Reconstituted phosphatidylserine synthase from Escherichia coli is activated by anionic phospholipids and micelle-forming amphiphiles

The activity of phosphatidylserine (PS) synthase (CDP-1,2-diacyl-sn-glycerol: l-serine O-phosphatidyltransferase, EC 2.7.8.8) from Escherichia coli was studied after reconstitution with lipid vesicles of various compositions. PS synthase exhibited practically no activity in the absence of a detergent and with the substrate CDP-diacylglycerol (CDP-DAG) present only in the lipid vesicles. Inclusion of octylglucoside (OG) in the assay mixture increased the activity 20- to 1000-fold, the degree of activation depending on the lipid composition of the vesicles. Inclusion of additional CDP-DAG in the assay mixture increased the activity 5- to 25-fold. When the fraction of phosphatidylglycerol (PG) was increased from 15 to 100 mol% in the vesicles the activity increased 10-fold using the assay mixture containing OG. The highest activities were exhibited with the anionic lipids synthesized by E. coli, namely PG, diphosphatidylglycerol (DPG), and phosphatidic acid, while phosphatidylinositol gave a lower activity. Cryotransmission electron microscopy showed that transformation of the vesicles to micelles brings about an activation of the enzyme that is proportional to the degree of micellization. Thus, the activity of PS synthase is modulated by the lipid aggregate structure and by the fraction and type of anionic phospholipid in the aggregates. The increase in the activity caused by PG and DPG is physiologically relevant; it may be part of a regulatory mechanism that keeps the balance between phosphatidylethanolamine, and the sum of PG and DPG, nearly constant in wild-type E. coli cells.     

Shear treatment of starter culture medium improves separation behavior of Streptococcus thermophilus cells

A central step in the production of starter cultures is the separation of the cells from the fermentation medium, which is usually achieved by disk centrifuges. In case of microorganisms which produce exopolysaccharides (e.g., various strains of lactic acid bacteria), the properties of the respective exopolysaccharides may interfere with this separation step. By using six strains of Streptococcus thermophilus the hypothesis was tested that a shear treatment of the fermented culture medium improves subsequent cell separation markedly. Depending on the type of exopolysaccharides (freely present in the medium, or as capsules around the cells) an energy input of up to 2.5 kJ/mL generated with an Ultra‐Turrax affected cell chain length of the strains and viscosity of fermentation medium differently. For bacteria producing capsular exopolysaccharides, space‐ and time‐resolved centrifugation experiments revealed an increase of sedimentation velocity after shear treatment. In general, viability of the microorganisms, detected by flow cytometry measurements and fermentation experiments, was not affected by the shearing procedure. The results therefore indicate that strain‐targeted shearing is helpful to improve the separability of cells from the fermented media.     

Spermatophore formation and sperm ultrastructure of Sundathelphusa philippina (Crustacea: Brachyura: Gecarcinucidae)

We investigated the morphology of spermatozoa, spermatophores and the anterior vas deferens (AVD) of the gecarcinucid freshwater crab Sundathelphusa philippina. The morphology of the acrosome (proportions, structure and arrangement of acrosomal layers) and the spermatophores complies with the known sperm and spermatophore morphology of the brachyuran family Gecarcinucidae. The sperm cells are packed within coenospermic spermatophores that are of a mucous type, lacking a complex spermatophore wall. Spermatophore formation takes place in the distal part of the AVD. The strongly proliferated inner epithelium of the vas deferens releases vesicles via apocrine secretion. These vesicles fuse with the incipient spermatophores that subsequently coalesce, thus forming the coenospermic aggregates that represent the mature spermatophores.     

Molecular mimicry in the recognition of glycosphingolipids by Gal{alpha}3Gal{beta}4GlcNAc{beta}-binding Clostridium difficile toxin A, human natural anti {alpha}-galactosyl IgG and the monoclonal antibody Gal-13: characterization of a binding-active human glycosphingolipid, non-identical with the animal receptor

Glycoconjugates with terminal Gal     

Metabolic effects of influenza virus infection in cultured animal cells: Intra- and extracellular metabolite profiling

Background  

Many details in cell culture-derived influenza vaccine production are still poorly understood and approaches for process optimization mainly remain empirical. More insights on mammalian cell metabolism after a viral infection could give hints on limitations and cell-specific virus production capacities. A detailed metabolic characterization of an influenza infected adherent cell line (MDCK) was carried out based on extracellular and intracellular measurements of metabolite concentrations.     

Campylobacter jejuni Colonization in Wild Birds: Results from an Infection Experiment

Campylobacter jejuni is a common cause of bacterial gastroenteritis in most parts of the world. The bacterium has a broad host range and has been isolated from many animals and environments. To investigate shedding patterns and putative effects on an avian host, we developed a colonization model in which a wild bird species, the European Robin Erithacus rubecula, was inoculated orally with C. jejuni from either a human patient or from another wild bird species, the Song Thrush Turdus philomelos. These two isolates were genetically distinct from each other and provoked very different host responses. The Song Thrush isolate colonized all challenged birds and colonization lasted 6.8 days on average. Birds infected with this isolate also showed a transient but significant decrease in body mass. The human isolate did not colonize the birds and could be detected only in the feces of the birds shortly after inoculation. European Robins infected with the wild bird isolate generated a specific antibody response to C. jejuni membrane proteins from the avian isolate, which also was cross-reactive to membrane proteins of the human isolate. In contrast, European Robins infected with the human isolate did not mount a significant response to bacterial membrane proteins from either of the two isolates. The difference in colonization ability could indicate host adaptations.     

Identification of Growth Phases and Influencing Factors in Cultivations with AGE1.HN Cells Using Set-Based Methods

Production of bio-pharmaceuticals in cell culture, such as mammalian cells, is challenging. Mathematical models can provide support to the analysis, optimization, and the operation of production processes. In particular, unstructured models are suited for these purposes, since they can be tailored to particular process conditions. To this end, growth phases and the most relevant factors influencing cell growth and product formation have to be identified. Due to noisy and erroneous experimental data, unknown kinetic parameters, and the large number of combinations of influencing factors, currently there are only limited structured approaches to tackle these issues. We outline a structured set-based approach to identify different growth phases and the factors influencing cell growth and metabolism. To this end, measurement uncertainties are taken explicitly into account to bound the time-dependent specific growth rate based on the observed increase of the cell concentration. Based on the bounds on the specific growth rate, we can identify qualitatively different growth phases and (in-)validate hypotheses on the factors influencing cell growth and metabolism. We apply the approach to a mammalian suspension cell line (AGE1.HN). We show that growth in batch culture can be divided into two main growth phases. The initial phase is characterized by exponential growth dynamics, which can be described consistently by a relatively simple unstructured and segregated model. The subsequent phase is characterized by a decrease in the specific growth rate, which, as shown, results from substrate limitation and the pH of the medium. An extended model is provided which describes the observed dynamics of cell growth and main metabolites, and the corresponding kinetic parameters as well as their confidence intervals are estimated. The study is complemented by an uncertainty and outlier analysis. Overall, we demonstrate utility of set-based methods for analyzing cell growth and metabolism under conditions of uncertainty.     

Calling Biomarkers in Milk Using a Protein Microarray on Your Smartphone

Here we present the concept of a protein microarray-based fluorescence immunoassay for multiple biomarker detection in milk extracts by an ordinary smartphone. A multiplex immunoassay was designed on a microarray chip, having built-in positive and negative quality controls. After the immunoassay procedure, the 48 microspots were labelled with Quantum Dots (QD) depending on the protein biomarker levels in the sample. QD-fluorescence was subsequently detected by the smartphone camera under UV light excitation from LEDs embedded in a simple 3D-printed opto-mechanical smartphone attachment. The somewhat aberrant images obtained under such conditions, were corrected by newly developed Android-based software on the same smartphone, and protein biomarker profiles were calculated. The indirect detection of recombinant bovine somatotropin (rbST) in milk extracts based on altered biomarker profile of anti-rbST antibodies was selected as a real-life challenge. RbST-treated and untreated cows clearly showed reproducible treatment-dependent biomarker profiles in milk, in excellent agreement with results from a flow cytometer reference method. In a pilot experiment, anti-rbST antibody detection was multiplexed with the detection of another rbST-dependent biomarker, insulin-like growth factor 1 (IGF-1). Milk extract IGF-1 levels were found to be increased after rbST treatment and correlated with the results obtained from the reference method. These data clearly demonstrate the potential of the portable protein microarray concept towards simultaneous detection of multiple biomarkers. We envisage broad application of this ‘protein microarray on a smartphone’-concept for on-site testing, e.g., in food safety, environment and health monitoring.     

The main thylakoid membrane lipid monogalactosyldiacylglycerol (MGDG) promotes the de-epoxidation of violaxanthin associated with the light-harvesting complex of photosystem II (LHCII)

In higher plants, the major part of the xanthophyll cycle pigment violaxanthin (Vx) is non-covalently bound to the main light-harvesting complex of PSII (LHCII). Under saturating light conditions Vx has to be released from its binding site into the surrounding lipid phase, where it is converted to zeaxanthin (Zx) by the enzyme Vx de-epoxidase (VDE). In the present study we investigated the influence of thylakoid lipids on the de-epoxidation of Vx, which was still associated with the LHCII. We isolated LHCII with different concentrations of native, endogenous lipids and Vx by sucrose gradient centrifugation or successive cation precipitation. Analysis of the different LHCII preparations showed that the concentration of LHCII-associated Vx was correlated with the concentration of the main thylakoid lipid monogalactosyldiacylglycerol (MGDG) associated with the complexes. Decreases in the MGDG content of the LHCII led to a diminished Vx concentration, indicating that a part of the total Vx pool was located in an MGDG phase surrounding the LHCII, whereas another part was bound to the LHCII apoproteins. We further studied the convertibility of LHCII-associated Vx in in-vitro enzyme assays by addition of isolated VDE. We observed an efficient and almost complete Vx conversion in the LHCII fractions containing high amounts of endogenous MGDG. LHCII preparations with low concentrations of MGDG exhibited a strongly reduced Vx de-epoxidation, which could be increased by addition of exogenous, pure MGDG. The de-epoxidation of LHCII-associated Vx was saturated at a much lower concentration of native, endogenous MGDG compared with the concentration of isolated, exogenous MGDG, which is needed for optimal VDE activity in in-vitro assays employing pure isolated Vx.     

Binding of Haemophilus ducreyi to carbohydrate receptors is mediated by the 58.5-kDa GroEL heat shock protein

The bacterium Haemophilus ducreyi causes the sexually transmitted disease chancroid, which is characterized by the appearance of mucocutaneous, persistent ulcers on the external genitals. To identify carbohydrate receptors that mediate the attachment of this pathogen to host cells, we investigated the binding of 35S-methionine-labeled H. ducreyi strains to a panel of defined glycosphingolipids that were separated on thin layer chromatography plates. H. ducreyi bound to lactosylceramide, gangliotriaosylceramide, gangliotetraosylceramide, neolactotetraosylceramide, the GM3 ganglioside, and sulfatide. To elucidate the role of the surface-located 58.5-kDa GroEL heat shock protein (HSP) of H. ducreyi in attachment, we investigated the binding of purified HSP to the same panel of glycosphingolipids. Our results suggest that the 58.5-kDa GroEL HSP of H. ducreyi is responsible for the attachment of this bacterium to the majority of the tested glycosphingolipids, and thus represents a potential bacterial adhesin.