Effects of an azasteroid on growth, development and reproduction of the free-living nematodes Caenorhabditis briggsae and Panagrellus redivivus

The azasteroid, 25-azacoprostane (ASA-6), was evaluated for its effects on the growth, development and reproduction of the free-living nematodes, Caenorhabditis briggsae and Panagrellus redivivus. The axenic culture medium for either species of nematode consisted of Caenorhabditis briggsae Maintenance Medium (CbMM): formalin-killed Escherichia coli (1:1) with or without the addition of 5 micrograms cholesterol per ml and/or 25 micrograms ASA-6 per ml medium. All cultures also contained 50 micrograms Tween 80 per ml medium. After two generations of growth in sterol-deficient media, both species displayed a decrease in mean length, a decrease in the percent development to the adult stage and an inhibition of reproductive capability. These effects were more apparent in the sterol-deficient medium containing ASA-6. In the presence of cholesterol and ASA-6, growth and reproduction of C. briggsae, but not of P. redivivus, was inhibited after five generations. Morphologic abnormalities of azasteroid-inhibited worms were similar to those shown by worms cultured in sterol-deficient medium. These results suggest that different species of nematodes may exhibit different responses to azasteroid and that sterol utilization and metabolism may vary between nematode species. In addition, the similarities between the known effects of azasteroid inhibition in insects and those presented in this study on nematodes suggest a similar mechanism of action by the inhibitor in both groups of organisms.     

Mitotic responses of the anterior pericardial wall of Biomphalaria glabrata (Mollusca) subjected to challenge

Using light microscope autoradiography and electron microscopy we studied the effect of juvenile hormone III (JHIII) and β-ecdysone insect molting hormone (β-ecd) on the replication of Tipula iridescent virus (TIV) in suspension cultured cells of Estigmene acrea. JHIII at a concentration of 87.5 μg/ml completely inhibited viral DNA synthesis, but upon removal of JHIII, [³H]thymidine was incorporated into the cytoplasm as detected by autoradiography and virions in developmental stages from the same cell samples were-readily seen by electron microscopy. β-ecd at a concentration of 17.5 μg/ml, unlike JHIII, permitted viral DNA synthesis in the presence of the hormone although at a reduced level when compared to TIV-infected cells. But the presence of β-ecd seemed to prevent capsid formation, although islands similar in fine structure to those of viroplastic centers were seen by electron microscopy. Once β-ecd was removed from the medium, TIV-inoculated cells appeared to synthesize new virions in a normal pattern. Both hormones inhibited host cell DNA synthesis in noninfected cells.     

Inhibitory Effects of Dichlorophenoxyacetones on Auxin-induced Growth of Avena Coleoptile Sections

Six dichlorophenoxyacetones were synthesized and examined as potential metabolic antagonists utilizing Avena coleoptile sections and the straight growth assay procedure. Supplements of indoleacetic acid promoted growth of the sections which were inhibited by the analogs; the most inhibitory derivatives were 2,3-; 2,4-; 2,5-; and 3,4-dichlorophenoxyacetone which produced half-maximal growth responses (relative to the unaug-mented control growth) at concentrations of 106, 86, 80, and 62 μg/ml, respectively. A Lineweaver-Burk plot of the data for the inhibition by 2,4-dichlorophenoxyacetone and its reversal by indoleacetic acid appeared to represent an uncompetitive-like inhibition.     

Action of two juvenile hormone antagonists on lepidopteran epidermis

The juvenile hormone antagonist ETB (ethyl-4-2(t-butylcarbonyloxy)-butoxybenzoate) caused formation of precocious larval-pupal intermediates after the 4th (penultimate)-larval instar of the tobacco hornworm, Manduca sexta, when 50 μg were applied to any 3rd stage larvae or to 4th stage larvae within 12 hr after ecdysis. This dose was most effective within 12 hr after ecdysis to the 3rd stage. In the black mutant larval assay for juvenile hormone, ETB had activity, 0.75 μg per larva giving half-maximal score. In vitro ETB acted as a juvenile hormone to prevent the ecdysteroid-induced change in commitment at concentrations above 0.1 μg/ml with an ED₅₀ at 2.8 μg/ml and as a partial juvenile hormone antagonist to 0.1 μg/ml juvenile hormone I at concentrations between 10^?3 and 10^?2 μg/ml. By contrast, EMD (ethyl-E-3-methyl-2-dodecenoate) had little juvenile hormone-like activity in vitro up to its limits of solubility (100 μg/ml) and exhibited sporadic partial juvenile hormone antagonistic activity in vitro at concentrations between 1 and 100 μg/ml. Since these concentrations were 10–1000 times that of juvenile hormone I in the medium, EMD apparently is not an efficient competitor.     

Inhibition of cell wall-associated enzymes in vitro and in vivo with sugar analogs

Sugar analogs were used to study the inhibition of cell wall-associated glycosidases in vitro and in vivo. For in vitro characterization, cell walls were highly purified from corn (Zea mays L.) root cortical cells and methods were developed to assay enzyme activity in situ. Inhibitor dependence curves, mode of inhibition, and specificity were determined for three sugar analogs. At low concentrations of castanospermine (CAS), 2-acetamido-1,5-imino-1,2,5-trideoxy-d-glucitol, and swainsonine, these inhibitors showed competitive inhibition kinetics with β-glucosidase, β-GIcNAcase, and α-mannosidase, respectively. Swainsonine specifically inhibited α-mannosidase activity, and 2-acetamido-1,5-imino-1,2,5-trideoxy-d-glucitol specifically inhibited β-N-acetyl-hexosamindase activity. However, CAS inhibited a broad spectrum of cell wall-associated enzymes. When the sugar analogs were applied to 2 day old corn seedlings, only CAS caused considerable changes in root growth and development. To ensure that the concentration of inhibitors used in vitro also inhibited enzyme activity in vivo, an in vivo method for measuring cell wall-associated activity was devised.     

F-244 specifically inhibits 3-hydroxy-3-methylglutaryl coenzyme A synthase

A beta-lactone isolated from Scopulariopsis sp. shows a potent inhibition of cholesterogenesis. The structure of this beta-lactone, termed F-244, is 3,5,7-trimethyl-12-hydroxy-13-hydroxymethyl-2,4-tetradecadiendioic acid 12,14-lactone. The inhibition site of F-244 in cholesterol synthesis was studied. The growth of Vero cells was inhibited at 6.25-12.5 micrograms/ml of F-244. The inhibition of growth was overcome by the addition of mevalonate to the culture medium, but not by the addition of acetate. In a rat liver enzyme system, the incorporations of [14C]acetate and [14C]acetyl-CoA into digitonin-precipitable sterol were 50% inhibited by 0.58 microgram/ml of F-244. The incorporation of [14C]mevalonate was not affected. Studies on the effects of F-244 on the three enzymes involved in mevalonate biosynthesis demonstrated that the drug specifically inhibits HMG-CoA synthase with IC50 value of 0.065 microgram/ml. The effect of analogs of F-244 on HMG-CoA synthase was also investigated.     

Distribution of allatostatin in the adult cockroach,Diploptera punctata and effects on corpora allata in vitro

Direct radiochemical measurements of juvenile hormone synthesis showed that corpora allata from adult female Diploptera punctata can be inhibited in vitro by neuropeptides extracted from several ganglia of the central nervous system of females at many stages of the reproductive cycle. Extracts of protocerebra, corpora cardiaca, suboesophageal, thoracic and ventral ganglia all elicited dose-depedent reductions in juvenile hormone synthesis. On a ‘per organ’ basis, the protocerebrum contains the most extractable material. Inhibitory activity of extracts of suboesophageal, thoracic and 6th abdominal ganglia, like that of protocerebra (Rankin et al., 1986) was trypsin sensitive.Glands of high activity were less sensitive to protocerebral extract than those of low activity. The inhibitory effect on glands of low activity was maximal within 1 h, persisted in the presence of protocerebral extract for at least 46 h, and was abolished within 1 h after corpora allata were placed in normal medium. The inhibitory effect of protocerebral extract was not altered by the addition of magnesium to the medium. The extract had a specific effect on synthetic step(s) prior to methylation and epoxidation as demonstrated by enhanced juvenile hormone synthesis in the presence of inhibitory factor and the juvenile hormone precursor, farnesoic acid.     

Effect of Plant Growth Regulators on Calcium-stimulated Serine Transport into Tobacco Cells

The transport of serine into tobacco cells (Nicotiana tabacum L.) cultured in liquid medium was examined. Transport was inhibited approximately 50% by 2,4-dichlorophenoxyacetic acid, indoleacetic acid, α-naphthalene acetic acid, and kinetin at a concentration of 10 micrograms per milliliter. Transport was not inhibited by 2,6-dichlorophenoxyacetic acid and inhibited less than 25% by p-chlorophenoxyacetic acid at this concentration. Removal of 2,4-dichlorophenoxyacetic acid from the transport medium resulted in an alleviation of inhibition. Gibberellic acid at concentrations from 2 to 20 micrograms per milliliter stimulated transport.

It was previously shown that inhibition of transport by La³⁺ was due to removal of Ca²⁺ from surface sites and inhibition of Ca²⁺ uptake by cells. None of the growth regulators tested had any significant effect on Ca²⁺ binding and/or transport.

A contributing factor to the low transport rates in the absence of Ca²⁺ is the increased rate of serine efflux. None of the growth regulators tested had any significant effect on the rate of serine efflux.

     

Bongkrekic acid and the adenosinetriphosphate requirement of malaria parasites

The avian malaria parasite Plasmodium lophurae, when removed from its host erythrocytes into an appropriate medium, develops extracellularly in vitro. This development was inhibited by bongkrekic acid at concentrations down to 2 μg/ml. Adenosine triphosphate at high concentrations partly reversed the inhibition. Bongkrekic acid also inhibited intraerythrocytic development in vitro of the human malaria P. falciparum.     

A culture and biochemical assay system for Trypanosoma lewisi

Trypanosoma lewisi was cultivated as forms which appeared to be physiologically similar to those found in vivo. The medium consisted of 1.0 g peptone, 1.0 g glucose, 10 ml rat serum, 10,000 units penicillin G, 10,000 μg streptomycin and 90 ml Hank's Balanced Salt Solution. It was supplemented with 8.0 × 10⁸ rat erythrocytes per milliliter. In the complete medium trypanosomes multiplied for 48–72 hr. Cultured forms were lethal to newborn rats and infective to adults.Adsorbed early immune serum inhibited the growth of the trypanosomes in vitro and the percentage of reproductives declined from 66 to 45%. The cultured trypanosomes were also susceptible to both trypanocidal antibodies.     

Allelopathic potential of two sesquiterpene lactones from Magnolia grandiflora L.

The allelopathic effects of the two sesquiterpene lactones, costunolide and parthenolide, isolated from the leaves of Magnolia grandiflora L. were evaluated on the wheat (Triticum aestivum L.), lettuce (Lactuca sativa L.), radish (Raphanus sativus L.) and onion (Allium cepa L.). Seed germination of the test species was significantly reduced at 500 μg/ml by both compounds. Both sesquiterpenes showed pronounced inhibition of root length of the test species and the inhibitory effect was concentration-dependent. In addition, shoot growth of the four species was significantly inhibited at all the concentrations tested (10–500 μg/ml). Parthenolide reduced germination and inhibited seedling growth more than costunolide. Inhibition of root growth was generally greater than that of shoot growth. The results encourage the use of these sesquiterpenes as models for development of new herbicides.     

Effect of Monensin and Lasalocid-Sodium on the Growth of Methanogenic and Rumen Saccharolytic Bacteria

It is thought that monensin increases the efficiency of feed utilization by cattle by altering the rumen fermentation. We studied the effect of monensin and the related ionophore antibiotic lasalocid-sodium (Hoffman-LaRoche) on the growth of methanogenic and rumen saccharolytic bacteria in a complex medium containing rumen fluid. Ruminococcus albus, Ruminococcus flavefaciens, and Butyrivibrio fibrisolvens were inhibited by 2.5 μg of monensin or lasalocid per ml. Growth of Bacteroides succinogenes and Bacteroides ruminicola was delayed by 2.5 μg of monensin or lasalocid per ml. Populations of B. succinogenes and B. ruminicola that were resistant to 20 μg of either drug per ml were rapidly selected by growth in the presence of each drug at 5.0 μg/ml. Selenomonas ruminantium was insensitive to 40 μg of monensin or lasalocid per ml. Either antibiotic (10 μg/ml) inhibited Methanobacterium MOH, Methanobacterium formicicum, and Methanosarcina barkeri MS. Methanobacterium ruminantium PS was insensitive to 40 μg of monensin or 20 μg of lasalocid per ml. The methanogenic strain 442 was insensitive to 40 μg of monensin but sensitive to 10 μg of lasalocid per ml. The results suggest that monensin or lasalocid acts in the rumen by selecting for succinate-forming Bacteroides and for S. ruminantium, a propionate producer that decarboxylates succinate to propionate. The selection could lead to an increase in rumen propionate formation. Selection against H₂ and formate producers, e.g. R. albus, R. flavefaciens, and B. fibrisolvens, could lead to a depression of methane production in the rumen.     

Isolation and Characterization of Coumaphos-Metabolizing Bacteria from Cattle Dip

Coumaphos, an organophosphate insecticide, is used for tick control in cattle dipping vats along the U.S.-Mexican border. Recently, several vats (problem vats) have experienced a loss of efficacy because of microbial degradation. Three morphologically distinct bacteria (designated B-1, B-2, and B-3) that metabolized coumaphos were isolated from enrichment cultures that were initiated from problem vat dip material. In general, amino acids, pyrimidines, and acetate supported growth; carbohydrates were not utilized. Only B-2 required growth factors. In resting cell experiments, coumaphos was hydrolyzed to diethylthiophosphoric acid and chlorferon by all three isolates. Chlorferon was subsequently metabolized by B-1 and B-2 to α-chloro-β-methyl-2,3,4-trihydroxy-trans-cinnamic acid. Only B-1 produced additional metabolites. Experiments with [benzo ring-labeled U-¹⁴C]coumaphos or chlorferon demonstrated that B-1 was capable of both mineralizing and incorporating into biomass the aromatic portion of the molecule. The majority of label, however, was recovered in the form of soluble products, including α-chloro-β-methyl-2,3,4-trihydroxy-trans-cinnamic acid. Although B-1 had the capacity to use chlorferon as a carbon source at low concentrations (100 μg/ml), visible growth at higher concentrations (1,000 μg/ml) was not observed. The addition of 400 μg of chlorferon per ml to B-1 cells in the mid-log phase of growth resulted in complete inhibition of growth, while the addition of 100 to 200 μg of chlorferon per ml resulted in partial inhibition. The growth of B-2 and B-3 was inhibited by 100 μg of chlorferon per ml. These data suggest that, although B-1 and, to a lesser extent, B-2 and B-3 are responsible for the primary degradation of coumaphos, other organisms in the enrichment culture may play a secondary role in coumaphos degradation by removing inhibitory products of coumaphos metabolism.     

Use of Epifluorescence Microscopy in Studies of the Germination and Recovery of Thermoactinomycetes

Spore germination and growth of thermoactinomycetes were observed by epifluorescence microscopy. Each of the principal stages in germination was recognized and found to correspond to changes in phase-contrast appearance commonly monitored during endospore germination. The effects of novobiocin and nalidixic acid on germination of Thermoactinomyces vulgaris and T. thalpophilus were studied by using epifluorescence microscopy. Outgrowth but not initiation was inhibited by 100 μg of nalidixic acid per ml and 50 μg of novobiocin per ml. When each of the compounds, at various concentrations, was incorporated into a medium for thermoactinomycete recovery, the antibiotics were found to inhibit colony development. Samples of water and sediment incubated in a growth medium containing novobiocin and selective for thermoactinomycete species were examined by epifluorescence microscopy for total numbers of outgrown spores. Direct viable counts of outgrown spores indicated that the standard plate count enumerated less than 10% of the viable population of thermoactinomycetes.     

Effects of steroid hormones in fetal bovine serum on plating and cloning of human cells in vitro

Summary Fetal bovine sera from each of three different commercial sources were tested for their ability to support cloning of human fibroblastoid cells in vitro. Cloning efficiencies varied according to serum source. Serum (10 samples) from company A did not support growth, while sera (10 samples) from companies B and C provided adequate to excellent conditions for cloning and growth. Cells from neonatal foreskin or embryonic lung responded to each serum similarly. Bovine serum albumin type H7 from company C supported cell growth in media without serum. Sera containing 1.0 ng per ml or more of progesterone inhibited growth, whereas sera containing less than 1.0 ng per ml supported cloning and growth. In the low progesterone sera, the concentration of 17-β-estradiol exceeded 100 pg per ml. Growth supporting sera could be made non-supportive by adding 0.1 μg per ml of progesterone. The addition to non-supportive sera of 0.1 μg per ml of 17-β-estradiol or hydrocortisone made these sera supportive of cell growth. Addition of estrogen or hydrocortisone to a culture medium that inhibits growth, with subsequent reversal of the inhibitory effect, implies that these hormones competitively regulate growth of responsive cells in vitro. Supported in part by NIH-NCI-EC2074.     

Effects of amino acids on polysaccharide synthesis ofEscherichia coli K-12lon + andlon − strains

Ultraviolet-sensitivelon ^? mutant ofEscherichia coli K-12 produced abundant polysaccharide when grown in a minimal medium at 37 C, but not when grown in a broth medium. The repression of polysaccharide synthesis in the broth-grownlon ^? andlon ⁺ cells was studied. The effects were largely dependent on the amino acid concentrations and on the requirements of the strain used. At 200 μg per ml of each of the essential amino acids, histidine, proline, and threonine, there was complete inhibition of polysaccharide synthesis. At 200 μg per ml the required amino acids, tryptophane and tyrosine promoted polysaccharide synthesis. Most amino acids inhibited cell growth at 200 μg per ml but the inhibiting effect was smaller at 400 μg per ml. Polysaccharide synthesis of cells was not correlated with the growth rate, and occurred even under non-growing conditions.     

Regeneration of Centella asiatica plants from non-embryogenic cell lines and evaluation of antibacterial and antifungal properties of regenerated calli and plants

Background

The threatened plant Centella asiatica L. is traditionallyused for a number of remedies. In vitro plant propagation and enhanced metabolite production of active metabolites through biotechnological approaches has gained attention in recent years.

Results

Present study reveals that 6-benzyladenine (BA) either alone or in combination with 1-naphthalene acetic acid (NAA) supplemented in Murashige and Skoog (MS) medium at different concentrations produced good quality callus from leaf explants of C. asiatica. The calli produced on different plant growth regulators at different concentrations were mostly embryogenic and green. Highest shoot regeneration efficiency; 10 shoots per callus explant, from non-embryogenic callus was observed on 4.42 μM BA with 5.37 μM NAA. Best rooting response was observed at 5.37 and 10.74 μM NAA with 20 average number of roots per explant. Calli and regenerated plants extracts inhibited bacterial growth with mean zone of inhibition 9-13 mm diameter when tested against six bacterial strains using agar well diffusion method. Agar tube dilution method for antifungal assay showed 3.2-76% growth inhibition of Mucor species, Aspergillus fumigatus and Fusarium moliniformes.

Conclusions

The present investigation reveals that non-embryogenic callus can be turned into embryos and plantlets if cultured on appropriate medium. Furthermore, callus from leaf explant of C. asiatica can be a good source for production of antimicrobial compounds through bioreactor.     

Comparative studies on the allelopathic effects of two different strains of Ulva pertusa on Heterosigma akashiwo and Alexandrium tamarense

Allelopathic effects of fresh tissue and dry powder of a nonsexual and a sexual strain of the macroalga Ulva pertusa on the growth of the microalgae Heterosigma akashiwo and Alexandium tamarense were evaluated using long-term coexistence culture systems in which several concentrations of macroalga fresh tissue and dry powder were used. The effects of macroalga culture medium filtrate on the two HAB algae were also investigated. Moreover, isolation co-culture systems were built to confirm the existence of allelochemicals and preclude the growth inhibition by direct contact. Short-term algicidal effect assays of macroalgae on H. akashiwo were carried out to measure the rate of algal cell lysis. The results of the coexistence assays showed that the growth of H. akashiwo and A. tamarense was strongly inhibited by fresh tissue and by dry powder of both strains of U. pertusa. The allelochemicals were lethal to H. akashiwo at relatively higher concentrations. The macroalga culture medium filtrate exhibited no apparent growth inhibitory effect on the two HAB algae under initial or semicontinuous filtrate addition, which suggested that continuous release of small quantities of rapidly degradable allelochemicals from the fresh tissue of both strains of U. pertusa was essential to effectively inhibit the growth of H. akashiwo and A. tamarense.     

Inhibition by heparin and dextran sulfate of stimulated rat pancreatic adenylate cyclase

Heparin inhibited the adenylate cyclase activity of semipurified rat pancreatic plasma membranes stimulated by hormones and by Gpp(NH)p but not by fluoride or when in the persistently active state. When observed, the inhibition was rapid and sustained. It was of a noncompetitive type and never exceeded 20% for secretin. The inhibition of Gpp(NH)p-stimulated activity was more pronounced (48% inhibition at a heparin concentration of 50 μg/ml). For the C-terminal octapeptide of pancreozymin (CCK-8)-stimulated adenylate cyclase, the inhibition amounted to 93% at 50 μg/ml. This inhibition was competitive at low heparin concentration and of a mixed type above 10 μg/ml. Besides, heparin inhibited (I₅₀ = 6 μg/ml) the binding of peptides of the CCK family to their specific receptors without affecting the apparent K_d value of binding. Taken together, these relatively specific effects of heparin gave evidence in favor of the existence of CCK spare receptors. Dextran sulfate was more potent than heparin as an inhibitor of adenylate cyclase activation while chondroitin-4-sulfate and chondroitin-6-sulfate were ineffective. Dansylated pancreatic plasma membranes exhibited characteristics of adenylate cyclase activation by CCK-8 which were similar to those found for untreated membranes exposed to heparin.     

Development and application of a radioimmunoassay for the juvenile hormones

A radioimmunochemical method for the quantification of juvenile hormones from hemolymph and whole body extracts is described. Rabbit polyclonal antiserum developed against a JH III-bovine thyroglobulin conjugate displayed minimal cross-reactivity with juvenile hormone metabolites including juvenile hormone acids, juvenile hormone diols and analogs but substantial cross-reactivity between juvenile hormone homologs. Minimum sensitivity of the assay toward racemic juvenile hormone III was 65 pg. The degree and relative order of cross-reactivities for juvenile hormones I, II and III varied according to the identity of the radioligand used. A method for isolating juvenile hormones from whole body and hemolymph for radioimmunoassay was developed utilizing organic solvent extraction followed by thin-layer chromatography. Noninterfering dyes were used to bracket the position of the hormone on thin-layer chromatography plates. Hemolymph extracts known to contain no JH did not interfere with the assay when this procedure was employed. Radioimmunoassay analysis of hemolymph samples containing known amounts of JH and corrected for recovery yielded the expected results. Quantification of total juvenile hormone in whole body and hemolymph extracts of Manduca sexta was in good agreement with total mass of JH determined by a GC/MS method.