Kinetics of cell growth and heterologous glucoamylase production in recombinant Aspergillus nidulans

In the work, a study of cell growth and the regulation of heterologous glucoamylase synthesis under the control of the positively regulated alcA promoter in a recombinant Aspergillus nidulans is presented. We found that similar growth rates were obtained for both the host and recombinant cells when either glucose or fructose was employed as sole carbon and energy source. Use of the potent inducer cyclopentanone in concentrations greater than 3 mM resulted n maximum glucoamylase concentration and maximum overall specific glucoamylase concentration over 80 h of batch cultivation. However, cyclopentanone concentrations in excess of 3 mM also showed an inhibitory effect on spore germination as well as fungal growth. In contrast, another inducer, threonine, had no negative effect on spore germination even when concentrations of up to 100 mM were used with either glucose or fructose as carbon source. Glucoamylase synthesis in the presence of glucose plus either inducer did not begin until glucose was totally depleted, suggesting strong catabolite repression. Similar results were obtained when fructose was employed, although low levels of glucoamylase were detected before fructose depletion, suggesting partial catabolite repression. The highest enzyme concentration (570 mg/L) and overall specific enzyme concentration (81 mg/g cell) were observed in batch culture when cyclopentanone was the inducer and fructose the primary carbon source. A maximum glucoamylase concentration of 1.1 g/L and an overall specific glucoamylase concentration of 167 mg/g cell were obtained in a bioreactor using cyclopentanone as the inducer and limited-fructose feeding strategy, which nearly doubles the glucoamylase productivity from batch cultures. (c) 1993 John Wiley & Sons, Inc.     

Chemical composition and larvicidal activity of essential oils of three Artemisia species

Aedes aegypti L. (Diptera: Culicidae) is a vector for serious diseases in tropical regions. This pest is mainly controlled by commercial larvicides but the application of such products has led to environmental problems. Essential oils (EO) have been consistently reported as molecules with insecticidal activity and can be used to produce more environmentally friendly larvicides in the control of A. aegypti. In this study, the larvicidal effect of essential oils (EO) from the leaves of three Artemisia species was evaluated against A. aegypti. The oils were obtained from steam distillation and their chemical composition was determined by gas chromatography–mass spectrometry. The EO of Artemisia camphorata was the most active in the screening bioassay and presented LC₅₀ and LC₉₅ of 64.95 and 74.18 μg ml⁻¹, respectively. In addition, we found that germacrene D-4-ol was the constituent responsible for the toxicity of this EO. Artemisia camphorata EO and its major constituent, germacrene D-4-ol, are promising for the development of natural larvicides against A. aegypti.     

Dissimilarity between prostaglandin E1 and nitric oxide donors as potentiators of plasma exudation in the rabbit skin in vivo

The ability of prostaglandin E₁ (PGE₁) and nitric oxide (NO) donor compounds such as sodium nitroprusside (SNP), glyceryl trinitrate (GTN), and 3-morpholino-sydnonimine (SIN-1) to modulate the histamine- and bradykinin-induced increase in microvascular permeability have been investigated in rabbit skin. The effect of the NO synthesis inhibitor N^ω-nitro- -arginine methyl ester ( -NAME) on the plasma exudation induced by histamine and bradykinin was also studied. Local edema formation was evaluated using [¹²⁵I]human serum albumin. New Zealand white rabbits received an intravenous injection of [¹²⁵I]human albumin followed immediately by the intradermal injection of edematogenic agents into the shaved dorsolateral skin. PGE₁ (0.1 nmol/site) significantly potentiated both histamine- and bradykinin-induced edema. In contrast, SNP (0.4–400 nmol/site), SIN-1 (0.4–400 nmol/site), and GTN (0.4–40 nmol/site) did not affect the edematogenic response induced by either histamine or bradykinin. GTN (0.4–40 nmol/site) also had no effect on the increase in plasma exudation induced by histamine and bradykinin in the presence of PGE₁. -NAME (50–400 nmol/site), but not its enantiomer -NAME, dose-dependently reduced the edema formation induced by a combination of either histamine or bradykinin with PGE₁. This inhibition was significantly reversed by SNP (4–400 nmol/site) and by high doses (2.5 μmol/site) of -arginine (but not by -arginine). Our results thus demonstrate that PGE₁, but not nitrovasodilators, can actually potentiate histamine- and bradykinin-induced edema in rabbit skin. This discrepancy cannot be explained by the lack of vasodilator activity of the nitrovasodilators since these were able to reverse the -NAME-induced inhibition of the edema provoked by histamine. Rather, this difference most likely reflects the ability of PGE₁ to modulate vascular permeability by mechanism(s) other than an increase in arterial flow.     

Stereoselective action of acylated crude papain toward mandelic and atrolactic hydrazides

Acylated crude papain has been shown to exert stereoselective behavior toward racemic hydrazides devoid of an amino acid residue, namely, (RS)-mandelic and (RS)-atrolactic hydrazides. These hydrazides functioned as nucleophiles to yield N¹,N²-diacylhydrazines. Several achiral acylating agents for the enzyme were chosen, including Z-glycine, BOC-glycine, AOC-glycine, and hippuric acid. With the exception of hippuric acid as the acylating agent, the reaction product, in every instance for these achiral hydrazides, consisted of an excess of the (+)-N¹,N²-diacylhydrazine. The relative rates of product formation for the mandelic hydrazides were considerably greater than for corresponding reactions with racemic atrolactic hydrazide. When chiral Z-l-alanine was employed to acylate crude papain, the stereoselective action was most pronounced, with the formation of a mixture of diastereoisomers consisting of 73% N¹-(Z-l-alanyl)-N²-[(R)-mandelyl]hydrazine. The relative reactivities for the electrophiles was Z-l-alanine ? Z-glycine ? hippuric acid ? AOC-glycine > BOC-glycine. The hydrazides of (R)-, (S)-mandelic, and (RS)-atrolactic acids were prepared by conversion of the corresponding acids to their esters by means of a catalytic dehydrating agent and subsequent treatment with a methanolic solution of hydrazine.     

Beyond the green: assessing quarry restoration success through plant and beetle communities

In assessing the effectiveness of ecological restoration actions, outcomes evaluation using a multi‐taxa approach can greatly contribute to a clearer understanding of their success/failure. Since comprehensive biodiversity assessments are rarely possible, choosing taxa groups that are indicative of the ecosystem's structural and functional recovery is of major importance. Our goal was to evaluate the success of revegetation actions performed in a Mediterranean limestone quarry, using plants and epigean beetles as indicators. We compared their abundance, diversity, and community composition between revegetated sites aged 5, 13, and 19 years and a natural reference. Total plant cover significantly increased with restoration age and quickly reached reference values. However, native woody species cover dropped in the oldest site, while non‐native species became dominant. The abundance of beetles was always lower in restoration sites when compared to the reference, increasing with age, although not significantly. The richness of both plant species and beetle families was lower in restoration sites and did not show any trend towards the reference values. Finally, using nonmetric multidimensional scaling, the composition of plant and beetle communities from restoration sites showed a clear separation from the reference. Restoration efforts have successfully modified post‐quarry sites, but considerable differences remain, probably largely related to the use of the non‐native species Pinus halepensis in restoration plans. P. halepensis high cover in restoration sites greatly affects the structure of the ecosystem, and most likely its functioning too, as well as related ecosystem services, causing divergence from the reference values and compromising restoration success.