Physiological consequence of disruption of the VMA1 gene in the riboflavin overproducer Ashbya gossypii.

The vacuolar ATPase subunit A structural gene VMA1 of the biotechnologically important riboflavin overproducer Ashbya gossypii was cloned and disrupted to prevent riboflavin retention in the vacuolar compartment and to redirect the riboflavin flux into the medium. Cloning was achieved by polymerase chain reaction using oligonucleotide primers derived form conserved sequences of the Vma1 proteins from yeast and filamentous fungi. The deduced polypeptide comprises 617 amino acids with a calculated molecular mass of 67.8 kDa. The deduced amino acid sequence is highly similar to that of the catalytic subunits of Saccharomyces cerevisiae (67 kDa), Candida tropicalis (67 kDa), and Neurospora crassa (67 kDa) with 89, 87, and 60% identity, respectively, and shows about 25% identity to the beta-subunit of the FoF1-ATPase of S. cerevisiae and Schizosaccharomyces pombe. In contrast to S. cerevisiae, however, where disruption of the VMA1 gene was conditionally lethal, and to N. crassa, where viable disruptants could not be isolated, disruption of the VMA1 gene in A. gossypii did not cause a lethal phenotype. Disruption of the AgVMA1 gene led to complete excretion of riboflavin into the medium instead of retention in the vacuolar compartment, as observed in the wild type.

Identification of mechanosensitive ion channels in the cytoplasmic membrane of Corynebacterium glutamicum.

Patch-clamp experiments performed on membrane fragments of Corynebacterium glutamicum fused into giant liposomes revealed the presence of two different stretch-activated conductances, 600 to 700 pS and 1,200 to 1,400 pS in 0.1 M KCl, that exhibited the same characteristics in terms of kinetics, ion selectivity, and voltage dependence.

[Amputation or reconstruction of IIIB and IIIC open tibial fracture. Decision criteria in the acute phase and late functional outcome]. / Amputation oder Rekonstruktion bei der IIIB und IIIC Offenen Unterschenkelfraktur. Entscheidungskriterien in der Akutphase und funktionelles Spätergebnis.

In IIIB and IIIC type open tibial fractures (according to Gustilo) the primary decision that has to be made regarding therapy is wether or not the limb can be salvaged. To standardize the criteria for amputation different salvage scores have been established in recent years. In this study the Hannover Fracture Scale (HFS), the Predictive Salvage Index (PSI), the Mangled Extremity Severity Score (MESS) and the NISSSA score were evaluated regarding their clinical relevance. When ROC Analysis was performed for all these scores in our patients the HFS revealed the highest sensitivity (0.91), but low specificity (0.71). The highest specificity was noted for the MESS (0.97), which in parallel showed the lowest sensitivity (0.59). In general it seems to be essential to make the right decision initially in order to avoid secondary amputation. All the scores mentioned here appear to be helpful in decision making. Salvaged limbs in IIIB and IIIC fractures presented a comparable good outcome, whereas salvaged IIIC injuries with a high score presented an outcome which was as bad as in secondary amputations. Secondary amputated patients required not only significant longer hospitalization but also resulted in poor outcome compared with the patients having received reconstruction or primary amputation.

Efflux of compatible solutes in Corynebacterium glutamicum mediated by osmoregulated channel activity.

Bacteria respond to hypoosmotic stress by releasing low-molecular-mass solutes in order to maintain constant turgor pressure. We have studied the function of osmoregulated channel(s) in Corynebacterium glutamicum, which are responsible for efflux of various solutes upon sudden decrease in osmotic pressure. The channels preferentially mediated efflux of compatible solutes such as glycine betaine and proline. The release of molecules of similar size, e.g. glutamate or lysine, was restricted, ATP was completely retained even after severe osmotic shock. The cells maintained high cytoplasmic K+ and Na+ concentrations under hypoosmotic shock. Several results suggest that the solute efflux is mediated by a channel and not by a carrier, e.g. by reversal of the glycine betaine uptake systems of C. glutamicum: the release of glycine betaine and proline was extremely fast reaching an efflux rate of 6000 micromol x min(-1) x g dm(-1) or higher; the efflux was not significantly influenced by addition of external transport substrate, e.g. glycine betaine; in spite of an extremely high chemical gradient, no significant efflux under isoosmolar conditions was observed; efflux of solutes was unchanged after full uncoupling of membrane energetics by carbonylcyanide m-chlorophenylhydrazone (CCCP). These results indicate the presence of an osmoregulated channel in C. glutamicum similar to the mechanosensitive channel(s) of Escherichia coli. The activity of the channel did not depend on the growth conditions, but we observed a tight regulation on the level of activity, i.e. the mechanosensitive channel behaved as a perfect osmometer. By monitoring release of glycine betaine under slow and continuous decrease of the external osmolality, we observed continous efflux whithout a stepwise release of solutes. This resulted in a significant steady-state decrease of the membrane potential.

Increased prevalence of antibrain antibodies in the sera from schizophrenic patients.

The pathogenesis of schizophrenia is still unknown. In a previous study we found antibrain antibodies in the sera of schizophrenic patients, but not in normal controls. Therefore we have further examined the sera of schizophrenic patients versus normal controls, increasing the number of brain areas, to explore whether certain areas were involved more often than others in the antibody binding process. The sera of 50 patients suffering from an acute episode of schizophrenia (classified by DSM III-criteria) were tested. 70% of the patients showed antibody binding, while only 12% of the age- and sex-matched controls were positive. The binding was mediated by IgG- as well as IgM-antibodies. Amygdala, frontal cortex, cingulate gyrus, and septal area were the prominent targets, while hippocampus, parahippocampal gyrus, entorhinal cortex, putamen, mamillary bodies and head of the caudate nucleus were involved to a lesser degree. Binding was not present to nucleus olivaris, to the thyroid gland or to HEp-2 cells, which we included to test for unspecific antinuclear factors. Longterm studies of schizophrenic patients and biochemical analyses of the antigen(s) involved are in progress.

Antibodies to brain tissue in sera of schizophrenic patients--preliminary findings.

The sera of 30 patients suffering from schizophrenia (DSM III) and 30 neurological controls were tested for antibrain antibodies in a blind indirect immunofluorescence assay. We found IgG- and IgM-binding in the sera of 22 patients, but only 4 out of the 30 age- and sex-matched controls. The binding was mainly directed to neurons from the frontal cortex and septal areas, areas, which are regarded as important in the development of schizophrenic illness. These preliminary data are presented, to encourage other immunological studies in schizophrenia research.