The site of action of the F transfer inhibitor

Summary The mechanism of inhibition of F transfer from E. coli K12 cells containing the fin⁺ R factor R100 was studied. For this, a series of Flac double mutants carrying both a traO^-mutation, which prevents function of the transfer inhibitor, and a suppressible mutation in one of ten genes required for conjugational DNA transfer, were constructed. The levels of retransfer of these elements from cells carrying a wild-type Fhis element and R100 showed that of the ten transfer genes, only traJ was directly affected by the transfer inhibitor. Furthermore, in the case of R100 and therefore probably in the case of F itself, it was shown that the products of the other nine genes are absent from the cell during transfer inhibition, suggesting that the traJ product is required for their synthesis.D.F. acknowledges the support of a George Murray Scholarship from the University of Adelaide, Australia.     

The molecular mechanism of action of the proton ionophore FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone).

We propose a simple model that accounts for the ability of the weak acid FCCP (Carbonylcyanide-p-trifluoromethoxyphenylhydrazone) to both transport protons across phospholipid bilayer membranes and uncouple oxidation from phosphorylation in mitochondria. Four parameters are required to characterize this model: the rate constant for the movement of A- across the membrane, kA, the rate constant for the movement of HA across the membrane, kHA, the adsorption coefficient of A- onto the membrane-solution interface, beta A, and the surface pK. These four parameters were determined from kinetic measurements on planar bilayer membranes using the charge-pulse and voltage-clamp techniques. We confirmed the adequacy of the model by determining each of these parameters independently, utilizing equilibrium dialysis, zeta potential, membrane potential, spectrophotometric, and conductance measurements. For a phosphatidylethanolamine bilayer the values of the parameters are kHA = 10(4)S-1, beta A = 3 10(-3) cm, and 6.0 less than pK less than 6.4. As predicted theoretically, the value of KA depends on both the applied voltage, V, and dielectric constant of the membrane, epsilon r; when V approaches zero and the membrane contains chlorodecane (epsilon r congruent to 2.7) kA = 700 s-1. If oxidation is coupled to phosphorylation by means of a delta microH+, and V er congruent to 2.7 for the inner membrane of the mitochondrion, the model predicts that FCCP should exert maximal uncoupling activity at a pH congruent to pK. This prediction agrees with the published experimental results.     

In vivo action of the anticoccidial diclazuril (Clinacox) on the developmental stages of Eimeria tenella: an ultrastructural evaluation

A single 5-mg/kg oral dose of diclazuril affected both the asexual and sexual development of Eimeria tenella in experimentally inoculated chickens. In second-generation schizonts, early growth and nuclear divisions progressed normally, but a marked inhibition of merozoite formation was observed. Exogenesis of merozoites was largely prevented, whereas production of micronemes, amylopectin granules, and dense bodies and the formation of rhoptries, conoid, and pellicle continued. All these subcellular organelles accumulated, together with differentiated nuclei, within the main cytoplasmic mass. In the end, complete necrosis of the schizonts occurred. In macrogamonts, dilation of the rough endoplasmic reticulum around type II wall-forming bodies, fusion of type II wall-forming body contents, disturbance of the normal parallel arrangement of rough endoplasmic reticulum, and disruption of row formation of amylopectin granules became evident. In the microgamonts, normal evagination of microgametes was prevented; the flagellar complex formed within the main cytoplasmic mass and the differentiated nuclei remained present within the parasite body. The macro- and microgamonts also ended up in a stage of complete necrosis. These data indicate that diclazuril treatment primarily affects the normal differentiation of the respective endogenous stages during parasite development. This leads to complete degeneration of schizonts and gamonts indicating the lethal effect of this new anticoccidial compound.     

In vivo action of the anticoccidial diclazuril (Clinacox) on the developmental stages of Eimeria tenella: a histological study

Diclazuril, a new benzeneacetonitrile anticoccidial, has potent activity against various stages of Eimeria tenella. A single treatment of experimentally infected chickens during the prepatent phase (up to day 5) results in a complete interruption of the life cycle and oocyst shedding. The first- and second-generation schizonts show extensive degenerative changes that finally result in a complete loss of the parasitic stage. The degeneration is characterized by loss of internal structure, the appearance of many intracytoplasmic vacuoles, and incomplete merogony. The merozoites themselves show similar degenerative changes, including the presence of numerous small vacuoles in the cytoplasm. Diclazuril is also effective against both the micro- and macrogametocytes that have a ballooned appearance and loose their internal structure completely. In the macrogametocytes, wall-forming bodies either do not develop or disappear rapidly. Development of typical caecal lesions is prevented when treatment with diclazuril is initiated before large numbers of second-generation schizonts appear, i.e., day 3. It is concluded that diclazuril is lethal against both the asexual and the sexual stages of E. tenella. At the proposed use level of 1 ppm in the feed, the life cycle is interrupted at a very early stage and lesion development and oocyst shedding are completely prevented.     

The ligandin (non-substrate) binding site of human Pi class glutathione transferase is located in the electrophile binding site (H-site).

Glutathione S -transferases (GSTs) play a pivotal role in the detoxification of foreign chemicals and toxic metabolites. They were originally termed ligandins because of their ability to bind large molecules (molecular masses >400 Da), possibly for storage and transport roles. The location of the ligandin site in mammalian GSTs is still uncertain despite numerous studies in recent years. Here we show by X-ray crystallography that the ligandin binding site in human pi class GST P1-1 occupies part of one of the substrate binding sites. This work has been extended to the determination of a number of enzyme complex crystal structures which show that very large ligands are readily accommodated into this substrate binding site and in all, but one case, causes no significant movement of protein side-chains. Some of these molecules make use of a hitherto undescribed binding site located in a surface pocket of the enzyme. This site is conserved in most, but not all, classes of GSTs suggesting it may play an important functional role.     

Problems concerning the biochemical action of superoxide dismutase (erythrocuprein).

The decay of the tetraperoxochromate- (V) complex (CrO83theta) was examined to study the substrate specificity of erythrocuprein (super-oxide dismutase). The decay of CrO83theta proved rather complex in aqueous solutions. Apart from the two known oxygen species O2theta and singlet oxygen (1 deltagO2), H2O2 and probably OH radicals were formed. No unequivocal evidence for the appearance of superoxide was obtained. The possible electron transfer from Cr5 to Fe3 (cytochrome c) was also discussed. In Tris buffer, pH 7.8, there were absolutely no signs of superoxide or OH radical formation. In fact, pulse radiolysis measurements employing a homogeneous OH source demonstrated that the Tris and OH radicals react with each other. One mol of H2O2 was generated from 1 mol of CrO83theta in Tris buffer. By contrast, only 0.5 mol H2O2 could be determined when the CrO83theta decay was carried out in 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES) buffer, pH 7.8. The phenomenon of reducing oxidized cytochrome c could not fully be assigned to a superoxide-mediated reduction, since erythrocuprein was unable to inhibit this cytochrome c reduction efficiently. The energetic oxygen species (1deltag O2, OH etc.) appearing during the CrO83theta decay gave rise to a clearly detectable chemiluminescence. In this system, erythrocuprein was very active regardless of which buffer was used. Even in the absence of a chemiluminescent mediating agent, which might have interferred with the enzyme, erythrocuprein proved capable of inhibiting the CrO83theta-induced chemiluminescence in a rather specific way. No such specificity was seen in the presence of low molecular weight Cu-chelates including Cu(Tyr)2, Cu(Lys)2 and Cu(His)2. The ability to suppress chemiluminescence was approximately 3 orders of magnitude less pronounced than that of the native enzyme. It is presumed that erythrocuprein reacts with oxygen species other than the superoxide radical.     

Eimeria tenella: anticoccidial action of drugs in birds with surgically closed ceca.

Surgical ligation of chick ceca was used to study the role of absorption and extraintestinal transport in the action of anticoccidial drugs. The administration of drugs in the feed was started after ligation of one of the paried ceca. Birds were inoculated orally with oocysts of Eimeria tenella before cecal ligation or were given bilateral cecal injections of sporozoites after ligation. Cecal lesions caused by the coccidia were evaluated and compared on day 6 postinoculation. Lesions in ligated and unligated ceca were reduced by feeding robenidine (33 ppm), arprinocid (70 ppm), zoalene (125 ppm), aklomide (250 ppm), clopidol (125 ppm), nicarbazin (125 ppm), monensin (120 ppm), salinomycin (60 ppm), and lasalocid (75 ppm). The lesions were more severe in the ligated cecum than in the intact cecum, whether in nonmedicated or medicated birds, but the differences were statistically significant only upon treatment with amprolium, aklomide, robenidine, and clopidol. Generally, however, all drugs except amprolium, significantly reduced the lesions in the ligated cecum in comparison with the control, nonmedicated ligated cecum. Therefore, we concluded that the systemic absorption of most anticoccidial drugs contributes significantly to their efficacy against coccidia in the intestinal mucosa.     

The cytoplasmic membrane as the site of the antimicrobial action of N-octylethanolamine

On agar spread plates, N-octylethanolamine was biocidal at comparable minimum inhibitory concentration (MIC) values (3–4mm) against Pseudomonas aeruginosa (two strains), Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Candida tropicalis, and Acremonium sp. which had been grown on a number of different media. The inhibition was greater at higher pH values. In liquid culture, growth inhibition by 3mm N-octylethanolamine was accompanied by cell lysis. Both effects could be prevented by the presence of 1mm spermine or spermidine, but only in bacteria, and not at high pH values. These effects of the polyamines were shown to be non-specific, being shared by other polycations and Mg²⁺ ions. N-Octylethanolamine at concentrations above its MIC caused total inhibition of the oxidation of 1mm glucose by Ps. aeruginosa (CAS1 and PAO1), E. coli, or C. tropic an effect that was partially reversible by Mg²⁺ ions. At concentrations below the MIC, there was little inhibit ion of glucose oxidation but a potent inhibition of the extrusion of ethidium bromide from intact cells of E. coli, suggesting that at such concentrations N-octylethanolamine is uncoupling oxidative phosphorylation. The data presented confirm the view that the biocidal effects are due to action on the cytoplasmic membrane.     

The selectivity of action of the aspartic-proteinase inhibitor IA3 from yeast (Saccharomyces cerevisiae).

The ability of the aspartic-proteinase inhibitor IA3 from yeast (Saccharomyces cerevisiae) to affect the activities of a range of mammalian and microbial aspartic proteinases was examined. The inhibitor appeared to be completely selective in that only the aspartic proteinase A from yeast was inhibited to any significant extent. IA3 thus represents the first example of a totally specific, naturally occurring, aspartic-proteinase inhibitor.     

The site of action of N-alpha-tosyl-L-lysyl-chloromethyl-ketone (TLCK) on cloned cytotoxic T lymphocytes

N-alpha-tosyl-L-lysyl-chloromethyl-ketone (TLCK), an irreversible inhibitor of trypsin-like serine proteases, is a potent, nontoxic inhibitor of cytotoxic T lymphocyte (CTL) activity with half-maximal inhibition of an alloreactive CTL clone occurring at [TLCK] = 30 microM. We have utilized TLCK as an affinity probe for functionally important CTL surface molecules by raising rabbit antibodies specific for the tosyl group and employing them as immunoprecipitating reagents. When 125I-labeled cloned CTL were treated with TLCK, immunoprecipitation with rabbit anti-tosyl antibodies and analysis by polyacrylamide gel electrophoresis revealed a small number of TLCK-binding proteins. Prior alkylation of radiolabeled CTL with iodoacetamide inhibited TLCK binding only slightly, suggesting that TLCK binding did not occur via free sulfhydryl groups. Thymocytes and a second CTL clone both had very similar patterns of TLCK-binding proteins; in contrast the TLCK-binding proteins of B cells differed greatly. Sequential immunoprecipitation experiments identified the predominant CTL TLCK-binding protein as T200. Lymphocyte function-associated antigen-1 also reacted with TLCK but to a lesser extent. The inhibitory role of cell-surface bound TLCK (vs intracellular TLCK) was demonstrated by protection experiments using Concanavalin A, a reversible ligand of the CTL cell surface. These experiments suggest that T200 may be required for cytotoxic activity of CTL.     

Transport of ABA from the site of biosynthesis to the site of action

There is substantial evidence that abscisic acid (ABA) moves within plants. ABA has been considered as a root-derived signaling molecule that induces stomatal closure in response to dry soil conditions. It has been also reported that ABA synthesized in vegetative tissues is translocated to the seeds. The transport of ABA is an important factor in determining the endogenous concentrations of the hormone at the site of action, and hence, it is an important process in physiological responses. However, the molecular mechanisms that regulate ABA transport are not fully understood. Recent studies using Arabidopsis indicate that ABA is actively synthesized in leaf vascular tissues in response to drought, and that ABA is subsequently transported to the guard cells to close stomata. Identification of the transporters that mediate ABA export from the inside to the outside of the cells at the site of ABA biosynthesis (vascular tissues) and ABA uptake into the cells at the site of action (guard cells), respectively, in this species indicates an active mechanism to regulate ABA transport. Although Arabidopsis represents only one model plant, these findings are useful to discuss common or different regulatory mechanisms among different species and to improve our total understanding of the regulation of ABA transport.