Effect of tryptophan on the production of mucidin in cultures of basidiomyceteOudemansiella mucida

Addition ofL-tryptophan to cultures of the basidiomyceteOudemansiella mucida brought about a pronounced increase of production of the antibiotic mucidin. The highest increase was reached in the presence of 0.15–0.20 % tryptophan and after its addition to a 1-d culture. The methyl ester of tryptophan exhibited the same effect. Mycelium growing during the initial phases in the presence of tryptophan synthesized mucidin powerfully during later phases of the fermentation. Part VII of the series Antifungal antibiotic of the basidiomyceteOudemansiella mucida; part VI:Folia Microbiol. 27, 35 (1982).     

Effects of the new antifungal antibiotic mucidin

The morphological changes in 19 fungal species (16 filamentous fungi and 3 yeasts) caused by the antifungal antibiotic mucidin (trade mark Mucidermin Spofa) were examined. The filamentous fungi showed an undulation and ramification of hyphae and thickening of cells. The yeastCandida pseudotropicalis changes the shape and size of cells and the structure of cytoplasm.     

Effect of glucitol on the production of mucidin inOudemansiella mucida

When studying the biosynthesis of mucidin under production (glucose as the main carbon source) and non-production (glucitol as the main carbon source) conditions it could be shown that the producer,Oudemansiella mucida, utilizes glucitol both for growth and for mucidin biosynthesis. However, the production of mucidin is 10 times ower than on glucose. When the culture was preincubated on glucose and transferred to non-production conditions the negative effect of glucitol could not be demonstrated. Biosynthesis of mucidin is influenced by the used carbon source already at an early stage of the cultivation     

Effects of the new antifungal antibiotic mucidin

The antibiotic activity of the antifungal substance mucidin was compared with the activity of nystatin and pimaricin. The antibiotics were tested by the plate method using 19 fungal species, mainly phytopathogenic ones. Toward 14 species, mucidin was ten times more active than nystatin and pimaricin, toward 5 species the activities were roughly the same. The antibiotics differed also in the sharpness of the inhibition zone boundaries.     

Composition of lipids and production of mucidin in a submerged culture of the basidiomyceteOudemansiella mucida

Mycelial lipids of the submerged culture ofOudemansiella mucida contain acylglycerols, free and esterified sterols. Free fatty acids are not present. Development of the culture is associated with an increased content of unsaturated fatty acids and, on the contrary, with a decreased content of saturated fatty acids. Content of total lipids depends on age of the culture and is inversely related with production of the antibiotic mucidin.     

Identification of three distinct spectral species of yeast mitochondrial cytochrome b using a combination of respiratory inhibitors

Appropriate combination of specific inhibitors of electron transport in the cytochrome bc₁ segment of the respiratory chain of Saccharomyces cerevisiae allows the rapid resolution of three spectral forms of mitochondrial cytochrome b. (1) Addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to aerobic yeast submitochondrial particles preincubated with cyanide and mucidin in the presence of NADH reveals cytochrome b-561.5. (2) Addition of funiculosin to aerobic yeast submitochondrial particles preincubated with cyanide, mucidin and n-heptylhydroxyquinolineN-oxide in the presence of NADH reveals cytochrome b-558 independently of cytochrome b-561.5 and cytochrome b-565. (3) Specific resolution of cytochrome b-565 can be obtained either by addition of mucidin to aerobic submitochondrial particles preincubated with cyanide, DCMU and NADH, or by addition of antimycin plus an oxygen pulse to NADH-reduced particles, preincubated with cyanide, in the presence of ascorbate plus TMPD, or by addition of antimycin A in the presence of oxidized TMPD to aerobically NADH-reduced particles.     

Mucidin resistance in yeast. Isolation, characterization and genetic analysis of nuclear and mitochondrial mucidin-resistant mutants of Saccharomyces cerevisiae.

Mutants of Saccharomyces cerevisiae resistant to the antibiotic mucidin, a specific inhibitor of electron transport between cytochrome b and c, were isolated and divided into three phenotypic groups, as follows. Class 1 mutants were cross-resistant to a variety of mitochondrial inhibitors and exhibited no resistance at the mitochondrial level. Class 2 mutants were specifically resistant to mucidin exhibiting resistance also at the level of isolated mitochondria. Biochemical studies indicated that the mucidin resistance in class 2 mutants involved a modification of mucidin binding of inhibitory sites on the mitochondrial inner membrane without a significance change in the sensitivity of mitochondrial oxygen uptake to antimycin A, 2-heptyl-4-hydroxyquinoline-N-oxide, and 2,3-dimercaptopropanol. Class 3 was represented by a mutant which showed a high degree of resistance to mucidin and was cross-resistant to a variety of mitochondrial inhibitors at the cellular level but exhibited only a resistance to mucidin at the mitochondrial level. Genetic analysis of mucidin-resistant mutants revealed the presence of both nuclear and mitochondrial genes determining mucidin resistance/sensitivity in yeast. Resistance to mucidin in class 1 mutants was due to a single-gene nuclear recessive mutation (mucPR) whereas that in class 2 mutants was caused by mutations of mitochondrial genes. Resistance in class 3 mutant was determined both by single-gene nuclear and mitochondrial mutations. In the mitochondrial mutants the mucidin resistance segregated mitotically and the resistance determinant was lost upon induction of petite mutation by ethidium bromide. Allelism tests indicated that the mucidin resistance mutations fell into two genetic loci (MUC1 and MUC2) which were apparently not closely linked in the mitochondrial genome. Recombination studies showed that the two mitochondrial mucidin loci were not allelic with other mitochondrial loci RIB1, RIB2 and OLI1. An extremely high mucidin resistance at the cellular level was shown to arise from synergistic interaction of the nuclear gene mucPR and the mitochondrial mucidin-resistance gene (MR) in a cell. The results suggest that at least two mitochondrial gene products, responsible for mucidin resistance/sensitivity in yeast, take part in the formation of the cytochrome bc1 region of the mitochondrial respiratory chain.     

Drug resistance inYarrowia lipolytica

Six strains ofYarrowia lipolytica tested here were resistant to 10–20 g erythromycin or chloramphenicol per L glycerol-agar medium. Cells tolerating 4 g chloramphenicol per L were very rare and reverted rapidly to the highest resistance. In analogy with Ery^R mutants ofKluyveromyces lactis, our strains did not grow at 36°C but did not lose their viability at that temperature. Two levels of resistance were found with oligomycin and antimycin A,i.e. 10 and 3 mg/L in the former and 10 and 2 mg/L in the latter. The higher resistance levels segregated mitotically and were, therefore, controlled extrachromosomally. The lower resistance levels showed very frequent changes from sensitivity to resistance that prevented the genetic analysis of this resistance. An almost continuous range of tolerance to <5–400 μg mucidin per L was found in populations of the strains analyzed. Newly formed Muc^R cells were established only in the presence of the antibiotic. Pure cultures of Muc^R cells showed an extremely high instability caused by their lower viability and very low growth rate in the absence of mucidin. No loss of resistance to antimycin A was found, although Ant^R cells revealed similar negative selection. Mutability Muc^S»Muc^R and Muc^R»Muc^S was higher in Ant^R cells than in Ant^S ones.     

Comparison of the mutagenic activity of N-ethyl- N-nitrosourea and N-methyl- N’- nitro- N- nitrosoguanidine inLens esculenta

Mutagenic activity of N-ethyl-N-nitrosourea (ENU) and of N-methyl-N’-nitro-N--nitrosoguanidine (MNG) in lentil was studied. The highest proportion of segregating progenies with chlorophyll mutants and chimeric plants was 34.8% from the total number of analysed offsprings, ENU being applied in this case in the concentration of 0.005% for 20 h at 18 to 19 °C. When MNG was applied in the concentration of 0.001 % for 10h at 22 to 23 °C the proportion was 5.1%. Progenies segregating two or more chlorophyll mutants originated with ENU only; their relative frequencies varied from 1.4% to 7.1%. The number of different types of mutants or of their combinations segregating at the same time in the same progeny was shown to be dependent with the two agent tested on the mutagenic activity of the concentration used. The most efficient concentration of ENU induced the total of 8 different mutants at the same time, together with a combination of two or three mutant types in the same progeny. With MNG no combination of chlorophyll mutants in the same progeny was ever found simultaneously. The greatest number of mutants corresponding to 1 progeny M₁ was 0.53 when ENU was applied; with MNG the maximum values were approximately ten times lower. The maximum number M₂ of chlorophyll mutants and chimeric plants was 3.58% with ENU and 0.23 with MNG.     

The action of 1-alkyl-1-nitrosoureas and 1-alkyl-3-nitro-1-nitrosoguanidines on the M1 generation of barley andArabidopsis thaliana (L.) heynh.

The activity of 1-methyl-1-nitrosourea (MNH), 1-ethyl-1-nitrosourea (ENH), 1-methyl-3-nitro-1-nitrosoguanidine (MNG) and 1-ethyl-3-nitro-1-nitrosoguanidine (ENG) was tested on seeds of barley andArabidopsis. The activity of nitrosoamides tested was expressed by the germination and M₁ seedling height reduction of barley and M₁ root length reduction ofArabidopsis.

1. 1)
   After the action of both nitrosoureas (MNH and ENH) the germination of barley is at the same level as that of controls, even at concentrations, leading to a maximal reduction in the height of seedlings. After the action of both nitrosoguanidines (MNG and ENG) germination decreases in parallel with the decreasing seedling height. InArabidopsis no such differences in the relation germination to root length reduction were observed after nitrosoureas and nitrosoguanidines treatment. The differences in the M₁ generation of barley andArabidopsis after nitrosoguanidines treatment may be the reason for the non-mutagenic action of MNG and ENG in barley.     
   
   

The effect of visible light on mutagenic activity of 1-methyl-1-nitrosourea and 1-methyl-3-nitro-1-nitrosoguanidine

Irradiation with visible light reduced the mutagenic activity of 1-methyl-1-nitrosourea (MNH) and 1-methyl-3-nitro-1-nitrosoguanidine (MNG) solutions during their application onArabidopsis thaliana seeds for 24 hours. The antimutagenic effect of light was stronger with MNG than with MNH and in solutions buffered to pH 5 than in aqueous solutions. The decrease of activity of both the mutagens corresponded with the rate and degree of their photolysis. In the paper are presented the curves for the dependence of the decomposition of both substances and the formation of nitrous acid on pH and temperature of the solutions, amount of seeds.     

Metabolism of aromatic acids in the antibiotic-producing basidiomyceteOudemansiella mucida

Aromatic acids were determined in the mycelium and fermentation medium ofOudemansiella mucida. Coumaric acids (bothm- andp-),p-hydroxybenzoic acid (salicylic acid) and benzoic acid were found to predominate in the mycelium. Phenylacetic acid represents the main component in the medium. Phenylalanine ammonia-lyase catalyzing conversion of phenylalanine to cinnamie acid which is further metabolized to benzoic acid was detected in the mycelium. The results are discussed with respect to the synthesis of the antibiotic mucidin.     

Regulation of biosynthesis of secondary metabolites

Auxotrophic mutants ofStreptomyces aureofaciens, necessary for a further genetic analysis, were obtained by treating standard strains and their mutants (differing by their biosynthetic activities) with UV-radiation and N-methyl-N′-nitro-N-nitrosoguanidine (MNG). Mean frequencies of induced mutations varied within 0.001–0.18% depending on the strain and experimental conditions. Doses of UV radiation and MNG yielding 0.1–0.9% and 0.001–0.0009% survival, respectively, were found to be roost effective. The technique of subsequent enrichment was found to be more useful for the detection of mutants than the technique of total isolation. Most isolated auxotrophs required arginine. Among other growth factors, methionine, purine bases and serine (or glycine) were frequently required. A new modification of the enrichment technique utilizing a synthetic arginine-free medium was worked out for the exclusion of thearg mutants. The nutritional deficiency was in most auxotrophs associated with a decrease or a complete loss of the ability to produce secondary metabolites. Difficulties caused by unequal mutation patterns and high unstability of mutants as well as presumable loci of certain genetic blocks leading to auxotrophy are discussed.     

Condensed tannin,an antibiotic chemical from Gossypium hirsutum

Antibiotic activity against Heliothis virescens was found in flower buds of Gossypium hirsutum, experimental stock Texas 254. Relatively low antibiotic activity was found in hexane extract, high activity in methanolic extract and residue, and no activity in acetone and water extracts. A condensed tannin having a molecular weight of 4850 was isolated from methanolic extract by column chromatography on Sephadex G-25. It was the major antibiotic component, 3.4% of the dried flower bud. The condensed tannin at 0.2% in the diet retarded larval growth by 84%.     

A Three-Dimensional Skeletal Reconstruction of the Stem Amniote Orobates pabsti (Diadectidae): Analyses of Body Mass,Centre of Mass Position,and Joint Mobility

Orobates pabsti, a basal diadectid from the lower Permian, is a key fossil for the understanding of early amniote evolution. Quantitative analysis of anatomical information suffers from fragmentation of fossil bones, plastic deformation due to diagenetic processes and fragile preservation within surrounding rock matrix, preventing further biomechanical investigation. Here we describe the steps taken to digitally reconstruct MNG 10181, the holotype specimen of Orobates pabsti, and subsequently use the digital reconstruction to assess body mass, position of the centre of mass in individual segments as well as the whole animal, and study joint mobility in the shoulder and hip joints. The shape of most fossil bone fragments could be recovered from micro-focus computed tomography scans. This also revealed structures that were hitherto hidden within the rock matrix. However, parts of the axial skeleton had to be modelled using relevant isolated bones from the same locality as templates. Based on the digital fossil, mass of MNG 10181 was estimated using a model of body shape that was varied within a plausible range to account for uncertainties of the dimension. In the mean estimate model the specimen had an estimated mass of circa 4 kg. Varying of the mass distribution amongst body segments further revealed that Orobates carried most of its weight on the hind limbs. Mostly unrestricted joint morphology further suggested that MNG 10181 was able to effectively generate propulsion with the pelvic limbs. The digital reconstruction is made available for future biomechanical studies.     

Evaluation of the antibiotic activity of extracellular compounds produced by the Pseudomonas strain against the Xanthomonas citri pv. citri 306 strain

In this work, we evaluated the antibiotic activity of metabolites produced by the Pseudomonas sp. LV strain and their effects on the cell morphology of the Xanthomonas citri pv. citri 306 strain (Xcc 306), which causes citrus canker lesions. The LV strain was cultivated, centrifuged, a cell-free supernatant was treated with dichloromethane and then concentrated, frozen in liquid nitrogen and lyophilized. The dichloromethane phase (DP) was fractionated by vacuum liquid chromatography (VLC) using six organic solvents with a crescent polarity. The antibiotic activity of the DP and all the fractions from VLC were tested against Xcc 306 and only the F3 fraction showed antimicrobial activity. The antibiotic activity of F3 was determined by minimum inhibitory concentration and the action on the cell morphology of Xcc 306 carried out in glass tubes with cell suspensions plus F3 solution sampled at three different times (one, three and six hours). The effects were analyzed by electron microscopy. Both the DP and F3 showed antibiotic activity against Xcc 306 in in vitro experiments. Electron microscopy showed that the F3 fraction completely disrupted the cell integrity after six hours. In a greenhouse experiment, the DP and F3 fraction (highly effective in in vitro experiments), reduced the formation of lesions by approximately 80% and 94%, respectively.     

Should FNA be Performed in Patients with a Multinodular Goiter and Compressive Symptoms?

Objective: In this study, we aimed to determine whether preoperative thyroid fine-needle aspiration (FNA) in patients with multinodular goiter (MNG) and compressive symptoms influences the type of thyroid surgery performed, the incidence of recurrent thyroid cancer, or the need for successive surgery.Methods: We retrospectively reviewed the charts of 431 patients who underwent thyroidectomy at our institution from 2008 to 2011. Patients who presented with compressive symptoms and no prior FNA at initial presentation were included in this study.Results: Eighty patients met the criteria for our study, of which 46 (57.5%) underwent FNA prior to surgery and 34 (42.5%) were referred to surgery without FNA. The prevalence rates of malignancy (>1 cm) on surgical pathology in the FNA and non-FNA groups were 41% (n = 19) and 38% (n = 13), respectively. There was no statistically significant difference between the rate of total/subtotal thyroidectomies (71.7% in FNA vs. 79.4% in non-FNA, P = .31), lobectomies/partial thyroidectomies (28.3% in FNA vs. 20.5% in non-FNA, P = .43), neck lymph node dissections (P = .89) or subsequent surgeries (P = .72) between the 2 groups.Conclusion: Our findings show that preoperative FNA in patients with an MNG and compressive symptoms does not influence the type of surgery performed, short-term outcomes, or the need for subsequent surgeries. Further studies are needed to validate the need for preoperative FNA in such patients.Abbreviations:FNA = fine-needle aspirationMNG = multinodular goiterWHO = World Health Organization     

The cytochrome b of the sea urchin Paracentrotus lividus is naturally resistant to myxothiazol and mucidin

The ubiquinol:cytochrome c reductase activity of Paracentrotus lividus mitochondria is relatively insensitive to the specific inhibitors myxothiazol and mucidin. The I50 of myxothiazol and mucidin are three and two orders of magnitude higher, respectively, in P. lividus than in bovine heart mitochondria. The natural resistance of the P. lividus reductase to these inhibitors can be correlated with a single amino replacement, an alanine for a glycine at position 143, in the sequence of cytochrome b. This position is located in a conserved region of the molecule, believed to be important in the oxidation of ubiquinol by the reductase.     

Plasmid pUPI126-encoded pyrrolnitrin production by Acinetobacter haemolyticus A19 isolated from the rhizosphere of wheat

An Acinetobacter species identified as A. haemolyticus A19 produces an antibiotic and the enzyme chitinase. The antibiotic produced by A. haemolyticus A19 was extracellular and inducible by co-cultivation with Klebsiella pneumoniae in the optimum ratio 2:1, respectively. pH 7, temperature 28 °C, and addition of 2 % (w/v) NaCl are the most suitable environmental conditions for production and activity of the antibiotic. The antibiotic was produced in the early stationary growth phase (48 h) of A. haemolyticus A19. It has a very broad spectrum of antimicrobial activity against plant and human pathogenic bacteria and fungi. The antibiotic was extracted with ethyl acetate and purified by column chromatography with further purification by preparative thin-layer chromatography. Yield of the antibiotic was 15 mg/l. The antibiotic was active at very low concentrations, for example 50 μg/ml, and was water-soluble. It was stable at room temperature for up to 7 days. ¹H NMR analysis revealed the antibiotic was a pyrrolnitrin. It was found that pyrrolnitrin production by A. haemolyticus A19 was encoded by plasmid pUPI126 of molecular weight 25.7 kb. Plasmid pUPI126 was transferred to E. coli HB101 at a frequency of 5 × 10^?5 per μg DNA. It was also conjugally transformed to E. coli HB101 rif ^r mutants at a frequency of 5.9 × 10^?8 per recipient cell. Plasmid pUPI126 was 100 % stable in Acinetobacter and 95 % stable in E. coli HB101. Transconjugants and transformants both produced the antibiotic. This is the first report of plasmid-mediated pyrrolnitrin production by A. haemolyticus A19 isolated from wheat rhizosphere.     

Biosynthesis of fatty acids and sterols in relation to the antibiotic formation inOudemansiella mucida

The production of mucidin by the basidiomyceteOudemansiella mucida was negatively influenced by the application of D-glucitol as the main carbon source, the effect being independent of the growth rate of the mycelium. The rate of fatty acid synthesis was measured by incorporation of 1-¹⁴C-acetate. After 8 days of cultivation, the amount of fatty acids was approximately half that synthetized during cultivation on glucose. The specific rate of incorporation reached its maximum after seven days of cultivation. Incorporation of 2-¹⁴C-mevalonate into sterols was the same under the two sets of cultivation conditions. Acetate units from the degraded fatty acids are probably also utilized for antibiotic synthesis.