Deletion analysis of gene minE which encodes the topological specificity factor of cell division in Escherichia coli

Division inhibition caused by the minCD gene products of Escherichia coli is suppressed specifically at mid-cell by MinE protein expressed at physiological levels. Excess MinE allows division to take place also at the poles, leading to a minicell-forming (Min⁻) phenotype. In order to investigate the basis of this topological specificity, we have analysed the ability of truncated derivatives of MinE to suppress either minCD -dependent division inhibition in a chromosomal Δ( minB ) background, or the division inhibition exerted by MinCD at the cell poles in a minB,+ strain. Our results indicate that these two effects are not mediated by identical interactions of MinE protein. In addition, gel filtration and the yeast two-hybrid system indicated that MinE interacts with itself by means of its central segment. Taken together, our results favour a model in which wild-type MinE dimer molecules direct the division inhibitor molecules to the cell poles, thus preventing polar divisions and allowing non-polar sites to divide. This model explains how excess MinE, or an excess of certain MinE derivatives which prevent the accumulation of the division inhibitor at the poles, can confer a Min⁻ phenotype in a minB ⁺ strain.     

Studies on the formation of long-chain dicarboxylic acids from puren-alkanes by a mutant ofCandida tropicalis

Summary Individualn-alkanes, from C₁₁–C₁₆, were metabolized by a mutant ofCandida tropicalis. This strain was selected for its inability to grow in the presence of dodecanedioic acid and dodecane as the sole carbon source. Transformations were studied in fed-batch cultures. Undecane was only poorly transformed, but from dodecane to hexadecane high transformation yields were achieved. Maximum yield of acid-precipitable long-chain dioic acids was obtained with tridecane as substrate. All the products were mixtures of different acids. Besides the ,-alkanedioic acids, the 3-hydroxy derivatives of long-chain ,-alkanedioic acids and dioic acids with a shortened carbon chain were found.     

Production of Staphylococcal Enterotoxins A, B, and C in Colloidal Dispersions

Larger amounts of enterotoxin were produced when Staphylococcus aureus S-6 was grown under still (nonshaken) conditions in a medium that was a paste or gel than were produced in a liquid dispersion with the same colloidal ingredient or in control basal broth (4% NZ Amine-NAK containing 50 mug of thiamine per 100 ml and 1 mg of niacin per 100 ml). Four colloidal ingredients were used which had been previously demonstrated to not support enterotoxin production in buffer. The effect of the type of dispersion occurred earlier than that of the colloidal ingredient, but interactions were found. This effect was not observed when the cells were grown with aeration (shaken). Four other strains of S. aureus followed a similar pattern for enterotoxins A, B, and C, although liquid and paste with cornstarch and carrageenan were the only media compared to the control broth. Enterotoxins A and B were produced earlier by S. aureus S-6, and much greater quantities of enterotoxins were produced for all strains when incubated shaken.     

Biomechanische Grundfragen

Ohne ZusammenfassungAnm. des Herausgebers. Ausnahmsweise wird mit dieser Abhandlung einer rein theoretischen Erörterung hier Raum gegeben. Der Autor vertritt in ihr Bestrebungen, die ich s. Z. als die der Probiologie bezeichnet habe. Da wir dies noch unbekannte Forschungsgebiet erst durch geistige Analyse in die eventuellen Möglichkeiten und scheinbaren Notwendigkeiten zerlegen und so vorläufig erst geistig etwas aufhellen müssen, ehe wir auf experimentellem Wege exakte Kenntnis gewinnen und auch die jetzt verbreiteten Selbsttäuschungen mehrerer Autoren, daß ihnen bereits eine künstliche Biogenesis gelungen sei, deutlich als Irrtümer charakterisieren können, so scheint es nützlich, das Endergebnis vieljährigen Nachdenkens unsres hochbejahrten Autors hier aufzunehmen. Dies geschieht, wie sonst auch, ohne Rücksicht darauf, daß ich in manchem speziellen Wesentlichen abweichende Auffassungen vertrete. Doch erlaube ich mir, auf meine früheren bezüglichen äußerungen hinzuweisen. Sie befinden sich in: Der Kampf der Teile im Organismus. 1881. Kapitel V »über das Wesen des Lebens« (auch Gesamm. Abh. I. S. 387 u. f.); ferner in Ergebnisse der Anatomie und Entwicklungsgeschichte. 1892. Bd. II. S. 430 ff. (oder Gesamm. Abh. II. S. 76ff.), da findet sich das von mir aufgestellte System einander superordinierter Probionten und niederster Bionten: Isoplasson, Autokineon, Antomerizon und Idioplasson (Naegeli-Weismanns). Später wurde das Psychoplasson von mir noch dazu gefügt. Der Artikel »über die Selbstregulation der Lebewesen« (NB. als eine charakteristische Eigenschaft derselben, dies Archiv. Bd. 13. S. 610–661) enthält frühere äußerungen und ihre Verteidigung. »Die angebliche künstliche Erzeugung lebender Wesen« (Wochenschrift »Die Umschau« 1906. Nr. 2) enthält zugleich die funktionelle Minimaldefinition des Lebens, welche allen solchen Erörterungen zugrunde gelegt werden sollte. Die Kapitel über Probiologie und künstliche Biogenesis in Vortrag I über Entwicklungsmechanik (1905. S. 105–132, S. 270 Nr. 144) führen das Thema etwas weiter. Ferner ist einzusehen über die Verwendbarkeit des anorganischen Experiments in der Biologie dies Arch. Bd. 5. S. 251 ff., über Psychomorphologie Arch. Bd. 24. S. 684–692, Bd. 25. S. 715–725, überStreckers Urzeugungshypothese Arch. Bd. 27. S. 310; schließlich mein auf dem 7. internat. Zool.-Kongreß in Boston 1907 verlesener Vortrag: Können wir die Faktoren und die gestaltenden Wirkungsweisen der typischen Entwicklungsvorgänge der Lebewesen ermitteln? (erschienen Cambridge 1909). Auch sei auf den Artikelvon Nathusius über dieHartingschen Körperchen in Bd. 6. S. 365-393 dieses Archivs hingewiesen.     

Mitochondrial dynamics in model organisms: What yeasts,worms and flies have taught us about fusion and fission of mitochondria

Mitochondrial fusion and fission are important for a great variety of cellular functions, including energy metabolism, development, aging and cell death. Many of the core components mediating mitochondrial dynamics in human cells have been first identified and mechanistically analyzed in model organisms, such as Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster. In particular, the functions of FZO/mitofusin and Mgm1/EAT-3/OPA1 in fusion and Dnm1/DRP1 in fission have been remarkably well conserved in yeasts, worms, flies and mammals. On the other hand, mechanisms to coordinate and regulate the activity of these molecular machines appear to be more diverse in different organisms. Here, I will discuss how S. cerevisiae, C. elegans and Drosophila have contributed to our current understanding of the cellular machineries mediating the dynamic behaviour of mitochondria.     

Molecular Mechanisms of Glutamine Synthetase Mutations that Lead to Clinically Relevant Pathologies

Glutamine synthetase (GS) catalyzes ATP-dependent ligation of ammonia and glutamate to glutamine. Two mutations of human GS (R324C and R341C) were connected to congenital glutamine deficiency with severe brain malformations resulting in neonatal death. Another GS mutation (R324S) was identified in a neurologically compromised patient. However, the molecular mechanisms underlying the impairment of GS activity by these mutations have remained elusive. Molecular dynamics simulations, free energy calculations, and rigidity analyses suggest that all three mutations influence the first step of GS catalytic cycle. The R324S and R324C mutations deteriorate GS catalytic activity due to loss of direct interactions with ATP. As to R324S, indirect, water-mediated interactions reduce this effect, which may explain the suggested higher GS residual activity. The R341C mutation weakens ATP binding by destabilizing the interacting residue R340 in the apo state of GS. Additionally, the mutation is predicted to result in a significant destabilization of helix H8, which should negatively affect glutamate binding. This prediction was tested in HEK293 cells overexpressing GS by dot-blot analysis: Structural stability of H8 was impaired through mutation of amino acids interacting with R341, as indicated by a loss of masking of an epitope in the glutamate binding pocket for a monoclonal anti-GS antibody by L-methionine-S-sulfoximine; in contrast, cells transfected with wild type GS showed the masking. Our analyses reveal complex molecular effects underlying impaired GS catalytic activity in three clinically relevant mutants. Our findings could stimulate the development of ATP binding-enhancing molecules by which the R324S mutant can be repaired extrinsically.     

Anisotropic cross-bands in peritrophic membranes of Diptera

In agreement with previous findings in adult Calliphora erythrocephala a periodic incorporation of glucose-¹⁴C and methionine-S35 has been observed into the peritrophic membranes of C. erythrocephala and Lucilia sericata larvae as well as in peritrophic membranes of adult L. sericata and Musca domestica. The width of the periodic units (=labelled band + unlabelled interval) is of the order of 50 to 80 μm.After staining with the dichroitic dye Congo red, peritrophic membranes exhibit a periodic pattern of anisotropic bands in polarized light, resembling the pattern in autoradiographs. If stained membranes are rotated between crossed polars, every 90° the extinction is shifted from a ‘band’ into an ‘interval’ and vice versa, indicating that in these peritrophic membranes two anisotropic sections are arranged perpendicular to each other in series. There are no differences between band patterns of males and females.A relation between width of the bands and the diameter of the peritrophic membranes seems to be possible, but since correlation factors are only of the order of 0·83 it is not significant. Besides the macrounits (band+interval) with a width of about 50 to 80 μm, smaller units with a width of 10 to 14 μm have been detected only in the peritrophic membranes of adult C. erythrocephala and L. sericata, as well as in the inner membranes of adult M. domestica. The anisotropic structure of these microbands is arranged about 45° with respect to that in macrobands.Peritrophic membranes of a blackfly larva, Odagmia ornata, after staining with Congo red show in polarized light a complicated zig-zag pattern of periodic units with a width of 15 to 20 μm, or patches with a regular orthogonal network.     

Switching on the lights for gene therapy

Strategies for non-invasive and quantitative imaging of gene expression in vivo have been developed over the past decade. Non-invasive assessment of the dynamics of gene regulation is of interest for the detection of endogenous disease-specific biological alterations (e.g., signal transduction) and for monitoring the induction and regulation of therapeutic genes (e.g., gene therapy). To demonstrate that non-invasive imaging of regulated expression of any type of gene after in vivo transduction by versatile vectors is feasible, we generated regulatable herpes simplex virus type 1 (HSV-1) amplicon vectors carrying hormone (mifepristone) or antibiotic (tetracycline) regulated promoters driving the proportional co-expression of two marker genes. Regulated gene expression was monitored by fluorescence microscopy in culture and by positron emission tomography (PET) or bioluminescence (BLI) in vivo. The induction levels evaluated in glioma models varied depending on the dose of inductor. With fluorescence microscopy and BLI being the tools for assessing gene expression in culture and animal models, and with PET being the technology for possible application in humans, the generated vectors may serve to non-invasively monitor the dynamics of any gene of interest which is proportionally co-expressed with the respective imaging marker gene in research applications aiming towards translation into clinical application.