Versatile multi-functionalization of protein nanofibrils for biosensor applications.

Protein nanofibrils offer advantages over other nanostructures due to the ease in their self-assembly and the versatility of surface chemistry available. Yet, an efficient and general methodology for their post-assembly functionalization remains a significant challenge. We introduce a generic approach, based on biotinylation and thiolation, for the multi-functionalization of protein nanofibrils self-assembled from whey proteins. Biochemical characterization shows the effects of the functionalization onto the nanofibrils' surface, giving insights into the changes in surface chemistry of the nanostructures. We show how these methods can be used to decorate whey protein nanofibrils with several components such as fluorescent quantum dots, enzymes, and metal nanoparticles. A multi-functionalization approach is used, as a proof of principle, for the development of a glucose biosensor platform, where the protein nanofibrils act as nanoscaffolds for glucose oxidase. Biotinylation is used for enzyme attachment and thiolation for nanoscaffold anchoring onto a gold electrode surface. Characterization via cyclic voltammetry shows an increase in glucose-oxidase mediated current response due to thiol-metal interactions with the gold electrode. The presented approach for protein nanofibril multi-functionalization is novel and has the potential of being applied to other protein nanostructures with similar surface chemistry.

An F-actin-depleted zone is present at the hyphal tip of invasive hyphae of Neurospora crassa.

The distribution of filamentous actin (F-actin) in invasive and noninvasive hyphae of the ascomycete Neurospora crassa was investigated. Eighty six percent of noninvasive hyphae had F-actin in the tip region compared to only 9% of invasive hyphae. The remaining 91% of the invasive hyphae had no obvious tip high concentration of F-actin staining; instead they had an F-actin-depleted zone in this region, although some F-actin, possibly associated with the Spitzenkörper, remained at the tip. The size of the F-actin-depleted zone in invasive hyphae increased with an increase in agar concentration. The membrane stain FM 4-64 reveals a slightly larger accumulation of vesicles at the tips of invasive hyphae relative to noninvasive hyphae, although this difference is unlikely to be sufficient to account for the exclusion of F-actin from the depleted zone. Antibodies raised against the actin filament-severing protein cofilin from both yeast and human cells localize to the tips of invasive hyphae. The human cofilin antibody shows a more random distribution in noninvasive hyphae locating primarily at the hyphal periphery but with some diffuse cytoplasmic staining. This antibody also identifies a single band at 21 kDa in immunoblots of whole hyphal fractions. These data suggest that a protein with epitopic similarity to cofilin may function in F-actin dynamics that underlie invasive growth. The F-actin-depleted zone may play a role in the regulation of tip yielding to turgor pressure, thus increasing the protrusive force necessary for invasive growth.

[Pleuropulmonary aspergillosis with defective neutrophil functions--G-CSF effects on neutrophil functions].

A 66-year-old man was admitted to our hospital with productive cough and fever. Chest X-ray films showed pleural thickening of the right apex and calcification of the lung field. Antibiotic therapy was administered but no improvement was obtained. Chest CT scan revealed pleural thickening with effusion in the right apex. Needle aspiration biopsy was performed and Aspergillus fumigatus was isolated from pleural fluid. We began pleural drainage and intrapleural infusion of miconazole, with systemic administration of miconazole, amphotericin B and fluconazole. In spite of normal bone marrow findings, the neutrophil functions, NAP score and phagocytosis, of the patient were found to be subnormal. We commenced to treatment with granulocyte colony stimulating factor and his neutrophil functions improved (NAP score: 156-->309.5, phagocytosis: 64.9-->70.0%). However, his fever persisted and he died following cardiopulmonary arrest. Autopsy revealed Aspergillus mycelia invading the visceral pleura. No intrapulmonary lesions caused by Aspergillus were found.

[Clinical studies on lung disease caused by atypical mycobacteria in our hospital--especially in relation to drug susceptibility].

From 1987 to 1991, 27 patients were diagnosed as atypical mycobacteriosis in our hospital. Some strains of M. avium complex were found to be M. avium and M. intracellulare by means of a DNA probe test. 1. Total cases consisted of 14 males (the average age was 66.7 years) and 13 females (65.7 years). 2. M. avium complex was observed in 24 patients (9 cases of M. avium and 5 cases of M. intracellulare): M. kansasii and M. chelonae were found in 2 patients and 1 patient, respectively. 3. The findings of sputum cultures became negative three months after the chemotherapy treatment in 3 out of 25 patients. Two male patients were operated on and cured. Three patients died, and all of them had respiratory infections. 4. To determine the susceptibility of mycobacteria strains isolated from patients to various antimicrobial agents, an investigation was carried out. There were 88 strains of M. tuberculosis, 52 strains of M. avium complex (23 strains of M. avium and 17 strains of M. intracellulare), 3 strains of M. kansasii, 2 strains of M. gordonae and 2 strains of M. chelonae. 5. Strains of M. tuberculosis, M. kansasii and M. gordonae were susceptible to various antituberculous agents and ciprofloxacin. Strains of M. chelonae were susceptible to ciprofloxacin, and one strain was susceptible to streptomycin and ethionamide. 6. The M. avium strains were more susceptible to cycloserine and ciprofloxacin than were the M. intracellulare strains. Conversely, the M. intracellulare strains were more susceptible to ethambutol than were the M. avium strains.