Recovery of a bacterial sub-population from sewage using immunofluorescent flow cytometry and cell sorting

Abstract The effectiveness of immunofluorescence flow cytometry and cell sorting to detect, quantify and separate indigenous bacterial populations present in low concentrations in sewage outflow was investigated. Preparatory experiments for targeted recovery revealed indigenous, immunoglobulin-G-binding particles present at low levels in sewage outflow samples taken from Coniston Water. Fluorescence-activated cell sorting of this population was employed to enrich for these particles, which were confirmed as bacterial cells. This cell population comprised approximately 23% of the total plate count on MacConkey agar before cell sorting, rising to approximately 95% after sorting. These results corresponded to cell densities of less than 5% of the total plate count on R2A agar. Taxonomic tests suggested the bacterium to be Ochrobactrum anthropi .     

Point Mutations in Dimerization Motifs of the Transmembrane Domain Stabilize Active or Inactive State of the EphA2 Receptor Tyrosine Kinase

The EphA2 receptor tyrosine kinase plays a central role in the regulation of cell adhesion and guidance in many human tissues. The activation of EphA2 occurs after proper dimerization/oligomerization in the plasma membrane, which occurs with the participation of extracellular and cytoplasmic domains. Our study revealed that the isolated transmembrane domain (TMD) of EphA2 embedded into the lipid bicelle dimerized via the heptad repeat motif L⁵³⁵X₃G⁵³⁹X₂A⁵⁴²X₃V⁵⁴⁶X₂L⁵⁴⁹ rather than through the alternative glycine zipper motif A⁵³⁶X₃G⁵⁴⁰X₃G⁵⁴⁴ (typical for TMD dimerization in many proteins). To evaluate the significance of TMD interactions for full-length EphA2, we substituted key residues in the heptad repeat motif (HR variant: G539I, A542I, G553I) or in the glycine zipper motif (GZ variant: G540I, G544I) and expressed YFP-tagged EphA2 (WT, HR, and GZ variants) in HEK293T cells. Confocal microscopy revealed a similar distribution of all EphA2-YFP variants in cells. The expression of EphA2-YFP variants and their kinase activity (phosphorylation of Tyr⁵⁸⁸ and/or Tyr⁵⁹⁴) and ephrin-A3 binding were analyzed with flow cytometry on a single cell basis. Activation of any EphA2 variant is found to occur even without ephrin stimulation when the EphA2 content in cells is sufficiently high. Ephrin-A3 binding is not affected in mutant variants. Mutations in the TMD have a significant effect on EphA2 activity. Both ligand-dependent and ligand-independent activities are enhanced for the HR variant and reduced for the GZ variant compared with the WT. These findings allow us to suggest TMD dimerization switching between the heptad repeat and glycine zipper motifs, corresponding to inactive and active receptor states, respectively, as a mechanism underlying EphA2 signal transduction.     

Application of muonic X-rays for elemental analysis

Because of the large μ mass compared to the electron mass, the muonic X-rays have energies very suitable for standard γ-ray spectroscopy (Ge detectors), so every element is easily recognized. By selecting the primary μ energies appropriately any part of the specimen, also well inside, can be nondestructively investigated. On the other hand, surface layers may be analyzed. Accuracies of quantitative analyses are 1% of the atomic abundance of the element in question in favorite cases. Results on applications in nuclear medicine and surface physics are presented, and ways of improving the muon flux density are discussed.     

Monoclonal antibodies to type D retrovirus (SRV-2)

Monoclonal antibodies (MoAbs) to the major gag core protein p27 and a viral protein p44 of type D retrovirus (SRV-2) were produced and used in the detection of SRV-2 antigens in infected Raji cells and in tissues from macaques with simian acquired immunodeficiency syndrome (SAIDS) and retroperitoneal fibromatosis (RF). Anti-p44 MoAb showed inhibition of syncytium formation by both SRV-1- and SRV-2-infected Raji cells.     

Flow cytometric screening and isolation of Escherichia coli clones which express surface antigens of the oil-degrading microorganism Acinetobacter calcoaceticus RAG-1

Flow cytometry (FCM) in conjunction with immunocytochemical-labeling was used to analyze and screen a population of Escherichia coli clones containing a genomic library from the oil-degrading microorganism Acinetobacter calcoaceticus RAG-1 for the isolation of clones which expressed specific RAG-1 surface antigens. Reconstruction experiments using mixed populations indicated that RAG-1 cells could be clearly distinguished at a ratio of one RAG-1 cell to 500 Escherichia coli cells. Using this technique two clones, WM143 and WM191, were isolated and shown by restriction endonuclease cleavage and Southern hybridization to contain plasmids carrying inserts of RAG-1 DNA of 9.4 and 9.8 kb respectively.Non-common abbreviations FCM flow cytometry - FITC fluorescein-iso-thiocyanate - LB Luria broth - MM minimal salt medium - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride     

Response to self-motion in waterstriders: visual discrimination between rotation and translation

Waterstriders (Gerris paludum) often try to maintain a nearly stationary position on a moving water surface. Passive motion is restricted to 3 degrees of freedom: yaw, longitudinal, and transverse displacement. They correct for passive rotation and translation by distinct behavioral sequences. The compensatory behavior is predominantly visually controlled.

Effects of serine protease inhibitors on oxyradical burst from phagocytizing neutrophils—analysis by chemiluminescence counting and its microscopic imaging

Reaction difference of oxyradical generation and luminol-dependent photoemission of zymosan- and phorbol ester-treated neutrophils were investigated using a conventional photomultiplier and ultrasensitive photonic imaging technique. Zymosan-treated cells released a concentrated photonic burst corresponding to the cellular distribution. In contrast, phorbol ester-treated cells produced a negligible level of photoemission, and the additional application of Ca²⁺ ionophore enhanced the photonic burst, which was gradually spread out into extracellular space. Serine protease inhibitors did not attenuate PMA-induced chemiluminescence but did attenuate zymosan-induced chemiluminescence. This suggests the involvement of serine protease in the respiratory burst of phagocytizing neutrophils.     

Buoyancy effect on forced convection in the leaf boundary layer

Abstract. Mixed convection (forced convection plus free convection) in the leaf boundary layer was examined by air flow visualization and by evaluation of the boundary layer conductance at different leaf-air temperature differences ( T _L- T _A) under low wind velocities. The visualized air flow was found to become more unstable and buoyant at higher T _L- T _A. An ascending longitudinal plume was induced along the upper surface, and the air flow along the lower surface ascended after passing the trailing leaf edge. The air flow modified by buoyancy was considered to result in an increase in boundary layer conductance ( G _A) for mixed convection, which became higher with higher T _L- T _A as compared with the conductance for pure forced convection without buoyancy. This increase in G _A appeared larger at larger Grashof number (Gr) and at smaller Reynolds number (Re). The dependences of buoyancy effect on Gr and Re were related to 'edge-effects'.     

N-α-biotinylated-neuropeptide Y analogs: Syntheses, cardiovascular properties, and application to cardiac NPY receptor visualization

Two monobiotinylated analogs of neuropeptide Y (NPY) were synthesized by coupling the N-hydroxysuccinimidyl esters of biotin and (6-biotinylamido)-hexanoic acid, respectively, to the free alpha-NH2 group of the side chain protected NPY peptide resin. Crude peptides obtained by HF cleavage were purified by RPLC and their integrities were confirmed by amino acid and mass spectral analysis. As with NPY, both biotinylated analogs inhibited 125I-NPY binding and adenylate cyclase activity of rat cardiac ventricular membranes in a dose-dependent manner. N-alpha-[(6-biotinylamido)-hexanoyl]-NPY exhibited potencies comparable to that of NPY whereas N-alpha-biotinyl-NPY was slightly less potent. In the in vivo experiments, however, both the biotinylated analogs exhibited responses comparable to NPY in increasing arterial blood pressure and decreasing heart rate in anesthetized rats. The responses of the biotinyl analogs were longer lasting than those of NPY. Histochemical studies revealed that N-alpha-[(6-biotinylamido)-hexanoyl]-NPY could label the NPY receptors in rat cardiac ventricular tissues. This labeling was specific since intact NPY inhibited the staining. These studies show that biotinyl-NPY analogs exhibit biological potencies comparable to intact NPY and can therefore be used to further probe the NPY-receptor interaction.     

MNNG对细胞DNA合成及其分布的影响

本文用流式细胞光度术(FCM)等方法研究了MNNG,ENNG和DMS对HeLa细胞DNA含量分布的影响。经MNNG(6.8μmol/L)处理后,细胞分裂减少,DNA合成速率下降,S期细胞的比例随处理时间的延长而增加。DMS显示有类似的现象而ENNG的效应则较小。