John Cazin

Toxicity of terpenoid deterrents to the leafcutting antAtta cephalotes and its mutualistic fungus.

Four natural products, of varying activity as deterrents of leafcutter attack, were tested for their effects on ant survival and on the growth of the mutualistic attine fungus. The substances were incorporated into an artificial liquid diet for bioassays on the ants or included in an agar culture medium for fungus growth-inhibition studies. Three of the four compounds exhibited deleterious effects on either adult leafcutting ants or their mutualistic fungus, and there appeared to be some correlation between deterrency and activity in these toxicity assays. The implications of these findings for leafcutting ant foraging patterns are discussed.

Isolation and characterization of highly purified streptavidin obtained in a two-step purification procedure from Streptomyces avidinii grown in a synthetic medium

A method is described for isolation of streptavidin from cultures of Streptomyces avidinii grown in a synthetic culture medium for 6-10 days. Streptavidin is precipitated directly from culture supernatant fluid using 80% ammonium sulfate, and the precipitate is dialyzed against water and centrifuged at 40,000 X g for 60 min. The absorbency coefficient at 280 nm of purified streptavidin was estimated to be 31.7142 +/- 0.1806 for a 1% solution. The protein appeared to be greater than 90% homogeneous by gel permeation chromatography and polyacrylamide gel electrophoresis. No biotin-binding molecules less than 70 kDa in size were detected at any step during the purification of streptavidin. Streptavidin was able to maintain a stable crosslink between two biotinylated molecules in a solid-phase assay. Streptavidin purified by this method was stable in 50% glycerol/water at -20 degrees C for more than 1 year. Lyophilization or iodination did not produce apparent damage to the protein.

Induction of humoral antibody response by soluble polysaccharide of Cryptococcus neoformans

Mice were immunized with purifiedCryptococcus neoformans soluble polysaccharide, or with the polysaccharide coupled to methylated bovine serum albumin or methylated bovine gamma globulin. The polysaccharide-methylated protein complexes were no more immunogenic than the purified polysaccharide when used without Freunds incomplete adjuvant; however, the methylated protein complexes induced higher levels of antibody than purified polysaccharide when emulsified in the adjuvant. Sera from mice were titrated by passive hemagglutination, and maximum antibody titers were observed 14 to 21 days after immunization. Antibody titers declined rapidly after 14 to 21 days in mice immunized with antigen alone; whereas, animals immunized with cryptococcal antigen emulsified in adjuvant remained at peak levels throughout a six week experimental period. All antigens were immunogenic over a wider dosage range when contained in adjuvant. Individual mice immunized with an adjuvant emulsion of purified polysaccharide varied widely in the amount of antibody produced, with some of the animals producing no detectable antibody.

Cissampentin: A new bisbenzylisoquinoline alkaloid from Cissampelos fasciculata

A new bisbenzylisoquinoline alkaloid, cissampentin, has been isolated from the aerial parts of Cissampelos fasciculata. Detailed interpretation of various spectra allowed identification of most structural features, including a rare methyleneoxy bridge. Although attempted methylation of this alkaloid led to complex mixtures, reaction with diethyl phosphorochloridate gave a single diethyl phosphate derivative and allowed assignment of a 7′-11 ether linkage. Bioassays indicate significant activity as a repellent to the leafcutter and Acromyrmex octospinosus, and limited antifungal activity.

Production of streptavidin in a synthetic medium

A simple, inexpensive procedure for producing streptavidin has been described. The biotin-binding protein was produced by growing Streptomyces avidinii in a synthetic liquid culture medium containing L-asparagine as the sole nitrogen source. With this procedure, extraneous proteinaceous substances inherently present in culture media prepared with yeast extract or with peptones were not present to interfere with isolation and purification of streptavidin. When harvested after 7-8 days of incubation, the culture fluid was relatively free of contaminating cell breakdown products. Maximal production of streptavidin (100-120 mg/l) was obtained in 8-10 day cultures. For some applications, the culture fluid can be used directly as a source of streptavidin. Under the same conditions used to grow S. avidinii, 11 other actinomycete strains and 134 eumycetes were found to lack the capacity to produce detectable amounts of an extracellular biotin-binding protein.

The comparative virulence of thermotolerant Mucorales species in mice

The comparative virulence of thermotolerant Mucorales was determined for cortisone-treated and untreated Swiss mice by intravenous administration of spores. The measure of virulence was based on an LD50 value, calculated after the 30-day observation period. Of the known etiological agents of mucormycosis, Mucor meihei, M. pusillus, Rhizopus arrhizus, R. chinensis, R. cohnii, R. microsporus, R. oryzae, R. rhizopodiformis and Cunninghamella elegans were able to produce fatal infections in mice; whereas, Mucor alternons, M. ramosissimus and Syncephalastrum racemosum were avirulent at dosages of up to 105 spores. Of those thermotolerant species which have not been reported to cause mucormycosis in human beings, Radiomyces embreei, R. spectabilis, Rhizopus oligosporus and Thermomucor indicae-seudaticae were found to produce fatal infections in mice; whereas, an isolate of Mycotypha africana was avirulent. Cortisone treatment of mice was found to lower their resistance to infection at a given spore dosage as measured by ET50 values.

Serological diagnosis of petriellidiosis (allescheriosis). I. Isolation and characterization of soluble antigens from Allescheria boydii and Monosporium apiospermum.

Soluble antigens in culture filtrates of three strains of Petriellidium boydii and three strains of Monosporium apiospermum were examined. Antigens were separated from concentrated crude filtrates by anion-exchange chromatography. A single major peak (Antigen 1), constituting a significant proportion of the total recoverable carbohydrate, was the only product isolated from each of four chromatographed filtrates. Depending on the fungus strain, Antigen 1 consisted of 90–96% carbohydrate, 3–4% protein, and 2–4% nucleic acid. Antigen 1 was found to consist of a population of molecules with a heterogeneous molecular size when assayed by gel filtration chromatography; however, isolated fractions of Antigen 1 proved to be immunologically identical when examined by Ouchterlony immunodiffusion. In addition, Antigen 1 from each strain was immunologically identical to similar preparations of Antigen 1 from the other five fungus strains. Chromatography of culture filtrates from two strains of M. apiospermum revealed a second peak (Antigen 2), which was found to consist of 70% carbohydrate, 16% protein, and 4% nucleic acid. Although Antigen 2 contained four times as much protein as Antigen 1, the two preparations were immunologically identical by immunodiffusion tests. Ion-exchange chromatography proved to be a useful procedure for isolating antigens of P. boydii and M. apiospermum from culture filtrates.

Serological diagnosis of petriellidiosis (allescheriosis). II. Indirect (passive) hemagglutination assay for antibody to polysaccharide antigens of Pertriellidium (Allescheria) boydii and Monosporium apiospermum.

The indirect (passive) hemagglutination assay (IHA) was utilized for detection of humoral antibody resulting from an experimental or a natural petriellidiosis. Polysaccharide antigens from three strains ofP. boydii and three strains ofM. apiospermum were isolated from culture filtrates of these fungi by anion-exchange chromatography and were coupled to sheep erythrocytes using chromium chloride. Rabbits were inoculated by the subcutaneous route with viable spores of these fungi and were bled periodically to obtain serum samples. IHA assay of serial specimens revealed varying levels of humoral antibody. Antibody stimulation by the subcutaneous route of inoculation appeared advantageous inasmuch as high IHA titers (1∶1024 to 1∶4096) could be achieved without endangering the life of the animal or exposing investigators to draining lesions. When serum specimens from six human beings with culturally proven petriellidiosis were tested by the IHA method, five were shown to have antibody levels of 1 ∶ 16 to 1 ∶ 512. IHA assay of serum samples obtained from normal human beings showed measurable levels (1 ∶ 4 to 1 ∶ 32) of antibody in 29 of 30 specimens. A serum pool composed of the higher titered specimens from the normal group was adsorbed with killed whole spores ofM. apiospermum, and the attached serum components were eluted by altering the pH. The immune globulin that had adsorbed to the spores was found, by immunoelectrophoresis, to be IgG. An indirect hemagglutination inhibition assay, developed from the IHA assay, provided a means for determining whether heterologous fungus antigens were capable of cross-reacting with petriellidium antibody. Soluble antigens from several other genera of fungi failed to react with petriellidium antibody in the IHA assay, suggesting that the reaction of petriellidium antibody with homologous antigen was genus specific.

MORPHOLOGICAL AND PHYSIOLOGICAL STUDIES OF THE GENUS RADIOMYCES

The range of morphological and physiological variation of ten isolates of Radiomyces embreei and four of R. spectabilis was examined. Scanning electron microscopy revealed that a single R. embreei isolate (1186) produced significantly smaller sporangiospores than nine other R. embreei isolates grown under four different culture conditions. The effect of temperature on hyphal growth and sporangiospore germination revealed that R. spectabilis isolates grew from 15 to 40 C, and two of the four isolates grew at 45 C, whereas nine of the 10 R. embreei isolates grew from 15 to 45 C, and five of these nine grew at 50 C. The unusual R. embreei isolate (1186) grew only at temperatures between 15 and 35 C. Sporangiospore germination occurred in eight of 10 isolates of R. embreei at temperatures between 25 and 50 C, and nine of the 10 isolates germinated between 25 and 45 C, whereas germination did not occur with isolate 1186 at 40 C or above. Sporangiospore germination of R. embreei isolates usually was 90% or greater in as few as 4 to 6 h, while only 1 to 5% of 1186 sporangiospores germinated in up to 24 h. Slow sporangiospore germination also was seen with R. spectabilis isolates, with two reaching only I to 3% germination in 16 to 24 h, and one strain attaining 31% germination in 10 h. Spore germination of R. spectabilis isolates and R. embreei 1186 continued over the period of up to one wk. The genus Radiomyces contains two species, Radiomyces spectabilis Embree (1959) and Radiomyces embreei Benjamin (1960). These fungi are Zygomycetes, members of the Mucorales, and were placed in the family Radiomycetaceae by Ellis and Hesseltine (1974). Both species have been isolated from dung of rodents and lizards collected from the southwestern United States and Mexico, and R. embreei also has been isolated from soil in Arizona (Ranzoni, 1968). Recently, an isolate of R. spectabilis was obtained from India. Both species of Radiomyces are homothallic, produce smooth-walled zygospores (FIG. 1), and sporangiophores bearing either multispored sporangioles (R. spectabilis, FIG. 2), or singlespored sporangioles (R. embreei, FIG. 3). The ultrastructure of R. embreei sporangioles was demonstrated by Dykstra (1974), who reported that each sporangiospore is surrounded by a distinct outer sporangiole wall which fragments at the time of germination. Embree (1964) determined the rate of asexual spore germination and hyphal growth in relation to temperature with the type isolates of the genus. He reported both species to be thermotolerant, and capable of growth at 45 C or greater. Since the ability to

A simplified routine method for determining carbohydrate fermentation by yeasts.

The use of commercially available materials in a minimal medium contributes to the simplicity, reliability, and reproducibility of a method described to demonstrate carbohydrate fermentation reactions by yeasts. The medium consists of 1% yeast extract and 2% test carbohydrate in distilled water, dispensed in a modified Durham fermentation tube. Carbohydrate fermentation patterns can usually be obtained within a period of 7 days. The ability of yeasts to ferment carbohydrates is determined strictly on the basis of gas production from the substrate. The method proved reliable in reproducing established fermentation patterns for 112 different yeast strains representing 13 separate genera.The use of commercially available materials in a minimal medium contributes to the simplicity, reliability, and reproducibility of a method described to demonstrate carbohydrate fermentation reactions by yeasts. The medium consists of 1% yeast extract and 2% test carbohydrate in distilled water, dispensed in a modified Durham fermentation tube. Carbohydrate fermentation patterns can usually be obtained within a period of 7 days. The ability of yeasts to ferment carbohydrates is determined strictly on the basis of gas production from the substrate. The method proved reliable in reproducing established fermentation patterns for 112 different yeast strains representing 13 separate genera. Der Gebrauch von handelsüblichen Grundbestandteilen in einem geringen Medium trägt zu der Einfachheit, Zuverlässigkeit und Reproduzierbarkeit der beschriebenen Methode, um die Gärungsreaktionen der Karbohydrate durch Hefen zu veranschaulichen, bei. Das Medium besteht aus einem 1 prozent. Hefenauszug und aus einem 2 prozentigen Testkarbohydrat in destilliertem Wasser, die auf modifiziertes Durham Gärungsreagensglas verteilt sind. Das Karbohydrat-Gärungsmodell kann gewöhnlich innerhalb einer Periode von sieben Tagen erhalten werden. Die Fähigkeit von Hefen, Karbohydrate zu vergären, ist ausschließlich auf Grund der Gasentwicklung aus den Produkten bestimmt. Die Methode erwies sich zuverlässig in der Erzeugung festgestellter Gärungsmodelle aus 112 Hefen, die 13 verschiedene Gattungen darstellten.