Electron spin resonance evaluation of pigment accumulation during asexual sporulation in Aspergillus flavus

The association between pigment accumulation and a free radical electron spin resonance (ESR) signal produced during sporulation in Aspergillus flavus was investigated.     

Malic acid accumulation by Aspergillus flavus

Summary Scanning electron microscopy revealed that Aspergillus flavus produced unusual crystals and hair-like processes during its l-malic acid production phase. Crystallinic dendritic aggregates were formed on the hyphae growing as pellets. The size and number of crystal aggregates increased during the fermentation in parallel with l-malic acid accumulation. The crystals (composed of calcium malate as well as small amounts of calcium succinate and calcium fumarate) were removed from the hyphae, after incubation with 6N HCl. On day 5 of the fermentation, about 9% of the total amount of l-malic acid produced was accounted for by the attached crystals. In addition to crystal formation we observed the appearance of hair-like processes during the early phase (2 days) of malic acid production only.     

Malic acid accumulation by Aspergillus flavus

Summary ¹³C Nuclear magnetic resonance and fumarase and NAD-malate dehydrogenase isoenzyme studies were carried out in a strain of A. flavus which produces relatively high levels of l-malic acid from glucose. The results of the ¹³C NMR showed that the ¹³C label from [1-¹³C] glucose was incorporated only to C-3 (-CH₂-) of l-malic acid and indicated that this acid must be synthesized from pyruvate mainly via oxaloacetate. Electrophoretic analysis has established the presence of unique mitochondrial and cytosolic isoenzymes for fumarase and malate dehydrogenase. Changes in the isoenzyme pattern were observed for malate dehydrogenase but not for fumarase during acid production. Cycloheximide inhibited profoundly both l-malic acid production and the increase in the major isoenzyme of malate dehydrogenase, without affecting either the total activity of fumarase or its isoenzyme pattern. The results suggested that de novo protein synthesis is involved in the increase in the activity of the major isoenzyme of malate dehydrogenase and that this isoenzyme is essential for l-malic acid production and accumulation.     

Coordinate control of secondary metabolite production and asexual sporulation in Aspergillus nidulans

Microbial secondary metabolite production is frequently associated with developmental processes such as sporulation, but there are few cases where this correlation is understood. Recent work with the filamentous fungus Aspergillus nidulans has provided new insights into the mechanisms coordinating production of the toxic secondary metabolite sterigmatocystin with asexual sporulation. These processes have been shown to be linked through a common need to inactivate a heterotrimeric G protein dependent signaling pathway that, when active, serves to stimulate growth while blocking both sporulation and sterigmatocystin biosynthesis.     

Electron spin resonance dating in paleoanthropology

Many archeological and paleoanthropological sites cannot be dated by well established and common dating techniques such as uranium series (U-series) or argon-argon (⁴⁰Ar/³⁹Ar) because of the lack of materials that are suitable for these techniques. Most sites, however, contain bones and teeth, and the latter can be used to obtain electron spin resonance (ESR) age estimates. The theoretical age range of ESR dating accuracy lies between a few thousand and more than a million years. In practice, continuing uranium accumulation increases the uncertainty of ESR age assessments in such a way that most age assignments beyond 300,000 years are very uncertain.     

Electron spin resonance of calmodulin-vanadyl complexes

X-band (9.2 GHz) electron spin resonance spectroscopy was used to investigate the binding of vanadyl to calmodulin. Solution spectra, obtained at ambient temperature with various VO2+:calmodulin molar ratios, suggested a binding stoichioimetry of 4 mol of VO2+/mol of protein and the possibility of two classes of binding sites. The latter was confirmed by using frozen solutions of calmodulin-VO2+ complexes that gave splitting of the spectral bands corresponding to the parallel components, which was particularly pronounced with the three high-field peaks. Competition of Ca2+ for the VO2+ binding sites was investigated, and the results indicated that two of the VO2+ sites corresponded to two of the Ca2+ sites; the other two VO2+ binding sites may have a higher affinity for VO2+ than for Ca2+ or they may correspond to Ca2+-independent sites. These results demonstrate that electron spin resonance spectroscopy can be used advantageously to probe subtle differences in the microenvironments of metal-binding sites in calmodulin.     

Electron spin resonance studies on anthocyanins

The ESR characteristics of anthocyanins and their congeners have been determined using both crystalline and non-crystalline preparations. For the latter, a micro-technique has been proposed. The results show that red and violet pigments specifically exhibit a distinct signal at g=2·057 being different from blue anthocyanins. It has been suggested that the g=2·057 signal arises from the unpaired electron of pyrylium ring. Comparison of ESR spectra of isolated anthocyanins with those in fresh petals has further shown that there is no difference in the g=2 region of ESR spectra and that the appearance of the g=2·003 signal is thus not due to anthocyanins alone.     

Suppressor mutations bypass the requirement of fluG for asexual sporulation and sterigmatocystin production in Aspergillus nidulans

Asexual sporulation (conidiation) in the filamentous fungus Aspergillus nidulans requires the early developmental activator fluG. Loss of fluG results in the blockage of both conidiation and production of the mycotoxin sterigmatocystin (ST). To investigate molecular mechanisms of fluG-dependent developmental activation, 40 suppressors of fluG (SFGs) that conidiate without fluG have been isolated and characterized. Genetic analyses showed that an individual suppression is caused by a single second-site mutation, and that all sfg mutations but one are recessive. Pairwise meiotic crosses grouped mutations to four loci, 31 of them to sfgA, 6 of them to sfgB, and 1 each to sfgC and sfgD, respectively. The only dominant mutation, sfgA38, also mapped to the sfgA locus, suggesting a dominant negative mutation. Thirteen sfgA and 1 sfgC mutants elaborated conidiophores in liquid submerged culture, indicating that loss of either of these gene functions not only bypasses fluG function but also results in hyperactive conidiation. While sfg mutants show varying levels of restored conidiation, all recovered the ability to produce ST at near wild-type levels. The fact that at least four loci are defined by recessive sfg mutations indicates that multiple genes negatively regulate conidiation downstream of fluG and that the activity of fluG is required to remove such repressive effects.     

Electron spin resonance of vanadium in Amanita muscaria

Electron spin resonance shows that vanadium(IV) is present as the same compound in the skin, gills, volva and flesh of the fungus Amanita muscaria.     

Electron spin resonance studies of starch-water-probe interactions

Interactions between starch, water and stable nitroxide radicals were studied by electron spin resonance. The motional properties of TEMPO, 4-(2-bromoacetamido) TEMPO (BrAcTEMPO), 5-DOXYL-stearic acid and 16-DOXYL-stearic acid probes as well as a label covalently attached to amylopectin were investigated in concentrated (10–50%) starch-water systems as a function of temperature, concentration of polymer and storage period. Compared with the free probes in solution, TEMPO and BrAcTEMPO showed slower tumbling rates in starch-water dispersions or gels, suggesting a higher microviscosity in the probe's environment. The spectra, however, remained motionally narrowed. In contrast, the three line spectra of the fatty acid probes in solution became highly anisotropic in the presence of starch. The results indicated that these probes were highly immobilized at room temperature by the starch granules or by the polysaccharide gel matrix. These interactions are weakened at elevated temperatures where the spectra revealed the presence of both motionally narrowed and motionally slowed spin populations. The nitroxide label on the amylopectin exhibited a much slower mobility than the corresponding free probe as well as being found to be more motionally sensitive to temperature changes; such motional behavior was interpreted as reflecting contributions from rotation of the label around the chain backbone as well as local segmental motion of the polymer chain itself. Starch gels doped with free probes or the spin labelled amylopectin displayed no change in the motion of the nitroxide group upon storage, i.e. the tumbling rates did not follow the time-dependent conformational changes associated with the retrogradation phenomenon.