Biosynthesis of fusarochromanone and its monoacetyl derivative by Fusarium equiseti

One fluorescent compound previously named TDP-2 was isolated and purified from a rice culture of Fusarium equiseti (Alaska 2-2). Mass spectral and nuclear magnetic resonance data indicated that it is a C-3'-N-acetyl derivative of fusarochromanone, a newly discovered mycotoxin. Time course studies of synthesis of these two compounds on autoclaved rice and Czapek-Dox medium enriched with soybean peptone indicated that fusarochromanone was converted to TDP-2 in the cultures. A high concentration of peptone in the liquid medium may stimulate both fusarochromanone synthesis and its conversion to TDP-2.     

Immunomodulatory effects of fusarochromanones TDP-1 and TDP-2.

An in vitro peripheral lymphocyte blastogenesis system was used to investigate the biological activities of the fungal toxin fusarochromanone (TDP-1) and its monoacetyl derivative TDP-2. Briefly, cultures of human or bovine peripheral lymphocytes were exposed to TDP-1 or TDP-2 and a mitogen (PHA, Con A or PWM). After a standard incubation time, cell proliferation was quantified using the MTT bioassay. Human and bovine lymphocyte proliferation was inhibited by high concentrations of TDP-1; however, bovine lymphocyte proliferation was significantly increased at low concentrations of TDP-1. TDP-2 has similar but less pronounced effects on lymphocyte proliferation.     

Isolation and Structural Identification of a New Metabolite of Fusarium equiseti

A fluorescent compound was isolated and purified from rice cultures of Fusarium equiseti (Alaska 2-2). Mass spectrometry and nuclear magnetic resonance data indicated that its structure is 2,2-dimethyl-5-amino-6-(3′-hydroxyl-4′-methoxyl-butyryl)-4-chromone. It is an analog of the mycotoxin fusarochromanone, in which the amino group on C-3′ is replaced by a hydroxyl group and the hydroxyl group on C-4′ is replaced by a methoxyl group.     

Peptone Induction and Rifampin-Insensitive Collagenase Production by Vibrio alginolyticus

Vibrio alginolyticus produces an extracellular collagenase which requires specific induction by collagen or its high-molecular-weight fragments. Peptone also induces collagenase during the late exponential and early stationary growth phases. The peptone inducers have been shown to have a broad molecular weight range between 1,000 and 60,000. The peptone inducers supported slow growth of V. alginolyticus when supplied as the sole nitrogen source in minimal medium. Digestion of the peptone inducers with purified V. alginolyticus collagenase resulted in a decrease in their inducing ability, whereas digestion with trypsin or alpha-chymotrypsin did not. This indicated that induction by the inducers required the presence of collagenase-sensitive bonds. Prolonged digestion of the inducers with collagenase did not completely eliminate the inducing ability of the inducers. The peptone inducers acted as inhibitors of collagenase. A minimal medium induction system has been developed which involves resuspending cells at high density in a medium containing succinate, (NH(4))(2)SO(4), KH(2)PO(4), and the peptone inducer. Cells grown in minimal medium induce earlier than cells grown on peptone, Casamino Acids, or tryptone. Collagenase production was shown to occur for 30 to 60 min in the presence of rifampin at levels which completely inhibit the incorporation of [(3)H]uracil into trichloroacetic acid-precipitable material. Chloramphenicol completely and immediately abolished collagenase production, which together with labeling studies has confirmed that collagenase production involves de novo synthesis of the enzyme. Both glucose and Casamino Acids repressed collagenase production, although synthesis of the enzyme continued for 30 to 60 min after their addition. The repression of collagenase production by glucose and Casamino Acids was more severe than the inhibition of enzyme formation due to addition of rifampin.     

Mycotoxin production by Fusarium oxysporum and Fusarium sporotrichioides isolated from Baccharis spp. from Brazil

Fusarium oxysporum isolated from roots of and soil around Baccharis species from Brazil produced the trichothecenes T-2 toxin, HT-2 toxin, diacetoxyscirpenol, and 3'-OH T-2 (TC-1), whereas Fusarium sporotrichioides from the same source produced T-2 toxin, HT-2 toxin, acetyl T-2, neosolaniol, TC-1, 3'-OH HT-2 (TC-3), iso-T-2, T-2 triol, T-2 tetraol, and the nontrichothecenes moniliformin and fusarin C. Several unknown toxins were found but not identified. Not found were macrocyclic trichothecenes, zearalenone, wortmannin, and fusarochromanone (TDP-1).     

Mycotoxin production by Fusarium oxysporum and Fusarium sporotrichioides isolated from Baccharis spp. from Brazil.

Fusarium oxysporum isolated from roots of and soil around Baccharis species from Brazil produced the trichothecenes T-2 toxin, HT-2 toxin, diacetoxyscirpenol, and 3'-OH T-2 (TC-1), whereas Fusarium sporotrichioides from the same source produced T-2 toxin, HT-2 toxin, acetyl T-2, neosolaniol, TC-1, 3'-OH HT-2 (TC-3), iso-T-2, T-2 triol, T-2 tetraol, and the nontrichothecenes moniliformin and fusarin C. Several unknown toxins were found but not identified. Not found were macrocyclic trichothecenes, zearalenone, wortmannin, and fusarochromanone (TDP-1).     

Natural occurrence of the mycotoxin fusarochromanone, a metabolite of Fusarium equiseti, in cereal feed associated with tibial dyschondroplasia.

The mycotoxin fusarochromanone, a metabolite of Fusarium fungi, is able to induce tibial dyschondroplasia (TD) in chickens under experimental conditions. On the basis of health surveillance data on TD, two broiler farms with TD prevalence rates of up to 56% were identified. In the corresponding pelleted feed samples, fusarochromanone was detected in all 12 samples analyzed by column purification and TLC, with concentrations 4 to 59 micrograms/kg. No Fusarium fungi were available from the feed because of the pelleting process, but seven Fusarium equiseti strains previously isolated from Danish cereals were checked for fusarochromanone production, and all produced fusarochromanone at 57 to 1,435 mg/kg. Thus, the potential for fusarochromanone production by F. equiseti is considerable. The identification of fusarochromanone from feed and F. equiseti was confirmed by mass, infrared, and nuclear magnetic resonance spectral analyses. This is the first report of fusarochromanone as a naturally occurring contaminant.     

Natural occurrence of the mycotoxin fusarochromanone, a metabolite of Fusarium equiseti, in cereal feed associated with tibial dyschondroplasia

The mycotoxin fusarochromanone, a metabolite of Fusarium fungi, is able to induce tibial dyschondroplasia (TD) in chickens under experimental conditions. On the basis of health surveillance data on TD, two broiler farms with TD prevalence rates of up to 56% were identified. In the corresponding pelleted feed samples, fusarochromanone was detected in all 12 samples analyzed by column purification and TLC, with concentrations 4 to 59 micrograms/kg. No Fusarium fungi were available from the feed because of the pelleting process, but seven Fusarium equiseti strains previously isolated from Danish cereals were checked for fusarochromanone production, and all produced fusarochromanone at 57 to 1,435 mg/kg. Thus, the potential for fusarochromanone production by F. equiseti is considerable. The identification of fusarochromanone from feed and F. equiseti was confirmed by mass, infrared, and nuclear magnetic resonance spectral analyses. This is the first report of fusarochromanone as a naturally occurring contaminant.     

Production of pigments byMonascus purpureus in solid culture

Summary The effect of physical and nutritional factors on the production of pigments byMonascus purpureus FRR 2190 was studied using cultures grown on both rice and a synthetic medium that was solidified with carrageenan and extruded into rice-like particles. Pigment yield was highly sensitive to physical parameters. Optimal pigment formation in rice cultures occurred at an initial pH of 6 and an initial moisture content of 56%. Lower moisture contents led to a large decrease in pigment concentration. Red and yellow pigment production on solidified gel media was increased up to three-fold compared to that of liquid cultures of the same medium composition, particularly when peptone was used as the sole nitrogen source. Solid state rice cultures gave the highest pigment yields.     

Effect of rice--glycerol complex medium on the production of Lovastatin by Monascus ruber

Response surface methodology (RSM) was employed to study the effect of the composition of the rice-glycerol complex medium on the production of lovastatin (Lvs) by the ascomyceteMonascus ruber in mixed solid-liquid (or submerged) cultures at 25°C. Four components (rice powder, peptone, glycerol, glucose) were studied to evaluate, the approximate polynomial for all dependent variables, explaining their effects on the production of Lvs. The best composition derived from RSM regression was (in g/L) rice powder 34.4, peptone 10.8, , glucose 129, KNO₃ 8.0, MgSO₄·7H₂O 4.0 and glycerol 36.4 mL/L. With this composition, the Lvs production was 157 mg/L after 10 d of cultivation. In comparison with glycerol and glucose, the rice powder becomes a more suitable carbon source and represents a great potential for the production of Lvs.     

Fusarochromanone production by Fusarium isolates

Sixty two Fusarium isolates representing nine species from many parts of the world were screened for fusarochromanone production. A simplified method for the detection of fusarochromanone in culture filtrates or grain cultures was used. Under UV irradiation (364 nm) the chloroform phase from fusarochromanone-positive culture extracts fluoresced a characteristic bright blue color. Results were confirmed by thin-layer-chromatography comparison with pure fusarochromanone standards. Detection was possible in cultures as young as 1 week old. Biosynthesis of fusarochromanone was rare in Fusarium spp. and was only detected in three isolates of Fusarium equiseti, namely R-4482 (barley [Federal Republic of Germany]), R-6137 (barley [Alaska]), and R-8508 (potato [Denmark]), among all the isolates tested from various geographic sources.     

Role of Ca2+ in t-PA production by human embryonic lung diploid fibroblast, IMR-90 cells, stimulated by proteose peptone

Proteose peptone (p.peptone) remarkably induced tissue plasminogen activator (t-PA) activity in the conditioned medium of confluently cultured human embryonic lung diploid fibroblast, IMR-90 cells, in a dose-dependent manner. t-PA activity correlated well with the amount of t-PA antigen found in the conditioned medium of IMR-90 cells stimulated by p.peptone. t-PA production by IMR-90 cells stimulated by p.peptone was dependent on extracellular Ca2+ concentration and maximum t-PA production required approximately 3.6 mM extracellular Ca2+. Conversely, elimination of Ca2+ from the culture medium by EGTA, Ca2+ chelate agent, strongly inhibited t-PA production induced by p.peptone. t-PA production induced by p.peptone was inhibited in a dose-dependent manner by Verapamil, which inhibits Ca2+ uptake through the slow channels and also by W-7, an inhibitor of calmodulin. These results suggested that influx of extracellular Ca2+ into IMR-90 cells was caused by p.peptone and induced t-PA production by the cells.     

Use of response surface methodology to optimize protease synthesis by a novel strain of Bacillus sp. isolated from Portuguese sheep wool

Aims: To investigate the influence of yeast extract, peptone, temperature and pH upon protease productivity by Bacillus sp. HTS102 – a novel wild strain isolated from wool of a Portuguese sheep breed (Merino). Methods and Results: A 2⁴ full factorial, central composite design together with response surface methodology was used to carry out the experiments and analyse the results, respectively. Among the individual parameters tested, temperature and peptone concentration produced significant effects upon protease productivity. A high correlation coefficient (R² = 0·994, P < 0·01) indicated that the empiric second‐order polynomial model postulated was adequate to predict said productivity, with the optimum loci characterized by: temperature of 43°C, peptone content of 1·4 g l^?1, pH of 5·1 and yeast extract concentration of 10·0 g l^?1. Conclusions: Protease synthesis depends chiefly on temperature and peptone level. The maximum protease activity was more than twice that obtained with the basal medium, so the experimental design and analysis undertaken were effective towards process optimization. Significance and Impact of the Study: Rational choice of processing conditions for maximum protease productivity will be relevant if an economically feasible fermentation process based on Bacillus sp. HTS102 is intended.     

Fusarochromanone production by Fusarium isolates.

Sixty two Fusarium isolates representing nine species from many parts of the world were screened for fusarochromanone production. A simplified method for the detection of fusarochromanone in culture filtrates or grain cultures was used. Under UV irradiation (364 nm) the chloroform phase from fusarochromanone-positive culture extracts fluoresced a characteristic bright blue color. Results were confirmed by thin-layer-chromatography comparison with pure fusarochromanone standards. Detection was possible in cultures as young as 1 week old. Biosynthesis of fusarochromanone was rare in Fusarium spp. and was only detected in three isolates of Fusarium equiseti, namely R-4482 (barley [Federal Republic of Germany]), R-6137 (barley [Alaska]), and R-8508 (potato [Denmark]), among all the isolates tested from various geographic sources.     

A MUCILAGE-FORMING LACTOBACILLUS SPECIES ISOLATED FROM CIDER. PART II. POLYSACCHARIDE FORMATION

SUMMARY: Cultures grown at 25° under an atmosphere of CO₂ in a medium containing maltose, inorganic salts and ethanolic extracts of peptone and yeast autolysate produced a polysaccharide, which was isolated and purified. Examination by a variety of chemical and biological methods indicated that it was a dextran of low molecular weight.     

Effect of environmental conditions on the production of two extracellular proteolytic enzymes byVibrio SA1

The production of two extracellular proteases, an endopeptidase and an aminopeptidase, by the marine bacteriumVibrio SA1 was studied in batch cultures. The production of the proteases was induced during growth of the organism in peptone media and by several amino acids during growth in minimal media. It was repressed by easily metabolisable carbon compounds such as glucose, lactate and succinate during growth in peptone media. During growth in a lactate basal medium, phenylalanine was one of the best inducers and this amino acid was therefore used in further experiments. That lactate dit not repress the synthesis of the proteases during growth in the lactate basal medium supplemented with 2mm phenylalanine as an inducer, appeared to be a consequence of the low iron content of this medium. Growth curves ofVibrio SA1 on such media showed a period of linear growth during which protease production was observed. When the iron concentration was made sufficiently high to prevent linear growth, the synthesis of the proteases remained repressed. Apparently by imposing an iron limitation on the organism, catabolite repression by lactate was relieved. Similarly, when growth was limited by very low values of the dissolved oxygen tension in the medium, a high rate of protease synthesis was found which was immediately repressed when the oxygen limitation was released. The results indicate that the growth rate and/or a factor associated with the energy metabolism play a role in the regulation of the synthesis of the enzymes.This study was supported by the Foundation for Fundamental Biological Research (BION), which is subsidised by the Netherlands Organization for the Advancement of Pure Research (ZWO).     

NUCLEOLAR AND BIOCHEMICAL CHANGES DURING UNBALANCED GROWTH OF TETRAHYMENA PYRIFORMIS

Numerous nucleoli can be observed in the macronucleus of the logarithmically growing ciliated protozoan Tetrahymena pyriformis; at late log phase the nucleoli aggregate and fuse. In stationary phase this fusion process continues, leaving a very few large vacuolated nuclear fusion bodies in the nucleus. When these stationary phase cells are placed into fresh enriched proteose peptone medium, the large fusion bodies begin to disaggregate during the 2.5-hour lag phase before cell division is initiated. By 3 to 6 hours after inoculation the appearance of the nucleoli in many cells returns to what it was in logarithmic cells. In view of the possible role of nucleoli in ribosome synthesis, attempts were made to correlate the morphological changes to changes in RNA and protein metabolism. The beginning of an increased RNA synthesis was concomitant with the beginning of disaggregation of the large fusion bodies into nucleoli, which was noticed in some cells by 1 hour after the return to fresh enriched proteose peptone medium. Increased protein synthesis then followed the increased RNA synthesis by 1 hour. The supply of RNA precursors (essential pyrimidines) were removed from cultures which were grown on a chemically defined synthetic medium, in order to study the relation between nucleolar fusion and synthesis of RNA and protein. Pyrimidine deprivation drastically curtailed RNA and protein synthesis, but did not cause fusion of nucleoli. When pyrimidines were added back to this culture medium, RNA synthesis was immediately stimulated and again preceded an increased protein synthesis by 1 hour. These studies suggest the involvement of unfused nucleoli in RNA and protein synthesis and demonstrate the extreme plasticity of nucleoli with respect to changes in their environment.     

高羊肚菌菌丝球液体培养的研究

本文探讨了高羊肚菌菌丝球在不同液体培养基中的生长情况。结果表明,在黄豆芽玉米玢培养基中菌丝球最多,大小均匀,菌丝生长量最大,在麦麦夫蛋白胨培养基上菌丝的糖含量最高。     

Ubiquilin-2 (UBQLN2) binds with high affinity to the C-terminal region of TDP-43 and modulates TDP-43 levels in H4 cells: Characterization of inhibition by nucleic acids and 4-aminoquinolines

Recently, it was reported that mutations in the ubiquitin-like protein ubiquilin-2 (UBQLN2) are associated with X-linked amyotrophic lateral sclerosis (ALS), and that both wild-type and mutant UBQLN2 can co-localize with aggregates of C-terminal fragments of TAR DNA binding protein (TDP-43). Here, we describe a high affinity interaction between UBQLN2 and TDP-43 and demonstrate that overexpression of both UBQLN2 and TDP-43 reduces levels of both exogenous and endogenous TDP-43 in human H4 cells. UBQLN2 bound with high affinity to both full length TDP-43 and a C-terminal TDP-43 fragment (261–414 aa) with K_D values of 6.2 nM and 8.7 nM, respectively. Both DNA oligonucleotides and 4-aminoquinolines, which bind to TDP-43, also inhibited UBQLN2 binding to TDP-43 with similar rank order affinities compared to inhibition of oligonucleotide binding to TDP-43. Inhibitor characterization experiments demonstrated that the DNA oligonucleotides noncompetitively inhibited UBQLN2 binding to TDP-43, which is consistent with UBQLN2 binding to the C-terminal region of TDP-43. Interestingly, the 4-aminoquinolines were competitive inhibitors of UBQLN2 binding to TDP-43, suggesting that these compounds also bind to the C-terminal region of TDP-43. In support of the biochemical data, co-immunoprecipitation experiments demonstrated that both TDP-43 and UBQLN2 interact in human neuroglioma H4 cells. Finally, overexpression of UBQLN2 in the presence of overexpressed full length TDP-43 or C-terminal TDP-43 (170–414) dramatically lowered levels of both full length TDP-43 and C-terminal TDP-43 fragments (CTFs). Consequently, these data suggest that UBQLN2 enhances the clearance of TDP-43 and TDP-43 CTFs and therefore may play a role in the development of TDP-43 associated neurotoxicity.