ABSTRACT
As the Bacillus Calmette-Guérin (BCG) vaccine does not confer long-lasting protection against lung Mycobacterium tuberculosis infection, the development of more efficient vaccines is greatly needed. Here, we used mycobacterial low-molecular weight proteins of the 6-kDa Early Secreted Antigenic Target (ESAT-6) protein family (ESX) antigens for the evaluation of a novel vaccine delivery strategy that enables versatile in vivo targeting of antigens into specialized dendritic cell (DC) subsets. ESX antigens were genetically fused to the tetramerizing core of streptavidin (SA) to form high-affinity complexes with biotin (biot)-conjugated antibodies recognizing DC surface receptors. When directed through the CD11b or CD11c ß2-integrins or diverse C-type lectins, the ESX-SA:biot-antibody complexes were efficiently captured and presented on major histocompatibility complex molecules of DCs to specific T-cell receptors. Robust ESX-specific T-cell responses were induced by immunization with as little as several picomoles of ESX-SA targeted to DC subsets. Moreover, directing of TB10.4-SA to airway CD205(+) cells enabled the induction of mucosal T-cell responses and provided significant protection against virulent M. tuberculosis.
Subject(s)
Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Dendritic Cells/immunology , Mycobacterium tuberculosis/immunology , Tuberculosis Vaccines/immunology , Tuberculosis, Pulmonary/prevention & control , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antigen Presentation , Antigens, CD/metabolism , CD11b Antigen/immunology , CD11c Antigen/immunology , CD18 Antigens/immunology , Cells, Cultured , Female , Humans , Immunity, Active , Lectins, C-Type/metabolism , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , Minor Histocompatibility Antigens , Receptors, Cell Surface/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Respiratory System/pathology , T-Lymphocytes/immunologyABSTRACT
The surface shear viscosity of the liquid expanded phase in Langmuir monolayers of fluorinated alcohols differs by orders of magnitude from the corresponding surface shear viscosity of Langmuir monolayers of their non-fluorinated counterparts. The line tension between the liquid expanded and the gaseous phase on the other hand is of similar magnitude for both fluorinated and non-fluorinated surfactants. The difference of fluorinated versus non-fluorinated monolayers is measured by active microrheology and by observing the shape relaxation of distorted liquid expanded domains toward a circular shape. Our microrheology measurements are supported by measurements of the relaxation rates of distortions, which in fluorinated liquid expanded phases are proportional to the deviation of the curvature from the mean curvature, whereas they are proportional to the square of the deviation of the curvature from the mean curvature in non-fluorinated monolayers.
ABSTRACT
For the production of a chemically inactivated Parapoxvirus ovis (PPVO), an adherent bovine kidney cell line was cultivated on Cytodex-3 microcarriers in suspension culture. The inactivated and purified virus particles have shown immune modulatory activity in several animal models. PPVO was produced by a biphasic batch process at the 3.5 and 10 L scale. Aeration was realised by bubble-free membrane oxygenation via a tube stator with a central two-blade anchor impeller. In order to increase efficiency, process robustness and safety, the established process was optimised. The cell line was adapted to a protein-free medium (except recombinant insulin) in order to increase biosafety. A scale up to a 50 L pilot plant with direct cell expansion was performed successfully. In parallel, the biphasic batch process was optimised with special emphasis on different operating conditions (cell number, Multiplicity of Infection (MOI), etc.) and process management (fed-batch, dialysis, etc.). The quality and concentration of the purified virus particles was assessed by quantitative electron microscopy, residual host cell protein and DNA-content and, finally, biologic activity in a transgenic mouse model. This integrated approach led to a new, safe, robust and highly productive large-scale production process, called "Volume-Expanded-Fed" Batch with cell densities up to 6-7e06 cells/mL. By subsequent dilution of infected cells into the next process scale, an increase in total productivity by a factor of 40 (related to an established biphasic batch process) was achieved.
Subject(s)
Parapoxvirus/growth & development , Parapoxvirus/immunology , Vaccines, Inactivated/biosynthesis , Animals , Cattle , Cell Count , Cell Survival , Cells, Cultured , Culture Media , DNA, Viral/biosynthesis , DNA, Viral/immunology , Hepatitis B/immunology , Kidney/cytology , Kidney/metabolism , Mice , Mice, Transgenic , Microscopy, Electron , Trypsin/chemistry , Vaccines, Inactivated/immunologyABSTRACT
Fatty acid profiles were used to characterize nutritional pathways in Chlamydomonas sp. isolated from an acidic mining lake (pH 2.7). Surprisingly, profiles of Chlamydomonas sp. grown in the lab under photoautotrophic, mixotrophic, and heterotrophic conditions at in situ deep strata lake water temperatures (8 degrees C) were very similar, polyunsaturated fatty acids including alpha-linolenic acid (18:3omega3) and 16:4omega3 along with palmitic acid (16:0) being most abundant. Therefore, heterotrophic growth of Chlamydomonas sp. at low temperatures can result in high concentrations of polyunsaturated fatty acids, as previously only described for some psychrophilic bacteria. By contrast, the cultivation of isolated Chlamydomonas sp. at 20 degrees C, reflecting surface water temperatures, provided fatty acid patterns characteristic of the nutrition strategy applied: the concentration of polyunsaturated fatty acids decreased when the growth pathway changed from photoautotrophic via mixotrophic to heterotrophic. Total fatty acid concentration also diminished in this order. Principal component analysis confirmed the significance of FA profiling to mirror nutritional pathways. Lake-water analysis revealed low concentrations of dissolved organic carbon, mainly consisting of polymeric fulvic acids that are unable to support heterotrophic growth of Chlamydomonas sp. Polymeric fulvic acids present in the deeper strata of the lake turned out to be formed in situ on the basis of organic monomers including reduced sulfur-containing ones, as revealed by thermochemolysis and pyrolysis. Growth of Chlamydomonas sp. in the deep chlorophyll maximum is therefore assumed to mainly result from photosynthesis, despite very low photon densities. Phytol-including metabolites proved to be significant biomarkers to indicate the nutritional pathway of Chlamydomonas sp. alpha, omega-Dicarboxylic acids-light-induced degradation products of unsaturated fatty acids-appeared to be good indicators of photooxidative alterations to the algal species under study.
Subject(s)
Chlamydomonas/isolation & purification , Chlamydomonas/metabolism , Fatty Acids/metabolism , Animals , Benzopyrans/metabolism , Chromatography, Ion Exchange , Cold Temperature , Culture Media , Fresh Water/analysis , Gas Chromatography-Mass Spectrometry , Hydrogen-Ion Concentration , Mining , Principal Component AnalysisABSTRACT
Mutations in gene encoding presenilin 1 (PS1) are responsible for the majority of familial Alzheimer's disease (FAD) cases. We studied PS1 localization in HEK293 cells and in primary neurons obtained from rat cortex and hippocampus. We first demonstrated that PS1-CTF, but neither PS1-FL nor PS1-NTF, is released into the medium as a soluble and membrane-associated form. After induction of apoptosis with staurosporine (Sts), we observed a dramatic increase in the level of PS1-CTF in the medium, both in HEK293 and in primary neurons. Immunocytochemical analysis suggested that the release of PS1-CTF might occur via membrane shedding. Abeta(1-42) treatment reduced PS1-CTF extracellular levels. This decrease was strongly associated to an impaired secretion of sAPP fragments, thus suggesting a role of PS1-CTF in the control of trafficking and generation of APP fragments.
Subject(s)
Membrane Proteins/chemistry , Alzheimer Disease/metabolism , Amyloid beta-Peptides/pharmacology , Animals , Apoptosis , Blotting, Western , Cell Line , Cells, Cultured , Cerebral Cortex/metabolism , Enzyme Inhibitors/pharmacology , Extracellular Matrix/metabolism , Hippocampus/metabolism , Humans , Immunohistochemistry , In Situ Nick-End Labeling , Microscopy, Fluorescence , Neurons/metabolism , Peptide Fragments/pharmacology , Peptides/pharmacology , Presenilin-1 , Protein Binding , Protein Structure, Tertiary , Rats , Rats, Wistar , Staurosporine/pharmacologyABSTRACT
Using high-resolution solid-state (15)N CMAS NMR, X-ray crystallography, and ab initio calculations, we have studied the structure of solid pyrazole-4-carboxylic acid (1). The crystal structure was determined at 295 and 150 K. Molecules of 1 are located on a two-fold axis, implying proton disorder of the NH and OH groups; no phase transition was observed between these two temperatures. The compound forms quasi-linear ribbons in which the molecules are linked by cyclic hydrogen bonds between pyrazole and carboxylic acid groups with disordered hydrogen-bonded protons. Crystallography is unable to decide whether the disorder is dynamic or static. NMR shows that this disorder is dynamic, that is, consisting of very fast degenerate double proton transfers between two rapidly interconverting O-H.N and O.H-N hydrogen bridges. However, at low temperature, NMR shows a proton disorder-order transition where the protons are preferentially localized on given nitrogen and oxygen atoms. An amorphous phase exhibiting proton order is observed when the compound is precipitated rapidly. In this case, the defects are annealed by moderate heating. Ab initio calculations performed on oligomers of 1 show that the O-H.N hydrogen bridge is about 0.064 A shorter and less bent ( approximately 171 degrees ) than the O.H-N hydrogen bridge ( approximately 150 degrees ). For an isolated ribbon, this result leads to structures with localized protons, either to a cycle with about 200 molecules, or to a quasi-linear ribbon involving an undulated structure, or to a combination of both motifs. Only the undulated structure is compatible with the linear ribbon observed by X-ray crystallography, where the fast proton transfer in the high-temperature phase is assisted by the motions of the undulated chain. A disordered structure is assigned to the amorphous phase, which exhibits the combination of the curved and the undulated motifs.
ABSTRACT
Microbial biomass carbon (Cmic) and soil enzyme activities were measured at 12 sites along a gradient of former emissions of phosphate fertilizer production. Seven years after close down of operation, still moderate to high total concentrations of the dust constituents cadmium (up to 33 mg kg-1 dw), fluoride (5300 mg kg-1 dw) and phosphorous (120,000 mg kg-1 dw) were found in topsoils of contaminated sites. Accumulation of partially decomposed plant matter, soil respiration and dehydrogenase activity paralleled the increase of dust deposits, whereas microbial biomass decreased along the gradient. A significant negative correlation was obtained between the Cmic-to-Corg-ratio and the concentration of contaminants. In contrast, the Cmic-specific respiration (qCO2) and the dehydrogenase activity-to-Cmic-ratio were positively correlated. The low Cmic-values and the enhanced activities in the contaminated soils are suggested as a response of microbial communities to environmental stress or ecosystem disturbances. The apparently missing detrimental effects of the alkaline deposits on soil microbial activities are probably due to the low bioavailability of contaminants in the calcareous soil.
Subject(s)
Alkalies/toxicity , Fertilizers/adverse effects , Industrial Waste/adverse effects , Phosphates/adverse effects , Soil Microbiology , Soil/analysis , Arylsulfatases/metabolism , Biomass , Cadmium/analysis , Dust , Environmental Pollution , Fertilizers/analysis , Fluorides/analysis , Germany , Oxidoreductases/metabolism , Phosphates/analysis , Phosphorus/analysis , Poaceae/growth & development , Soil PollutantsSubject(s)
Friends , Publications , Social Behavior , Women's Health , Women, Working , Anthropology, Cultural/education , Anthropology, Cultural/history , Authorship , France/ethnology , Friends/ethnology , Friends/psychology , History, 17th Century , Interpersonal Relations , Interprofessional Relations , Language , Publications/economics , Publications/history , Social Conditions/economics , Social Conditions/history , Social Identification , Women's Health/economics , Women's Health/ethnology , Women's Health/history , Women's Health/legislation & jurisprudence , Women, Working/education , Women, Working/history , Women, Working/legislation & jurisprudence , Women, Working/psychologyABSTRACT
Purified Bacillus subtilis DnaG primase (predicted molecular mass 68.8 kDa) behaves as a monomer in solution. We demonstrate that DnaG physically interacts with bacteriophage SPP1 hexameric helicase G40P (G40P6) in the absence of ATP. G40P6-ATP forms an unstable complex with ssDNA, and by itself carries out ATP-driven translocation along a ssDNA template with low processivity. The presence of DnaG in the reaction mixture increased the helicase activity of G40P6 about 3-fold, but not the ATPase activity. The results presented here suggest that the DnaG protein stabilises the G40P6-ssDNA complexes.
Subject(s)
Bacillus subtilis/enzymology , DNA Helicases/metabolism , DNA Primase/metabolism , DNA, Single-Stranded/metabolism , Viral Proteins , Adenosine Triphosphatases/metabolism , Bacillus subtilis/metabolism , Bacteriophages/enzymology , Bacteriophages/metabolism , DNA Primase/chemistry , DNA Primase/isolation & purificationABSTRACT
To examine the effects of the combined muscarinic ml-agonist/m2-antagonist Lu 25-109 on regulated processing of the amyloid protein precursor (APP), we used both transfected cells expressing human muscarinic m1 or m2 acetylcholine receptors, and fresh rat hippocampal slices. Lu 25-109 readily stimulated APPs secretion from HEK 293 cells overexpressing m1, but not m2, receptors, as well as from the hippocampal brain slices. Time-course analyses revealed a rapid (5-35 minutes), and a delayed (55-75 minutes) secretory response to Lu 25-109 with distinct concentration profiles suggesting two distinct cell biological mechanisms. Both responses appeared to reflect post-translational mechanisms because levels of APP message were unchanged after 60 minutes of stimulation with Lu 25-109. In comparison to carbachol, Lu 25-109 had a significantly lower intrinsic activity at muscarinic m1 receptors, compatible with a pharmacological profile as a partial agonist at recombinantly expressed m1 receptors. In as much as stimulation of APPs secretion is associated with reduced formation of A beta peptides, Lu 25-109 may be useful to reduce A beta generation, and thus, slow amyloid plaque formation. Moreover, Lu 25-109 may be useful in promoting the known neurotrophic and neuroprotective biological functions of secreted APPs.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Protein Precursor/metabolism , Muscarinic Agonists/pharmacology , Muscarinic Antagonists/pharmacology , Protein Processing, Post-Translational/drug effects , Pyridines/pharmacology , Tetrazoles/pharmacology , Animals , Cell Line , Hippocampus/metabolism , Humans , In Vitro Techniques , Male , Rats , Rats, Inbred Lew , TransfectionABSTRACT
We probed the complex between the replication origin, oriC, and the initiator protein DnaA using different types of mutations in the five binding sites for DnaA, DnaA boxes R1-R4 and M: (i) point mutations in individual DnaA boxes and combinations of them; (ii) replacement of the DnaA boxes by a scrambled 9 bp non-box motif; (iii) positional exchange; and (iv) inversion of the DnaA boxes. For each of the five DnaA boxes we found at least one type of mutation that resulted in a phenotype. This demonstrates that all DnaA boxes in oriC have a function in the initiation process. Most mutants with point mutations retained some origin activity, and the in vitro DnaA-binding capacity of these origins correlated well with their replication proficiency. Inversion or scrambling of DnaA boxes R1 or M inactivated oriC-dependent replication of joint replicons or minichromosomes under all conditions, demonstrating the importance of these sites. In contrast, mutants with inverted or scrambled DnaA boxes R2 or R4 could not replicate in wild-type hosts but gave transformants in host strains with deleted or compromised chromosomal oriC at elevated DnaA concentrations. We conclude that these origins require more DnaA per origin for initiation than does wild-type oriC. Mutants in DnaA box R3 behaved essentially like wild-type oriC, except for those in which the low-affinity box R3 was replaced by the high-affinity box R1. Apparently, initiation is possible without DnaA binding to box R3, but high-affinity DnaA binding to DnaA box R3 upsets the regulation. Taken together, these results demonstrate that there are finely tuned DnaA binding requirements for each of the individual DnaA boxes for optimal build-up of the initiation complex and replication initiation in vivo.
Subject(s)
Bacterial Proteins/genetics , DNA-Binding Proteins/genetics , Escherichia coli/genetics , Mutation , Replication Origin , Bacterial Proteins/metabolism , Base Sequence , Binding Sites/genetics , Codon, Nonsense/genetics , DNA, Bacterial/genetics , DNA-Binding Proteins/metabolism , Escherichia coli/metabolism , Phenotype , Point MutationABSTRACT
Derivatives of Bacillus subtilis plasmid pUB110 lacking the major lagging strand replication origin (ssoU-) accumulate intracellular single-strand circular (SS(c)) DNA intermediates and are unable to propagate in dnaB and dnaD hosts. DnaA-dependent priming requires a DnaA box in a stable hairpin form; a higher copy number of a DnaA box is not sufficient as a signal for the conversion of the SS(c) into its dsDNA form. The introduction into the plasmid of a hairpin structure, whose stem carries a DnaA box, mediates conversion of SS(c) into dsDNA and makes plasmid replication independent of the B. subtilis dnaB function. This conversion signal has been termed ssoA.
Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/physiology , DNA Replication , DNA, Bacterial/biosynthesis , DNA-Binding Proteins/physiology , Plasmids , Base Sequence , Molecular Sequence DataABSTRACT
We probed the complex between oriC and DnaA protein using two types of mutants in oriC. Base changes in the DnaA binding sites, DnaA boxes, had little effect on origin function. Mutations which change the distance between DnaA boxes R3 and R4, on the other hand, inactivated oriC unless the mutation deleted or inserted one complete helical turn. Origins with other 10 base pair insertions in the interval between DnaA boxes R2 and R3 were functional, but not insertions in the R1-R2 interval. FIS protein binds to a bipartite site in oriC between DnaA boxes R2 and R3. A model for the oriC/DnaA complex based on these results suggests an array of DnaA monomers with a 34 A spacing upon which oriC is arranged.