Pathways involved in fluid phase and adsorptive endocytosis in neuroblastoma

The endocytosis of ricin, horseradish peroxidase (HRP), and a conjugate of ricin-HRP by monolayer cultures of murine neuroblastoma was studied using morphological and biochemical techniques. The binding of (125)I-ricin and (125)I-ricin-HRP to cells at 4 degrees C, as a function of ligand concentration, was a saturable process. The apparent affinity constants, determined at equilibrium, were 2.8 X 10(6) M(-1) for ricin and 1 x 10(6) M(-1) for ricin-HRP. The number of binding sites per cell was 8 x 10(7) and 3 x 10(7) for the lectin and the conjugate, respectively. The binding of (125)I-ricin to monolayers as not proportional to cell density. We found reduced binding at higher cell concentrations, suggesting a decrease in the accessibility of the ligand for the receptor site or fewer sites with increasing cell population. Neuroblastoma cells have an acid-phosphatase-positive network of cisternae and vesicles near the Golgi apparatus (GERL). Ricin-HRP undergoes endocytosis in vesicles and cisternae corresponding to GERL, and in residual bodies (dense bodies). The cellular uptake of ricin-HRP was 100-200 times greater than free HRP and there was no stimulation of fluid phase endocytosis by ricin. When monolayers were exposed to concentrations of native HRP 100-fold that of the conjugate, cellular uptake of peroxidase was comparable, but HRP was localized only in residual bodies and never in elements of GERL. These results support the conclusion that GERL is involved in the adsorptive endocytosis of ricin-HRP, while residual bodies are involved in the bulk uptake of HRP. In addition, the binding, uptake, and possible recycling of (125)I- subunit B (the binding subunit) of ricin and of (125)I-ricin was examined by quantitative electron microscope autoradiography. Both ricin and its binding subunit displayed similar autoradiographic grain distributions at 4 degrees C, and there was no evidence of their breakdown or recycling to the plasma membrane during endocytosis for 2 h.     

Glutamate detection from nerve cells using a planar electrodes array integrated in a microtiter plate

There is an increasing interest in new strategies for replacing animal tests in research. The use of cell cultures and integrated electrodes is seen as a promising alternative that could potentially solve this problem. In this work, we present a L-glutamate sensor based on a bienzyme redox hydrogel, capable of detecting the release of this excitatory neurotransmitter from adherently growing cells upon stimulation. The low working potential required for the operation of the sensor decreases the possibility of interference by easily oxidizable compounds always present in complex biological samples. A low detection limit of 0.5 microM L-glutamate, a response time of about 35 s, and a linear range of up to 60 microM are the main characteristics of the sensor. The system has been successfully employed to monitor the release of l-glutamate from HN10 and C6 cells upon stimulation with K(+)-ions. The developed integrated electrochemical platform will be used in future for drug screening and potentially for replacing animal models in neurological experiments.     

Imaging immobilised ssDNA and detecting DNA hybridisation by means of the repelling mode of scanning electrochemical microscopy (SECM)

The supposed repelling mode of scanning electrochemical microscopy (SECM) allows truly label-free electrochemical recognition of the presence and hybridisation of nucleic acids that are immobilised on conducting DNA chips. Basically, the SECM-based detection of single- and double-stranded DNA profits from the electrostatic repulsion between deprotonated phosphate groups at the backbone of the oligonucleotides and a free-diffusing negatively charged redox mediator (e.g. [Fe(CN)(6)](3-/4-)). In electrolytes of proper pH and ionic strength, this coulomb interaction is heavily influencing the diffusion properties of the mediator in the vicinity of the surface-anchored DNA strands. This charge interaction modulates the diffusional mass transport for the charged redox species in the DNA modified regions, and thus locally decreases the positive feedback currents measured with a SECM tip placed within the electrochemical nearfield of the chip surface. This approach was used to study arrays of synthetic 20-base oligonucleotide probes that were immobilised on monolayer-modified gold surfaces. Evidence is provided that the density of probes, the ionic strength of solution and the tip-to-sample distance have a strong impact on the capability of the repelling mode of SECM to visualise probe spots and hybridisation while the concentration of the chosen mediator did not significantly affect detection.     

Visualization of micropatterned complex biosensor sensing chemistries by means of scanning electrochemical microscopy

Redox hydrogel-based micropatterned complex biosensor architectures, used as sensing chemistries in amperometric ethanol or glucose biosensors, were deposited on gold, graphite or glass. Well-localized immobilization of active hydrogels with variable compositions was achieved by dispensing 100 pl droplets of cocktails containing alcohol or glucose dehydrogenase, redox polymer (PVI(13)dmeOs) and crosslinker (PEGDGE) while moving the target surface relative to the position of the nozzle of a piezo-actuated microdispenser. The resulting structures were microscopic patterns of enzyme-containing lines of a redox hydrogel with a line width of about 100 microm. Scanning electrochemical microscopy (SECM) in the amperometric feedback mode was used to visualize the immobilized enzyme microstructures and their localized biochemical activity was observed with high lateral resolution by detecting the enzymatically consumed substrate using K(4)[Fe(CN)(6)] as a free-diffusing electron-transfer mediator.     

The family of Deg/HtrA proteases in plants

ABSTRACT: BACKGROUND: The Deg/HtrA family of ATP-independent serine endopeptidases is present in nearly all organisms from bacteria to human and vascular plants. In recent years, multiple deg/htrA protease genes were identified in various plant genomes. During genome annotations most proteases were named according to the order of discovery, hence the same names were sometimes given to different types of Deg/HtrA enzymes in different plant species. This can easily lead to false inference of individual protease functions based solely on a shared name. Therefore, the existing names and classification of these proteolytic enzymes does not meet our current needs and a phylogeny-based standardized nomenclature is required. RESULTS: Using phylogenetic and domain arrangement analysis, we improved the nomenclature of the Deg/HtrA protease family, standardized protease names based on their well-established nomenclature in Arabidopsis thaliana, and clarified the evolutionary relationship between orthologous enzymes from various photosynthetic organisms across several divergent systematic groups, including dicots, a monocot, a moss and a green alga. Furthermore, we identified a "core set" of eight proteases shared by all organisms examined here that might provide all the proteolytic potential of Deg/HtrA proteases necessary for a hypothetical plant cell. CONCLUSIONS: In our proposed nomenclature, the evolutionarily closest orthologs have the same protease name, simplifying scientific communication when comparing different plant species and allowing for more reliable inference of protease functions. Further, we proposed that the high number of Deg/HtrA proteases in plants is mainly due to gene duplications unique to the respective organism.     

Improved specificity of reagentless amperometric PQQ-sGDH glucose biosensors by using indirectly heated electrodes

Indirectly heated electrodes operating in a non-isothermal mode have been used as transducers for reagentless glucose biosensors. Pyrroloquinoline quinone-dependent soluble glucose dehydrogenase (PQQ-sGDH) was entrapped on the electrode surface within a redox hydrogel layer. Localized polymer film precipitation was invoked by electrochemically modulating the pH-value in the diffusion zone in front of the electrode. The resulting decrease in solubility of an anodic electrodeposition paint (EDP) functionalized with Osmium complexes leads to precipitation of the redox hydrogel concomitantly entrapping the enzyme. The resulting sensor architecture enables a fast electron transfer between enzyme and electrode surface. The glucose sensor was operated at pre-defined temperatures using a multiple current-pulse mode allowing reproducible indirect heating of the sensor. The sensor characteristics such as the apparent Michaelis constants K(M)(app) and maximum currents I(max)(app) were determined at different temperatures for the main substrate glucose as well as a potential interfering co-substrate maltose. The limit of detection increased with higher temperatures for both substrates (0.020 mM for glucose, and 0.023 mM for maltose at 48 degrees C). The substrate specificity of PQQ-sGDH is highly temperature dependent. Therefore, a mathematical model based on a multiple linear regression approach could be applied to discriminate between the current response for glucose and maltose. This allowed accurate determination of glucose in a concentration range of 0-0.1mM in the presence of unknown maltose concentrations ranging from 0 to 0.04 mM.     

Disproportionation of inorganic sulfur compounds by the sulfate-reducing bacterium Desulfocapsa thiozymogenes gen. nov., sp. nov.

A new strictly anaerobic, gram-negative bacterium was isolated from the sediment of a freshwater lake after enrichment with thiosulfate as the energy source. The strain, named Bra2 (DSM 7269), is able to grow by disproportionation of thiosulfate or sulfite to sulfate plus sulfide. Elemental sulfur is also disproportionated to sulfate and sulfide, but this only supports growth if free sulfide is chemically removed from the culture, e.g., by precipitation with amorphous ferric hydroxide. Growth is also possible by coupling the reduction of sulfate to sulfide with the oxidation of ethanol, propanol, or butanol to the corresponding fatty acid. The cells are rod-shaped, motile, and have genomic DNA with a mol% G+C content of 50.7. Cytochromes are present, but desulfoviridin is not. The new strain was shown to be related to, but distinct from members of the genus Desulfobulbus on the basis of physiological characteristics and by comparative sequence analysis of its 16S rDNA. Strain Bra2 is described as the type strain of a new taxon, Desulfocapsa thiozymogenes gen. nov., sp. nov. Received: 29 January 1996 / Accepted: 31 May 1996     

Interaction of yeast 3-phosphoglycerate kinase with negatively charged carriers

The aim of this study was to investigate the possibility of an interaction of yeast 3-phosphoglycerate kinase with negatively charged carriers such as polyanionic agents or a polarized electrode. Various polyanions were found to promote enzyme aggregation as judged by ultracentrifugation measurements and chemical modification. The data obtained suggest that these interactions are mediated through the N-terminal domain of the protein. However, the most striking property of 3-phosphoglycerate kinase described here is concerned with its significant dipolar moment as evidenced by electrocapillary measurements, which allows an orientation of the macromolecule in an electric field. Further, the enzyme could be absorbed by a negatively charged surface, first by hydrophobic links and then oriented perpendicularly to the surface. Therefore, the intrinsic properties of yeast 3-phosphoglycerate kinase agree with the formation of an enzyme-membrane complex and afford the ability for a specific orientation of the molecule at the lipid bilayer surface or in the cytoplasm.