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.     

Iron utilization studies in Citrobacter species

Abstract Seventy-one strains of Citrobacter were screened for iron scavenging mechanisms by biologic and chemical assays. Essentially all citrobacteria (70 / 71) were found to elaborate enterobactin-like siderophores by both biologic and chemical assays, however only C. koseri (C. diversus) was found to produce aerobactin. The concentration of ethylenediamine di( o -hydroxyphenylacetic acid) (EDDA) required to inhibit the growth of individual Citrobacter strains by depleting free iron ranged from 250 μg/ml to 1000 μg/ml. Iron utilization studies of selected Citrobacter isolates indicated that hemin and hematin could reverse the effects of iron limitation on growth under iron-stressed conditions (1000 μg/ml of EDDA). Two C. koseri strains grown under iron-restricted conditions showed similar changes in their whole cell protein profiles including induction of high molecular mass proteins (72–83 kDa) which may play a role in iron acquisition under iron-stressed conditions. The collective results support an additional virulence-associated mechanism for C. koseri strains which may help explain the greater pathogenic potential this group has for causing serious extraintestinal disease in humans.     

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.     

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.     

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 .     

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.     

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     

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.     

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

Mycobacterium neoaurum was grown with a range of iron concentrations from 0.01 to 4.0 micrograms/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 micrograms/ml) were added to the medium and were repressed when the iron concentration was increased to 0.5 micrograms/ml and above. These results re-inforce the conclusion that the iron-regulated proteins do fulfil an essential function in iron metabolism.     

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.     

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.     

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.     

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.     

Response of Campylobacter jejuni to combinations of ferrous sulphate and cadmium chloride

On Mueller Hinton (MH) agar, Campylobacter jejuni showed 20.0 and 30.9mm zones of inhibition surrounding discs impregnated with 2.5 and 20 μg CdCl₂ respectively. The minimal inhibitory concentration (MIC) ranged from 0.64 to 3.2 μg CdCl₂/ml of MH agar for four C. jejuni strains. In the presence of 23 μg FeSO₄/ml of MH the MIC increased to a range of 1.5–5.4 μg CdCl₂/ml of MH. Moreover, the numbers of colonies present on MH supplemented with FeSO₄ were greater than on MH without iron. The growth response of C. jejuni in the presence of 0.025% (w/v) FeSO₄ in MH broth was increased about 10000 fold in three of four strains when compared with the growth in unsupplemented MH broth. Zones of inhibition surrounding 20 μg discs of CdCl₂ were 50.6 and 24.4 mm on MH and Campy-BAP media respectively, with cells grown on MH. These results suggest that the blood-containing medium 'neutralized' the biocidal influence of the CdCl₂. In comparison, C. jejuni inoculum from fluid thioglycollate (FT) medium showed smaller zones of inhibition. These decreased from 34.9 mm on MH agar to 19.6 mm on Campy-BAP agar, suggesting that components in the FT growth medium ameliorated the toxic influence of CdCl₂. Atomic absorption spectroscopy analysis indicated mean values (mg/100 g dry weight) of selected metals bound by C. jejuni as: Cu, 10.4; Mg, 146; Na, 2385; Fe, 45.1; Zn, 13.0; and K, 172.     

Mycotoxin production in a carbendazim-resistant strain of fusarium sporotrichioides

Mycelial yield and production of three trichothecenes, namely T-2 toxin, diacetoxyscirpenol (DAS) and neosolaniol (NEO) were compared in control (CS) and carbendazim-resistant strains (RS) ofFusarium sporotrichioides. Each strain was exposed to graded concentrations of carbendazim (0, 1, 2, and 4 μg/ml media) for 2, 5 and 7 days under shake-culture conditions at an incubation temperature of 25°C. Mycelial yield was significantly (P<0.001) affected by strain, carbendazim concentration and incubation time. The strain differences in mycelial mass at 2 days (P<0.05) became more pronounced at 5 and 7 days of incubation (P<0.001). However, mycelial growth differences between the two strains were greatest following exposure to carbendazim, with the effects becoming more divergent with time. Combined results for the three incubation times showed dose related effects in carbendazim inhibition of T-2 toxin production by CS isolates. In contrast, RS cultures exposed to the 2 μg/ml addition of carbendazim significantly increased T-2 toxin production (P<0.05 or better). At 1 and 4 μg/ml additions, T-2 toxin inhibition occurred but the effect was less marked than in the CS series. RS yielded more DAS than CS at 5 days (P<0.05) and at 7 days (P<0.01) of incubation. The major component of this strain difference arose from the effects of the 2 μg/ml addition of carbendazim (P<0.01). NEO production was also higher in RS than in CS, with the difference becoming progressively more pronounced from day 5 (P<0.05) to day 7 (P<0.01) of incubation. However, these differences reflected enhanced NEO output with carbendazim addition of 4 μg/ml (P<0.05) in day 5 extracts and of both 2 μg/ml (P<0.01) and 4 μg/ml additions (P<0.05) in day 7 samples. Moreover, the ratio of NEO to T-2 toxin production was affected by an interaction involving incubation time, strain and carbendazim dose (P<0.05 or better). On day 5, this ratio was greater in CS exposed to 2 μg/ml, but at 4 μg/ml, the ratio was higher in RS. It is concluded that carbendazim resistance induced genuine differences in the synthesis of T-2 toxin and NEO. It is suggested that the strain difference may reside in the conversion of NEO to T-2 toxin which may be sensitive to fungicide concentration. This would imply that carbendazim resistance induces changes in the terminal rather than initial phases of trichothecene biosynthesis.     

Studies on the effect of toxin T-514 on the integrity of peroxisomes in methylotrophic yeasts

Abstract We have studied the response of two methylotrophic yeasts ( Hansenula polymorpha and Candida boidinii ) to toxin T-514, a toxin lethal to man, extracted from the shrub Karwinska homboldtiana . Growth experiments indicated a dose-response effect; at enhanced concentrations (50 μg/ml) the different subcellular organelles rapidly disintegrated resulting in death of the cultures. At non-lethal concentrations (<2 μg / ml ) growth ceased initially, but resumed after a lag period of 4 h. At the subcellular level a specific effect was observed on peroxisomal integrity. Distinct holes appeared in the peroxisomal membranes, resulting in leakage of matrix proteins from these organelles. In addition, import of newly synthesized proteins appeared to be blocked since cytosolic aggregates of matrix proteins were formed. The peroxisomal damage was probably irreversible since affected organelles were degraded at later stages of incubation. Upon restoration of growth on methanol, new peroxisomes developed from those which had escaped degradation.     

Larvicidal toxicity of Japanese Bacillus thuringiensis against the mosquito Anopheles stephensi

Japanese isolates of Bacillus thuringiensis were screened for larvicidal activity against the mosquito Anopheles stephensi , the urban malaria vector of the Indian subcontinent. Among more than 30 strains identified, larvicidal activity causing >80% mortality in 72 h was demonstrated for 41/1449 (2.8%) isolates. The majority of strains and isolates (97.2%) exhibited little or no larvicidal activity. Anopheles -active strains belonged to more than 12 H serotypes, especially H3ade (serovar fukuokaensis ) and H44 (serovar higo ). SDS-PAGE profiles of inclusion proteins showed 4 distinct types among 6 active strains examined. The most active Japanese isolates were H20 strain 89-T-34-14 (LC₅₀ 4.4 μg/ml) and H44 serovar higo strain 74-E-45-24 (LC₅₀ 7.6 μg/ml), respectively, 13-fold and 23-fold less active than the international standard H14 serovar israelensis (LC₅₀ 0.33 μg/ml).     

Transformation of the entomopathogenic fungus Metarhizium anisopliae to benomyl resistance

Abstract Protoplasts of the entomopathogenic fungus Metarhizium anisopliae were transformed to benomyl resistance using cosmid pSV50 which harbours a β-tubulin gene cloned from a Neurospora crassa benomyl-resistant mutant. Transformant colonies, which appeared at a frequency of 4 per 50 μg DNA, grew and sporulated on 10 μg/ml benomyl, whereas the wild type was inhibited by 3 μg/ml. Southern blot hybridization of DNA from transformants showed that, in each case, tandem repeats of the cosmid had integrated at several chromosomal loci. The transformants were mitotically stable when subcultured on non-selective agar and retained the ability to infect and kill larvae of Manduca sexta . Two transformants were less virulent than the wild type and one of them showed slower in vitro spore germination. The benomyl-resistant phenotype persisted in reisolates from insect cadavers.     

A carbon source-dependent antimetabolite sensitivity test in Bacillus megaterium B71 for isolation of ethionine resistant mutants

Alteration of carbon sources significantly altered the analogue sensitivity of Bacillus megaterium B71. DL-Ethionine (ETN) was highly inhibitory with glucose, mannitol, sucrose, citrate, glycerol and arabinose. DL-Norleucine, L-homoserine and S(2'-aminoethyl)-L-cysteine were either highly inhibitory, slightly inhibitory or non-inhibitory depending on the carbon sources used. Maltose markedly overcame the inhibitory effect of ETN in liquid culture. Uninhibited growth was poor on citrate and arabinose when compared with other carbon sources. Six carbon sources showing comparable growth were used to determine minimum inhibitory concentrations (MIC) of the analogues. The MIC of ETN was highest (450 μg/ml) with maltose and lowest (4 μg/ml) with mannitol. ETN sensitivity was inversely related to the endogenous L-methionine pool size, and was relatively low with mannitol which was used to isolate ETN resistant mutants of B. megaterium B71. The best mutant BUE-118 produced 435 μg/ml of L-methionine.