Thymineless death inLactobacillus acidophilus R-26

Thymineless death (TLD) was studied inLactobacillus acidophilus R-26. Thymine synthesis was inhibited with 5-fluorouracil (FU) or deoxyadenylate (dAMP) or by the absence of folic acid. In the case of FU, the maximum rate of dying was obtained at concentrations exceeding 0.1 μg/ml. This concentration did not affect the growth of the bacteria in the presence of thymine (4 μg/ml) and uracil (10 μg/ml). At higher FU concentrations up to 10 μg/ml, the course of TLD was unaltered, but the growth of bacteria in complete medium was slower. In the case of dAMP, the same course of TLD was obtained at a concentration of 150 μg/ml. If 1,500 μg dAMP/ml was used, the pre-death lag phase was shortened the rate of dying being unaltered. These concentrations of dAMP retarded the growth of bacteria even in a complete medium. If the thymine synthesis was prevented by the absence of folic acid the rate of dying was much lower than that caused by the presence of FU or dAMP. This was true even if the aminopterin was added. The authors conclude that the folic acid starvation did not inhibit completely the synthesis of thymine.     

Effect of soil solution phosphorus in Volcanic Ash soils on the response of tomato (Lycopersicum esculentum) to added phosphate

Summary Sorption isotherms of six Volcanic Ash soils were used to determine Optimum P, i.e. the amount of added P required to produce a soil solution concentration of 0.2 µg P per ml. In a pot test, application of these amounts of P to the soils led to maximum or near maximum growth of tomato plants, (Lycopersicum esculentum), harvested at 35 days. After harvest the soil solution concentration of P in all the soils had fallen below 0.2 µg per ml whatever the rate of P added initially. The amount of P required to restore the soil solution to 0.2 µg P per ml was linearly related to the fertilizer P originally applied to the soil; the higher the P applied originally the lower was the future P requirement. The regression coefficients suggested that the applied P would be twice as effective at reducing subsequent P requirement in a Recent Volcanic Ash soil than in the other more developed soils. Measurement of extractable Al (NH₄OAc at pH 4.2) showed that lack of growth at low levels of added P on a high P fixing soil could be caused by insufficient P due to high levels of Al, although with a soil of pH 6 this would not have been expected. re]19760217     

Rapid and extensive vacuolation of the budding yeastsSaccharomyces cerevisiae andCandida albicans by amphotericin B

The effect of amphotericin B (AMPH) on vacuolation in the budding yeastsSaccharomyces cerevisiae andCandida albicans was studied. The minimum inhibitory concentration of AMPH for growth ofS. cerevisiae andC. albicans was 1 µg/ml. In untreated control cultures, mature cells had large central vacuoles in the exponential phase, which hampered the detection of vacuolation effect. Small buds in untreated exponential phase cells, however, only rarely showed vacuoles under the light microscope. Treatment with 0.2 µg/ml of AMPH for 20–30 min induced extensive vacuolation not only in mothers but also buds ofS. cerevisiae. Extensive vacuolation lasted 4 h or more, and growth rate of the cells was much reduced for 8 h or more. Vacuolation itself was not fatal: on removal of the drug most cells gradually recovered from vacuolation and eventually multiplied. A similar effect of AMPH was also observed inC. albicans but at a higher concentration (0.5 µg/ml).     

Production,partial purification and bioassay of toxic metabolites of three plant pathogenic species ofColletotrichum in Nigeria

Colletotrichum lindemuthianum, C. truncatum andC. graminicola, produced metabolites in culture which induced necrotic lesions on susceptible hosts. The highest production was obtained from Richard's medium under shake incubation. The toxic metabolites of these pathogens fluoresce at 254 nm and 366 nm under ultraviolet light. The crude metabolites of theseColletotrichum species inhibited seed germination at the concentration of 100 µg/ml while the potency of the metabolites decreases with increase in dilution to 0.1 µg/ml. Similarly, the metabolites also inhibited growth of seedlings of hosts to the pathogen at 100 µg/ml and the potency again reduced with increasing dilution to 0.10 µg/ml.     

Influence of the mycotoxins aflatoxin B1, rubratoxin B,patulin and diacetoxyscirpenol on the fermentation activity of baker's yeast

The fermentation activity of baker's yeast (measured by the amount of produced CO₂) is inhibited by 100µg/ml and 10µg/ml aflatoxin B₁, and by 100µg/ml and 10µg/ml diacetoxyscirpenol. Lower concentrations of these mycotoxins as well as of rubratoxin B enhance the fermentation. Only 0.001µg/ml aflatoxin B₁, 0.00001µg/ml diacetoxyscirpenol and 0.01µg/ml rubratoxin B are without effect or slightly inhibitory. Patulin in all concentrations tested does not influence the CO₂ production significantly. Cytochemical studies show that the enzyme alcohol dehydrogenase is inhibited by 100µg/ml and enhanced by 1µg/ml and 0.1µg/ml aflatoxin B₁. It is suggested that the influence of at least aflatoxin B₁ on the fermentation activity of the yeast cells is due to an interaction with alcohol dehydrogenase. It is possible that the activity of other enzymes of yeast is also influenced by mycotoxins.     

Über die Physiologischen Eigenschaften von Gewebekulturen aus Tumor- und normalem Gewebe vonPicea glauca

Summary In contrast to other plant tumor tissues (crown gall and genetic tumors), that fromPicea glauca can be cultivated in vitro only in the presence of IAA. The auxin requirement for maximal growth of the tumor is appreciably higher (10^–5 g/ml IAA) than that of normal Picea cultures (10^–8 g/ml). A similar marked difference is also found in the ascorbic acid requirement, which is 10^–5 g/ml for neoplastic, but 10^–7 g/ml for normal tissue. The significance of these results for the characterisation and the in vitro growth of plant tumors is discussed.

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Efficacy of 6-amino-2-n-pentylthiobenzothiazole onTrichophyton in vitro and in vivo

In vitro and in vivo antifungal activities of synthetically parepared 6-animo-2-n-pentylthiobenzothiazole (APB) againstTrichophyton strains were studied. APB inhibited the growth of 3Trichophyton strains at 65 µg/ml. 2-Mercaptobenzothiazole was not effective at 125 µg/ml and ketoconazole inhibited the growth at 20–30 µg/ml. Treatment of experimental dermatophytosis in guinea pigs using 2.5% APB cream was studied in comparison to Canesten cream (1% clotrimazole). Dermatophytosis was considerably reduced after both APB and Canesten therapies.     

Studies on the synergistic effect of amphotericin B and 5-fluorocytosine on the growth rate of single hyphae ofAspergillus fumigatus by a biocell-tracer system

The biocell-tracer system is a microscopical system to measure the growth rate of a single fungal hypha. The synergistic effect of amphotericin B (AMPH) and 5-fluorocytosine (5-FC) on the growth of hyphae ofAspergillus fumigatus was studied by using this system. Although neither 2µg/ml of AMPH nor 250µg/ml of 5-FC alone showed any effect on the hyphal growth, their combination at these concentrations showed a distinct inhibitory activity. The biocell-tracer system is useful for antifungal activity testing in filamentous fungi.     

Promoting effect of wood vinegar compounds on fruit-body formation ofPleurotus ostreatus

The promoting effect of wood vinegar compounds on the fruiting ofPleurotus ostreatus (Japanese name, Hiratake) was investigated. Not only crude wood vinegar but its components, 3,5-dimethylphenol, 2-methoxyphenol, butanoic acid and 1-pentanol, had the ability to promote fruit-body formation on liquid medium. For use of these promoters industrially, a test for practical cultivation was carried out using a commercial sawdust medium. The addition of 100 µg/ml butanoic acid and 100 µg/ml 2-methoxyphenol into the sawdust medium after removal of the surface mycelial layer (kinkaki in Japanese) produced 29 and 23% higher yields of fruit-bodies than the control cultures (137.2 g/bottle), respectively. The addition of the crude wood vinegar as a medium component into sawdust substrates in the concentration range of 0.1–6% increased yields of fruit-bodies by 21–42% over the control.     

Effect of some antibiotics on Sclerotium cepivorum Berk., the cause of white rot of onion

The effect of some antibiotics onSclerotium cepivorum, the cause of white rot of onion was studied in agar culture and soil. The growth ofS. cepivorum was inhibited in Czapek Dox yeast agar containing 50µg of gliotoxin, viridin, actidione and 100µg of patulin per ml of the medium. Lower concentrations of the antibiotics retarded the growth of the fungus. In soil, patulin had no effect in the control ofS. cepivorum infection of onion seedlings. Concentration of actidione of 5µg/g of soil completely controlled white rot infection but severely stunted the growth of onion seedlings; 40µg/g of actidione killed the seedlings. Despite the importance of actidione as a fungistatic agent its use on onion is limited by its phytotoxicity.     

The modulation of growth of normal rat liver epithelial cells in calcium-poor medium by epidermal growth factor,phenobarbital, phorbol ester,and retinoic acid

Summary The ability of a normal rat liver epithelial cell line with phenotypic characteristics of “oval” cells to grow in calcium-poor medium has been investigated. The growth of these cells could be arrested in medium containing 0.03 mM Ca²⁺, a concentration below which cell necrosis began to occur 24 h postexposure. With increasing calcium concentration, progressive cell proliferation was observed. Epithelial growth factor (EGF) (10 ng/ml) increased the survival and proliferation of cells in calcium-poor medium and the response was inversely correlated with the extracellular calcium concentration. In contrast, phenobarbital (0.2 to 2 mM), 12-0-tetradecanoylphorbol-13-acetate (0.01 to 1 μg/ml), or retinoic acid (0.001 to 0.1 μg/ml) depressed growth of cells in calcium-poor medium. The results confirm the ability of EGF to lower the calcium requirement for proliferation of normal cells, but such an effect does not seem to be a universal property of tumor promoters. This research was supported by National Institutes of Health Grant CA 29323.     

Aflatoxins in mutants of Aspergillus flavus

Mutants ofAspergillus flavus were recovered following the irradiation of conidia with ultraviolet light. Analysis of the mutants for aflatoxins B₁, B₂, G₁, and G₂ indicated a wide range of variability in aflatoxin levels. None of the isolates produced the G toxins, and four produced little or no aflatoxin B₂. Production of B₁ and B₂ by the mutants ranged from 1.3 µ;g/ml to 967 µg/ml and zero to 30 µg/ml, respectively. The correlation between production of B₁ and B₂ was statistically significant. There was no apparent correlation between nutritional requirement or conidial color and aflatoxin production.     

Effects of organic compounds on the degradation ofp-nitrophenol in lake and industrial wastewater by inoculated bacteria

Many microorganisms fail to degrade pollutants when introduced in different natural environments. This is a problem in selecting inocula for bioremediation of polluted sites. Thus, a study was conducted to determine the success of four inoculants to degradep-nitrophenol (PNP) in lake and industrial wastewater and the effects of organic compounds on the degradation of high and low concentrations of PNP in these environments.Corynebacterium strain Z4 when inoculated into the lake and wastewater samples containing 20 µg/ml of PNP degraded 90% of PNP in one day. Addition of 100 µg/ml of glucose as a second substrate did not enhance the degradation of PNP and the bacterium utilized the two substrates simultaneously. Glucose used at the same concentration (100 µg/ml), inhibited degradation of 20 µg of PNP in wastewater byPseudomonas strain MS. However, glucose increased the extent of degradation of PNP byPseudomonas strain GR. Phenol also enhanced the degradation of PNP in wastewater byPseudomonas strain GR, but had no effect on the degradation of PNP byCorynebacterium strain Z4.Addition of 100 µg/ml of glucose as a second substrate into the lake water samples containing low concentration of PNP (26 ng/ml) enhanced the degradation of PNP and the growth ofCorynebacterium strain Z4. In the presence of glucose, it grew from 2×10⁴ to 4×10⁴ cells/ml in 3 days and degraded 70% of PNP as compared to samples without glucose in which the bacterium declined in cell number from 2×10⁴ to 8×10³ cells/ml and degraded only 30% PNP. The results suggest that in inoculation to enhance biodegradation, depending on the inoculant, second organic substrate many play an important role in controlling the rate and extent of biodegradation of organic compounds.Abbreviations PNP p-nitrophenol     

Cocarcinogenesis in vitro using Balb/3T3 cells and aromatic hydrocarbon cocarcinogens

The mouse skin cocarcinogens fluoranthene, pyrene, and undecane were used with the indirect-acting carcinogen, benzo(a)pyrene (BP), and the direct-acting alkylating carcinogen, ß-propiolactone (BPL), in an in vitro transformation assay. Dose response, cytotoxicity, and transformation studies with these compounds were performed with a subclone (A31-1-1) of the Balb/3T3 cell line. Transformation frequencies were found to increase with increasing concentrations of BP used up to 1.0 µg/ml or when BPL was used up to 4.0 µg/ml. A significant increase (P<0.05) in the transformation frequency over that seen with carcinogen alone was observed when cells were exposed to a combination of fluoranthene (4.0 µg/ml) and BP (0.063 µg/ml) or pyrene (5.0 µg/ml) and BP (0.063 µg/ml). Thus, the transformation frequency obtained with BP + fluoranthene was 3.8 × 10^–4 compared to 1.2 × 10^–4 when BP was tested alone. Similarly, the transformation frequency using BP + pyrene was 2.8 × 10^–4 vs 1.2 × 10^–4 when BP was tested alone. Undecane did not exert any cocarcinogenic effect with BP in the dose range tested. In this in vitro assay, no cocarcinogenic effect was observed when BPL was used with any of the above mouse skin cocarcinogens. All cells isolated from transformed foci showed characteristics of transformed cells including anchorage-independent growth.Abbreviations BP benzo(a)pyrene - BPL ß-propiolactone - CE cloning efficiency - CE₅₀ median CE - RCE relative CE Department of Cell Biology, New York University Medical CenterInstitute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA.Contribution No. L217 from the Laboratory of Organic Chemistry and Carcinogenesis.     

Growth factor requirements of two strains ofSphaerotilus discophorus

The relatively complex growth-factor requirements of two strains ofSphaerotilus discophorus, strains 31 and 32, have been elucidated. In addition to thiamin and biotin which are required by other strains ofS. discophorus, the two strains must be supplied also with adenine or guanine and with cyanocobalamin for growth in a glucose — Casamino Acids — mineral salts medium. The cyanocobalamin can be replaced by methionine but only if relatively large amounts of this amino acid, 100 µg or more per ml, are added to the medium. The methionine requirement of the two strains is approximately 3 times greater than that of otherS. discophorus strains.     

Aflatoxin M1 removal from aqueous solutions by Flavobacterium aurantiacum

In liquid cultures growing and stationary phase cells ofFlavobacterium aurantiacum NRRL B-184 eliminated aflatoxin M₁. Toxin concentrations of 15µg/ml and 37.5µg/ml interfered with bacterial growth, and at the higher level 4.4µg M₁ was removed from the growth medium by a milligram (dry weight) of bacteria. Toxin was completely removed from the liquid medium by incubating 5 × 10¹⁰ resting cells per milliliter with 8µg/ml of aflatoxin M₁ for 4 h. Attempted recovery of M₁ from cells following incubation of the bacteria with the toxin demonstrated that the M₁ was essentially nonextractable. Bacterial cells also removed aflatoxin M₁ from toxin-contaminated milk.     

Tourmaline ceramic balls stimulate growth and metabolism of three fermentation microorganisms

Effects of tourmaline ceramic balls on growth and metabolism of Saccharomyces cerevisiae, Lactobacillus acidophilus and Aspergillus oryzae were studied. Treatments with 3, 6, 9 or 12 g of tourmaline ceramic balls in a 50 ml culture showed significant stimulation of the growth of the three microorganisms. In optimal treatments with 12 g of tourmaline balls, the growth of S. cerevisiae, L. acidophilus, and A. oryzae was increased by 34, 32 and 10%, respectively. After 72 h fermentation of S. cerevisiae, total carbohydrate content in the culture medium was decreased by 65% and ethanol production was increased by 150%. Total carbohydrate content was decreased by 80% and the pH value was decreased by 0.3, as a result of organic acid production in the medium of L. acidophilus after 72 h fermentation. In the case of A. oryzae, enzyme activities of protease and amylase were increased by 90 and 31%, respectively, after 96 h fermentation. Results indicated that tourmaline stimulates initiation of growth in the early lag stage and increases production of metabolites at a later stage of fermentation. The strong stimulatory effect of tourmaline on growth, utilization of substrates and production of metabolites in the three microorganisms suggests a potential application in the fermentation industry.     

In vitro susceptibilities of clinical yeast isolates to three antifungal agents determined by the microdilution method

A comparative evaluation of the in vitro susceptibilities of 597 clinical yeast isolates to amphotericin B, fluconazole, and 5-fluorocytosine (5FC) was conducted. The broth macrodilution reference method of the National Committee for Clinical Laboratory Standards (NCCLS, M27-P) was adapted to the microdilution method. Microdilution endpoints for amphotericin B were scored as the lowest concentration in which a score of 0 (complete absence of growth) was observed and for 5FC and fluconazole as the lowest concentration in which a score of 2 (prominent decrease in turbidity; MIC-2) was observed compared to the growth control. The MIC values were read after 24 and 48 h incubation. A broad range of MIC values was observed with each antifungal agent. Amphotericin B was very active (MIC₉₀1.0 µg/ml) against all of the yeast isolates with the exception ofC. lusitaniae (MIC₉₀2.0 µg/ml). Fluconazole was most active againstC. parapsilosis (MIC₉₀ of 1.0 µg/ml) and least active againstC. krusei (MIC₉₀ of 32 µg/ml). 5FC was most active againstC. albicans, C. parapsilosis, C. tropicalis, andT. glabrata (MIC₉₀1.0 µg/ml) and was least active againstC. krusei andC. lusitaniae (MIC₉₀16 µg/ml). These data indicate that the microdilution method, performed in accordance with M27-P, provides a means of testing larger numbers of yeast isolates against an array of antifungal agents and allows this to be accomplished in a reproducible and standardized manner. Given these results, it appears that the microdilution method may be a useful alternative to the macrodilution reference method for susceptibility testing of yeasts.     

Analysis of functional properties of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis>

Metabolites from Lactobacillus acidophilus were analysed. The results showed that Lactobacillus acidophilus Ind-1 and Lactobacillus acidophilus Lakcid produced respectively 12.73 g and 13.33 g lactic acid l⁻¹ after incubating in skim milk at 37 °C for 36 h; and 2.229 unit and 1.808 unit β-galactosidase l⁻¹ in an MRS medium. The proteolytic activity of Lactobacillus acidophilus was high and the content of 17 free amino acids in the fermented milk of Lactobacillus acidophilus Ind-1 and Lactobacillus acidophilus Lakcid was 394.4 mg l⁻¹ and 563.2 mg l⁻¹, respectively. Meantime, Lactobacillus acidophilus reduced cholesterol level in an MRS medium supplemented with cholesterol. Furthermore, Lactobacillus acidophilus Ind-1 and Lactobacillus acidophilus Lakcid showed antimicrobial activity against Bacillus anthracis and Escherichia coli.     

Effect of water soluble vitamins and their analogues on the growth of candida albicans. I. Biotin,pyridoxamine, pyridoxine and fluorinated pyrimidines

Summary C. albicans showed an absolute dependency for biotin in shaker cultures in a basal mineral synthetic medium free of vitamin-precursors and vitamin-sparing amino acids. Diminished growth activity was observed with biotin sulfone and biotin diamine sulfate, but not with biocytin, N-biotinyl--alanine, N-biotinyl-L-aspartic ethyl ester, D-desthiobiotin or biotin-D-sulfoxide. The ability of the organism to utilize desthiobiotin indicates that its block in biosynthesis of biotin occurs at a step prior to desthiobiotin biosynthesis. Pyridoxamine and pyridoxine were both highly growth stimulatory at 1000 and 2056 µg/ml but not in the vitamin range at 1 to 10 µg/ml. Since desoxypyridoxine compounds failed to inhibit growth in the absence of B₆, it was concluded thatC. albicans has no dependency for vitamin B₆, although it actively metabolizes it. Pyridoxamine shortened the lag phase of the organism and reversed the toxicity of 5-fluorouracil and 5-fluoro-2-deoxyuridine, pointing to a new role of vitamin B₆ in nucleic acid metabolism of the organism. Inhibition indices for pyridoxamine and pyridoxine versus FU and FUDR were inconstant, indicating that the antagonism with the fluoropyrimidines was non-competitive in nature and the B₆ competes with these compounds at more than one site on the cell.