Protein-sensing assay formats and devices.

Proteins are used as biocatalysts, therapeutic or diagnostic agents, and as such they are biotechnological products. Moreover, they are biomarkers for health states, diseases or toxic or other adverse effects, and the intracellular protein network is essential for the adaptation of an organism to its environment. Thus, there is a strong need for analytical methods for protein determination, which allow not only to indicate the presence of a protein, but also its concentration, covalent modification and activity, and corresponding developments of new methods experienced strong support.

Controlling biofilms of gram-positive pathogenic bacteria.

Many bacteria can form aggregates on interfaces, called biofilms, where they are much more protected against toxic agents such as antibiotics or antibodies. Bacteria organized in biofilms are therefore very difficult to control and often even high dosages of antibiotics cannot clear infectious biofilms. To form biofilms bacteria have to start a complex genetic program to switch from planktonic to sessile lifestyle.

Development of an enzyme-linked immunoreceptor assay (ELIRA) for quantification of the biological activity of recombinant human bone morphogenetic protein-2.

Human bone morphogenetic protein-2 is a representative of the transforming growth factor-beta (TGF-beta) superfamily of cytokines. It was produced in high-cell-density cultivations of recombinant Escherichia coli leading to the formation of inclusion bodies with aggregated inactive protein so that the protein had to be solubilized and renatured. Thus, the biological activity of the recombinant protein had to be determined.

Development of a rapid, quantitative glucosyltransferase assay based on a screen-printed fructose enzyme electrode and application to optimization studies on gtfD expression in recombinant Escherichia coli.

A biosensor for fructose determination was used as basis of an assay for the determination of glucosyltransferase (GTF) activities and applied to monitoring recombinant enzyme production. GTFs catalyze the synthesis of glucans from sucrose leading to the release of fructose. Specific fructose determinations in the microM concentration range were achieved with a fructose electrode based on fructose dehydrogenase, which was immobilized on a screen-printed platinum electrode.

Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. II. Dynamic response to famine and feast, activation of the methylglyoxal pathway and oscillatory behaviour.

The metabolic dynamics of the Escherichia coli K-12 strain TG1 to feast and famine were studied in glucose-limited steady-state cultures by up- and downshifts of the dilution rate, respectively. An uncoupling of anabolic and catabolic rates was observed upon dilution rate upshifts, apparent through immediately increased glucose uptake rates which were not accompanied by an immediate increase of the growth rate but instead resulted in the temporary excretion of methylglyoxal, D- and L-lactate, pyruvate and, after a delay, acetate. The energetic state of the cell during the transient was followed by measuring the adenylate energy charge, which increased within 2 min after the upshift and declined thereafter until a new steady-state level was reached.

Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. I. Growth-rate-dependent metabolic efficiency at steady state.

The Escherichia coli K-12 strain TG1 was grown at 28 degrees C in aerobic glucose-limited continuous cultures at dilution rates ranging from 0.044 to 0.415 h(-1).

Application of an SPR-based receptor assay for the determination of biologically active recombinant bone morphogenetic protein-2.

Human bone morphogenetic protein-2 (hBMP-2) is a member of the human transforming growth factor-beta superfamily. Biologically active bone morphogenetic protein-2 (BMP-2) is a dimeric protein that binds in the first step of the signal transduction cascade to specific receptors on the cell-surface. This specific interaction of the dimeric protein with the extracellular ligand-binding domain (ECD) of the receptor was used to develop a receptor-based assay based on an optical biosensor system (Biacore 2000, Biacore AB, Uppsala, Sweden).

Role of host genetic factors in susceptibility to group A streptococcal infections.

Background & Objectives: Epidemiological evidences indicate that host genetic factors might be critical in determining susceptibility to infection with group A streptococci (GAS). The objective of the present study was to determine the extent to which the genetic background of the mouse strain affected induction and resolution of GAS infection.

Methods: Several inbred mouse strains were intravenously infected with Streptococcus pyogenes strain A20 and the mean survival times of mice was recorded overtime.

Characterization of the interaction of the pneumococcal surface protein SpsA with the human polymeric immunoglobulin receptor (hpIgR).

Background & Objectives: The polymeric immunoglobulin receptor (pIgR) is produced by mucosal epithelial cells and plays a crucial role in mucosal immunity. At the basolateral surface of mucosal cells, the pIgR binds predominantly polymeric immunoglobulins, such as dimeric IgA and polymeric IgA (pIgA) and mediates their transport across the polarized cells. This results in apical release of secretory component (SC), either free or bound covalently to IgA, forming secretory IgA (SIgA).

Immunodetection and quantification of vascular endothelial growth factor receptor-3 in human malignant tumor tissues.

Vascular endothelial growth factor receptor-3 (VEGFR-3) and its ligands, vascular endothelial growth factor-C (VEGF-C) and -D (VEGF-D), are the major molecules involved in developmental and pathological lymphangiogenesis. Here we describe for the first time the development of a specific indirect enzyme-linked immunosorbent assay (ELISA) for the quantification of VEGFR-3 in different human cell and tissue lysates. A combination of the goat polyclonal anti-VEGFR-3 antibody and the mouse monoclonal anti-human VEGFR-3 antibody was used.

Selective leakage of host-cell proteins during high-cell-density cultivation of recombinant and non-recombinant Escherichia coli.

Significant leakage of host-cell proteins into the culture medium occurred during high-cell-density cultivation of E. coli. Identification of these medium proteins revealed almost exclusively a periplasmic origin.

Quantification of vascular endothelial growth factor-C (VEGF-C) by a novel ELISA.

Lymphangiogenesis plays an important role in several normal and pathological conditions such as wound healing, inflammation or metastasis formation in several malignancies. VEGF-C and VEGF-D are important and specific regulatory factors for lymphatic endothelial proliferation and lymphangiogenesis. In order to develop a highly sensitive and specific detection system for VEGF-C, we produced soluble binding proteins and antibodies for a microtiterplate-based assay.

Thalassolituus oleivorans gen. nov., sp. nov., a novel marine bacterium that obligately utilizes hydrocarbons.

An aerobic, heterotrophic, Gram-negative, curved bacterial strain, designated MIL-1T, was isolated by extinction dilution from an n-tetradecane enrichment culture that was established from sea water/sediment samples collected in the harbour of Milazzo, Italy. In the primary enrichment, the isolate formed creamy-white, medium-sized colonies on the surface of the agar. The isolate did not grow in the absence of NaCl; growth was optimal at 2.

A dual lethal system to enhance containment of recombinant micro-organisms.

Active containment systems based on the controlled expression of a lethal gene are designed to increase containment of recombinant micro-organisms used for environmental applications. A major drawback in containment is the existence of mutations that generate surviving cells that cease to respond to the toxic effect of the lethal function. In this work the authors have developed for the first time a strategy to reduce the problem of mutations and increase the efficiency of containment based on the combination of two lethal functions acting on different cellular targets of major concern in containment, DNA and RNA, and whose expression is under control of different regulatory signals.

Degradation of alkanes and highly chlorinated benzenes, and production of biosurfactants, by a psychrophilic Rhodococcus sp. and genetic characterization of its chlorobenzene dioxygenase.

Rhodococcus sp. strain MS11 was isolated from a mixed culture. It displays a diverse range of metabolic capabilities.

Thermoleophilum album and Thermoleophilum minutum are culturable representatives of group 2 of the Rubrobacteridae (Actinobacteria).

Analysis of the morphological and genotypic properties of three obligately thermophilic strains of Thermoleophilum album and Thermoleophilum minutum, originally described as green non-sulfur bacteria, indicates that these taxa belong to the Rubrobacter subdivision of the Actinobacteria. EM of the cell wall clearly showed morphology typical of Gram-positive bacteria. A comparison of 16S rRNA gene sequences, including signature nucleotide pairs and secondary structural features considered diagnostic for the subclass Rubrobacteridae, revealed that the three strains of Thermoleophilum were highly similar and should be considered as members of group 2 of this subclass, represented up to now only by uncultured organisms.

Induction of NF-kappaB nuclear translocation in human respiratory epithelial cells by group A streptococci.

The activation of the transcription factor NF-kappaB and the production of inflammatory mediators play an essential role in the host response to pathogenic organisms. The objective of this study was to investigate the ability of group A streptococci (GAS) to stimulate the nuclear translocation of NF-kappaB in cultured human epithelial (HEp-2) cells. Infection of HEp-2 cells with a strain of Streptococcus pyogenes capable to efficiently internalize HEp-2 cells (strain A40) resulted in translocation of NF-kappaB during the first 15 min of infection, reaching a peak after 30 min that persisted at slightly lower levels 1h thereafter.

Phagocytosis of bacille Calmette-Guérin-infected necrotic macrophages induces a maturation phenotype and evokes antigen-presentation functions in dendritic cells.

The interaction of pathogens with dendritic cells (DCs) seems to play a critical role in the initiation of the immune response. Tissue damage and induction of an inflammatory reaction are events frequently associated with the progression of the infection. Although DCs are very efficient at phagocytosing pathogens, the capacity of these cells to uptake microbes from a necrotic environment has not yet been proven.

Novel insights into the interplay between peripheral reactions encoded by xyl genes and the chlorocatechol pathway encoded by tfd genes for the degradation of chlorobenzoates by Ralstonia eutropha JMP134.

Many bacteria can grow on chloroaromatic pollutants because they can transform them into chlorocatechols, which are further degraded by enzymes of a specialized ortho-cleavage pathway. Ralstonia eutropha JMP134 is able to grow on 3-chlorobenzoate by using two pJP4-encoded, ortho-cleavage chlorocatechol degradation gene clusters (tfdC(I)D(I)E(I)F(I) and tfdD(II)C(II)E(II)F(II)). Very little is known about the acquisition of new catabolic genes encoding enzymes that lead to the formation of chlorocatechols in R.

Influence of proteins Bsp and FemH on cell shape and peptidoglycan composition in group B streptococcus.

Group B streptococcus (GBS) is surrounded by a capsule. However, little is known about peptidoglycan metabolism in these bacteria. In the present study, a 65 kDa protein was isolated from the culture supernatant of GBS and N-terminally sequenced, permitting isolation of the corresponding gene, termed bsp.

Expression of recombinant cytoplasmic yeast pyruvate carboxylase for the improvement of the production of human erythropoietin by recombinant BHK-21 cells.

Recently, a recombinant yeast pyruvate carboxylase expressed in the cytoplasm of BHK-21 cells was shown to reconstitute the missing link between glycolysis and TCA, thus increasing the flux of glucose into the TCA and resulting in a higher intracellular ATP content. Now, these metabolically engineered cells have been additionally transfected with a plasmid bearing the gene for human erythropoietin. EPO yield and substrate-specific productivity of the recombinant BHK-21 cells have been compared to control cells without the PYC2-gene but transfected with the plasmid coding for the expression of the selection genes and EPO.

Genetic control of susceptibility to group A streptococcal infection in mice.

The influence of genetic background on the ability to control infection with group A streptococci was investigated in different inbred strains of mice. Whereas BALB/c, C57BL/10, and DBA/2 mice were the most resistant strains, with lower bacteria loads and higher survival times, C3H/HeN and CBA/J mice exhibited substantially higher bacterial growth and 100% mortality. Differences in susceptibility were not dependent on the inoculum size.

Molecular characterization of a deletion/duplication rearrangement in tfd genes from Ralstonia eutropha JMP134(pJP4) that improves growth on 3-chlorobenzoic acid but abolishes growth on 2,4-dichlorophenoxyacetic acid.

Ralstonia eutropha JMP134(pJP4) is able to grow on minimal media containing the pollutants 3-chlorobenzoate (3-CB) or 2,4-dichlorophenoxyacetate (2,4-D). tfd genes from the 88 kb plasmid pJP4 encode enzymes involved in the degradation of these compounds. During growth of strain JMP134 in liquid medium containing 3-CB, a derivative strain harbouring a approximately 95 kb plasmid was isolated.