Cytokinesis of Candida albicans by Diethylstilbestrol

In vitro effects of the synthetic oestrogenic hormone diethylstilbestrol (DS) and diethylstilbestrol dipropionate (DSP) on Candida albicans have been assessed. At a concentration of 5–20 μg/ml. these compounds suppressed the growth of C. albicans even though the multiplication of the organism was not influenced immediately. When C. albicans was grown for approximately 4 h in tryptic soy broth, its multiplication was rapidly retarded by these two substances. Since C. albicans must grow on a suitable culture medium in order to absorb DS and DSP, it was not surprising that respiration, the uptake, and incorporation of nutrients by C. albicans was not influenced when the cells were 'resting'. Such plasma steroids as androsterone (0·5 μg/ml), 5α-androstan-3 β-diol (0·25 μg/ml), dehydroisoandrosterone (2 μg/ml), epiandrosterone (0·1 μg/ml), oestrone (0·1 μg/ml), progesterone (0·4 μg/ml), cortisol (0·2 μg/ml), cholesterol (10 μg/ml) in combination with DSP did not antagonize the retardive action of DSP for C. albicans .     

β-Lactamase production and in vitro antimicrobial susceptibility of Porphyromonas gingivalis

Abstract β-Lactamase production by 98 Porphyromonas strains was investigated by the nitrocefin (chromogenic cephalosporin) test. Human isolates of P. gingivalis (91), P. endodontalis (2), and P. asaccharolytica (1) were tested, with four closely related Porphyromonas spp. of animal origin and four reference strains. The in vitro susceptibility of 64 P. gingivalis strains was investigated on Brucella blood agar by the E test. None of the human Porphyromonas isolates tested produced β-lactamase, but one Porphyromonas strain of animal origin, most closely resembling P. endodontalis , produced β-lactamase. P. gingivalis was susceptible to almost all of the drugs tested: benzylpenicillin, ampicillin, cefaclor, cefuroxime, erythromycin, clindamycin, tetracycline, doxycycline, metronidazole and ciprofloxacin; all strains were inhibited at 0.016 μg/ml, 0.023 μg/ml, 0.315 μg/ml, 0.064 μg/ml, 0.19 μg/ml, 0.016 μg/ml, 0.094 μg/ml, 0.047 μg/ml, 0.023 μg/ml, and 0.75 μg/ml of these drugs, respectively. Cotrimoxazole exhibited variable efficacy against P. gingivalis ; the range of MICs was 0.1095-32.0 μg/ml. The results indicate that β-lactamase production is currently not a problem amongst clinical isolates of P. gingivalis and strains are susceptible to most antimicrobial agents.     

Inhibitory effect of oregano and thyme essential oils on moulds and foodborne bacteria

The essential oil of oregano ('origanum oil'; thymol type oil from Origanum vulgare) inhibited completely the mycelial growth of Aspergillus niger and A. flaous at 400 μg/ml, while A. ochraceus was inhibited at 600 μg/ml. At 700 μg/ml, thyme oil inhibited the mycelial growth of A. flavus and A. niger but not that of A. ochraceus . Fungal spore germination was inhibited by 600 μg/ml of origanum oil and (with the exception of A. ochraceus) by 700 μg/ml of thyme oil. Under aerobic conditions, the essential oils of oregano (250 μg/ml) and thyme (350 μg/ml) inhibited to some extent the growth of Staphylococcus aureus and Salmonella typhimurium. Pseudomonas aeruginosa was not affected by either oregano or thyme oil at concentrations up to 500 μg/ml. The origanum oil was very effective against Campylohacter jejuni and Clostridiurn sporogenes and thyme oil was very effective against C. jejuni. The antagonistic effect of the two oils on Staph. aureus and Salm. typhimuriutn was greatly enhanced when those organisms were incubated in atmospheres of low oxygen tensions     

Efficacy of Novel Antimicrobials Against Clinical Isolates of Opportunistic Amebas

ABSTRACT We examined the effects of the macrolide antimicrobial agent azithromycin and phenothiazine compounds against clinical isolates of Acanthamoeba spp. and Balamuthia mandrillaris , opportunistic pathogens of human beings and other animals. Acanthamoeba growth was inhibited in vitro at 1,5, and 10 μg/ml of azithromycin, but not the macrolides, erythromycin, and clarithromycin. In experiments attempting to simulate in vivo conditions, azithromycin protected monolayers of rat glioma cells from destruction by Acanthamoeba at a concentration of 0.1 μg/ml, and delayed destruction at concentrations of 0.001 and 0.01 μg/ml. We concluded that the minimal inhibitory concentration of azithromycin was 0.1 μg/ml. Our results, however, suggested that the drug was amebastatic but not amebicidal, since ameba growth eventually resumed after drug removal. The phenothiazines (chlorpromazine, chlorprothixene, and triflupromazine) inhibited Acanthamoeba growth by 70-90% at 5 and 10 μg/ml, but some of these compounds were toxic for rat glioma cells at 10 μg/ml. Azithromycin was not very effective against B. mandrillaris in an in vitro setting, but was amebastatic in tissue culture monolayers at concentrations of 0.1 μg/ml and higher. Balamuthia amebas showed in vitro sensitivity to phenothiazines. Ameba growth was inhibited 30-45% at 5 μg/ml in vitro, but completely at 5 μg/ml in the rat glioma model. In spite of their potential as antiamebic drugs in Balamuthia infections, toxicity of phenothiazines limits their use in clinical settings.     

The toxicity of a number of different bactericides to Clavibacter michiganense subsp. michiganense (Smith 1910) Jensen 1934 comb. nov. [basonym Corynebacterium michiganense pv. michiganense (AL)] and to the tomato plant, Lycopersicon esculentum

Twenty-seven proprietary products and pure chemicals were tested in vitro against cells of Clavibacter michiganense subsp. michiganense (Smith 1910) Jensen 1934 comb. nov. [basonym Corynebacterium michiganense pv. michiganense (AL)] (the cause of bacterial canker of tomato) and also for their phytotoxicity to tomato plants. The most bactericidal of these, with a minimum cidal concentration (MCC) range of > 10-< 100 μg/ml, were a phenolic product called Applied 3–78, two quaternary ammonium compounds (benzalkonium chloride and cetrimide), and a silver colloid compound. Of these, only Applied 3–78 was not phytotoxic at values of 10 μg/ml or less, although it was phytotoxic at 10000 μg/ml. Copper oxychloride and sodium hypochlorite were amongst the group with a middle range of bactericidal properties, their MCC range being from > 1000 to < 10000 μg/ml. They were phytotoxic at 1000 μg/ml or less. When organic matter, a dead yeast suspension, was added to Applied 3–78, Kohrsolin and Panacide, only the activity of Applied 3–78 was relatively unchanged. The MCC ranges were: Applied 3–78, >80–< 100 μg/ml; Kohrsolin, > 800-< 1000 μg/ml; and Panacide, > 1000 μg/ml. Phytotoxicity tests on 10 different tomato cultivars confirmed that Applied 3–78 was the least phytotoxic of these three products. Field trials on tomato crops showed that when Applied 3–78 was sprayed on the plants once, and Kohrsolin was either sprayed on or they were drenched with it once at 1000 μg/ml, no phytotoxicity symptoms developed.     

Growth and Differentiation of Primary Cultures of Mouse Mammary Epithelium Embedded in Collagen Gel

Mammary glands from BALB/cfC3H midpregnant (9–11 days) mice were dissociated with collagenase and pronase, separated on a Percoll gradient, and the epithelial cells were cultured inside collagen gel. The cell number increased three-to five-fold when cultured for 6–8 days in DME/F12 (1: 1) medium containing 3% swine serum, insulin (10 μg/ml), cortisol (1.0 μg/ml), prolactin (10 μg/ml), transferrin (10 μg/ml), and epidermal growth factor (0.01 μg/ml). The casein level, as determined by radioimmunoassay, at the end of this growth phase, was much lower than that present in freshly dissociated cells. In order to stimulate casein production, the gels were released from the sides of the plastic dish and allowed to float for eight days in Waymouth's medium, containing insulin (10 μg/ml), cortisol (5 μg/ml), prolactin (10 μg/ml), and 0.25% bovine serum albumin. The casein level at the end of this differentiation phase was found to be comparable to that seen in the original freshly dissociated cells. Cells grown in DME/F12 (1: 1) medium containing 3% swine serum, insulin (10 μg/ml), and transferrin (10 μg/ml) were still capable of undergoing casein production, indicating that the presence of exogenous lactogenic hormones such as cortisol and prolactin, as well as exogenous growth factors such as epidermal growth factor, is not necessary during the growth phase for subsequent casein production during the differentiation phase. Two factors that seemed more important for subsequent casein stimulation were: (1) releasing collagen gels at the beginning of the differentiation phase, and (2) switching to'differentiation' medium. This present two-step protocol has allowed primary cultures of dissociated midpregnant mouse mammary epithelial cells to undergo several rounds of division inside a collagen gel matrix and to be subsequently stimulated to produce the mammary-specific protein, casein.     

The Effect of Monensin on Pure and Mixed Cultures of Rumen Bacteria

The antibiotic monensin was added to pure cultures of Bacteroides ruminicola, Selenomonas ruminantium, Anaerovibrio lipolytica and Megasphaera elsdenii. These organisms, representing succinate- and propionate-producing rumen bacteria, were not affected by monensin up to 10 μg/ml. Methanobacterium ruminantium was slightly inhibited by monensin, Butyrivibrio fibrisolvens, Ruminococcus albus and Streptococcus bovis were inhibited to differing extents by monensin at concentrations between 0.1 and 10 μg/ml. Bacteroides succinogenes was inhibited at first by monensin at >0.5 μg/ml but after a prolonged lag phase adapted to grow in the presence of monensin at concentrations below 5 μg/ml.
Monensin (1 μg/ml) almost completely stopped the digestion of chopped straw and dewaxed cotton fibres by rumen contents incubated in vitro. The digestion of grass and powdered filter paper was not significantly reduced under these conditions, but when the concentration of monensin was increased to between 3 and 5 μg/ml, the digestion of these substrates was reduced.     

In vitro cytotoxicity of lipopolysaccharides for chinese hamster ovary cells

Abstract From our survey of various lipopolysaccharide (LPS) preparations, we demonstrated that three out of five commercial LPS preparations of Salmonella typhimurium were not cytotoxic for Chinese hamster ovary (CHO) cell monolayers at a concentration of 1000 μg/ml. One commercial LPS preparation produced cellular damage at a concentration of 1000 μg/ml and another at 400 μg/ml. Two S. typhimurium LPS preparations made in our laboratory were also cytotoxic at a concentration of 1000 μg/ml but not at lower concentrations. Cell-free sonic lysates of S. typhimurium TML R66 were cytotoxic when tested undiluted and up to a dilution of 1:20. Based on the 2-Keto-3-deoxyoctonate (KDO) content of all preparations, sonic lysateas were cytotoxic at KDO concentrations of 0.42 μg/ml while the KDO content of the most cytotoxic LPS preparation was 15.2 μg/ml. There was no apparent correlations between KDO content of the LPS preparations and cell detachment, leading to the conclusion that cell detachment activity of Salmonella cell lysates cannot be attributed to their LPS content.     

A Note on the Sensitivity of Chlorine-stressed Bifidobacteria to Nalidixic Acid

Bifidobacterium adolescentis , normally resistant to 200 μg/ml nalidixic acid, became sensitive to 10 μg/ml nalidixic acid after 15 min exposure to chlorinated sewage effluent (0–08 mg/1000 ml of residual chlorine). After exposure to 0.7 mg/1000 ml of residual chlorine, Bifidobacterium adolescentis became sensitive to 5 μg/ml nalidixic acid.     

Critical concentrations of lincomycin and polymyxin B in the Biken assay for detection of the heat labile enterotoxin of Escherichia coli

Reproducibility of the Biken test for detection of the LT-enterotoxin of Escherichia coli was evaluated at various concentrations of lincomycin and polymyxin B sulphate. Since 14 out of 65 clinical isolates could not grow at the recommended concentration (90 μ g/ml) of lincomycin, the standard test was modified by reducing its concentration from 90 μ g/ml to 60 μ g/ml and by reducing the concentration of polymyxin B from 500 IU to 300 IU.     

Ferritin-iron increases killing of Chinese hamster ovary cells by X-irradiation

Abstract. Stationary-phase Chinese hamster ovary cells were cultured in medium containing ferritin (-19% iron by weight) added at concentrations ranging from 0 to 128 μ g/ml. One set of cultures was unirradiated, and another set was exposed to 4.0 Gy of X-ray. Clonogenic cell survival was assessed in each set of cultures. In the absence of added ferritin, 4.0 Gy killed approximately 50% of the cells. In the absence of radiation, ferritin was not toxic at less than 48 μ g/ml; above 48 μ g/ml, toxicity increased with concentration. Apoferritin was not toxic at any concentration tested (up to 1000 μ g/ml). Although 32 μg/ ml ferritin, reflecting only a 3–6 fold increase in iron concentration over normal serum, was not toxic, it reduced the survival of X-irradiated cells by an additional 75%. These results indicate that a sublethal concentration of ferritin can be a potent radiosensitizer. This suggests the possibility that high body iron stores may increase susceptibility to radiation injury in humans.     

Detection of inducible β-lactamase by an agar dilution technique

Abstract An agar dilution technique for the detection of inducible β-lactamase-mediated resistance to the newer cephalosporins is described. Cefoxitin (16 μg/ml) was incorporated in agar plates together with cefamandole (8 μg/ml) or cefotaxime (8 μg/ml). The susceptibility of 35 strains of Enterobacter cloacae to these combinations was compared with their susceptibility to the individual antimicrobial agents. Of 31 strains which could be evaluated, 18 (58%) produced an inducible β-lactamase which inactivated cefamandole, while 10 (32%) produced an inducible cefotaxime-inactivating enzyme. The technique has the advantages of being 24 h faster than the alternative disc-diffusion induction test, and of being suitable for testing large numbers of strains simultaneously.     

Co-ordinated expression of the components of iron transport (mycobactin, exochelin and envelope proteins) in Mycobacterium neoaurum

Abstract Mycobacterium neoaurum was grown with a range of iron concentrations from 0.01 to 4.0 μg/ml. Synthesis of the extracellular siderophore, exochelin, the intracellular iron storage compound, mycobactin and the iron-repressible envelope proteins were co-ordinately expressed. All three components of the iron transport system were synthesized when low amounts of iron (0.01 to 0.2 μg/ml) were added to the medium and were repressed when the iron concentration was increased to 0.5 μg/ml and above. These results re-inforce the conclusion that the iron-regulated proteins do fulfil an essential function in iron metabolism.     

Isolation of a cytotoxin from L-form Salmonella typhimurium

Abstract A cytotoxin protein was isolated from the sodium dodecyl sulphate (SDS)-solubilized extract of the stable L forms of Salmonella typhimurium by ion-retardation chromatography, ion-exchange chromatography, isoelectric focusing and gel filtration. The purified toxin, with a molecular mass of 32 kDa and with isoelectric point of 6.4, was thermolabile and trypsin-sensitive. Against mouse macrophages, its cytolytic effect was detectable in vitro at concentrations higher than 0.7 μg/ml, with a complete lysis obtained at 5 μg/ml. In contrast, it stimulated C3H/HeJ macrophages in the dose range of 0.1–0.5 μg/ml to allow the cell to respond to endotoxin, resulting in the significant production of tumor necrosis factor α. By Northern blot analysis, this effect was detectable at a dose as low as 0.01 μg/ml. These findings suggest that the transformation of bacillary S. typhimurium into L forms in vivo may induce alterations in host resistance against murine typhoid.     

Aster Formation in Sea Urchin Eggs Induced by the injection of Enzyme-Treated Sperm Centrioles

To determine the responsible components of isolated sperm centrioles for the aster induction in sea urchin eggs, the sperm centriolar fraction was treated with various enzymes and was injected into the unfertilized eggs, then the aster formation in first division was observed after fertilization.
Treatment with 1 μg/ml or higher concentration of trypsin inhibited the centriolar activity for aster induction, whereas the treatment with 50 μg/ml of DNase 1, 80 μg/ml of RNase A, 40 μg/ml of RNase T1, or 0.1 μg/ml of trypsin had no inhibitory effect to induce asters. Injection of 0.5 μg/ml of RNase A or 1 mUg/ml of RNase T1 into the egg caused the detention of mitosis at the streak stage. To examine the temperature effect for aster induction, the centriolar fraction was pre-treated with boiling temperature, and it was found that the fraction became incapable to induce any aster.
Results obtained suggest that the effective components of the sperm centriolar fraction to induce asters in the fertilized sea urchin eggs are the proteins but not the nucleic acids. The aster inducing activity is destroyed by heat treatment.     

Fungicidal effect of hydrogen peroxide on fungal infection of rainbow trout eggs

Exposure to 1,500 μg/ml of hydrogen peroxide (H₂O₂) for 60 min at 13°C was found to be injurious to rainbow trout eggs. On the other hand, the concentration which effectively inhibited pathogenic fungi in vitro was substantially less than this toxic dosage; specifically, 500 μg/ml for 60 min at 20°C to inhibit the zoosporic stage and 1,000 μg/ml for 60 min at 20°C to inhibit the vegetative stage. From in vivo tests, treatment with 1,000 μg/ml of H₂O₂ for 60 min at 13°C was found to be the most effective procedure to control fungal infection and increase the hatching rate of rainbow trout eggs.     

Temperature-dependent phosphate solubilization by cold- and pH-tolerant species of Aspergillus isolated from Himalayan soil

Ten species of Aspergillus isolated from soil samples collected from different locations in the Indian Himalayan region have been studied for their growth requirements and tricalcium phosphate solubilization at different temperatures. The Aspergillus species could grow at low temperature and tolerated a wide range of pH. Phosphate solubilization by various Aspergillus species ranged between 374 μg/ml (A. candidus) to 1394 μg/ml (A. niger) at 28°C, 33 μg/ml (A. fumigatus) to 2354 μg/ml (A. niger) at 21°C, 93 μg/ml (A. fumigatus) to 1452 μg/ml (A. niger) at 14°C, and 21 μg/ml (A. wentii) to 83 μg/ml (A. niger) at 9°C. At 21 and 28°C, phosphate solubilization showed a decrease within 4 weeks of incubation, whereas at 9°C and 14°C, it continued further up to 6 weeks of incubation. In general, phosphate solubilization by different Aspergillus species was recorded at a maximum of 28°C or 21°C; biomass production was favored at 21°C or 14°C. Conversely, A. nidulans and A. sydowii exhibited maximum phosphate solubilization at 14°C and produced maximum biomass at 21°C. Data suggest that suboptimal conditions (higher or lower temperature) for fungal growth and biomass production were optimal for the production of metabolites involved in phosphate solubilization. Significant negative correlations were obtained between pH and phosphate solubilization for eight species at 28°C, for seven at 21°C, and for nine at 14°C. Extracellular phosphatase activity was exhibited only in case of A. niger, whreas intracellular phosphatase activity was detected in all species, the maximum being in A. niger. Statistically significant positive or negative correlations were obtained between phosphate solubilization and other parameters in most cases at different temperatures.     

SELECTIVE INHIBITION BY APHIDICOLIN OF THE ACTIVITY OF DNA POLYMERASE ALPHA LEADS TO BLOCKADE OF DNA SYNTHESIS AND CELL DIVISION IN SEA URCHIN EMBRYOS

Aphidicolin at 2 μg/ml caused 90% inhibition of mitotic cell division of sea urchin embryos at the I-cell stage. However, at 40 μg/ml it did not affect meiotic maturational divisions of starfish oocytes, which do not involve DNA replication. At 2 μg/ml it caused 90% inhibition of incorporation of tritiated thymidine into DNA of sea urchin embryos but did not affect protein or RNA synthesis even at a higher concentration. At 2 μg/ml it also caused 90% inhibition of the activity of DNA polymerase α, obtained from the nuclear fraction of sea urchin embryos, but did not affect the activity of DNA polymerase β or γ. These findings suggest that DNA polymerase α is responsible for replication of DNA in sea urchin embryos.     

Production, purification and characterization of a 50-kDa extracellular metalloprotease from Serratia marcescens

The extracellular metalloprotease (SMP 6.1) produced by a soil isolate of Serratia marcescens NRRL B-23112 was purified and characterized. SMP 6.1 was purified from the culture supernatant by ammonium sulfate precipitation, acetone fractional precipitation, and preparative isoelectric focusing. SMP 6.1 has a molecular mass of approximately 50 900 Da by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The following substrates were hydrolyzed: casein, bovine serum albumin, and hide powder. SMP 6.1 has the characteristics of a metalloprotease, a pH optimum of 10.0, and a temperature optimum of 42° C. The isoelectric point of the protease is 6.1. Restoration of proteolytic activity by in-gel renaturation after SDS-PAGE indicates a single polypeptide chain. SMP 6.1 is inhibited by EDTA (9 μg/ml) and not inhibited by antipain dihydrochloride (120 μg/ml), aprotinin (4 μg/ml), bestatin (80 μg/ml), chymostatin (50 μg/ml), E-64 (20 μg/ml), leupeptin (4 μg/ml), Pefabloc SC (2000 μg/ml), pepstatin (4 μg/ml), phosphoramidon (660 μg/ml), or phenylmethylsulfonyl fluoride (400 μg/ml). SMP 6.1 retains full activity in the presence of SDS (1% w/v), Tween-20 (1% w/v), Triton X-100 (1% w/v), ethanol (5% v/v), and 2-mercaptoethanol (0.5% v/v). The extracellular metalloprotease SMP 6.1 differs from the serratiopeptidase (Sigma) produced by S. marcescens ATCC 27117 in the following characteristics: isoelectric point, peptide mapping and nematolytic properties. Received: 22 November 1996 / Received revision: 27 February 1997 / Accepted: 7 March 1997     

Identification and Phytotoxicity of 3-methylthiopropanoic and Trans-3-methylthiopropenoic Acids Produced in Culture by Xanthomonas campestris pv. vitians

Two phytotoxic metabolites were isolated from culture filtrates of Xanthomonas campestris pv, vitians , the causal agent of lettuce leaf spots and headrot. The two compounds were identified as 3-methylthiopropanoic (1) and trans-3-methylthiopropenoic (2) acids by chemical and spectroscopic methods. Toxic effects of the two compounds on leaf tissues and protoplasts of lettuce and cabbage were investigated. Solutions of 1 and 2 induced chlorosis and necrosis on lettuce leaves at minimum concentrations of 300 and 50 μg/ml, respectively. Infiltration in cabbage leaves did not produce any symptoms. The LD₅₀ values for 1 and 2 against lettuce protoplasts were 15 and 16 μg/ml, respectively. Activity of the two metabolites against cabbage protoplasts was very low (LD₅₀ > 500 μg/ml).