction into M. aeruginosa culture (10⁵ cells mL^-1) at densities of 10⁷, 10⁵, and 10³ PA14 cells mL^-1, higher initial inoculations of PA14 removed correspondingly more M. aeruginosa cells (100%, 100%, and 92% at 15, 30, and 10?days, respectively) and degraded microcystin (extracellular MCs: 83.7%, 77.7%, and 51.6% at 30?days; total MCs: 91.0%, 86.9%, and 61.6% at 30?days, respectively). However, the activity of PA14 diminished when its density decreased to less than 10⁶ cells mL^-1. At three initial algal densities (10⁶, 10⁵, and 10³ cells mL^-1), PA14 at a density of 10⁵ cells mL^-1 easily and quickly removed algal cells (100%, 100%, and 97.3% at 8, 16, and 30?days, respectively). Host range assays showed that at lower initial PA14 inoculation (10⁵ cells mL^-1), the algicidal activity of PA14 was effective species-specifically on M. aeruginosa, while at higher initial inoculation (10⁷ cells mL^-1), a wider algicidal range regardless of the general taxonomical relationships was observed. These results indicate that inoculation with 10⁵ Pseudomonas aeruginosa PA14 cells mL^-1 into developing natural algal blooms can remove both M. aeruginosa and MCs without causing problems for other algae species.     

In vitro antitrypanosomal activity of plant terpenes against Trypanosoma brucei

During the course of screening to discover antitrypanosomal compounds, 24 known plant terpenes (6 sesquiterpenes, 14 sesquiterpene lactones and 4 diterpenes) were evaluated for in vitro antitrypanosomal activity against Trypanosoma brucei brucei. Among them, 22 terpenes exhibited antitrypanosomal activity. In particular, α-eudesmol, hinesol, nardosinone and 4-peroxy-1,2,4,5-tetrahydro-α-santonin all exhibited selective and potent antitrypanosomal activities in vitro. Detailed here in an in vitro antitrypanosomal properties and cytotoxicities of the 24 terpenes compared with two therapeutic antitrypanosomal drugs (eflornithine and suramin). This finding represents the first report of promising trypanocidal activity of these terpenes. Present results also provide some valuable insight with regard to structure–activity relationships and the possible mode of action of the compounds.     

Antiplasmodial and antitrypanosomal activity of bicyclic amides and esters of dialkylamino acids

Several bicyclic amides and esters of dialkylamino acids were prepared. Their activities against a multiresistant strain of Plasmodium falciparum and against Trypanosoma brucei rhodesiense (STIB 900) were examined. Structure–activity relationships were discussed. Particularly the ester compounds showed good antiplasmodial and antitrypanosomal activity and a single compound was tested in vivo against Plasmodium berghei.     

Effects of density on growth rates of four benthic diatoms and variations in biochemical composition associated with growth phase

Diatom cell quantity and their biochemical composition vary among species and are greatly affected by harvest stage or culture conditions. This study compares growth pattern, cell attachment, and biochemical composition of four diatoms suitable for abalone post-larvae: Navicula incerta, Proschkinia sp., Nitzschia sp., and Amphora sp. The four diatoms were grown in F/2 medium at 28.5?±?1.4°C, under 62?±?8?μmol?photons?m^?2?s^?1, at different original inoculating densities (0.05?×?10⁶, 0.10?×?10⁶, and 0.25?×?10⁶?cells?mL^?1) and were harvested in log and stationary phase of growth for biochemical analysis. Total protein, carbohydrate, lipid, and ash composition, as well as fatty acid composition, were determined. All diatoms grew better when inoculated at 0.10?×?10⁶?cell?mL^?1 with Proschkinia sp. reaching the highest cell density of 6.56?×?10⁶?cells?mL^?1 in log phase. Amphora sp. had the highest cell attachment capacity when inoculated at 0.10?×?10⁶?cell?mL^?1 (11,580?cells?mm^?2), whereas N. incerta had the lowest (7,750?cells?mm^?2). Protein and lipid (percent dry weight) contents were generally highest in cells during log phase of growth; Amphora sp. in log phase of growth had the highest lipid content of 9.74% DW, whereas significant differences in carbohydrate between the two growth phases were only observed for Proschkinia sp. Besides, all diatoms had higher energy contents in log phase of growth. There were no significant differences in ash content among the four diatoms. Polyunsaturated fatty acid (PUFA) content ranged from 23.25% to 38.62% of the total fatty acids, and the four diatoms tested were richer in n-3 PUFA than in n-6 PUFA. All the diatoms had significant quantities of 20:5n-3 (EPA) (between 12.69% and 17.68% of TFA), and Proschkinia sp., in log phase of growth, had the highest quantity of arachidonic acid (20:4n-6; ARA). The results highlight the influence of culture conditions and harvest protocols on diatom nutritive value and enabled a preliminary approach towards the selection of novel diatom species.     

Dietary administration of the probiotic,Lactobacillus plantarum,enhanced the growth,innate immune responses,and disease resistance of the grouper Epinephelus coioides

The percent weight gain (PWG) and feed efficiency (FE) of Epinephelus coioides were calculated, and the lactobacilli and total microbiota in the posterior intestines, and non-specific immune parameters of grouper, and its susceptibility to Streptococcus sp. and an iridovirus were determined when the fish were fed diets containing Lactobacillus plantarum at 0 (control), 10⁶, 10⁸, or 10¹⁰ colony-forming units (cfu) kg^?1 for 4 weeks. Results showed that grouper fed a diet containing L. plantarum at the levels of 10⁶, 10⁸, and 10¹⁰ cfu kg^?1 had significantly increased PGW and FE especially at 10⁸ cfu kg^?1 group which were 404.6% and 1.26, respectively. L. plantarum significantly increased in the fish posterior intestines during the L. plantarum feeding period, but decreased rapidly from the intestine within 1 week after changing to the control diet (without L. plantarum). Fish fed a diet containing L. plantarum at 10⁶ and 10⁸ cfu kg^?1 had significantly higher survival rates than those fed the control diet after challenge with Streptococcus sp., as well as those fed 10⁸ cfu kg^?1 after challenge with an iridovirus, causing increases in the survival rates of 23.3%, 20.0%, and 36.7%, respectively, compared to the control group. The alternative complement activity (ACH₅₀) level of fish fed diets containing L. plantarum after 4 weeks was significantly higher than that of fish fed the control diet, and that of the 10⁸ cfu kg^?1 group was significantly higher than those of the 10⁶ and 10¹⁰ cfu kg^?1 groups, which increased by 83.4% compared to the control group. The lysozyme activity and glutathione peroxidase (GPx) activity of fish fed the L. plantarum-containing diets at 10⁸ and 10¹⁰ cfu kg^?1 significantly increased compared to those fed the 10⁶ cfu kg^?1 L. plantarum diet and control diet, and had increased by 76.3% and 136.6%, and 57.1% and 113.3%, respectively, compared to those fed the control diet. The phagocytic activity (PA), phagocytic index (PI), and respiratory bursts of head kidney leucocytes of fish fed 10⁶, 10⁸, and 10¹⁰ cfu kg^?1 L. plantarum diets were significantly higher than those of fish fed the control diet after 4 weeks of feeding, and increased 2.2-, 2.2-, and 2.3-fold; 1.8-, 1.8-, and 2.0-fold; and 1.4-, 1.4-, and 1.4-fold, respectively, compared to the control group. We therefore recommend dietary L. plantarum administration at 10⁸ cfu kg^?1 to promote growth and enhance immunity and resistance against Streptococcus sp. and an iridovirus of E. coioides.     

Enantiospecific antitrypanosomal in vitro activity of eflornithine

The polyamine synthesis inhibitor eflornithine is a recommended treatment for the neglected tropical disease Gambian human African trypanosomiasis in late stage. This parasitic disease, transmitted by the tsetse fly, is lethal unless treated. Eflornithine is administered by repeated intravenous infusions as a racemic mixture of L-eflornithine and D-eflornithine. The study compared the in vitro antitrypanosomal activity of the two enantiomers with the racemic mixture against three Trypanosoma brucei gambiense strains. Antitrypanosomal in vitro activity at varying drug concentrations was analysed by non-linear mixed effects modelling. For all three strains, L-eflornithine was more potent than D-eflornithine. Estimated 50% inhibitory concentrations of the three strains combined were 9.1 μM (95% confidence interval [8.1; 10]), 5.5 μM [4.5; 6.6], and 50 μM [42; 57] for racemic eflornithine, L-eflornithine and D-eflornithine, respectively. The higher in vitro potency of L-eflornithine warrants further studies to assess its potential for improving the treatment of late-stage Gambian human African trypanosomiasis.     

Synthesis of antitrypanosomal 1,2-dioxane derivatives based on a natural product scaffold

A short practical synthesis of a new natural product based scaffold (6), based on antitrypanosomal and antimalarial compounds isolated from different Plakortis species is described. The scaffold contains a peroxide unit that is surprisingly stable to chemical manipulation elsewhere in the molecule, enabling it to be elaborated into a small library of derivatives. It is stable to ozonolysis, reductive work-up with dimethylsulfide and the Wittig reaction with stabilized phosphorus ylides. The scaffold along with its Wittig analogues has displayed low to sub-micro molar (0.2-3.3 μM) antitrypanosomal activity.