Insertional mutagenesis of Bordetella pertussis adenylate cyclase.

We developed an improved method of linker insertion mutagenesis for introducing 2 or 16 codons into the Bordetella pertussis cyaA gene which encodes a calmodulin-dependent adenylate cyclase. A recombinant kanamycin resistance cassette, containing oligonucleotide linkers, was cloned in plasmids which carried a truncated cyaA gene, fused at its 3' end to the 5' end of the Escherichia coli lacZ gene, specifying the alpha-peptide. This construction permitted a double selection for in-frame insertions by using screening for kanamycin resistance and for lactose-positive phenotype, resulting from alpha-complementation. We showed that most of the two-amino acid insertions within the N-terminal moiety of the catalytic domain of adenylate cyclase abolished enzymatic activity and/or altered the stability of the protein. All two-amino acid insertions within the C-terminal part of adenylate cyclase resulted in fully stable and active enzymes. These results confirm the modular structure of the catalytic domain of adenylate cyclase, previously proposed on the basis of proteolytic studies. Two-amino acid insertions between residues 247-248 and 335-336 were shown to affect the calmodulin responsiveness of adenylate cyclase, suggesting that the corresponding region in the enzyme is involved in the binding of calmodulin or in the process of calmodulin activation. In addition, we have identified within the primary structure of adenylate cyclase several permissive sites which tolerate 16-amino acid insertions without interfering with the catalytic activity or calmodulin binding. By inserting foreign antigenic determinants into these permissive sites the resulting recombinant adenylate cyclase toxin could be used to deliver specific epitopes into antigen-presenting cells.     

A Targeted LC‐MS Strategy for Low‐Abundant HLA Class‐I‐Presented Peptide Detection Identifies Novel Human Papillomavirus T‐Cell Epitopes

For rational design of therapeutic vaccines, detailed knowledge about target epitopes that are endogenously processed and truly presented on infected or transformed cells is essential. Many potential target epitopes (viral or mutation‐derived), are presented at low abundance. Therefore, direct detection of these peptides remains a challenge. This study presents a method for the isolation and LC‐MS³‐based targeted detection of low‐abundant human leukocyte antigen (HLA) class‐I‐presented peptides from transformed cells. Human papillomavirus (HPV) was used as a model system, as the HPV oncoproteins E6 and E7 are attractive therapeutic vaccination targets and expressed in all transformed cells, but present at low abundance due to viral immune evasion mechanisms. The presented approach included preselection of target antigen‐derived peptides by in silico predictions and in vitro binding assays. The peptide purification process was tailored to minimize contaminants after immunoprecipitation of HLA‐peptide complexes, while keeping high isolation yields of low‐abundant target peptides. The subsequent targeted LC‐MS³ detection allowed for increased sensitivity, which resulted in successful detection of the known HLA‐A2‐restricted epitope E7_11–19 and ten additional E7‐derived peptides on the surface of HPV16‐transformed cells. T‐cell reactivity was shown for all the 11 detected peptides in ELISpot assays, which shows that detection by our approach has high predictive value for immunogenicity. The presented strategy is suitable for validating even low‐abundant candidate epitopes to be true immunotherapy targets.     

Monitoring of the Cytoskeleton-Dependent Intracellular Trafficking of Fluorescent Iron Oxide Nanoparticles by Nanoparticle Pulse-Chase Experiments in C6 Glioma Cells

Iron oxide nanoparticles (IONPs) are used for various biomedical and therapeutic approaches. To investigate the uptake and the intracellular trafficking of IONPs in neural cells we have performed nanoparticle pulse-chase experiments to visualize the internalization and the fate of fluorescent IONPs in C6 glioma cells and astrocyte cultures. Already a short exposure to IONPs for 10 min at 4 °C (nanoparticle pulse) allowed binding of substantial amounts of nanoparticles to the cells, while internalization of IONPs into the cell was prevented. The uptake of bound IONPs and the intracellular trafficking was started by increasing the temperature to 37 °C (chase period). While hardly any cellular fluorescence nor any iron staining was detectable directly after the nanoparticle pulse, dotted cellular fluorescence and iron patterns appeared already within a few minutes after start of the chase incubation and became intensified in the perinuclear region during further incubation for up to 90 min. Longer chase incubations resulted in separation of the fluorescent coat from the core of the internalized IONPs. Disruption of actin filaments in C6 cells strongly impaired the internalization of IONPs, whereas destabilization of microtubules traped IONP-containing vesicles to the plasma membrane. In conclusion, nanoparticle pulse-chase experiments allowed to synchronize the cellular uptake of fluorescent IONPs and to identify for C6 cells an actin-dependent early and a microtubule-dependent later process in the intracellular trafficking of fluorescent IONPs.     

Phenological shifts of the alien colonizer Bunias orientalis: Image-based analysis of temporal niche separation

Abstract. Phenological development of the colonizer Bunias orientalis L. was investigated in permanent plots in herbaceous vegetation, using photography and image analysis. In all habitats but one, B. orientalis showed a two-phased rosette growth behaviour, with a first peak cover reached in spring, roughly coinciding with an early peak cover of the matrix vegetation, and a second autumn growth flush occurring in a phase of reduced matrix vegetation cover. This seasonally bimodal growth pattern of B. orientalis appears to partly compensate for its competitive inferiority in crowded herbaceous vegetation. Small or overshadowed individuals of this species particularly profit from higher above-ground performance or release from overlap in autumn. The significance of temporal niche separation for survival and growth of B. orientalis individuals varies with habitat conditions, being most apparent in occasionally disturbed habitats with a relatively low frequency of gap formation. Despite some limitations, image analysis proved to be useful for phenological investigations within herbaceous plant stands.     

Uptake and Toxicity of Copper Oxide Nanoparticles in C6 Glioma Cells

Copper oxide nanoparticles (CuO-NPs) are frequently used for many technical applications, but are also known for their cell toxic potential. In order to investigate a potential use of CuO-NPs as a therapeutic drug for glioma treatment, we have investigated the consequences of an application of CuO-NPs on the cellular copper content and cell viability of C6 glioma cells. CuO-NPs were synthesized by a wet-chemical method and were coated with dimercaptosuccinic acid and bovine serum albumin to improve colloidal stability in physiological media. Application of these protein-coated nanoparticles (pCuO-NPs) to C6 cells caused a strong time-, concentration- and temperature-dependent copper accumulation and severe cell death. The observed loss in cellular MTT-reduction capacity, the loss in cellular LDH activity and the increase in the number of propidium iodide-positive cells correlated well with the specific cellular copper content. C6 glioma cells were less vulnerable to pCuO-NPs compared to primary astrocytes and toxicity of pCuO-NPs to C6 cells was only observed for incubation conditions that increased specific cellular copper contents above 20 nmol copper per mg protein. Both cellular copper accumulation as well as the pCuO-NP-induced toxicity in C6 cells were prevented by application of copper chelators, but not by endocytosis inhibitors, suggesting that liberation of copper ions from the pCuO-NPs is the first step leading to the observed toxicity of pCuO-NP-treated glioma cells.     

The transition to reproductive phase inchenopodium murale L. ecotype 197 - Early flowering long-day plant

Five days of suitable continuous light induced flowering in the majority ofChenopodium murale L. ecotype 197 plants as early as at the phase of the first pair of leaves. At the time of initiation of the 2nd to 4th pairs of leaves the capacity of plants to flower was reduced, the number of flowering plants being significantly lower under the same inductive light treatment. The capacity to flower increased again at the phase of the 5th and the 6th pairs of leaves. Inductive light treatment brought about a marked growth activation of organs present before induction, shoot apex elongation, precocious formation of new leaves and activation of axillary meristems. The course of these changes in plants of different age is demonstrated. The terminal flower developed during 5 short days following inductive light treatment. The paper shows similarities and differences between long-daymutale L. ecotype 197 and short-day C.rubrum L. ecotype 374 grown under practically uniform conditions.     

Ethylene production and metabolism of 1-aminocyclopropane-1-carboxylic acid in a long-day plant,Chenopodium murale L., as influenced by photoperiodic flower induction

Chenopodium murale plants, induced to flower by 5 days of continuous light, produced 43% more ethylene than vegetative plants kept under short days (16 h darkness, 8 h light). The 1-aminocyclopropane-1-carboxylic acid (ACC)-induced ethylene production, using saturating ACC concentration (10 mol·m⁻³) was also 55% higher in induced plants. Their ACC and N-malonyl-ACC (MACC) levels were also higher, the former increasing by 56% in both shoots and roots, the latter by 288% and 108% in shoots and roots, respectively. Administration of labeled [2,3-¹⁴C]ACC produced a very similar relative content of ACC and MACC in both treatments. The only process influenced by flower induction was ACC conversion to ethylene. Induced plants converted 66% more ACC than the vegetative ones. The effects of photoperiod on ethylene formation and metabolism in a long-day plant (LDP)C. murale and a short-day plant (SDP)C. rubrum are compared. Ethylene formation seems to be under photoperiodic control in both species, but its role in flower induction remains obscure.     

Immunofluorescent Identification of Type 12 Group A Streptococci

The fluorescent antibody (FA) conjugate prepared by labeling streptococcal M type 12 antibody with fluorescein isothiocyanate was found to exhibit considerable nonspecific FA staining with other group A M-serotypes. The cross-reactions could be reduced sufficiently or eliminated by the addition of adsorbed homologous blocking serum (AHB) but not by preimmune serum. The AHB was prepared by adsorbing type 12 antiserum with untreated homologous cells. Comparative staining with unblocked and AHB-blocked FA conjugates enabled type 12 streptococci from clinical specimens to be rapidly and accurately identified.     

Regulation by Phospholipids and Kinetic Studies of Plant Membrane-Bound UDP-Glucose Sterol beta-d-Glucosyl Transferase

Solubilization and partial purification of the microsomal UDP-glucose sterol glucosyl transferase activity from maize coleoptiles by chromatography on DEAE-cellulose resulted in a highly delipidated (>95%) and inactive enzymic preparation. Addition of sterols revealed part of the activity and subsequent addition of phospholipids further increased the activity. Negatively charged phospholipids were shown to be by far the best activators. The purification step also produced the elimination of two interfering microsomal enzymic activities: UDPase and steryl glucoside acyl transferase. The removal of these two enzymic activities was a prerequisite for kinetic studies including product-inhibition studies, since the substrates of these two latter enzymes are the products of UDPG-SGTase activity. The results of the kinetic studies strongly suggest an ordered bi-bi mechanism for the glucosylation of sterols. Finally the effect of different phospholipids on the kinetic parameters of the reaction was studied. Both phosphatidylcholine and phosphatidylglycerol significantly decrease K_m-sterol (and not K_m-UDPglucose) and increase the reaction V_max. The decrease of K_m-sterol is similar with both phospholipids whereas the increase of V_max is much greater with phosphatidylglycerol than with phosphatidylcholine.