THE EFFECT OF BENZIMIDAZOLE ON CATION UPTAKE BY PLANT ROOTS

Klingensmith , M. J. (Colgate U., Hamilton, N.Y.) The effect of benzimidazole on cation uptake by plant roots. Amer. Jour. Bot. 48(8): 711–716. Illus. 1961.—A number of benzazole compounds were examined for their effect on cation uptake. Benzimidazole was found to almost double the uptake of potassium by excised barley roots in a 6-hr period. Chlorobenzimidazole also enhanced the absorption of potassium but not to the same extent. This stimulation of potassium accumulation was found to be insensitive to cyanide but was dependent on the temperature of the ambient solution. There was also an increased accumulation of potassium by roots of intact barley plants with benzimidazole treatment without interference with subsequent transport of the potassium. Benzimidazole also stimulated uptake of sodium and calcium by excised barley roots but not at identical levels. Results are discussed in the light of various theories of ion absorption.     

Cytological changes and DNA and protein synthesis in parthenogenetically activated sea urchin eggs

Summary The present study deals with cytological observations, DNA and protein synthesis in artificially activated sea urchin eggs. The eggs were activated by means of Loeb's double treatment with butyric acid and hypertonic sea water. Most of the eggs ofHemicentrotus pulcherrimus divided when the chromosomes duplicated after formation of the first monaster and other eggs divided at a later cell cycle. In the eggs ofTemnopleurus toreumaticus, however, haploid division at the first cell cycle was observed predominantly.Activated eggs that were treated for 25 min with hypertonic sea water showed a marked uptake of³H-thymidine during the two periods of 30–40 min and 90–100 min after the double treatment. These periodic changes in the³H-thymidine uptake paralleled morphological changes within the nucleus. However, these periods of increased uptake were not observed in the eggs treated with hypertonic sea water for 60 min. During exposure to hypertonic sea water, the³H-thymidine-uptake by eggs activated with butyric acid decreased gradually. When the uptake of¹⁴C-valine by eggs was measured, a very low level was seen in unfertilized eggs. The level of uptake increased strikingly when the eggs were activated with butyric acid but was suppressed by the hypertonic treatment. However, removal of the eggs to sea water allowed the uptake to return to the former high level. This pattern suggests that the hypertonic treatment has an inhibitory effect on the synthesis of protein (or enzymes) which obstruct cleavage induction.     

Efficiency of a genetic test to detect benzimidazole resistant Haemonchus contortus nematodes in sheep farms in Quebec,Canada

Haemonchus contortus is a hemophilic nematode which infects sheep and causes anemia and death to lambs. Benzimidazole drugs are used to remove these parasites, but the phenomenon of resistance has arisen worldwide. A sensitive test to detect resistance before treatment would be a useful tool to enable farmers to anticipate the efficiency of the drug before drenching the flock. In this study, we compared a test for benzimidazole resistance based on detection of genetic markers in H. contortus before treatment with the common method of fecal egg count reduction test (FECRT). We recruited 11 farms from different regions of Quebec for this study. Fecal samples from animals were collected per rectum before and after treatment in control and treated groups (10 animals per group). The 10 sheep were treated with fenbendazole at the recommended dose rate. Among the 11 farms participating in the study, we found H. contortus in 8 of them and it was the most predominant nematode species detected by egg count. Using the genetic test, we found benzimidazole resistance in each of these 8 farms. In 5 of these 8 farms there were sufficient sheep with an egg count for H. contortus above 150 eggs per gram to allow the FECRT test to be conducted. Benzimidazole resistance was observed in each of these 5 farms by the FECRT. When we compared the results from the genetic test for samples off pasture and from individual sheep, with the results from the FECRT, we concluded that the genetic test can be applied to samples collected off pasture to estimate benzimidazole resistance levels before treatment for H. contortus infections.     

Cytological effects of urethan on cleavage induction in parthenogenetically activated sea urchin eggs

The effect of urethan on artificial induction of cleavage in eggs of the sea urchin, Hemicentrotus pulcherrimus, was studied. When the eggs were exposed for 20 minutes to seawater containing urethan (final concentration, 0.08 M) after butyric acid-activation and then treated with hypertonic seawater, the cleavage rate was enchanced by about 1.5 times as compared with the nontreated eggs. In the eggs exposed to urethan–seawater for over 70 minutes many clear spots appeared throughout the cytoplasm. Simultaneously, the pigment granules, which had been embedded within the cortex, migrated to the inner cytoplasm and encircled a monastral centrosphere and clear spots. The clear spots were composed of microtubules much like cytasters, and in the central region of them centrioles were not yet found. The eggs in which the pigment granules disappeared from the cortex may be more susceptible to cleavage induction by succeeding hypertonic treatment.     

AN INDIRECT METHOD TO ASSAY FOR MITOTIC CENTERS IN SAND DOLLAR (DENDRASTER EXCENTRICUS) EGGS

It is possible consistently to induce sea urchin and sand dollar eggs to cleave directly from one cell into four cells. This is done by exposing the fertilized eggs to benzimidazole for 20 to 30 min beginning about early metaphase. The mitotic apparatus regresses, the cells do not cleave, and shortly after they are returned to normal sea water an early-prophase-appearing nucleus is present in each cell. Each cell then organizes a tetrapolar tetrahedral mitotic apparatus de novo, instead of transforming a bipolar mitotic apparatus into a tetrapolar figure, and cleaves one-to-four. In another type of experiment, it appears that sand dollar eggs exposed to mercaptoethanol during the first period of mitotic center duplication have only half as many centers by first cleavage metaphase as the normal controls. This is consistent with an earlier report by Mazia et al (1960). Using this same experimental technique, it was demonstrated that benzimidazole, on the contrary, does not interfere with mitotic center duplication in sand dollar eggs. A labeling experiment demonstrated that benzimidazole does not interfere markedly with the normal pattern of incorporation of C¹⁴-thymidine into the DNA of sea urchin eggs. The data reported here suggest that judicious treatment of sand dollar eggs (and probably sea urchin eggs, too) with benzimidazole can induce the eggs to cleave into as many cells as there were mitotic centers sometime earlier, for example at early metaphase of the first cleavage division. This provides a very useful tool for studies on the process of mitotic center duplication.     

Effect of benzimidazole and nickel ions in the control of Oryza sativa leaf senescence by red and far-red light

Changes in the levels of chlorophyll and protein were determined in detached rice leaves floated on water, benzimidazole (mM) and nickel chloride (mM) under continuous dark, red (R) and far-red (FR) illumination. Senescence was enhanced under FR and retarded under R illumination as compared to the dark treatment for leaves floated both on water and benzimidazole solution. Benzimidazole and nickel ions also delayed the senescence of leaves still further, although only to a limited extent under FR illumination. Protein changes showed similar trends to chlorophyll during senescence.     

A CYTOLOGICAL STUDY OF THE CENTRIFUGED WHOLE, HALF, AND QUARTER EGGS OF THE SEA URCHIN ARBACIA PUNCTULATA

While the ooplasmic components of centrifuged eggs of Arbacia punctulata do not stratify in homogeneous layers, we have obtained the following strata beginning with the centripetal end: lipid droplets, pronucleus, clear zone, mitochondria, yolk, and pigment. Whereas mitochondria may be found mingled with yolk bodies, we have never observed lipid droplets nor pigment bodies among any of the other inclusions. The so-called clear zone contains a heterogeneous population of inclusions: annulate lamellae, heavy bodies, Golgi complexes, and rod-containing vacuoles. The peripheral cortical granules of immature (germinal vesicle stage) and of mature eggs are not dislodged from the cortical ooplasm with the centrifugal force utilized. When the eggs are treated with urethane, prior to centrifugation, the cortical granules of mature eggs abandon their peripheral position. Further centrifugation of the initially stratified eggs produces nucleated and nonnucleated halves and the centrifugation of the halves results in quarters. The cytology of the halves and quarters is discussed. The halves and quarters have been activated with either sperm or hypertonic sea water. With the exception of the nucleated halves, we were unable to obtain plutei larvae from the other fractions (red halves and quarters). We believe that the lack of development of the various fragments is a function of the balance of particular inclusions necessary for differentiation.     

A CYTOLOGICAL STUDY OF ARTIFICIAL PARTHENOGENESIS IN THE SEA URCHIN ARBACIA PUNCTULATA

Eggs of the sea urchin Arbacia punctulata were artificially activated with hypertonic seawater. The artificially activated eggs undergo the cortical reaction which is not distinguished by a wavelike progression as in the case of inseminated eggs. The cortical granules are released at random loci at the surface of the egg and result in spaces separated by large cytoplasmic projections. Unreacted cortical granules and ribosomes are found within the matrix comprising the large cytoplasmic projections. No "fertilization cone" is formed. The subsequent release of additional cortical granules results in the formation of a continuous perivitelline space, 15 min following activation. 85 min postactivation, an organization of annulate lamellae, endoplasmic reticulum of the smooth variety, and microtubules around a centriole is observed prior to nuclear division. Before the breakdown of the nuclear envelope a streak stage is formed. The streak is composed of a central core of annulate lamellae and is encompassed by endoplasmic reticulum and vesicular components. Condensation of chromatin is followed by the establishment of the mitotic apparatus. Centrioles were not found in the mature egg; however, they are present after activation prior to the first nuclear division, in the four-cell embryo, multicellular embryo, and at blastula. Artificially activated eggs have been observed to develop to the pluteus stage in more than 50% of the eggs treated.     

Coagulation of sea urchin egg cytoplasm by high salt concentrations

Unfertilized sea urchin eggs exposed to hyperosmotic salt solutions in excess of 1.75 M undergo a form of intracellular coagulation known as black cytolysis, similar to that seen in eggs injured by freezing. The process can be simulated by the microinjection of hypertonic salt into the cell suspended in isotonic solution in the absence of volume reduction. Black cytolysis during hyperosmotic stress can be attributed to the entry of concentrated extracellular solution through a membrane made permeable by excessive osmotic stress.     

Effects of benzimidazole on the purine and pyrimidine metabolism of yeast.

Yeast cells inhibited by benzimidazole accumulate hypoxanthine with associated efflux of xanthine. Unlike control cells, inhibited cells contain no detectable free UMP and CMP. Benzimidazole decreases uptake of [8-14C]hypoxanthine into the intracellular pool of hypoxanthine and xanthine but causes radioactive xanthine to accumulate in the medium. In inhibited cultures there is a threefold increase in incorporation of [8-14C]hypoxanthine into the total (intracellular plus extracellular) xanthine. Uptake of [8-14C]hypoxanthine into free nucleotides and into bound adenine and guanine was inhibited by 70%. Uptake of [U-14C]glycine into IMP, AMP, GMP, DNA and RNA was also substantially decreased. Incorporation of [2-14C]uracil into the intracellular uracil pool was inhibited by 30% and into free uridine and cytidine by over 90%. Benzimidazole inhibited incorporation of [8-3H]IMP into AMP and GMP, and decreased substantially the activity of glutamine-amidophosphoribosyltransferase (EC 2.4.2.14). Yeast cultures were shown to N-ribotylate benzimidazole. Results are consistent with benzimidazole inhibiting yeast growth by competing for P-rib-PP and so depriving other ribotylation processes such as the 'salvage' pathways and de novo synthesis of purines and pyrimidines.     

The Influence of Benzimidazole on the Gametophyte of Thelypteris felix-mas

Benzimidazole has been studied previously by certain investigators as a growth regulator. This compound was found to promote various phases of growth in the gametophyte of the fern Thelypteris felix-mas. Concentrations of benzimidazole in the order of 0.01 mm, 0.02 mm, and 0.1 mm stimulated cell division, an increase in body size, and also rhizoid lengths of the gametophyte. A concentration of 1.0 mm was always inhibitory.     

Variation in resistance to benzimidizole in different biocontrol agents based on protein sequence homology

Benzimidazole resistance in thirty isolates of the biocontrol agents was studied with reference to specific mutations in the beta-tubulin genes. Our results suggest that apart from the correlation between specific mutations in the beta-tubulin gene and variation in resistance, the overall ratio of polar and non-polar amino-acids may also play a vital role in conferring benzimidazole resistance to these fungi.     

Effects of benzimidazole on the purine and pyrimidine metabolism of yeast

Yeast cells inhibited by benzimidazole accumulate hypoxanthine with an associated efflux of xanthine. Unlike control cells, inhibited cells contain no detectable free UMP and CMP. Benzimidazole decreases uptake of [8-¹⁴C]-hypoxanthine into the intracellular pool of hypoxanthine and xanthine but causes radioactive xanthine to accumulate in the medium. In inhibited cultures there is a threefold increase in incorporation of [8-¹⁴C]hypoxanthine into the total (intracellular plus extracellular) xanthine. Uptake of [8-¹⁴C]hypoxanthine into free nucleotides and into bound adenine and guanine was inhibited by 70%. Uptake of [U-¹⁴C]glycine into IMP, AMP, GMP, DNA and RNA was also substantially decreased. Incorporation of [2-¹⁴C]uracil into the intracellular uracil pool was inhibited by 30% and into free uridine and cytidine by over 90%. Benzimidazole inhibited incorporation of [8-³H]IMP into AMP and GMP, and decreased substantially the activity of glutamine-amidophosphoribosyltransferase (EC 2.4.2.14). Yeast cultures were shown to N-ribotylate benzimidazole. Results are consistent with benzimidazole inhibiting yeast growth by competing for P-rib-PP and so depriving other ribotylation processes such as the ‘salvage’ pathways and de novo synthesis of purines and pyrimidines.     

THE EFFECT OF CERTAIN BENZAZOLE COMPOUNDS ON PLANT GROWTH AND DEVELOPMENT

Klingensmith , M. J. (Colgate U., Hamilton, N. Y.) The effect of certain benzazole compounds on plant growth and development. Amer. Jour. Bot. 48(1): 40–45. Illus. 1961.—A number of benzazoles, in particular benzimidazole, benzothiazole, and benzotriazole, were examined for their effects on growth of seedlings and established plants. Benzothiazole was the most active in repressing elongation of the primary root of cucumber. Benzimidazole and benzotriazole were about 1/10 as active. Adenine was without effect in reversing the benzazole-induced inhibition of cucumber root elongation and, in fact, supplemented the inhibition caused by benzimidazole and benzothiazole. Application of benzotriazole to the root medium of bean, coleus, tomato, oat and wheat caused a pronounced inhibition of internodal elongation with a stimulation of axillary development. Distinct morphological changes were observed which did not correspond to those produced by other growth regulators. Application of benzimidazole to the root medium of several genera of plants resulted in injury to laminar tissue followed by desiccation, with no concomitant effect on the stem. Application of benzothiazole to the root medium induced development of adventitious roots in bean and tomato. This compound also caused initiation of roots on cultures of tobacco-stem segments, while not suppressing bud development. The benzazoles tested seem to be of a different class of compounds from other growth regulators which have been studied. The responses elicited by treatment with each of the 3 benzazoles are distinctly characteristic and are dependent on the structure of the azole portion of the molecule.     

EFFECT OF DNP ON ACCELERATION OF THE CLEAVAGE FOLLOWING INSUFFICIENT PARTHENOGENETIC ACTIVATION IN THE SEA URCHIN EGG*

Unfertilized eggs of sea urchins, Hemicentrotus pulcherrimus and Pseudocentrotus depressus, were treated with 4–5% butyric acid-sea water for 40–60 sec so that they were activated partheno-genetically without visible cortical changes. When these insufficiently activated eggs were inseminated 90–120 min after butyric acid-treatment, they divided much earlier than the control eggs in the first cleavage cycle. In the present paper, it becomes clear that if eggs are put into m /2,000-m /16,000 DNP-sea water at 60 min after insufficient activation and 30 min later, returned to normal sea water and then inseminated, they still show acceleration of the first cleavage in the same degree as the eggs which are not treated with DNP, while if eggs are exposed to DNP for 30 min prior to the insufficient activation or within 60 min after the activation, they do not show any acceleration of the cleavage. From these results, it may be concluded that some preparations for cleavage acceleration which are arrested by DNP become ready in the eggs at an early period in the first cleavage cycle and these preparations cannot be cancelled by DNP-treatment once they have been completed.     

The interaction of benzimidazole compounds with DNA: intercalation and groove binding modes.

Benzimidazole compounds (Fig. 1) have been synthesized to study their DNA-binding properties. Results obtained with spectroscopy and viscosity measurements indicate that the binding mode varies from intercalation to groove-binding, depending on the number of benzimidazole rings (conformation and size of compounds).     

Treatment of Toxocara canis infections in mice with liposome-incorporated benzimidazole carbamates and immunomodulator glucan

Benzimidazole carbamates (mebendazole, albendazole and fenbendazole) are the most commonly used anthelmintic drugs for the treatment of larval toxocariasis (Toxocara canis) in paratenic hosts. However, the bioavailability of these drugs for tissues is very low due to their extremely low solubility, resulting in the administration of relatively high doses over a long period. To overcome this problem, neutral, negatively or positively charged and stabilized liposome drug carriers were examined in the chronic phase of T. canis infections in mice each orally inoculated with 1000 eggs. Moreover, liposomized albendazole and fenbendazole were co-administered with liposomized immunomodulator glucan. The highest efficacy of both drugs, evaluated 4 weeks after treatment, was recorded after their subcutaneous administration (ten doses of 25 mg kg(-1)) in stabilized liposomes and intramuscular co-administration of liposomized glucan (two doses of 5 mg kg(-1)). Fenbendazole was more effective in muscles (91.5%) whereas albendazole was more effective in the brain (92.2%). Liposomes with incorporated benzimidazole carbamate anthelmintics provide sustained drug-release reservoirs and can considerably enhance drug efficacy. Moreover, despite suppression by T. canis antigens, stimulation of the immune system by the immunomodulator glucan potentiates the effects of these antiparasitic drugs.     

THE RELATION BETWEEN THE ELECTRICAL CONDUCTIVITY OF THE EXTERNAL MEDIUM AND THE RATE OF CELL DIVISION IN SEA URCHIN EGGS

Dilution of sea water with isotonic sugar solution leaves the rate of cleavage of Arbacia eggs almost unchanged until the proportion of sea water is decreased to 20 or 25 volumes per cent. From this point cleavage becomes progressively slower with further dilution. Many eggs fail to cleave at dilutions of 5 to 6 volumes per cent. No cleavage occurs in 2 volumes per cent sea water or in pure sugar solution. Eggs returned from these media to sea water resume cleavage and development. There is thus no relation between the rate of cleavage and the electrical conductivity of the medium, except possibly within the range of dilutions from 20 to 5 volumes per cent sea water. In this range cleavage rate decreases as conductivity decreases, but the relation is not a linear one.     

Prevalence of anthelmintic resistance in gastrointestinal nematodes of dairy goats under extensive management conditions in southwestern France

The occurrence of benzimidazole (BZ) and levamisole resistance was investigated in 18 randomly selected dairy goat herds located in southwestern France and characterized by extensive management. On each of the 18 farms, 45 adult goats were randomly allocated into three groups of 15 animals each: an untreated control group, a group that was orally administered fenbendazole (10 mg kg(-1) body weight) and a group that received orally a levamisole drench (12 mg kg(-1) body weight). Individual faecal egg counts and pooled larval cultures were done 10 days after anthelmintic treatment. Naive lambs were infected with larvae obtained from control and fenbendazole treated groups and were necropsied 35 days after infection for worm recovery. Faecal egg count reductions (FERC) were calculated for fenbendazole and levamisole and, when less than 95 per 100, were considered as indicative of anthelmintic resistance. An in vitro egg hatch test (EHT) was conducted with thiabendazole on eggs isolated from pooled faeces of fenbendazole treated goats in nine farms. Faecal egg count reductions indicated the occurrence of benzimidazole resistance in 15 out of 18 farms. Among these farms, nine had EHT values above 0.1 microg thiabendazole ml(-1) confirming the benzimidazole resistance status. Levamisole resistance was detected in two farms through FECR. Based on necropsy results, the prevalence of benzimidazole resistance was higher in Trichostrongylus colubriformis, medium in Haemonchus contortus and lower in Teladorsagia circumcincta. In nine farms the benzimidazole resistance was monospecific whereas multispecific resistance was found in the six remaining farms. A negative relationship was found between FECR for fenbendazole and the average number of anthelmintic treatments given per year on the farm. Despite extensive management including a low number of treatments, the prevalence of benzimidazole resistance was very high suggesting that the repeated and sometimes exclusive use of benzimidazole drugs, even at low frequency, is probably the main cause in developing nematode resistance in dairy goat herds. The importance of other factors such as under-dosing or buying animals already carrying resistant nematodes are discussed.