Proteome and transcriptome based analysis of Bacillus subtilis cells overproducing an insoluble heterologous protein.

Bacillus subtilis and related Bacillus species are frequently used as hosts for the industrial production of recombinant proteins. In this study the cellular response of B. subtilis to the overproduction of an insoluble heterologous protein was investigated. For this purpose PorA, an outer membrane protein from Neisseria meningitidis, which accumulates after overexpression in the cytoplasm of B. subtilis mainly in the form of inclusion bodies, was used. The molecular response to overexpression of porA has been analysed at the transcriptional level using the DNA macro array technique and at the translational level by two-dimensional polyacrylamide gel electrophoresis. It was found that the expression of the heat shock genes of class I (dnaK, groEL and grpE) and class III (clpP and clpC) are increased under overproducing conditions. Furthermore, the protein levels of the two ribosomal proteins RpsB and RplJ are increased in the PorA overproducing cells. The transcriptome analysis indicated that mRNA levels of genes encoding pyrimidine and purine synthesis enzymes but also from ribosomal protein genes have elevated levels under overproducing conditions. Finally, the association of the protease ClpP and its ATPase subunits ClpC and ClpX with the PorA inclusion bodies was demonstrated by means of the immunogold labelling technique.

Cut edges and surface characteristics produced by different microkeratomes.

PURPOSE: To determine the cutting characteristics of seven different microkeratomes and to compare the cut edges and surface characteristics of the corneas with respect to different keratome parameters, ie, blade oscillation frequencies and keratome speed. METHODS: Lamellar keratectomies were performed using each microkeratome on eight freshly enucleated porcine corneas. The freshly cut corneal bed was then examined using scanning electron microscopy. A scoring system was used to evaluate the serration of the cut edge and the regularity of the corneal wound bed. RESULTS: Serrated cut edges were produced by the Microtech microkeratome, the Automatic Corneal Shaper, the Draeger rotor keratome, and the Schwind microkeratome. The other tested cutting devices generally yielded a smooth cut edge. Smooth and regular wound surfaces were obtained with the Schwind microkeratome, the Automatic Corneal Shaper, the Microtech microkeratome, and the MKM set. The specimens cut with the Schwind microkeratome showed particularly regular surface characteristics. CONCLUSION: The relationship between keratome propulsion speed, blade oscillation frequency, and blade material appears crucial for the quality of the microkeratome cut. Our results favor a low advancement/oscillation ratio among automated microkeratomes.

The clp proteases of Bacillus subtilis are directly involved in degradation of misfolded proteins.

The presence of the heat stress response-related ATPases ClpC and ClpX or the peptidase ClpP in the cell is crucial for tolerance of many forms of stress in Bacillus subtilis. Assays for detection of defects in protein degradation suggest that ClpC, ClpP, and ClpX participate directly in overall proteolysis of misfolded proteins. Turnover rates for abnormal puromycyl peptides are significantly decreased in clpC, clpP, and clpX mutant cells. Electron-dense aggregates, most likely due to the accumulation of misfolded proteins, were noticed in studies of ultrathin cryosections in clpC and clpP mutant cells even under nonstress conditions. In contrast, in the wild type or clpX mutants such aggregates could only be observed after heat shock. This phenomenon supports the assumption that clpC and clpP mutants are deficient in the ability to solubilize or degrade damaged and aggregated proteins, the accumulation of which is toxic for the cell. By using immunogold labeling with antibodies raised against ClpC, ClpP, and ClpX, the Clp proteins were localized in these aggregates, showing that the Clp proteins act at this level in vivo.

[Potential of the water jet in cataract surgery]. / Was kann der Wasserstrahl in der Kataraktchirurgie?

UNLABELLED: Though cataract surgery is highly developed today, there are still problems such as endothelial cell loss after surgery and the occurrence of aftercataract. To reduce these complications we looked for techniques with a high degree of safety and precision. We found the water jet, an instrument already well established in liver surgery. We tested the possibility of improving the results of cataract surgery using the water jet method. METHODS: We performed cataract surgery--phacoemulsifikation and polishing of the capsule--on freshly enucleated porcine bulbs using water jet and by conventional procedures. By scanning electron microscope examination we compared the results. Additionally we emulsified human lens nuclei obtained by extracapsular cataract extraction using the water jet. RESULTS: The epithelial cells and lens fragments on the capsule were considerably reduced after the water jet procedure. CONCLUSION: The results show the possibility of improvement of cataract surgery by using the water jet. Further studies are necessary to adapt this technique to routine surgery in humans.

Differential expression of fructosyllysine-specific receptors on monocytes and macrophages and possible pathophysiological significance.

A differing individual expression of fructosyllysine-specific receptors has been found on the monocytes of 90 insulin-dependent diabetic patients and 101 healthy control subjects. The degree of receptor expression is neither age- nor sex-dependent; however, in the diabetic group it correlates significantly with the severity and age of onset of diabetic microangiopathy. To interpret the results of the human study, spontaneously diabetic and non-diabetic BB/OK rats were used to estimate tissue content of glucose-modified proteins and capillary basement membrane thickness in relation to the receptor expression on macrophages. In non-diabetic and diabetic rats no correlation was found between receptor expression and tissue content (i.e. artery, nerve) of fructosyllsine and fluorescent advanced glycation end products. However, animals which express the fructosyllysine receptor showed a greater increase in muscle capillary basement membrane thickness. There are indications that fructosyllysine receptor expression is positively associated with indices of diabetic complications such as microangiopathy and/or capillary basement membrane thickening.

Immunolocalization of cytokeratin 19 in bovine and human pulmonary microvascular endothelial cells in situ.

1. Immunocytochemical analysis of bovine and human lung sections revealed the presence of the 41 kD intermediate filament protein cytokeratin 19 in microvessel and subpleural lymphatic endothelial cells as well as the mesothelial cell layer of the lung visceral pleura. 2. Cytokeratin 19 was expressed by human and bovine pulmonary microvessels with diameters ranging from 5 to 50 microns. 3. Cytokeratin 19 was also found in microvessels of the rete mirabile, an oxygen exchange organ of the eel. 4. Immunoperoxidase electron microscopy demonstrated cytokeratin 19 associated with the lateral membranes of adjacent bovine alveolar capillary endothelial cells.