Growth of and fumitremorgin production by Neosartorya fischeri as affected by temperature, light, and water activity.

The effects of temperature, light, and water activity (aw) on the growth and fumitremorgin production of a heat-resistant mold, Neosartorya fischeri, cultured on Czapek Yeast Autolysate agar (CYA) were studied for incubation periods of up to 74 days. Colonies were examined visually, and extracts of mycelia and CYA on which the mold was cultured were analyzed for mycotoxin content by high-performance liquid chromatography. Growth always resulted in the production of the tremorgenic mycotoxins verruculogen and fumitremorgins A and C. The optimum temperatures for the production of verruculogen and fumitremorgins A and C on CYA at pH 7.0 were 25, 30, and 37 degrees C, respectively. The production of fumitremorgin C by N. fischeri has not been previously reported. Fumitremorgin production was retarded at 15 degrees C, but an extension of the incubation period resulted in concentrations approaching those observed at 25 degrees C. Light clearly enhanced fumitremorgin production on CYA (pH 7.0, 25 degrees C), but not as dramatically as did the addition of glucose, fructose, or sucrose to CYA growth medium (pH 3.5, 25 degrees C). Growth and fumitremorgin production was greatest at aw of 0.980 on CYA supplemented with glucose or fructose and at aw of 0.990 on CYA supplemented with sucrose. Growth and fumitremorgin production were observed at aw as low as 0.925 on glucose-supplemented CYA but not at aw lower than 0.970 on CYA supplemented with sucrose. Verruculogen was produced in the highest amount on all test media, followed by fumitremorgin A and fumitremorgin C.

Evaluation of a rapid tube assay for presumptive identification of Escherichia coli from veterinary specimens.

Three hundred sixty-six isolates of gram-negative, oxidase-negative bacteria from veterinary specimens were tested by a tube test for identification as Escherichia coli by production within 60 min of indole, beta-galactosidase, and beta-glucuronidase. The test correctly identified 255 of 269 isolates of E. coli (95% sensitivity) and correctly indicated that 97 of 97 isolates were not E. coli (100% specificity). We conclude that production of indole, beta-galactosidase, and beta-glucuronidase as measured by a rapid tube test is useful for identification of E. coli from veterinary specimens.

Determination of biotypes of Haemophilus influenzae and Haemophilus parainfluenzae a comparison of methods and a description of a new biotype (VIII) of H. parainfluenzae.

A total of 180 strains of Haemophilus influenzae and 119 strains of Haemophilus parainfluenzae were characterized with respect to biotype (i.e., production of indole, urease, and ornithine decarboxylase) using conventional biochemical methods and two commercially available biotyping systems: Trio-Tube Haemophilus system (Carr Microbiologicals) and the Rapid NH System (Inovative Diagnostic Systems). Concordance between the results of the Trio-Tube system and conventional biochemicals was achieved with 294 of the 299 test organisms (98.3%). With the Rapid NH System, concordance with the results of conventional biochemical tests was observed with 275 of the 299 tests strains (92.0%). One previously unrecognized biotype of H. parainfluenzae, designated biotype VIII, is described. Typical reactions of this biotype include indole production but no production of urease or ornithine decarboxylase.

Tyrosinase-catalyzed oxidation of dopa and related catechol(amine)s: a kinetic electron spin resonance investigation using spin-stabilization and spin label oximetry.

The oxidation of four catechol(amine)s by tyrosinase has been studied by electron spin resonance and optical methods. Rates of oxygen consumption and of dopaquinone and dopachrome formation during the oxidation of dopa have been measured, and compared with rates of dopasemiquinone production measured using spin-stabilization procedures. In the presence of spin-stabilizing metal ions, production of semiquinone is approximately quantitative. Time-dependent ESR spectra obtained from dopa and dopamine show a slow regeneration of semiquinone, suggesting that a semiquinone precursor is slowly reformed. In contrast, time-dependent spectra for 4-methylcatechol and N-acetyldopamine show decay of the primary semiquinone together with buildup of a secondary semiquinone apparently derived from the corresponding 6-hydroxy-catechol(amine). Thus, catecholamines that give rise to a cyclizable quinone show a pattern of behavior that differs from those that produce a non-cyclizable quinone. These results are discussed in terms of their possible significance to melanogenesis and the toxicity of catechol(amine)s, which has been attributed to production of semiquinones and/or other oxygen radicals.

Temporal separation of steps in the biosynthesis of verruculogen.

A mixture of 2[3H] mevalonate and U[14C] proline, given to Penicillium simplicissimum on days 2, 3, 4, 5 or 6 of stationary liquid culture, gave rise to radiolabelled verruculogen in which the ratio of 3H to 14C measured on day 8 increased from 2.0:1 to 9.5:1 over the period of administration of radiolabelled precursors. This suggests temporal separation of the formation of the diketopiperazine moiety and its subsequent prenylation. The putative precursor diketopiperazine, cyclo-L-tryptophanyl-L-proline, was isolated from 21 day culture broth.

Elicitor-mediated induction of tryptophan decarboxylase and strictosidine synthase activities in cell suspension cultures of Catharanthus roseus.

Treatment of one cell line (No. 615) of Catharanthus roseus c.v. Little Delicata with an elicitor preparation of autoclaved and homogenized Pythium aphanidermatum culture resulted in rapid accumulation of indole alkaloids. Alkaloid formation was preceded by rapid transient increases in the extractable activities of the enzymes tryptophan decarboxylase and strictosidine synthase. The induction of these two enzyme activities occurred when cells were transferred to alkaloid production medium or treatment with fungal elicitors. Treatment of this cell line with translational or transcriptional inhibitors prevented the Pythium-induced increases of enzyme activity as well as alkaloid accumulation. When cells were transferred to alkaloid production medium the induction of strictosidine synthase activity preceded that of tryptophan decarboxylase by many hours even when cells were also treated with Pythium elicitor. Results suggested that tryptophan decarboxylase induction proceeds only when endogenous tryptamine levels were decreased by two-third. The internal cellular level of tryptamine, therefore, could regulate expression of tryptophan decarboxylase, whereas induction of strictosidine synthase or of another enzyme in the biosynthetic pathway could control channeling of tryptamine into alkaloids. The results demonstrate that fungal elicitors can be used to facilitate studies of the factors which regulate expression of indole alkaloid pathway enzymes and their ultimate pathway products.

Favorece la Giardia lamblia la proliferación intestinal de bacterias? / Does giardia lamblia favor intestinal bacterial proliferation?

Con el propósito de estudiar si la G. lamblia se acompaña en el intestino de una gran población de bacterias productoras de indol, se investigó la concentración de sulfato de indoxil en la orina, antes y después de erradicar este parásito. Los resultados plantean, indirectamente, que la giardiasis se asocia a una proliferación de enterobacterias que sobrepasa en magnitud la que ordinariamente existe en el intestino

Effect of culture conditions on tremorgen production by some Penicillium species.

Four strains each of seven tremorgenic Penicillium species were grown under various conditions and tested for tremorgen production by intraperitoneal injection of mice and by chemical analysis. Half of the strains had previously been found to be tremorgenic on bioassay after growth on Czapek Dox yeast extract broth or potato-milk-sucrose broth for 3 weeks at 26 degrees C. In the tests reported here nearly all previously nontremorgenic strains were either tremorgenic to mice or produced tremorgens detectable by chemical analysis but did so after longer incubation periods than used in the original screening. Bioassay was not suitable for the estimation of absolute levels but was preferable to chemical analysis when the identity of the tremorgens was not known. Species and strains within species gave different responses to changes in culture medium, incubation temperature, light irradiation, and shaking. Overall, tremorgen production was maximal at 20 or 26 degrees C, increased with time, and was reduced in shaken culture.

Production and degradation of indole by gram-negative bacteria.

The indole reaction is performed in various modifications with regard both to the reagents and to the media used. Especially the medium has hitherto attracted little attention and there are divergent recommendations for its composition. The comparison of some broths without and with addition of tryptophan after incubation with different indole-positive species revealed that tryptone without tryptophan yielded stronger reactions than with tryptophan added. Other broths showed stronger reactions with tryptophan. Investigations revealed that numerous indole-positive as well as indole-negative species possess an enzyme system degrading indole. It is induced after one or two days of incubation and acts slowly. All species of the Morganella-Proteus-Providencia group possess it, as does Serratia. Some other species show different characteristics. The production of indole from peptone containing tryptophan and the degradation of indole by such species of bacteria are due to figures showing one or more peaks depending on time. Any false indole reactions described up to now may also be explained by the antagonism of the two enzyme systems. Therefore, the indole reaction should be standardized with regard to the medium used to avoid some of these difficulties.

Heterokaryosis between Aspergillus oryzae cyclopiazonic acid-defective strains: method for estimating the risk of inducing toxin production among cyclopiazonic acid-defective industrial strains.

Aspergillus oryzae strains are used extensively in the food industry. Some of these strains excrete alpha-cyclopiazonic acid (CPA), a mycotoxin which may provoke toxicoses in rats. Physicochemical methods may reveal the presence of this toxin, but they are inadequate to screen CPA-nonproducing (CPA-) strains. CPA production is revealed by either bacterial growth inhibition or alkalinization of the culture medium. This first biological property was used to devise a time-saving screening method to isolate mutants affected in their ability to produce CPA. The second method was used as a further test. After N-methyl-N'-nitro-N-nitrosoguanidine treatment, we isolated CPA- mutants from CPA producer strains (CPA+) and CPA+ mutants from CPA- strains. The mutants unable to produce CPA may be used in the food industry to reduce or eliminate the risk of intoxication in humans. Heterokaryon formation between different mutant strains was carried out to evaluate the risks of obtaining CPA from a mixture of mutants modified in their ability to synthesize this toxin. Pairings between two CPA+ strains always gave rise to CPA+ heterokaryons. Pairings between CPA+ and CPA- strains led, most often, to CPA+ heterokaryons. This could be directly correlated to the more frequent genotype (CPA+) in the heterokaryon. CPA hypoproducer and hyperproducer heterokaryons were obtained. Pairings between CPA- strains always gave rise to CPA- heterokaryons. These results suggest that the risks of producing this toxin from two CPA- individuals are not high.

Synthesis in vitro of 5,6-dihydroxyindole-2-carboxylic acid by dopachrome conversion factor from Cloudman S91 melanoma cells.

The oxidation of dopachrome to melanin occurs spontaneously in vitro but has been shown to be under enzymatic control in vivo. Dopachrome conversion factor, purified from Cloudman S91 melanoma cells, converts dopachrome, which is orange, to a colorless compound. We have purified this colorless compound and identified it as 5,6-dihydroxyindole-2-carboxylic acid. This compound is generated enzymatically from a solution containing dopa and dopachrome and, under these conditions, is not readily oxidized to melanin in solution at pH 6.8. The synthesis in vitro of 5,6-dihydroxyindole-2-carboxylic acid suggests that this compound may be an intermediate in the synthesis of melanin.

[Production of cyclopiazonic acid, its effect on the chick embryo and on 1-day-old cockerels]. / Produkce cyklopiazonové kyseliny, její ucinky na kurecí zárodek a na jednodenní kohoutky.

In 1979 to 1983, 148 strains of Penicillium cyclopium were isolated from wheat and from poultry feed mixtures; 11.5% of these strains produced cyclopiazonic acid at the rate of up to 500 mg per kg of wheat. Forty-seven percent of the 96 isolates of Aspergillus flavus and 56% of the nine isolates of Penicillium griseofulvum produced cyclopiazonic acid at the maximum rate of 80 and 10 mg per kg. The ED50 of cyclopiazonic acid for two, three and four days old chicken embryo was found to be 2.40, 4.70 and 2.70 micrograms, respectively. Teratogenic effects were observed only in the two days old embryos in which the caudal end of trunk was shortened at the frequency of 0.36 and microophthalmia occurred at the frequency of 0.33. The LD50 of cyclopiazonic acid in day-old cockerels was found to be 21.71 mg per kg of weight.

Inhibition of serum melatonin concentration and synthesis of brain indolealkylamines by monofluoromethyldopa in the rat.

The simultaneous effect of alpha-monofluoromethyldopa (MFMD) on the synthesis of rat brain indolealkylamines has been investigated both in the daytime and in the dark phase. The effect on serum melatonin concentration has also been determined in the dark period. MFMD inhibits 5-hydroxytryptophan decarboxylase in the pineal gland, hypothalamus, and the cerebral cortex. Simultaneous measurement of 5-hydroxytryptophan (5-HTP), 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) levels in the three brain areas revealed that MFMD caused large increases in pineal 5-HTP concentrations and substantial decreases in pineal 5-HT and 5-HIAA both in the light and dark phases; a significant decrease in daytime hypothalamic 5-HT content after 7-h pretreatment; and a large increase in dark-phase cortical 5-HT concentration after 4-h pretreatment. Serum melatonin levels were also reduced by the action of MFMD in the dark period. The evidence suggests that differences occur in the rate-limiting steps in the indolealkylamine biosynthesis in the three brain areas.

Rhythmic synthesis of various 5-methoxyindoles in the pineal gland of male adult golden hamsters, kept under the same artificial conditions throughout the year.

Until now the day/night and seasonal rhythmicity in the synthesis of 5-methoxyindoles (MI) is thought to be regulated by environmental factors, especially photoperiod and temperature. Endogenous factors are also implicated in the generation of N-acetyltransferase and hydroxyindole-O-methyltransferase activity rhythms. In the present experiments seasonal rhythmicity in the synthesis of MI in the pineal gland was investigated in hamsters kept under the same artificial conditions throughout the year. Though the environmental conditions were the same, day/night and seasonal rhythmicity in the production of MI in the pineal were observed indicating the existence of endogenous factors influencing the rhythmicities. In November, most of the MI showed the highest synthesis, MA and ML excepted, which were especially produced in July and September. The results obtained sustain the hypothesis that aMT is synthesized from MT rather than from aHT. Moreover, the rhythmicities in aMT synthesis are not identical to those found in aMT concentration as described in the literature. This indicates that synthesis and concentration of a compound are not comparable. At the end of the light period, when aMT injections have an antigonadotropic effect, a peak of aMT synthesis was always present. Although MI synthesis showed seasonal rhythmicity, no reproductive cycle occurred in the hamsters. At present, the concept that the pro- and/or antigonadal effects of the pineal are mediated by aMT seems to be the most acceptable. The present results, however, indicate that aMT and perhaps other MI, often regarded as factors influencing gonadal growth in golden hamsters, are not the only factors involved.

The effect of reduced neopterin on the synthesis of several methylated indoles in the pineal gland of adult male golden hamsters, kept under standardized conditions.

In the present study the effect was tested of reduced neopterin (RN) on the methylating capacity of the pineal gland of adult, male golden hamsters, housed under standardized conditions throughout the year. An effect of RN on the synthesis of a number of methylated compounds was, indeed, demonstrated. It is concluded that RN not only influences the indole metabolism by being the cofactor of tryptophan-hydroxylase, but that it might be involved in the regulation of other enzymes as well. Incubation with RN was most effective at the end of the light period. As this is also the time at which melatonin (aMT) injections cause gonadal atrophy in hamsters, kept under long photoperiod, this time of the day may be very important for aMT synthesis. A season-bound influence of RN was also demonstrated. The effect of RN was stimulatory in September, November and January for 5-methoxy-tryptamine (MT) and in November for 5-methoxytryptophan (MW) synthesis, but inhibitory in July. Furthermore, the effect of RN was stimulating for 5-methoxyindole-3-acetic acid (MA) and aMT in September, while the influence in the other months tested was absent or slightly inhibiting. These results suggest that the influence of RN in the pineal may be regulatory to various enzymes of the indole metabolism.

[Haemophilus influenzae isolated in Switzerland: antibiotic sensitivity and biotyping]. / Haemophilus influenzae isolés en Suisse: sensibilité aux antibiotiques et biotypisation.

208 H. influenzae (HI) strains originating from the whole of Switzerland have been analysed for capsular serotype, biotype, and susceptibility to the following antibiotics: ampicillin, chloramphenicol, tetracycline, co-trimoxazole, and ceftriaxone. Serotype b is the commonest of the encapsulated strains. Biotypes II and III (respiratory tract) and I (invasive diseases) are the biotypes most encountered. Ceftriaxone is the most active among the antibiotics tested: 0.03 microgram inhibits 100% of strains, whether penicillinase producers (PP) or not. To evaluate the rate of resistant HI in Switzerland, 1883 isolates, 206 of which originated from invasive diseases (meningitis, epiglottitis, septicemia) have been considered. The PP rate is about 4%, irrespective of the group considered. Among the isolates from the invasive diseases, 3 were resistant to chloramphenicol, and 1 to ampicillin and chloramphenicol. The value of 4% for the PP strains is not very high; however, because of its powerful antibacterial activity and its high penetration into the cerebrospinal fluid, it seems reasonable to consider the use of a third generation cephalosporin, such as ceftriaxone, for the early treatment of meningitis in infants. If such a drug is indicated as a first-choice antibiotic for this meningitis it should be confined to this use only, to avoid the emergence of resistant strains.

A new indole N-glycoside antibiotic SF-2140 from an Actinomadura. I. Taxonomy and fermentation of producing microorganism.

A new indole N-glycoside antibiotic SF-2140 which shows antiviral and weak antibacterial activity has been obtained from the cultured broth of an actinomycete strain. Strain SF-2140, designated Actinomadura albolutea sp. nov., was isolated from a soil sample collected in Hyogo Prefecture, Japan.