Aspergillus fumigatus can utilize chicken feather keratin as its sole carbon and nitrogen source. Because enzymatic conversion of native keratin into readily usable products is of economic interest, this fungus was studied for its capacity to produce and secrete keratin-hydrolyzing proteinases. Substantial keratin-azure hydrolyzing activity was present in the culture fluid of keratin-containing media. Considerably lower activity was present in cultures containing glucose and nitrate as the carbon and nitrogen sources, or keratin plus glucose and nitrate. Secretion of keratin-hydrolyzing activity in A. fumigatus was induced by keratin but repressed by low-molecular-weight carbon and nitrogen sources. The amount of keratinolytic enzyme present in the culture fluid was dependent on the initial pH of the culture medium. The crude enzyme also hydrolyzed native keratin and casein in vitro. Hydrolysis was optimal at pH 9 and 45 degrees C. The crude enzyme was remarkably thermostable. At 70 degrees C, it retained about 90% of its original activity for 1.5 h. The obtained results indicated that the A. fumigatus keratinolytic enzyme may be suitable for enzymatic improvement of feather meal.

No abstract

No abstract

During the last decades nitric oxide (•NO) has emerged as a critical physiological signaling molecule in mammalian tissues, notably in the brain. •NO may modify the activity of regulatory proteins via direct reaction with the heme moiety, or indirectly, via S-nitrosylation of thiol groups or nitration of tyrosine residues. However, a conceptual understanding of how •NO bioactivity is carried out in biological systems is hampered by the lack of knowledge on its dynamics in vivo. Key questions still lacking concrete and definitive answers include those related with quantitative issues of its concentration dynamics and diffusion, summarized in the how much, how long, and how far trilogy. For instance, a major problem is the lack of knowledge of what constitutes a physiological •NO concentration and what constitutes a pathological one and how is •NO concentration regulated. The ambient •NO concentration reflects the balance between the rate of synthesis and the rate of breakdown. Much has been learnt about the mechanism of •NO synthesis, but the inactivation pathways of •NO has been almost completely ignored. We have recently addressed these issues in vivo on basis of microelectrode technology that allows a fine-tuned spatial and temporal measurement •NO concentration dynamics in the brain.

Acetylcholine (ACh) modulates neuronal network activities implicated in cognition, including theta and gamma oscillations but the mechanisms remain poorly understood. Joint measurements of cholinergic activity and neuronal network dynamics with high spatio-temporal resolution are critical to understand ACh neuromodulation. However, current electrochemical biosensors are not optimized to measure nanomolar cholinergic signals across small regions like hippocampal sub-layers. Here, we report a novel oxidase-based electrochemical biosensor that matches these constraints. The approach is based on measurement of H2O2 generated by choline oxidase (ChOx) in the presence of choline (Ch). The microelectrode design consists of a twisted pair of 50µm diameter Pt/Ir wires (sensor and sentinel), which is scalable, provides high spatial resolution and optimizes common mode rejection. Microelectrode coating with ChOx in chitosan cross-linked with benzoquinone is simple, mechanically robust and provides high sensitivity (324±46nAµM(-1)cm(-2)), a limit of detection of 16nM and a t50 response time of 1.4s. Local field potential (LFP)-related currents dominate high-frequency component of electrochemical recordings in vivo. We significantly improved signal-to-noise-ratio compared to traditional sentinel subtraction by a novel frequency domain common mode rejection procedure that accounts for differential phase and amplitude of LFP-related currents on the two channels. We demonstrate measurements of spontaneous nanomolar Ch fluctuations, on top of which micromolar Ch increases occurred during periods of theta activity in anesthetized rats. Measurements were not affected by physiological O2 changes, in agreement with the low biosensor Km for O2 (2.6µM). Design and performance of the novel biosensor opens the way for multisite recordings of spontaneous cholinergic dynamics in behaving animals.