Differences in Elasticity of Vinculin-Deficient F9 Cells Measured by Magnetometry and Atomic Force Microscopy

We have investigated a mouse F9 embryonic carcinoma cell line, in which both vinculin genes were inactivated by homologous recombination, that exhibits defective adhesion and spreading [Collet al.(1995)Proc. Natl. Acad. Sci. USA92, 9161–9165]. Using a magnetometer and RGD-coated magnetic microbeads, we measured the local effect of loss and replacement of vinculin on mechanical force transfer across integrins. Vinculin-deficient F9Vin(−/−) cells showed a 21% difference in relative stiffness compared to wild-type cells. This was restored to near wild-type levels after transfection and constitutive expression of increasing amounts of vinculin into F9Vin(−/−) cells. In contrast, the transfection of vinculin constructs deficient in amino acids 1–288 (containing the talin- and α-actinin-binding site) or substituting tyrosine for phenylalanine (phosphorylation site, amino acid 822) in F9Vin(−/−) cells resulted in partial restoration of stiffness. Using atomic force microscopy to map the relative elasticity of entire F9 cells by 128 × 128 (n= 16,384) force scans, we observed a correlation with magnetometer measurements. These findings suggest that vinculin may promote cell adhesion and spreading by stabilizing focal adhesions and transferring mechanical stresses that drive cytoskeletal remodeling, thereby affecting the elastic properties of the cell.     

Inhibition of regeneration in the planarian Dugesia polychroa (Schmidt) by treatment with magnesium chloride: a morphological study of wound closure

At a concentration of 0.2% (21 m M) in culture water, magnesium chloride impaired muscle contraction and completely inhibited head regeneration in specimens of Dugesia polychroa cut prepharyngeally. The wound stayed open for nine days, with neoblasts accumulating beneath the wound without any signs of differentiation. Extremely delayed wound closure occurred by spreading epithelial cells, and was completed after 30 days in the magnesium chloride solution. Histological examination confirmed the absence of any regenerated head structures. Interestingly, the inhibitory effect was removed when such headless fragments were cut once more and kept in normal culture water: complete head regeneration then occurred at a normal rate. Among several possible explanations for the failure to regenerate, the following hypothesis is an attractive alternative: direct contact between parenchyma and epithelial cells during the period following injury seems to be an essential stimulus for the start of cell differentiation within the blastema, and the lack of such contact as a result of the drug action prevents normal regeneration. When the wound has eventually closed, a continuous basement membrane separates epithelium from parenchyma. Thus a direct contact between these tissues is never established. This revised version was published online in July 2006 with corrections to the Cover Date.     

Diversity of Cytochrome bc Complexes: Example of the Rieske Protein in Green Sulfur Bacteria

The Rieske 2Fe2S cluster of Chlorobium limicola forma thiosulfatophilum strain tassajara was studied by electron paramagnetic resonance spectroscopy. Two distinct orientations of its g tensor were observed in oriented samples corresponding to differing conformations of the protein. Only one of the two conformations persisted after treatment with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. A redox midpoint potential (E_m) of +160 mV in the pH range of 6 to 7.7 and a decreasing E_m (−60 to −80 mV/pH unit) above pH 7.7 were found. The implications of the existence of differing conformational states of the Rieske protein, as well as of the shape of its E_m-versus-pH curve, in green sulfur bacteria are discussed.     

Evidence of Two Oxidative Reaction Steps Initiating Anaerobic Degradation of Resorcinol (1,3-Dihydroxybenzene) by the Denitrifying Bacterium Azoarcus anaerobius

The denitrifying bacterium Azoarcus anaerobius LuFRes1 grows anaerobically with resorcinol (1,3-dihydroxybenzene) as the sole source of carbon and energy. The anaerobic degradation of this compound was investigated in cell extracts. Resorcinol reductase, the key enzyme for resorcinol catabolism in fermenting bacteria, was not present in this organism. Instead, resorcinol was hydroxylated to hydroxyhydroquinone (HHQ; 1,2,4-trihydroxybenzene) with nitrate or K₃Fe(CN)₆ as the electron acceptor. HHQ was further oxidized with nitrate to 2-hydroxy-1,4-benzoquinone as identified by high-pressure liquid chromatography, UV/visible light spectroscopy, and mass spectroscopy. Average specific activities were 60 mU mg of protein⁻¹ for resorcinol hydroxylation and 150 mU mg of protein⁻¹ for HHQ dehydrogenation. Both activities were found nearly exclusively in the membrane fraction and were only barely detectable in extracts of cells grown with benzoate, indicating that both reactions were specific for resorcinol degradation. These findings suggest a new strategy of anaerobic degradation of aromatic compounds involving oxidative steps for destabilization of the aromatic ring, different from the reductive dearomatization mechanisms described so far.     

Bacterial Community Dynamics during Start-Up of a Trickle-Bed Bioreactor Degrading Aromatic Compounds

This study was performed with a laboratory-scale fixed-bed bioreactor degrading a mixture of aromatic compounds (Solvesso100). The starter culture for the bioreactor was prepared in a fermentor with a wastewater sample of a car painting facility as the inoculum and Solvesso100 as the sole carbon source. The bacterial community dynamics in the fermentor and the bioreactor were examined by a conventional isolation procedure and in situ hybridization with fluorescently labeled rRNA-targeted oligonucleotides. Two significant shifts in the bacterial community structure could be demonstrated. The original inoculum from the wastewater of the car factory was rich in proteobacteria of the alpha and beta subclasses, while the final fermentor enrichment was dominated by bacteria closely related to Pseudomonas putida or Pseudomonas mendocina, which both belong to the gamma subclass of the class Proteobacteria. A second significant shift was observed when the fermentor culture was transferred as inoculum to the trickle-bed bioreactor. The community structure in the bioreactor gradually returned to a higher complexity, with the dominance of beta and alpha subclass proteobacteria, whereas the gamma subclass proteobacteria sharply declined. Obviously, the preceded pollutant adaptant did not lead to a significant enrichment of bacteria that finally dominated in the trickle-bed bioreactor. In the course of experiments, three new 16S as well as 23S rRNA-targeted probes for beta subclass proteobacteria were designed, probe SUBU1237 for the genera Burkholderia and Sutterella, probe ALBO34a for the genera Alcaligenes and Bordetella, and probe Bcv13b for Burkholderia cepacia and Burkholderia vietnamiensis. Bacteria hybridizing with the probe Bcv13b represented the main Solvesso100-degrading population in the reactor.Many branches of industry produce waste gases which contain odorous organic and inorganic components. Apart from the conventional thermal and physicochemical techniques for removal of pollutants from exhaust air, biological waste gas treatment is becoming more and more important. This kind of treatment is advantageous in cases in which the recovery of the components (e.g., absorption in liquids and adsorption in solids) or the utilization of a thermal process (thermal or catalytic combustion) is not economical. Today three different process variations for biological waste gas treatment are used: biofilters, bioscrubbers, and trickle-bed bioreactors. In biofilters and trickle-bed reactors, the pollutant-degrading microorganisms are immobilized on a carrier material, whereas in bioscrubbers the microorganisms are dispersed in the liquid phase. Biofilters and bioscrubbers are preferred in industry, while biofilters are common in compost production and sewage plants (10).Biological waste gas treatment has a long tradition. Already in 1953, a soil system was employed for the treatment of odorous sewer exhaust gases in Long Beach, Calif. (25), and although up to now a lot of efforts have been funneled into process engineering (14, 17, 18, 24), current knowledge of the involved microorganisms is still very limited. Diversity of the microbial communities in the bioreactors for the exhaust gas purification have mostly been analyzed by culture-dependent methods (9, 12, 28, 31). However, there is a large discrepancy between the total (direct) microscopic cell counts and viable plate counts in many ecosystems and every cultivation medium selects for certain microorganisms. Therefore, cultivation-based studies of bacterial populations may give wrong impressions of the actual community structure in an ecosystem (35). A possible means of avoiding qualitative and quantitative errors in the analysis of microbial community structure in complex ecosystems is the use of fluorescently labeled, rRNA-targeted oligonucleotides (5) for the in situ identification and enumeration of bacteria. This method has already been used successfully in complex microbial communities, such as multispecies biofilms (6, 22, 26), trickling filters (27), and activated sludge (37).The current study was performed with a laboratory-scale trickle-bed bioreactor degrading a mixture of aromatic compounds (Solvesso100). The starter culture for the inoculation of the bioreactor was an enrichment prepared in a fermentor which was itself started with a wastewater sample from a car painting factory as the inoculum and Solvesso100 as the sole carbon source. The goal of our study was to use for the first time fluorescent in situ hybridization (FISH) to investigate the microbial community structure and dynamics in the fermentor and the bioreactor during start-up. One of the open questions was whether the fermentor enrichment, which is done in suspension, indeed selects for those bacteria that later are immobilized in the bioreactor. In the course of this study, new 16S as well as 23S rRNA-targeted probes for phylogenetic groups within the beta subclass of the class Proteobacteria have been developed and applied in order to obtain a higher taxonomic resolution of the molecular techniques. The molecular data were compared to those obtained by traditional cultivation-dependent techniques.     

Evolution of Histone H4 and H3 Genes in Different Ciliate Lineages

The histones H4 are known as highly conserved proteins. However, in ciliates a high degree of variation was found compared both to other eukaryotes and between the ciliate species. To date, only H4 histones of species belonging to two distantly related classes have been investigated. In order to obtain more detailed information on histone H4 variation in ciliates we undertook a comprehensive sequence analysis of PCR-amplified internal H4 fragments from 12 species belonging to seven out of the nine currently recognized ciliate classes. In addition, we used PCR primers to amplify longer fragments of H3 and H4 genes including the intergenic region. The encoded amino acid sequences reveal a high number of differences when compared with those of other eukaryotes and the ciliate species investigated. Furthermore, in some species H4 gene variants were detected, which result in amino acid differences. The greatest number of substitutions and insertions found was in the amino terminal region of the H4 histones. However, all sequences possess a conserved region corresponding to those of all other eukaryotic H4 histones. The histone gene variations were used to reconstruct phylogenetic relationships. The tree from our data matches perfectly with the ribosomal RNA data: The heterotrichs, which were considered as a late branching lineage, diverge at the base of the ciliate tree and groups formerly thought to represent ancestral lineages now appear as highly derived ciliates. Received: 4 April 1997 / Accepted: 1 August 1997     

Characterization of a Dopamine D1 Receptor from Apis mellifera: Cloning, Functional Expression, Pharmacology, and mRNA Localization in the Brain

Abstract: The neurotransmitter dopamine is an important regulator of physiological and behavioral functions in both vertebrates and invertebrates. We have isolated a homologue of the vertebrate dopamine D1 receptor subfamily from the honeybee Apis mellifera . [³H]Lysergic acid diethylamide specifically binds to the heterologously expressed receptor with K _D∼5 n M . Dopaminergic receptor ligands compete for this high-affinity binding, with the following order of potency: R (+)-lisuride > chlorpromazine = cis ( Z )-flupentixol > dopamine > S (+)-butaclamol > R (+)-SCH 23390 > haloperidol. Activation of the heterologously expressed receptor of Apis mellifera leads to cyclic AMP production. Receptor mRNA is expressed in perikarya of different brain neuropils, including those of mushroom body intrinsic neurons. These results suggest that this dopamine receptor is involved in signal processing of visual and olfactory information in the honeybee.     

The Interleukin-17 Gene of Herpesvirus Saimiri

In comparison to wild-type herpesvirus saimiri, viral interleukin-17 gene knockout mutants have unaltered behavior regarding viral replication, T-cell transformation in vitro, and pathogenicity in cottontop tamarins. Thus, this gene is not required for T-cell lymphoma induction but may contribute to apathogenic viral persistence in the natural host, the squirrel monkey.