Decrease in cell surface sialic acid in etoposide-treated Jurkat cells and the role of cell surface sialidase

The present study investigated the mechanism underlying alterations of cell surface sugar chains of Jurkat cells by inducing apoptosis with etoposide, an inhibitor of topoisomerase II. Within 3[emsp4 ]h of etoposide treatment, flowcytometric analysis revealed a decrease in Maackia amurensis agglutinin recognized 2,3-linked sialic acid moieties and an increase in Ricinus communis agglutinin recognized galactose. The results suggested that asialo-sugar chains on glycoconjugates were rapidly induced on the etoposide-treated cell surface. To clarify the desialylation mechanism, we studied 2,3-sialyltransferase mRNA expression and the activity of sialidase on the cell surface during etoposide-induced apoptosis. The expression of hST3Gal III and hST3Gal IV mRNAs were down-regulated and sialidase activity on the cell surface increased threefold within 2[emsp4 ]h of etoposide treatment. Moreover, the decrease in 2,3-linked sialic acid levels was significantly suppressed in the presence of 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, an inhibitor of sialidase. These results suggested that activation or exposure of sialidase on the cell surface was induced by etoposide treatment and was the main cause of the decrease in sialic acids.     

Na+- and K+-dependent oligomeric interconversion among alphabeta-protomers, diprotomers and higher oligomers in solubilized Na+/K+-ATPase

Protein fractions of a higher-oligomer (H), (alphabeta)(2)-diprotomer (D) and alphabeta-protomer (P) were separated from dog kidney Na(+)/K(+)-ATPase solubilized in the presence of NaCl and KCl. Na(+)/K(+)-dependent interconversion of the oligomers was analysed using HPLC at 0 degrees C. With increasing KCl concentrations, the content or amount of D increased from 27.6 to 54.3% of total protein, i.e. DeltaC(max) = 26.7%. DeltaC(max) for the sum of D and H was equivalent to the absolute value of DeltaC(max) for P, regardless of the anion present, indicating that K(+) induced the conversion of P into D and/or H, and Na(+) had the opposite effect. When enzymes that had been denatured to varying degrees by aging were solubilized, DeltaC(max) increased linearly with the remaining ATPase activity. The magnitude of the interconversion could be explained based on an equilibrium of D <==> 2P, assuming 50-fold difference in the K(d) between KCl and NaCl, and coexistence of unconvertible oligomers, which comprised as much as 39% of the eluted protein. Oligomeric interconversion, determined as a function of the KCl or NaCl concentration, showed K(0.5)s of 64.8 microM and 6.50 mM for KCl and NaCl, respectively, implying that oligomeric interconversion was coupled with Na(+)/K(+)-binding to their active transport sites.     

Carabid beetle (Coleoptera: Carabidae) assemblages in the intermediate successional stage after Mt. Bandai eruption of 1888: Effects of environmental variables on carabid beetles in the Urabandai area

We studied carabid beetle assemblage structure and species diversity in an intermediate successional stage (seral) forest established in areas affected by the 1888 eruption of Mt. Bandai and a climax forest that had not been affected by the eruption at the Urabandai area, Fukushima Prefecture, Japan. In total, 2,131 carabid beetles representing 31 species were collected using pitfall traps without bait. A comparison of carabid beetle assemblages between the two forest types revealed that the number of species observed was comparable, but their abundance was greater in the seral forest. The assemblage structure clearly differed between the two forest types. In the seral forest, forest generalists, such as Synuchus arcuaticollis and Pterostichus prolongatus, along with forest specialists including Carabus vanvolxemi and Pterostichus asymmetricus, were collected. In the climax forest, forest generalists, such as Synuchus cycloderus and Carabus albrechti tsukubanus, were more abundant than forest specialists. This suggests that the current seral forest in the Urabandai area possesses environmental variables that enable the coexistence of both forest specialist and generalist beetle species. A redundancy analysis showed that six species from the genera Synuchus and Pterostichus were associated with high canopy openness and high understory vegetation cover, whereas species recorded only in the climax forest were associated with deep litter. Therefore, it seems likely that carabid beetles in the Urabandai area were affected by these three environmental variables.     

Slr2019, lipid A transporter homolog,is essential for acidic tolerance in Synechocystis sp. PCC6803

Photosynthesis Research - Living organisms must defend themselves against various environmental stresses. Extracellular polysaccharide-producing cells exhibit enhanced tolerance toward adverse...     

Development of a method for reconstruction of crowded NMR spectra from undersampled time-domain data

NMR is a unique methodology for obtaining information about the conformational dynamics of proteins in heterogeneous biomolecular systems. In various NMR methods, such as transferred cross-saturation, relaxation dispersion, and paramagnetic relaxation enhancement experiments, fast determination of the signal intensity ratios in the NMR spectra with high accuracy is required for analyses of targets with low yields and stabilities. However, conventional methods for the reconstruction of spectra from undersampled time-domain data, such as linear prediction, spectroscopy with integration of frequency and time domain, and analysis of Fourier, and compressed sensing were not effective for the accurate determination of the signal intensity ratios of the crowded two-dimensional spectra of proteins. Here, we developed an NMR spectra reconstruction method, “conservation of experimental data in analysis of Fourier” (Co-ANAFOR), to reconstruct the crowded spectra from the undersampled time-domain data. The number of sampling points required for the transferred cross-saturation experiments between membrane proteins, photosystem I and cytochrome b ₆ f, and their ligand, plastocyanin, with Co-ANAFOR was half of that needed for linear prediction, and the peak height reduction ratios of the spectra reconstructed from truncated time-domain data by Co-ANAFOR were more accurate than those reconstructed from non-uniformly sampled data by compressed sensing.     

Validation of a P-Glycoprotein (P-gp) Humanized Mouse Model by Integrating Selective Absolute Quantification of Human MDR1, Mouse Mdr1a and Mdr1b Protein Expressions with In Vivo Functional Analysis for Blood-Brain Barrier Transport

It is essential to establish a useful validation method for newly generated humanized mouse models. The novel approach of combining our established species-specific protein quantification method combined with in vivo functional studies is evaluated to validate a humanized mouse model of P-gp/MDR1 efflux transporter. The P-gp substrates digoxin, verapamil and docetaxel were administered to male FVB Mdr1a/1b(+/+) (FVB WT), FVB Mdr1a/1b(-/-) (Mdr1a/1b(-/-)), C57BL/6 Mdr1a/1b(+/+) (C57BL/6 WT) and humanized C57BL (hMDR1) mice. Brain-to-plasma total concentration ratios (K_p) were measured. Quantitative targeted absolute proteomic (QTAP) analysis was used to selectively quantify the protein expression levels of hMDR1, Mdr1a and Mdr1b in the isolated brain capillaries. The protein expressions of other transporters, receptors and claudin-5 were also quantified. The K_p for digoxin, verapamil, and docetaxel were 20, 30 and 4 times higher in the Mdr1a/1b(-/-) mice than in the FVB WT controls, as expected. The K_p for digoxin, verapamil and docetaxel were 2, 16 and 2-times higher in the hMDR1 compared to the C57BL/6 WT mice. The hMDR1 mice had 63- and 9.1-fold lower expressions of the hMDR1 and Mdr1a proteins than the corresponding expression of Mdr1a in C57BL/6 WT mice, respectively. The protein expression levels of other molecules were almost consistent between C57BL/6 WT and hMDR1 mice. The P-gp function at the BBB in the hMDR1 mice was smaller than that in WT mice due to lower protein expression levels of hMDR1 and Mdr1a. The combination of QTAP and in vivo functional analyses was successfully applied to validate the humanized animal model and evaluates its suitability for further studies.     

Immunization with dendritic cells loaded with alpha-galactosylceramide at priming phase, but not at boosting phase, enhances cytotoxic T lymphocyte activity against infection by intracellular bacteria

We evaluated the effect of immunization with dendritic cells (DCs) pulsed with alpha-galactosylceramide (alphaGalCer) and listeriolysin O (LLO) 91-99 peptide, a dominant cytotoxic T lymphocyte (CTL) epitope of Listeria monocytogenes by observing the responses of specific CD8(+) T cells and in vivo CTL activity. DCs were pulsed with various combinations of alphaGalCer and LLO91-99 peptide and administered to BALB/c mice. Immunization with DCs pulsed with alphaGalCer and LLO91-99 at priming phase and with DCs pulsed with LLO91-99 alone at boosting phase induced stronger in vivo CTL activity, reduced the bacterial load in spleens of Listeria-challenged mice and augmented CD62L(+) CD8(+) central memory T cells compared with other immunization protocols. The blockade of interferon-gamma (IFN-gamma) at boosting phase reversed the induction of CD8(+) central memory T cells and reduced the bacterial load in spleens of Listeria-challenged mice immunized with DCs pulsed with alphaGalCer and LLO91-99 at both phases, suggesting that alphaGalCer at boosting phase has deleterious effects through IFN-gamma production. These results indicate that immunization with DCs pulsed with CTL epitope peptide together with alphaGalCer at priming phase, but not at boosting phase, is feasible for eliciting a specific CTL activity and protective immunity against infection of intracellular bacteria.     

Stichopin-containing nerves and secretory cells specific to connective tissues of the sea cucumber

Stichopin, a 17-amino acid peptide isolated from a sea cucumber, affects the stiffness change of the body-wall catch connective tissues and the contraction of the body-wall muscles. The localization of stichopin in sea cucumbers was studied by indirect immunohistochemistry using antiserum against stichopin. Double staining was performed with both stichopin antiserum and 1E11, the monoclonal antibody specific to echinoderm nerves. A stichopin-like immunoreactivity (stichopin-LI) was exclusively found in the connective tissues of various organs. Many fibres and cells with processes were stained by both the anti-stichopin antibody and 1E11. They were found in the body-wall dermis and the connective tissue layer of the cloacae and were suggested to be connective tissue-specific nerves. Oval cells with stichopin-LI (OCS) without processes were found in the body-wall dermis, the connective tissue sheath of the longitudinal body-wall muscles, the connective tissue layer of the tube feet and tentacles, and the connective tissue in the radial nerves separating the ectoneural part from the hyponeural part. Electron microscopic observations of the OCSs in the radial nerves showed that they were secretory cells. The OCSs were located either near the well-defined neural structures or near the water-filled cavities, such as the epineural sinus and the canals of the tube feet. The location near the water-filled cavities might suggest that stichopin was secreted into these cavities to function as a hormone.     

Complanadines C and D, new dimeric alkaloids from Lycopodium complanatum

Two new dimeric Lycopodium alkaloids, complanadines C (1) and D (2), have been isolated from the club moss Lycopodium complanatum, and the structures and relative stereochemistry of 1 and 2 were elucidated on the basis of the spectral data. Complanadine D (2) enhanced mRNA expression for NGF.