Scale-up from shake flasks to fermenters in batch and continuous mode with Corynebacterium glutamicum on lactic acid based on oxygen transfer and pH

Scale-up from shake flasks to fermenters has been hampered by the lack of knowledge concerning the influence of operating conditions on mass transfer, hydromechanics, and power input. However, in recent years the properties of shake flasks have been described with empirical models. A practical scale-up strategy for everyday use is introduced for the scale-up of aerobic cultures from shake flasks to fermenters in batch and continuous mode. The strategy is based on empirical correlations of the volumetric mass transfer coefficient (k(L) a) and the pH. The accuracy of the empirical k(L) a correlations and the assumptions required to use these correlations for an arbitrary biological medium are discussed. To determine the optimal pH of the culture medium a simple laboratory method based on titration curves of the medium and a mechanistic pH model, which is solely based on the medium composition, is applied. The effectiveness of the scale-up strategy is demonstrated by comparing the behavior of Corynebacterium glutamicum on lactic acid in shake flasks and fermenters in batch and continuous mode. The maximum growth rate (micro(max) = 0.32 h(-1)) and the oxygen substrate coefficient (Y O2 /S= 0.0174 mol/l) of C. glutamicum on lactic acid were equal for shake flask, fermenter, batch, and continuous cultures. The biomass substrate yield was independent of the scale, but was lower in batch cultures (Y(X/S) = 0.36 g/g) than in continuous cultures (Y(X/S) = 0.45 g/g). The experimental data (biomass, respiration, pH) could be described with a simple biological model combined with a mechanistic pH model.     

Copulation behaviour of Glossina pallidipes (Diptera: Muscidae) outside and inside the female, with a discussion of genitalic evolution

If species-specific male genitalia are courtship devices under sexual selection by cryptic female choice, then species-specific aspects of the morphology and behaviour of male genitalia should often function to stimulate the female during copulation. The morphology and behaviour of the complex, species-specific male genitalia of the tsetse fly, Glossina pallidipes Austen, were determined from both direct observations and dissections of flash-frozen copulating pairs; we found that some male genitalic traits probably function to stimulate the female, while others function to restrain her. The male clamps the ventral surface of the female's abdomen tightly with his powerful cerci. Clamping does not always result in intromission. Clamping bends the female's body wall and her internal reproductive tract sharply, posteriorly and dorsally, and pinches them tightly. The male performed sustained, complex, stereotyped, rhythmic squeezing movements with his cerci that were not necessary to mechanically restrain the female and appeared instead to have a stimulatory function. Six different groups of modified setae on and near the male's genitalia rub directly against particular sites on the female during squeezing. The designs of these setae correlate with the force with which they press on the female and the probable sensitivity of the female surfaces that they contact. As expected under the hypothesis that these structures are under sexual selection by female choice, several traits suspected to have stimulatory functions have diverged in G. pallidipes and its close relative, G. longipalpis. Additional male non-genitalic behaviour during copulation, redescribed more precisely than in previous publications, is also likely to have a courtship function. The elaborate copulatory courtship behaviour and male genitalia may provide the stimuli that previous studies showed to induce female ovulation and resistance to remating.     

Quantitative determination of the macrolide antibiotic tulathromycin in plasma and broncho-alveolar cells of foals using tandem mass spectrometry

The long-acting antibiotic tulathromycin is on the marked for treatment of pulmonary infection of cattle, swine and horses. To measure disposition and distribution of tulathromycin in foals, a high throughput method was developed for horse plasma (calibration range: 0.006-0.8 microg/mL) and broncho-alveolar cells (calibration range: 0.1-4.0 microg/10(9)cells) using tandem mass spectrometry. Tulathromycin was extracted from plasma and broncho-alveolar fluid using cation exchange cartridges with acetonitrile/ammonia (95:5, v/v). The chromatography was performed isocratically with a mobile phase consisting of 5 mM ammonium formiate buffer/acetonitrile (30:70, v/v). The mass spectrometer operated in selected ion mode with atmospheric pressure chemical ionization to monitor the respective MH+ ions m/z 577.3 for tulathromycin and m/z 679.3 for the internal standard roxithromycin. All quality parameters fulfilled the international criteria for bioanalytical method validation and were successfully applied to the determination of tulathromycin in plasma of foals and broncho-alveolar cells. In foals, tulathromycin after intramuscular administration was rapidly absorbed, widely distributed and slowly eliminated. It cumulated manifold in broncho-alveolar cells.     

Vitellogenin cleavage products as indicators for toxic stress in zebra fish embryos: a proteomic approach

Vitellogenins (Vtgs) are the major yolk proteins in all oviparous animals. Systematic and regulated processing of these during embryogenesis is crucial for embryonic development. In the present study, toxicant-induced disturbance of Vtg degradation processes during Danio rerio (DR) embryogenesis was analysed to establish a sensitive tool for monitoring toxic stress at the molecular level. A 2-DE-based proteomic approach for whole DR embryos was established to study Vtg cleavage products (lipovitellin (Lv) derivatives). Ethanol was chosen as a positive control for a toxicity related change in the proteome of whole zebra fish embryos. Protein extracts from embryos treated with two ethanol concentrations, 0.5 and 2% v/v, showing either no or very strong visible effects, like absent heartbeat and blood circulation, were examined. Significant changes in the Lv pattern were detected for both conditions. The results are interpreted as scope for the use of the high abundant Lv derivatives as sensitive stress indicators in zebra fish embryos reflecting the overall fitness of the intact organisms.     

Proteome analysis of chemically induced mouse liver tumors with different genotype

Mouse liver tumors frequently harbor mutations in Ha-ras, B-raf, or Ctnnb1 (encoding beta-catenin). We conducted a proteome analysis with protein extracts from normal mouse liver and from liver tumors which were induced by a single injection of N-nitrosodiethylamine (DEN) as initiator followed by multiple injections of two different polychlorinated biphenyls (PCBs) as tumor promoters, or corn oil as a control. Liver tumors were stratified into two classes: they were either mutated in Ctnnb1 and positive for the marker glutamine synthetase (GS(+)), or they lacked Ctnnb1 mutations and were therefore GS-negative (GS(-)). Proteome analysis by 2-DE and MS revealed 98 significantly deregulated proteins, 44 in GS(+) and 54 in GS(-) tumors. Twelve of these proteins showed expression changes in both tumor types, but only seven of them were deregulated in the same direction. Several of the identified enzymes could be assigned to fundamental metabolic or other cellular pathways with characteristically different alterations in GS(+) and GS(-) tumors such as ammonia and amino acid turnover, cellular energy supply, and calcium homeostasis. Our data suggest that GS(+) and GS(-) tumor cells show a completely different biology and use divergent evolutionary strategies to gain a selective advantage over normal hepatocytes.     

Structure of coenzyme F420H2 oxidase (FprA), a di-iron flavoprotein from methanogenic Archaea catalyzing the reduction of O2 to H2O

The di-iron flavoprotein F(420)H(2) oxidase found in methanogenic Archaea catalyzes the four-electron reduction of O(2) to 2H(2)O with 2 mol of reduced coenzyme F(420)(7,8-dimethyl-8-hydroxy-5-deazariboflavin). We report here on crystal structures of the homotetrameric F(420)H(2) oxidase from Methanothermobacter marburgensis at resolutions of 2.25 A, 2.25 A and 1.7 A, respectively, from which an active reduced state, an inactive oxidized state and an active oxidized state could be extracted. As found in structurally related A-type flavoproteins, the active site is formed at the dimer interface, where the di-iron center of one monomer is juxtaposed to FMN of the other. In the active reduced state [Fe(II)Fe(II)FMNH(2)], the two irons are surrounded by four histidines, one aspartate, one glutamate and one bridging aspartate. The so-called switch loop is in a closed conformation, thus preventing F(420) binding. In the inactive oxidized state [Fe(III)FMN], the iron nearest to FMN has moved to two remote binding sites, and the switch loop is changed to an open conformation. In the active oxidized state [Fe(III)Fe(III)FMN], both irons are positioned as in the reduced state but the switch loop is found in the open conformation as in the inactive oxidized state. It is proposed that the redox-dependent conformational change of the switch loop ensures alternate complete four-electron O(2) reduction and redox center re-reduction. On the basis of the known Si-Si stereospecific hydride transfer, F(420)H(2) was modeled into the solvent-accessible pocket in front of FMN. The inactive oxidized state might provide the molecular basis for enzyme inactivation by long-term O(2) exposure observed in some members of the FprA family.     

Gaseous NO2 as a regulator for ammonia oxidation of Nitrosomonas eutropha

Cells of Nitrosomonas eutropha strain N904 that were denitrifying under anoxic conditions with hydrogen as electron donor and nitrite as electron acceptor were unable to utilize ammonium (ammonia) as an energy source. The recovery of ammonia oxidation activity was dependent on the presence of NO₂. Anaerobic ammonia oxidation activity was observed in a helium atmosphere supplemented with 25 ppm NO₂ after 20 h. Ammonia oxidation activity was detected after 2–3 days using an oxic atmosphere with 25 ppm NO₂. In contrast, ammonia consumption started after 8–9 days under oxic conditions without the addition of NO₂; in this case, small amounts of NO and NO₂ were detected and their concentrations increased with increasing ammonia oxidation activities. Hardly any ammonia oxidation was detected when nitrogen oxides were removed by intensive aeration. It would seem, therefore, that NO₂ is the master regulatory signal for ammonia oxidation in Nitrosomonas eutropha. Anaerobic ammonia oxidation activity was inhibited by the addition of NO. This inhibition was partly compensated by either increasing the NO₂ concentration or by using 2,3-dimercapto-1-propane-sulfonic acid as a NO binding substrate. DMPS was inhibitory to nitrification under oxic conditions, while increased amounts of NO or NO₂ led to increased oxidation activities.     

Hydrogel modified uptake of salt ions and calcium in<Emphasis Type="Italic"> Populus euphratica</Emphasis> under saline conditions

The effects of hydrogel on growth and ion relationships of a salt resistant woody species, Populus euphratica , were investigated under saline conditions. The hydrogel used was Stockosorb K410, a highly cross-linked polyacrylamide with about 40% of the amide group hydrolysed to carboxylic groups. Amendment of saline soil (potassium mine refuse) with 0.6% hydrogel improved seedling growth (2.7-fold higher biomass) over a period of 2 years, even though plant growth was reduced by salinity. Hydrogel-treated plants had approximately 3.5-fold higher root length and root surface area than those grown in unamended saline soil. In addition, over 6% of total roots were aggregated in gel fragments. Tissue and cellular ion analysis showed that growth improvement appeared to be the result of increased capacity for salt exclusion and enhancement of Ca²⁺ uptake. X-ray microanalysis of root compartments indicated that the presence of polymer restricted apoplastic Na⁺ in both young and old roots, and limited apoplastic and cytoplastic Cl^– in old roots while increasing Cl^– compartmentation in cortical vacuoles of both young and old roots. Collectively, radical transport of salt ions (Na⁺ and Cl^–) through the cortex into the xylem was lowered and subsequent axial transport was limited. Hydrogel treatment enhanced uptake of Ca²⁺ and microanalysis showed that enrichment of Ca²⁺ in root tissue mainly occurred in the apoplast. In conclusion, enhanced Ca²⁺ uptake and the increased capacity of P. euphratica to exclude salt were the result of improved Ca²⁺/Na⁺ concentration of soil solution available to the plant. Hydrogel amendment improves the quality of soil solutions by lowering salt level as a result of its salt-buffering capacity and enriching Ca²⁺ uptake, because of the polymers cation-exchange character. Accordingly, root aggregation allows good contact of roots with a Ca²⁺ source and reduces contact with Na⁺ and Cl^–, which presumably plays a major role in enhancing salt tolerance of P. euphratica.     

Mechanical anisotropy of adherent cells probed by a three-dimensional magnetic twisting device

We describe a three-dimensional magnetic twisting device that is useful in characterizing the mechanical properties of cells. With the use of three pairs of orthogonally aligned coils, oscillatory mechanical torque was applied to magnetic beads about any chosen axis. Frequencies up to 1 kHz could be attained. Cell deformation was measured in response to torque applied via an RGD-coated, surface-bound magnetic bead. In both unpatterned and micropatterned elongated cells on extracellular matrix, the mechanical stiffness transverse to the long axis of the cell was less than half that parallel to the long axis. Elongated cells on poly-L-lysine lost stress fibers and exhibited little mechanical anisotropy; disrupting the actin cytoskeleton or decreasing cytoskeletal tension substantially decreased the anisotropy. These results suggest that mechanical anisotropy originates from intrinsic cytoskeletal tension within the stress fibers. Deformation patterns of the cytoskeleton and the nucleolus were sensitive to loading direction, suggesting anisotropic mechanical signaling. This technology may be useful for elucidating the structural basis of mechanotransduction. cytoskeleton; prestress; stress fibers; mechanotransduction; mechanical deformation