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Acetic acid bacteria such as Gluconobacter oxydans are used in several biotechnological processes due to their ability to perform rapid incomplete regio- and stereo-selective oxidations of a great variety of carbohydrates, alcohols, and related compounds by their membrane-bound dehydrogenases. In order to understand the growth physiology of industrial strains such as G. oxydans ATCC 621H that has high substrate oxidation rates but poor growth yields, we compared its genome sequence to the genome sequence of strain DSM 3504 that reaches an almost three times higher optical density. Although the genome sequences are very similar, DSM 3504 has additional copies of genes that are absent from ATCC 621H. Most importantly, strain DSM 3504 contains an additional type II NADH dehydrogenase (ndh) gene and an additional triosephosphate isomerase (tpi) gene. We deleted these additional paralogs from DSM 3504, overexpressed NADH dehydrogenase in ATCC 621H, and monitored biomass and the concentration of the representative cell components as well as O2 and CO2 transfer rates in growth experiments on mannitol. The data revealed a clear competition of membrane-bound dehydrogenases and NADH dehydrogenase for channeling electrons in the electron transport chain of Gluconobacter and an important role of the additional NADH dehydrogenase for increased growth yields. The less active the NADH dehydrogenase is, the more active is the membrane-bound polyol dehydrogenase. These results were confirmed by introducing additional ndh genes via plasmid pAJ78 in strain ATCC 621H, which leads to a marked increase of the growth rate.

AbstractThe mutant M301A of the acetylcholinesterase B from Nippostrongylus brasiliensis (NbAChE) was produced in a high‐cell‐density fermentation of a recombinant methylotrophic yeast Pichia pastoris. Dissolved oxygen (DO) spikes were used as an indicator for feeding the carbon source. Wet cell weight (WCW) reached after 8 days a maximum value of 316 g/L and the OD600 at this time was 280. The acetylcholinesterase activity increased up to 6,600 U/mL corresponding to an expression rate of 2 g of NbAChE per liter supernatant. The specific activity of the mutant NbAChE was determined after purification as 3,300 U/mg. Active site titration with chlorpyrifos, a strong AChE inhibitor, yielded in a specific activity of 3,400 U/mg. The enzyme was secreted by Pichia pastoris. Therefore, it could be concentrated from culture broth by cross‐flow‐filtration (50 kDa cut‐off membrane). It was further purified in one‐step anion‐exchange chromatography, using a XK 50/20 column filled with 125 mL Q Sepharose HP. Mutant NbAChE was purified 1.9‐fold up to a purity of 97% and a yield of 87%. The isolated enzyme was nearly homogenous, as seen on the silver stained SDS–PAGE as well as by a single peak after gel filtration. This extraordinary high expression rate and the ease of purification is an important prerequisite for their practical application, for example in biosensors for the detection of neurotoxic insecticides. © 2005 Wiley Periodicals, Inc.

AbstractThe aim of this study was to determine if extracorporeal shock wave therapy (ESWT) in vivo affects the structural integrity of articular cartilage. A single bout of ESWT (1500 shock waves of 0.5 mJ/mm2) was applied to femoral heads of 18 adult Sprague–Dawley rats. Two sham‐treated animals served as controls. Cartilage of each femoral head was harvested at 1, 4, or 10 weeks after ESWT (n = 6 per treatment group) and scored on safranin‐O‐stained sections. Expression of tenascin‐C and chitinase 3‐like protein 1 (Chi3L1) was analyzed by immunohistochemistry. Quantitative real‐time polymerase chain reaction (PCR) was used to examine collagen (II)α1 (COL2A1) expression and chondrocyte morphology was investigated by transmission electron microscopy no changes in Mankin scores were observed after ESWT. Positive immunostaining for tenascin‐C and Chi3L1 was found up to 10 weeks after ESWT in experimental but not in control cartilage. COL2A1 mRNA was increased in samples 1 and 4 weeks after ESWT. Alterations found on the ultrastructural level showed expansion of the rough‐surfaced endoplasmatic reticulum, detachment of the cell membrane and necrotic chondrocytes. Extracorporeal shock waves caused alterations of hyaline cartilage on a molecular and ultrastructural level that were distinctly different from control. Similar changes were described before in the very early phase of osteoarthritis (OA). High‐energy ESWT might therefore cause degenerative changes in hyaline cartilage as they are found in initial OA. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1050–1056, 2010

The glutamine transporter SLC38A3 (SNAT3) plays an important role in the release of glutamine from brain astrocytes and the uptake of glutamine into hepatocytes. It is related to the vesicular GABA (gamma-aminobutyric acid) transporter and the SLC36 family of proton-amino acid cotransporters. The transporter carries out electroneutral Na+-glutamine cotransport-H+ antiport. In addition, substrate-induced uncoupled cation currents are observed. Mutation of asparagine 76 to glutamine or histidine in predicted transmembrane helix 1 abolished all substrate-induced currents. Mutation of asparagine 76 to aspartate rendered the transporter Na+-independent and resulted in a gain of a large substrate-induced chloride conductance in the absence of Na+. Thus, a single residue is critical for coupled and uncoupled ion flows in the glutamine transporter SNAT3. Homology modeling of SNAT3 along the structure of the related benzyl-hydantoin permease from Microbacterium liquefaciens reveals that Asn-76 is likely to be located in the center of the membrane close to the translocation pore and forms part of the predicted Na+ -binding site.

Lipase‐catalyzed transesterification of triglycerides and alcohols to obtain biodiesel is an environmentally friendly and sustainable route for fuels production since, besides proceeding in mild reaction conditions, it allows for the use of low‐cost feedstocks that contain water and free fatty acids, for example non‐edible oils and waste oils. This review article reports recent advances in the field and focus in particular on a major issue in the enzymatic process, the inactivation of most lipases caused by methanol, the preferred acyl acceptor used for alcoholysis. The recent results about immobilization of enzymes on nano‐materials and the use of whole‐cell biocatalysts, as well as the use of cell‐surface display technologies and metabolic engineering strategies for microbial production of biodiesel are described. It is discussed also insight into the effects of methanol on lipases obtained by modeling approaches and report on studies aimed at mining novel alcohol stable enzymes or at improving robustness in existing ones by protein engineering.

Ethnopharmacological relevance Phaseaoli pericarpium (bean pods) is a pharmacopeial plant material traditionally used as a diuretic and antidiabetic agents. Diuretic activity of pod extracts was reported first in 1608. Since then Phaseoli pericarpium tea figures in many textbooks as medicinal plant material used by patients. Aim of the study Despite the traditional use of extracts from Phaseolium vulgaris pericarp, limited information is available on bioactivity, chemical composition, and bioavailability of such preparations. The following study aimed to investigate the phytochemical composition, the in vitro permeability of selected extract's constituents over the Caco-2 permeation system, and potential antivirulence activity against uropathogenic Escherichia coli of a hydroalcoholic Phaseoli pericarpium extract (PPX) in vitro to support its traditional use as a remedy used in urinary tract infections. Material and methods The chemical composition of the extract PPX [ethanol:water 7:3 (v/v)] investigated by using UHPLC-DAD-MSn and subsequent dereplication. The permeability of compounds present in PPX was evaluated using the Caco-2 monolayer permeation system. The influence of PPX on uropathogenic E. coli (UPEC) strain NU14 proliferation and against the bacterial adhesion to T24 epithelial cells was determined by turbidimetric assay and flow cytometry, respectively. The influence of the extract on the mitochondrial activity of T24 host cells was monitored by MTT assay. Results LC-MSn investigation and dereplication, indicated PPX extract to be dominated by a variety of flavonoids, with rutin as a major compound, and soyasaponin derivatives. Rutin, selected soyasaponins and fatty acids were shown to permeate the Caco-2 monolayer system, indicating potential bioavailability following oral intake. The extract did not influence the viability of T24 cells after 1.5h incubation at 2 mg/mL and UPEC. PPX significantly reduced the bacterial adhesion of UPEC to human bladder cells in a concentration-dependent manner (0.5–2 mg/mL). Detailed investigations by different incubation protocols indicated that PPX seems to interact with T24 cells, which subsequently leads to reduced recognition and adhesion of UPEC to the host cell membrane. Conclusions PPX is characterised by the presence of flavonoids (e.g. rutin) and saponins, from which selected compounds might be bioavailable after oral application, as indicated by the Caco-2 permeation experiments. Rutin and some saponins can be considered as potentially bioavailable after the oral intake. The concentration-dependent inhibition of bacterial adhesion of UPEC to T24 cells justifies the traditional use of Phaseoli pericarpium in the prevention and treatment of urinary tract infections.

More and more microbes are discovered that are capable of extracellular electron transfer, a process in which they use external electrodes as electron donors or acceptors for metabolic reactions. This feature can be used to overcome cellular redox limitations and thus optimizing microbial production. The technologies, termed microbial electrosynthesis and electro-fermentation, have the potential to open novel bio-electro production platforms from sustainable energy and carbon sources. However, the performance of reported systems is currently limited by low electron transport rates between microbes and electrodes and our limited ability for targeted engineering of these systems due to remaining knowledge gaps about the underlying fundamental processes. Metabolic engineering offers many opportunities to optimize these processes, for instance by genetic engineering of pathways for electron transfer on the one hand and target product synthesis on the other hand. With this review, we summarize the status quo of knowledge and engineering attempts around chemical production in bio-electrochemical systems from a microbe perspective. Challenges associated with the introduction or enhancement of extracellular electron transfer capabilities into production hosts versus the engineering of target compound synthesis pathways in natural exoelectrogens are discussed. Recent advances of the research community in both directions are examined critically. Further, systems biology approaches, for instance using metabolic modelling, are examined for their potential to provide insight into fundamental processes and to identify targets for metabolic engineering.

It has been estimated that more than 80% of alcoholics are also nicotine dependent and that, vice versa, the rate of alcoholism is substantially increased by a factor of 4-10 in the nicotine-dependent population. However, the cause for this very high degree of comorbidity is still largely unknown. At the molecular and cellular level, both drugs have very different mechanisms of action. Nicotine specifically activates ligand-gated ion channels in the brain, which are normally gated by acetylcholine, while alcohol interacts with various neurotransmitter receptors. Despite this diversity, both drugs seem to engage the endogenous opioid system as a modulator of some of its pharmacological effect. An acute exposure to nicotine or alcohol leads to a release of opioid peptides in specific brain regions, thus resulting in an activation of their corresponding receptors. If the brain is exposed repeatedly or chronically to these drugs, adaptive changes in the level and expression of opioid peptides and receptors occur. These adaptive changes are thought to contribute to the homeostatic or allostatic adaptations of the brain, which have been associated with drug dependence. This review summarizes pharmacological and genetic studies in animal models and in humans that have addressed the role of specific opioid peptides and receptors in various stages of the addiction process.

We present a protocol for the biosensor Cell-Fit-HD4D. It enables long-term monitoring and correlation of single-cell fate with subcellular-deposited energy of ionizing radiation. Cell fate tracking using widefield time-lapse microscopy is uncoupled in time from confocal ion track imaging. Registration of both image acquisition steps allows precise ion track assignment to cells and correlation with cellular readouts. For complete details on the use and execution of this protocol, please refer to Niklas et al. (2022).

The light-driven proton pump bacteriorhodopsin (bR) was functionally expressed in Xenopus laevis oocytes and in HEK-293 cells. The latter expression system allowed high time resolution of light-induced current signals. A detailed voltage clamp and patch clamp study was performed to investigate the DeltapH versus Deltapsi dependence of the pump current. The following results were obtained. The current voltage behavior of bR is linear in the measurable range between -160 mV and +60 mV. The pH dependence is less than expected from thermodynamic principles, i.e., one DeltapH unit produces a shift of the apparent reversal potential of 34 mV (and not 58 mV). The M(2)-BR decay shows a significant voltage dependence with time constants changing from 20 ms at +60 mV to 80 ms at -160 mV. The linear I-V curve can be reconstructed by this behavior. However, the slope of the decay rate shows a weaker voltage dependence than the stationary photocurrent, indicating that an additional process must be involved in the voltage dependence of the pump. A slowly decaying M intermediate (decay time > 100 ms) could already be detected at zero voltage by electrical and spectroscopic means. In effect, bR shows optoelectric behavior. The long-lived M can be transferred into the active photocycle by depolarizing voltage pulses. This is experimentally demonstrated by a distinct charge displacement. From the results we conclude that the transport cycle of bR branches via a long-lived M(1)* in a voltage-dependent manner into a nontransporting cycle, where the proton release and uptake occur on the extracellular side.