Automatic bioprocess control. 1. A general concept.

Automation of bioprocesses is presented and discussed. A general concept is applied to laboratory scale reactors as well as to large scale production facilities consisting of many unit operations with a hierarchical and highly modular structure. The implementation of non-dedicated and intelligent analytical subsystems is foreseen. Hard- and software requirements are discussed in view of the functional requirements of both scientific research and production engineering. Some practical experience is reported using several different components in parallel installations.     

Theory and application of fluorescence homotransfer to melittin oligomerization.

Fluorescence homotransfer (electronic energy transfer between identical fluorophores) has the potential to quantitate the number of subunits in membrane protein oligomers. Homotransfer strongly depolarizes fluorescence emission as a result of intermolecular excitation energy exchange between an initially excited, oriented molecule and a randomly oriented neighbor. We have theoretically treated fluorescein labeled subunits in an oligomer as a cluster of molecules that can exchange excitation energy back and forth among the subunits within that group. We find that the larger the number of subunits, the more depolarized is the emission. The general equations to calculate the expected anisotropy for complexes composed of varying numbers of labeled subunits are presented. Self-quenching of fluorophores, orientation, and changes in lifetime are also discussed and/or considered. To test this theory, we have specifically labeled melittin on its N-terminal with fluorescein and monitored its monomer to tetramer equilibrium both in solution and in lipid bilayers. The calculated anisotropies are close to the experimental values when non-fluorescent fluorescein dimers are taken into account. Our results show that homotransfer may be a promising method to study membrane-protein oligomerization.     

The intrinsically disordered region of coronins fine-tunes oligomerization and actin polymerization

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Resuscitation teaching room in a district general hospital: concept and practice.

Practical training in cardiopulmonary resuscitation presents a problem because of the shortage of teacher time and the many potential trainees. A resuscitation teaching room in district general hospital has been established, equipped with training mannikins and models, together with wall diagrams and cassette recordings. The arrangement enables many trainees to gain training in small groups with minimal demand on teachers. Experience with the room in use has suggested that the concept may have an application in many district general hospitals and possibly also in large industrial concerns.     

Defective oligomerization of arylsulfatase a as a cause of its instability in lysosomes and metachromatic leukodystrophy.

In one of the most common mutations causing metachromatic leukodystrophy, the P426L-allele of arylsulfatase A (ASA), the deficiency of ASA results from its instability in lysosomes. Inhibition of lysosomal cysteine proteinases protects the P426L-ASA and restores the sulfatide catabolism in fibroblasts of the patients. P426L-ASA, but not wild type ASA, was cleaved by purified cathepsin L at threonine 421 yielding 54- and 9-kDa fragments. X-ray crystallography at 2.5-A resolution showed that cleavage is not due to a difference in the protein fold that would expose the peptide bond following threonine 421 to proteases. Octamerization, which depends on protonation of Glu-424, was impaired for P426L-ASA. The mutation lowers the pH for the octamer/dimer equilibrium by 0.6 pH units from pH 5.8 to 5.2. A second oligomerization mutant (ASA-A464R) was generated that failed to octamerize even at pH 4.8. A464R-ASA was degraded in lysosomes to catalytically active 54-kDa intermediate. In cathepsin L-deficient fibroblasts, degradation of P426L-ASA and A464R-ASA to the 54-kDa fragment was reduced, while further degradation was blocked. This indicates that defective oligomerization of ASA allows degradation of ASA to a catalytically active 54-kDa intermediate by lysosomal cysteine proteinases, including cathepsin L. Further degradation of the 54-kDa intermediate critically depends on cathepsin L and is modified by the structure of the 9-kDa cleavage product.     

Presynaptic control as a mechanism of sensory-motor integration.

In studies of central nervous system networks, it is synaptic transmission to the postsynaptic soma-dendritic membrane that has received the most attention, in particular in relation to the analysis of sensory-motor integration. Sensory transmission is gated during ongoing movements in both invertebrates and vertebrates, such that it may be depressed in one phase of a cyclic movement and facilitated in another, in order to optimize the execution of the ongoing motor task. This presynaptic modulation is not limited to sensory afferents, but also occurs in synapses of both excitatory and inhibitory premotor interneurons. The modulation can be mediated by the release of different transmitters at axo-axonal synapses, which activate different types of receptors. In addition, presynaptic sensory axons can be coupled via gap junctions, which under certain conditions may mediate a presynaptic facilitation.     

The critical role of the stem region as a functional domain responsible for the oligomerization and Golgi localization of N-acetylglucosaminyltransferase V. The involvement of a domain homophilic interaction

We demonstrated that a region in the stem of N-acetylglucosaminyltransferase V (GnT-V), a Golgi resident protein, is not required for enzyme activity but serves as functional domain, responsible for intracellular localization. Deletion of the domain led to complete retention of the kinetic properties but resulted in the cell surface localization of the enzyme as well as its efficient secretion into the medium. The lack of this domain concomitantly abolished the disulfide-mediated oligomerization of GnT-V, which appears to confer the Golgi retention. When the domain was inserted into the stem region of a cell surface-localized type II membrane protein, the resulting chimeric protein was substantially oligomerized and predominantly localized in the intracellular organelle. Furthermore, it was found that the presence of this domain is exclusively responsible for homo-oligomer formation. This homophilic interaction appears to involve a hydrophobic cluster of residues in the alpha-helix of the domain, as indicated by secondary structure predictions. These findings suggest that the domain specifically participates in the Golgi retention of GnT-V, probably via inducing homo-oligomer formation, and would also provide a possible mechanism for the oligomerization, which is critical for localization in the Golgi.