Mitochondrial respiratory chain involvement in peroxiredoxin 3 oxidation by phenethyl isothiocyanate and auranofin

Mitochondrial peroxiredoxin 3 (Prx 3) is rapidly oxidized in cells exposed to phenethyl isothiocyanate (PEITC) and auranofin (AFN), but the mechanism of oxidation is unclear. Using HL-60 cells deplete of mitochondrial DNA we show that peroxiredoxin 3 oxidation and cytotoxicity requires a functional respiratory chain. Thioredoxin reductase (TrxR) could be inhibited by up to 90% by auranofin without direct oxidation of peroxiredoxin 3. However, inhibition of thioredoxin reductase promoted peroxiredoxin 3 oxidation and cytotoxicity in combination with phenethyl isothiocyanate or antimycin A. We conclude that rapid peroxiredoxin 3 oxidation occurs as a consequence of increased oxidant production from the mitochondrial respiratory chain.     

Schistosoma mansoni: lectin-dependent cytotoxicity of hemocytes from susceptible host snails, Biomphalaria glabrata

Normally benign hemocytes from a strain (M-line) of the snail, Biomphalaria glabrata, susceptible to Schistosoma mansoni, became cytotoxic toward the sporocyst stage if the parasite was first treated with the lectin, concanavalin A. Concanavalin A binding was inhibitable with alpha-methyl mannoside and killing was dose-dependent. Maximal levels of concanavalin A-induced cytotoxicity were comparable with levels observed when hemocytes from a resistant snail strain (13-16-R1) encountered untreated sporocysts. Induction of the cytotoxic response did not occur if hemocytes alone were pretreated with the lectin. A unique method incorporating ultraviolet microscopy and the vital fluorescent dye, eosin Y, was used for discriminating between live and dead sporocysts. This model may prove useful in understanding mechanisms used by invertebrate effector cells in recognition and killing of invading organisms.     

Incorporation of a Fluorescent Compound by Live Heterodera glycines

The incorporation of fluorescein isothiocyanate (FITC) by J2 of Heterodera glycines, the soybean cyst nematode, and the resulting effects on fitness were determined. Live soybean cyst nematode J2 incubated in FITC fluoresced, primarily in the intestinal region, beyond auto-fluorescence. Dissection of animals, as well as fluorescence-quenching techniques, indicated that FITC was not simply bound to the cuticle. FITC was also found to cross the egg shell. Fluorescence increased in relation to FITC concentration and incubation time. Nematodes incubated in FITC remained active and did not lose their fluorescence even after two weeks at room temperature. Fluorescence of nematodes was not stable through development. Males which developed from fluorescent juveniles did not retain the stain. Both FITC and the DMF solvent reduced the hatching rate. However, those individuals that successfully hatched remained viable and able to infect roots. Incorporation of FITC was found to occur in three other genera of nematodes. Rhodamine B isothiocyanate was also found to be incorporated by H. glycines.     

Genetic sexing in the mediterranean fruit fly,Ceratitis capitata,using the alcohol dehydrogenase locus

Summary Using the alcohol dehydrogenase (ADH) locus a genetic sexing system is being developed in the Mediterranean fruit fly Ceratitis capitata based on the sensitivity of ADH null mutations to environmental ethanol. A series of null mutants have been induced at this locus, however, none proved viable as homozygotes. One of these null mutants was translocated to the male determining chromosome and this line can be used for genetic sexing. When larvae from this line were reared on larval medium containing various concentrations of allyl alcohol, 97% of the emerging adults were males; in the absence of the allyl alcohol the sex ratio in the line is distorted in favour of the females. It is proposed that the higher ADH activity of the females (homozygous positive) in comparison with the males (heterozygous null) is responsible for their lower survival in larval medium containing allyl alcohol. ADH converts the allyl alcohol to the lethal ketone. The possible use of this line to sex large populations of medflies for use in sterile insect release programmes is discussed.     

Schistosoma mansoni: identification, characterization, and purification of the spine glycoprotein by monoclonal antibody

A tegumental surface membrane antigen of Schistosoma mansoni has been identified by use of a monoclonal antibody. The binding of ¹²⁵I-labeled monoclonal antibody showed that proteins sharing antigenic determinants recognized by this monoclonal antibody were present in cercariae and worms of both sexes, but were absent from schistosome egg extract. The protein molecules expressing these antigenic determinants differed in molecular weight: 120,000 in cercaria and 170,000 in male and female worms. The cercarial glycoprotein immunoprecipitated with the monoclonal antibody was also immunoprecipited by sera of infected humans, as shown by two-dimensional gel electrophoresis and tryptic peptide mapping. The location of the glycoprotein identified by the monoclonal antibody was restricted to the spines of the schistosomular surface, the tubercle-associated spines of the male worm, and the dorsal spines of the female worm. The spine glycoprotein was readily purified by immunoaffinity chromatography. These findings are discussed in relation to parasite development and the relevance of this antibody for serodiagnosis and immunoprophylaxis.     

Particle size influences fibronectin internalization and degradation by fibroblasts

The application of nanotechnology for drug targeting underlines the importance of controlling the kinetics and cellular sites of delivery for optimal therapeutic outcomes. Here we examined the effect of particle size on internalization and degradation of surface-bound fibronectin by fibroblasts using polystyrene nanoparticles (NPs; 51 nm) and microparticles (MPs; 1 μm). Fibronectin was strongly bound by NPs and MPs as assessed by immuno-dot blot analysis (5.1 ±0.4×10^{– 5} pg fibronectin per μm² of NP surface; 4.2±±0.3×10^–5 pg fibronectin per μm² of MP surface; p>0.2). We estimated that ~193 fibronectin molecules bound to a MP compared with 0.6 fibronectin molecules per NP, indicating that ~40% of nanoparticles were not bound by fibronectin. One hour after incubation, fibronectin-coated NPs and MPs were rapidly internalized by Rat-2 fibroblasts. MPs and NPs were engulfed partly by receptor-mediated endocytosis as indicated by decreased uptake when incubated at 4 °C, or by depletion of ATP with sodium azide. Pulse-chase experiments showed minimal exocytosis of NPs and MPs. Internalization of NPs and MPs was inhibited by jasplakinolide, whereas internalization of MPs but not NPs was inhibited by latrunculin B and by integrin-blocking antibodies. Extraction of plasma membrane cholesterol with methyl β-cyclodextrin inhibited internalization of fibronectin-coated NPs but not MPs. Biotinylated fibronectin internalized by cells was extensively degraded on MPs but not NPs. Particle size affects actin and clathrin-dependent internalization mechanisms leading to fibronectin degradation on MPs but not NPs. Thus either prolonged, controlled release or an immediate delivery of drugs can be achieved by adjusting the particle size along with matrix proteins such as FN.     

Inactivation of cytochrome P-450 and production of N-alkylated porphyrins caused in isolated hepatocytes by substituted dihydropyridines. Structural requirements for loss of haem and alkylation of the pyrrole nitrogen atom

Incorporation of 4.5 nmol fluorescein isothiocyanate/mg rabbit sarcoplasmic reticulum, or of 7.4 nmol/mg purified ATPase, was sufficient to inhibit the activity completely. These results are not consistent with the suggestion (Pick, U. and Karlish, S.J.D. (1980) Biochim. Biophys. Acta 626, 255-261) that 2 mol ATPase were inhibited by each mole of reagent incorporated. A single labelled peptide was purified from the inhibited ATPase and it was shown that Lys 3/190, 10 residues from the N-terminus of tryptic fragment B, was the reactive lysine residue. This site is close to a potential nucleotide-binding fold in the ATPase sequence. A similar peptide showing only 2 conservative replacements was isolated from the sarcoplasmic reticulum of the lobster.     

Anti-Barnacle Activities of Isothiocyanates Derived from β-Citronellol and Their Structure–Activity Relationships

Antifouling agents with low toxicity are in high demand for sustaining marine industries and the environment. This study aimed to synthesize 15 isothiocyanates derived from β-citronellol and evaluate their antifouling activities and toxicities against cypris larvae of the barnacle Amphibalanus amphitrite. The synthesized isothiocyanates exhibited effective antifouling activities (EC₅₀=0.10–3.33 μg mL⁻¹) with high therapeutic ratios (LC₅₀/EC₅₀ >30). Four isothiocyanates with an amide or isocyano group showed great potential as effective antifouling agents (EC₅₀=0.10–0.32 μg mL⁻¹, LC₅₀/EC₅₀=104–833). The enantiomers of the isothiocyanates only slightly differed in their antifouling activities. These results may serve as a basis for further research and development of β-citronellol-derived isothiocyanates as effective low-toxic antifouling agents. To the best of our knowledge, this study is the first to report the antifouling activities of isothiocyanates derived from accessible natural products.