The in vitro and in vivo effects of metronidazole and chloroquine on Trypanosoma brucei brucei

Bloodstream forms of Trypanosoma brucei brucei were grown over baby hamster kidney cells in minimum essential medium with various concentrations of metronidazole (Flagyl) and chloroquine. Both drugs inhibited the multiplication of the parasite in vitro. The least effective concentrations for metronidazole and chloroquine were 0.003 mg/ml and 0.0024 mg/ml, respectively. Groups of 12-day-old female CDI mice were treated with 1 of the 2 drugs at 24, 48, and 72 hr after T. brucei infection. The drugs administered stat or daily reduced the number of parasites in the mice but did not effect a cure; they prolonged the survival period of the animals. However, metronidazole (0.1 mg/kg body weight) and chloroquine (0.08 mg/kg body weight) combined and given daily for 4 consecutive days cleared the infection. No trypanosomes were detected in the blood of these mice 3 mo after treatment. The dosages for both the in vitro (metronidazole 0.003 mg/ml; chloroquine 0.0024 mg/ml) and in vivo (metronidazole 0.1 mg/kg body weight; chloroquine 0.08 mg/kg body weight) were well below those prescribed for humans.     

Fungistatic and fungicidal effects of the ionophores monensin and tetronasin on the rumen fungus Neocallimastix sp. LM1

The ionophore antibiotics monensin and tetronasin have been reported to inhibit anaerobic fungi in vitro, and are suitable for animal use. In this study, their effectiveness in removing the anaerobic fungus Neocallimastix sp. LM1 from the rumen was investigated in vitro. Both antibiotics were fungistatic: tetronasin at 0.5 microgram/ml and monensin at 1.0 microgram/ml; exposure for 24 h did not inhibit subsequent growth after removal of the ionophore. The ionophores were fungicidal at much higher concentrations, 1 microgram/ml for tetronasin and 16 micrograms/ml for monensin. It seems likely that the combination of relatively high inhibitory dose and the fungistatic nature of monensin would explain difficulties in using this compound to eliminate anaerobic fungi from the rumens of experimental animals.     

Transferrin coupled azanthraquinone enhances the killing effect on trypanosomes. The role of lysosomal mannosidase

Partially purified azanthraquinone (AQ) extract from Mitracarpus scaber was coupled to bovine transferrin (Tf) using azidophenyl glyoxal (APG). The AQ-APG-Tf conjugate was found to possess an enhanced in vitro trypanocidal activity against Trypanosoma congolense and T. brucei brucei. At low concentrations of 0.39-90 mg/ml, the conjugate diminished the growth of T. congolense and T. b. brucei dose dependently at the logarithmic phase. Both parasites were more sensitive to AQ-APG-Tf than to the free (AQ) extract. Growth inhibition on the parasites by the free extract was observed at 20-200 mg/ml. The total activity of the lysosomal enzyme a-mannosidase was reduced in the T. congolense cells treated with AQ-APG-Tf in a dose related pattern. However, the activity of the mannosidase in the T. b. brucei treated cells is less affected. The AQ-APG-Tf is more effective on a mannosidase than free AQ, eight and four fold for T. congolense and T. b. brucei respectively. The results are discussed as regards the potency of using transferrin as suitable drug carrier in the chemotherapy of Human sleeping sickness.     

Anomalies in mineralization of low concentrations of organic compounds in lake water and sewage

The rates of mineralization of nitrilotriacetic acid (NTA), 2,4-dichlorophenoxyacetic acid (2,4-D), p-nitrophenol, aniline, and isopropyl N-phenylcarbamate (IPC) at one or more concentrations ranging from 100 pg/ml to 1.0 microgram/ml were proportional to chemical concentrations in samples of three lakes. The rates at 100 pg of NTA, 2,4-D, p-nitrophenol, and aniline per ml in samples of one or more lakes were less than predicted, assuming the rates were linearly related to the concentration. Neither NTA nor 2,4-dichlorophenol at 2.0 ng/ml was mineralized in some lake waters, but higher levels of the two chemicals were converted to CO2 in samples of the same waters. In samples from two lakes, little or no mineralization of IPC or 2,4-D occurred at 1.0 microgram/ml, but 10 ng/ml or lower levels of the herbicides were mineralized. The mineralization in sewage of 1.0 microgram of NTA per ml was biphasic; about 20% of the substrate was mineralized in 20 h, and mineralization was only reinitiated after a period of 130 h. The biphasic transformation was not a result of the accumulation of organic products, and it was still evident if protozoan activity was inhibited. NTA also underwent a biphasic mineralization in lake waters, and the biphasic pattern was not altered by additions of growth factors and inorganic nutrients. From 40 to 60% of the carbon of aniline added to lake water at levels of 100 pg/ml to 1.0 microgram/ml was mineralized, but more than 90% of the carbon of NTA, 2,4-D, or p-nitrophenol added to lake water at 10 ng/ml or 1.0 microgram/ml was mineralized.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of anti-Vibrio activities of potassium sorbate, sodium benzoate, and glycerol and sucrose esters of fatty acids.

The effects of fatty acids and their glycerol and sucrose esters, potassium sorbate, and sodium benzoate on growth of Vibrio parahaemolyticus in laboratory media at pH 6.7 were evaluated. The minimum concentrations at which inhibition by esters of glycerol could be detected were lowest for monolaurin (5 microgram/ml) and monocaprin (40 microgram/ml); these concentrations were lower than those observed for inhibition by lauric and capric acids, respectively. Inhibitory action of sucrose caprylate was detected at 40 microgram/ml, whereas sucrose caprate was effective at 100 microgram/ml; sucrose esters of lauric, myristic, and palmitic acids were ineffective at 100 microgram/ml. Potassium sorbate and sodium benzoate inhibited growth at concentrations as low as 30 and 300 microgram/ml, respectively, and enhanced the rate of thermal inactivation of V. parahaemolyticus at slightly higher concentrations. Fatty acid esters of glycerol and sucrose offer potential as perservatives for slightly acid or alkaline low-fat foods which do not lend themselves to the full antimicrobial action of traditional food preservatives such as potassium sorbate and sodium benzoate.     

Anomalies in mineralization of low concentrations of organic compounds in lake water and sewage.

The rates of mineralization of nitrilotriacetic acid (NTA), 2,4-dichlorophenoxyacetic acid (2,4-D), p-nitrophenol, aniline, and isopropyl N-phenylcarbamate (IPC) at one or more concentrations ranging from 100 pg/ml to 1.0 microgram/ml were proportional to chemical concentrations in samples of three lakes. The rates at 100 pg of NTA, 2,4-D, p-nitrophenol, and aniline per ml in samples of one or more lakes were less than predicted, assuming the rates were linearly related to the concentration. Neither NTA nor 2,4-dichlorophenol at 2.0 ng/ml was mineralized in some lake waters, but higher levels of the two chemicals were converted to CO2 in samples of the same waters. In samples from two lakes, little or no mineralization of IPC or 2,4-D occurred at 1.0 microgram/ml, but 10 ng/ml or lower levels of the herbicides were mineralized. The mineralization in sewage of 1.0 microgram of NTA per ml was biphasic; about 20% of the substrate was mineralized in 20 h, and mineralization was only reinitiated after a period of 130 h. The biphasic transformation was not a result of the accumulation of organic products, and it was still evident if protozoan activity was inhibited. NTA also underwent a biphasic mineralization in lake waters, and the biphasic pattern was not altered by additions of growth factors and inorganic nutrients. From 40 to 60% of the carbon of aniline added to lake water at levels of 100 pg/ml to 1.0 microgram/ml was mineralized, but more than 90% of the carbon of NTA, 2,4-D, or p-nitrophenol added to lake water at 10 ng/ml or 1.0 microgram/ml was mineralized.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of pertussis toxin on mediator release from human basophils

We have found that basophils (n = 9) treated with pertussis toxin (1.0 microgram/ml) fail to respond to a subsequent challenge with either 1.0 microM f-Met peptide (p less than 0.0005) or 0.24 microgram/ml of C5a (p less than 0.0005) although their responses to anti-IgE (0.1 microgram/ml) and A23187 (1.0 microgram/ml) were unaltered. These results were confirmed in purified (average purity = 89 +/- 3%) basophils (n = 4). Leukotriene C4 release was also reduced to 15 +/- 5% of control (p less than 0.005) when pertussis toxin-treated basophils were exposed to 1.0 microM f-Met peptide, although no inhibition was noted when anti-IgE or A23187 were used as the stimuli. The effect of pertussis toxin on basophils appears to be independent of the presence of contaminating mononuclear cells. We found that pertussis toxin inhibited f-Met peptide-induced histamine release regardless of the magnitude of the stimulus (0.01 microM to 1.0 microM f-Met peptide), although anti-IgE-induced release was unaffected over a dose-response curve. The effect of pertussis toxin was found to be both time- and concentration-dependent. The maximum effects were obtained after a 3-hr incubation with 1 microgram/ml of toxin. Lower (0.01 to 0.05 microgram/ml) concentrations of toxin or shorter (30 to 60 min) incubation periods did not significantly (p greater than 0.05) inhibit mediator release.     

Gelatin of Limulus amoebocyte lysate by simple polysaccharides

The Limulus lysate gelatin activity of several simple polysaccharides including yeast mannans and bacterial dextrans was investigated. The mannans from Saccharomyces cerevisiae wild and mutant strains possessing dense branches showed positive gelatin activity at concentrations of 1 microgram/ml or more regardless of differences in their chemical structure. However, two synthetic mannans possessing linear structures with alpha 1 leads to 2 and alpha 1 leads to 6 linkages also gave positive reactions at concentrations of 10 microgram/ml or more and 500 microgram/ml or more, respectively. The dextran from Leuconostoc mesenteroides IAM 1046 consisting of a dense branching moiety displayed reactivity at concentrations of 100 microgram/ml or more, while the dextrans devoid of such branches were negative in this reaction. The optimal concentration for Limulus lysate gelatin could not be determined for any of the polysaccharides and lipopolysaccharides (LPS) tested in this study. The gelation activity of the polysaccharides was stable to treatment with 100 mM NaOH at 30 C for 72 hr. The minimum concentration for the gelation activity of LPS treated with 100 mM NaOH under the same conditions was reduced from 10(-6) approximately(-9) microgram/ml to 1-10 microgram/ml. The above findings demonstrate that the major part of Limulus lysate gelation activity of LPS depends on the alkali-degradable lipid A moiety, and that such simple polysaccharides are also able to participate in this activity even though the extent of participation is very low.     

Influences of zinc on fertilisation and development of bovine oocytes in vitro.

The effects of zinc (as ZnCl2) on in vitro production of bovine embryos (IVMFC) and components of the procedure, that is in vitro oocyte maturation (IVM), fertilisation (IVF) and embryo development in culture (IVC), and the effect of added zinc on sperm motility were studied. Immature cumulus oocyte complexes (COCs) were aspirated from ovarian follicles (2-5 mm diameter) at slaughter, and matured, fertilised and cultured in chemically defined conditions. The presence of zinc (10, 100 or 1000 micrograms added per millilitre) throughout IVMFC inhibited fertilisation. After addition of 10 micrograms zinc per millilitre separately to media for IVM and IVF, fertilisation was inhibited only when zinc was present for IVM. When present for IVF, 80% of oocytes selected for IVM reached 2- to 4-cell stages by 46 h after insemination whereas 67% of control oocytes (inseminated without added zinc) cleaved. Higher zinc concentrations (100 and 1000 micrograms added per millilitre) for IVF inhibited fertilisation. Sperm motility was reduced with addition of 10 micrograms per millilitre of zinc for sperm preparation (i.e. capacitation interval). Addition of 1.0 microgram zinc per millilitre to media used through IVMFC, or to the IVC medium alone, resulted in inhibition of development after 2- to 4-cell stages. When added to IVM or to both IVM and IVF media 1.0 microgram/ml of zinc compromised development to the morula stage and beyond. Maturing bovine oocytes may be more sensitive to 1.0 microgram ml of zinc in vitro than in vivo because a concentration of 3.0 micrograms/ml has been reported for bovine follicular fluid. Fertilisation was not adversely affected by 10 micrograms/ml of zinc; however, higher concentrations were inhibitory.     

Stabilization of the colchicine-binding activity of tubulin by organic acids

A number of carboxylic acids and organic phosphates were found to be highly effective in stabilizing the colchicine-binding activity of calf brain tubulin. The most active were glutamate, glutarate, delta-aminovalerate, glucose 1-phosphate, glucose 6-phosphate, fructose 1,6-(bis)phosphate, creatine phosphate and 6-phosphogluconate Maximum effects occurred at high concentrations. Combinations of agents were also examined, and the most effective mixture for stabilizing tubulin found thus far was the combination of 1.0 M glutamate, 100 mM glucose 1-phosphate, 1 mM GTP and 0.5 mg/ml of albumin. No loss of activity occurred over 48 h at 37 degrees C with tubulin was present at a concentration of 100 microgram/ml.     

Chromosomal aberrations and sister-chromatid exchanges in Chinese hamster cells treated in vitro with hexavalent chromium compounds.

Chinese hamster cells (CHO line) were treated in vitro for 30--39 h with hexavalent chromium compounds (K2Cr2O7 and Na2CrO7), at concentrations ranging from 0.1 to 1.0 microgram of Cr6+ per ml, in medium containing BUdr. Chromosomal aberrations and sister-chromatid exchanges were scored on BUdr-labelled 2nd division metaphases, collected at the end of treatment and stained with Giemsa. Treatment with mitomycin C 0.009--0.030 microgram/ml) was carried out as a control for the responsiveness of the cell system to chromosomal damage. Both chromium compounds induced marked mitotic delays. Chromosomal aberrations were increased about 10-fold by exposure to Cr6+ (1.0 microgram/ml). The principal aberrations observed were single chromatid gaps, breaks and interchanges, whose frequencies increased proportionally to the concentration of chromium. Dicentric chromosomes, isochromatid breaks, chromosome and chromatid rings were also induced. The frequenyc of sister-chromatid exchanges was hardly doubled 30 h after exposure to Cr6+ at 0.3 microgram/ml, whereas it was trebled 39 h after treatment, in the cells whose division cycle had been slowed down by chromium.     

In vitro acitivity of ribostamycin against Prototheca sp.

The in vitro activity of ribostamycin against algae of the genus Prototheca was evaluated by minimum inhibitory concentration (MIC) studies on solid media. Concentrations of 4 mcg/ml were required to inhibit 100% of the P. zopfii strains; 16 mcg/ml inhibited 100% of the P. stagnora strains and 95% of the P. wickerhamii strains. These values are inferior to plasma concentrations obtained after injection of ribostamycin. It is likely that this antibiotic could be effective in the treatment of protothecosis in man.     

Effects of phenytoin on acetylcholinesterase activity and cell protein in cultured chick embryonic skeletal muscle

Cultured pectoral muscle from 11-day-old chick embryos was treated for 48 h with phenytoin (diphenylhydantoin, DPH) in concentrations ranging from 15 to 270 microgram/ml on days 7-9 in vitro. Acetylcholinesterase (AChE, EC 3.1.1.7), creatine phosphokinase (CPK, EC 2.7.3.2), and lactic dehydrogenase (LDH, EC 1.1.1.27) activities, [3H]leucine incorporation into protein, and total protein of the cultures decreased in a dose-related manner with DPH concentrations of 30 microgram/ml and greater. Total AChE activity and AChE activity released into the medium were specifically decreased with 15 microgram DPH per millilitre. In cultures treated chronically with 15 microgram DPH per millilitre on days 5-13 in vitro, total AChE activity and AChE activity released into the medium were 66.0 +/- 13.2 and 64.7 +/- 11.8% of untreated controls, respectively, but cellular AChE activity, cell protein, and [3H]leucine incorporation into protein were unaffected. The results indicate that DPH specifically decreases the total net synthesis of AChE activity by a direct action on cultured chick embryo muscle.     

Effect of minocycline on subgingival plaque bacteria

The effects of minocycline on subgingival plaque samples from patients with chronic periodontitis were investigated in vitro. Minocycline concentrations as low as 1.0 microgram/ml inhibited 95.7% of the cultivable bacteria in the samples but 256 micrograms/ml was necessary to inhibit all of the cultivable bacteria in the samples. Although up to 99.9% of bacteria in the plaque samples were killed by a 6 h exposure to 8.0 micrograms/ml of minocycline, large numbers of viable bacteria remained. These results imply that adequate reductions in the numbers of viable subgingival plaque bacteria are unlikely to occur after exposure to minocycline at concentrations attainable in gingival crevicular fluid after systemic administration.     

Effects of daunomycin and radiation on cell-survival and repair of DNA single-strand breaks.

The combined action of Daunomycin and irradiation was investigated using mouse L-929 cells in culture. Survival of cells was measured with the colony assay. Sedimentation in alkaline sucrose gradients was used to study repair of DNA single-strand breaks (SSB) in the presence of various concentrations of Daunomycin. A small increase in radio-sensitivity, as measured by decreasing Do, was obtained for doses of Daunomycin that are considerably toxic to the cells (0.1 microgram/ml). However, the Dq values remained constant even at high concentrations indicating that Daunomycin does not interfere with recovery processes. The rate of rejoining of SSB remained constant up to 1.0 microgram/ml, whereas concentrations of Daunomycin as high as 10 microgram/ml reduced the velocity of repair by a factor of 13. Our data show that concentrations of Daunomycin similar to those required for other DNA-binding drugs are required to inhibit SSB repair. For clinical purposes, no increase in tumour-killing efficiency may be expected from a combined treatment with Daunomycin and radiation.     

SCE levels in Bloom-syndrome cells at very low bromodeoxyuridine (BrdU) concentrations: monoclonal anti-BrdU antibody

A stable staining procedure of sister-chromatid differentiation (SCD) using a monoclonal antibromodeoxyuridine (BrdU) antibody was newly established by combining it with the immunoperoxidase reaction (3,3'-diaminobenzidine, DAB reaction). This procedure permitted detection of SCD and SCE at very low BrdU concentrations. SCD was not usually observed below 2.0 micrograms/ml BrdU with flame-dried chromosome slides. When chromosome slides were prepared by air-drying over 37 degrees C warm water, SCD was detected at 10.0, 5.0, 1.0, 0.5, 0.3 and 0.2 micrograms/ml BrdU with FPG and even at 0.1 microgram/ml BrdU with the antibody technique. SCE levels were evaluated using the antibody technique and endomitotic analysis with FPG at low BrdU concentrations (1.0, 0.5, 0.3, 0.2 microgram/ml) in two BS B-lymphoblastoid cell lines (LCLs). Even though the BS SCE level was approximately 70 per cell at 10 micrograms/ml, the value decreased to the level of 20-30 SCE per cell at 0.1 microgram/ml with the antibody technique. In BrdU-labelled BS endomitoses, single SCEs highly decreased with BrdU concentrations (130-140 level at 10 micrograms/ml: 38-60 level at 0.2 microgram/ml), when compared to the rare twin SCE values (3-6 SCE level) at all BrdU concentrations. These findings conclusively indicate that the spontaneous baseline SCE in BS B-lymphoblastoid cells is low and most BS SCEs are caused by BrdU.     

Adsorption of 14C-labeled gramicidin S on cell of bacteria

Gramicidin S (GS) containing 14C-labeled proline was synthesized by a solid-phase method, and the labeled GS dihydrochloride was obtained as crystals. The labeled GS exhibited same antibacterial activity as natural GS. Strains sensitive to GS (B. subtilis and S. aureus) and an insensitive strain (E. coli) were treated with the labeled GS, and the amount of the labeled GS adsorbed on the cells was measured. GS was adsorbed rapidly on the cells of the sensitive strains; the amount adsorbed increased linearly with GS concentration up to 1-1.5 microgram/ml, and at a lower rate at above 1.5 microgram/ml. GS molecules covered most of the cell surface at the minimum inhibitory concentration of 1.5 microgram/ml; the number of molecules adsorbed per cell was 1.3-1.4 x 10(6). No GS was adsorbed by the insensitive strain.     

Effect of thyrotropin releasing hormone thyroxine on the activity of cytochrome oxidase in rat adenohypophysis]

Thyrotropin releasing-hormone (TRH) increased the activity of cytrochrome C oxidase in a concentration of 0.01 and 1.0 microgram/ml in the adenohypophysis of rats fed methylthiouracil for 6 weeks. This effect of TRH on the activity of the enzyme was blocked with T4 added to the incubation medium in a concentration of 20 microgram/ml. Actinomycin D (20 MICrogram/ml) prevented the block of the enzyme with thyroxin. In a concentration of 0.01 microgram/ml TRH, and in a concentration of 2.0 microgram/ml T4 failed to change the activity of cytochrome oxidase in the adenohypophysis of normal and partially thyroidectomized rats.     

Schistosoma mansoni: activity responses in vitro to praziquantel.

The effects of praziquantel, a novel antischistosomal compound, on the activity of adult Schistosoma mansoni (Liverpool strain) in vitro were investigated. Worm activity was modified at all concentrations of praziquantel. High concentrations (above 0.5 microgram/ml) produced rapid paralysis, whilst low concentrations of praziquantel stimulated worm activity. It is concluded that such activity modification could lead to worm displacement in vivo.     

Inhibition of rat heart ornithine decarboxylase by basic polypeptides.

Purified and partially purified ornithine decarboxylase (ODC) from rat heart was inhibited by basic polypeptides in vitro. Poly-L-arginine, the most effective, was inhibitory at a concentration as low as 0.1 microgram/ml; protamine and histone clearly inhibited ODC at concentrations higher than 2 micrograms/ml, but poly-L-lysine was less effective. The ability to inhibit ODC appeared to correlate with the arginine-residue content of basic polypeptides. The inhibition effect could be decreased by increasing substrate concentration and ionic strength.