Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes

Background information. Mercurials inhibit AQPs (aquaporins), and site‐directed mutagenesis has identified Cys¹⁸⁹ as a site of the mercurial inhibition of AQP1. On the other hand, AQP4 has been considered to be a mercury‐insensitive water channel because it does not have the reactive cysteine residue corresponding to Cys¹⁸⁹ of AQP1. Indeed, the osmotic water permeability (P_f) of AQP4 expressed in various types of cells, including Xenopus oocytes, is not inhibited by HgCl₂. To examine the direct effects of mercurials on AQP4 in a proteoliposome reconstitution system, His‐tagged rAPR4 (rat AQP4) M23 was expressed in Saccharomyces cerevisiae, purified with an Ni²⁺‐nitrilotriacetate affinity column, and reconstituted into liposomes with the dilution method. Results. The water permeability of AQP4 proteoliposomes with or without HgCl₂ was measured with a stopped‐flow apparatus. Surprisingly, the P_f of AQP4 proteoliposomes was significantly decreased by 5 μM HgCl₂ within 30 s, and this effect was completely reversed by 2‐mercaptoethanol. The dose‐ and time‐dependent inhibitory effects of Hg²⁺ suggest that the sensitivity to mercury of AQP4 is different from that of AQP1. Site‐directed mutagenesis of six cysteine residues of AQP4 demonstrated that Cys¹⁷⁸, which is located at loop D facing the intracellular side, is a target responding to Hg²⁺. We confirmed that AQP4 is reconstituted into liposome in a bidirectional orientation. Conclusions. Our results suggest that mercury inhibits the P_f of AQP4 by mechanisms different from those for AQP1 and that AQP4 may be gated by modification of a cysteine residue in cytoplasmic loop D.     

Construction of hybrid photosynthetic units using peripheral and core antennae from two different species of photosynthetic bacteria: detection of the energy transfer from bacteriochlorophyll a in LH2 to bacteriochlorophyll b in LH1

Typical purple bacterial photosynthetic units consist of supra-molecular arrays of peripheral (LH2) and core (LH1-RC) antenna complexes. Recent atomic force microscopy pictures of photosynthetic units in intact membranes have revealed that the architecture of these units is variable (Scheuring et al. (2005) Biochim Bhiophys Acta 1712:109–127). In this study, we describe methods for the construction of heterologous photosynthetic units in lipid-bilayers from mixtures of purified LH2 (from Rhodopseudomonas acidophila) and LH1-RC (from Rhodopseudomonas viridis) core complexes. The architecture of these reconstituted photosynthetic units can be varied by controlling ratio of added LH2 to core complexes. The arrangement of the complexes was visualized by electron-microscopy in combination with Fourier analysis. The regular trigonal array of the core complexes seen in the native photosynthetic membrane could be regenerated in the reconstituted membranes by temperature cycling. In the presence of added LH2 complexes, this trigonal symmetry was replaced with orthorhombic symmetry. The small lattice lengths for the latter suggest that the constituent unit of the orthorhombic lattice is the LH2. Fluorescence and fluorescence-excitation spectroscopy was applied to the set of the reconstituted membranes prepared with various proportions of LH2 to core complexes. Remarkably, even though the LH2 complexes contain bacteriochlorophyll a, and the core complexes contain bacteriochlorophyll b, it was possible to demonstrate energy transfer from LH2 to the core complexes. These experiments provide a first step along the path toward investigating how changing the architecture of purple bacterial photosynthetic units affects the overall efficiency of light-harvesting.     

Probing binding site of bacteriochlorophyll a and carotenoid in the reconstituted LH1 complex from Rhodospirillum rubrum S1 by Stark spectroscopy

Stark spectroscopy is a powerful technique to investigate the electrostatic interactions between pigments as well as between the pigments and the proteins in photosynthetic pigment–protein complexes. In this study, Stark spectroscopy has been used to determine two nonlinear optical parameters (polarizability change Tr(Δα) and static dipole-moment change |Δμ| upon photoexcitation) of isolated and of reconstituted LH1 complexes from the purple photosynthetic bacterium, Rhodospirillum (Rs.) rubrum. The integral LH1 complex was prepared from Rs. rubrum S1, while the reconstituted complex was assembled by addition of purified carotenoid (all-trans-spirilloxanthin) to the monomeric subunit of LH1 from Rs. rubrum S1. The reconstituted LH1 complex has its Q_y absorption maximum at 878 nm. This is shifted to the blue by 3 nm in comparison to the isolated LH1 complex. The energy transfer efficiency from carotenoid to bacteriochlorophyll a (BChl a), which was determined by fluorescence excitation spectroscopy of the reconstituted LH1 complex, is increased to 40%, while the efficiency in the isolated LH1 complex is only 28%. Based on the differences in the values of Tr(Δα) and |Δμ|, between these two preparations, we can calculate the change in the electric field around the BChl a molecules in the two situations to be E _Δ ≈ 3.4 × 10⁵ [V/cm]. This change can explain the 3 nm wavelength shift of the Q_y absorption band in the reconstituted LH1 complex.     

Four-wave mixing signals from β-carotene and its n = 15 homologue

The third-order nonlinear optical responses of β-carotene and its homologue having a conjugation-double bond n = 15 have been investigated using sub-20 fs ultra-short optical pulses in order to clarify the dissipation processes of excess energy. Using the four-wave mixing spectroscopy, we observed a clear coherent oscillation with a period of a few tens of femtoseconds. The spectral density of these molecules was estimated that allowed the theoretical linear and nonlinear optical signals to be directly compared with the experimental data. Calculations based on the Brownian oscillator model were performed under the impulsive excitation limit. We show that the memory of the vibronic coherence generated upon the excitation into the S₂ state is lost via the relaxation process including the S₁ state. The vibronic decoherence lifetime of the system was estimated to be 1 ps, which is about 5 times larger than the life time of the S₂ state (∼150 fs) determined in previous studies. The role of coherence and the efficient energy transfer in the light-harvesting antenna complexes are discussed.     

Lilliputians get into the limelight: Novel class of small peptide genes in morphogenesis

Generally, bioactive small peptides are derived from precursors with signal sequences at their N-terminal ends, which undergo modification and proteolysis through a secretory pathway. By contrast, small peptides encoded in short open reading frames (sORF) lack signaling sequences and therefore are released into the cytoplasm, which may result in their having functions distinct from those of secreted peptides. Several small peptides encoded by sORF are involved in the morphogenesis of multicellular organisms. POLARIS, ROTUNDIFOLIA4, and Enod40 are plant peptides that are involved, respectively, in root formation, leaf shape control, and cortical cell division during nodule formation. Brick1 / HSPC300 is an evolutionarily conserved component of the actin reorganization complex. polished rice / tarsal-less and mille-pattes encode related small peptides that are required for epithelial morphogenesis in Drosophila and segmentation in Tribolium . There are only a few known examples of small peptides encoded by sORF, and their molecular functions are still largely obscure. Nevertheless, an increasing number of sORF genes is being identified, and further research should reveal their roles in novel molecular mechanisms underlying developmental events.     

Nepmucin/CLM-9, an Ig domain-containing sialomucin in vascular endothelial cells, promotes lymphocyte transendothelial migration in vitro

Nepmucin/CLM-9 is an Ig domain-containing sialomucin expressed in vascular endothelial cells. Here we show that, like CD31, nepmucin was localized to interendothelial contacts and to vesicle-like structures along the cell border and underwent intracellular recycling. Functional analyses showed that nepmucin mediated homotypic and heterotypic cell adhesion via its Ig domain. Nepmucin-expressing endothelial cells showed enhanced lymphocyte transendothelial migration (TEM), which was abrogated by anti-nepmucin mAbs that block either homophilic or heterophilic binding. Notably, the mAbs that inhibited homophilic binding blocked TEM without affecting lymphocyte adhesion. These results suggest that endothelial nepmucin promotes lymphocyte TEM using multiple adhesion pathways.     

Microchamber arrays for the identification of individual cells exposed to an X-ray microbeam

To identify individual cells exposed to a X-ray microbeam in a cell population, we developed a biocompatible microchamber-array chip using UV lithography of photopolymer SU-8. The center-to-center distance between microchambers is 50 μm including a wall of 15 μm height. Using the microchamber-array chip, we performed tracking of individual exposed cells. Sample cells loaded in a microchamber array were selectively irradiated with the X-ray microbeam under microscopic observation. All the irradiated cells were indexed by the array arrangement of the microchambers. For about 24 h of post-irradiation incubation, the irradiated cells were identified successfully by time-lapse observation. In addition, the induction of radiation effects was observed in identified cells using immunofluorescence.     

Development of the reverse passive latex agglutination method for the detection and quantification of the genus Nitrospira in the wastewater treatment process

This report describes a new immunological method for the detection and quantification of Nitrospira populations using the reverse passive latex agglutination (RPLA). The numbers of the genus Nitrospira have been quantified only by molecular biological techniques such as FISH and quantitative PCR to date. Using high-density latex particles and a specific polyclonal antibody, Nitrospira populations in the wastewater treatment process were quantified in the shortest 4 h of incubation. The minimum detectable number of Nitrospira cells was 7.0x10(5) (log(10) 5.85) cells/ml. It is thought that the RPLA method can quantify Nitrospira populations more simply, economically, and speedily than molecular biological techniques or the culture method, because this procedure has a simple protocol and does not require the use of specialized equipment, expensive reagents, or technical skill. Therefore it is applicable for use in the everyday control and maintenance of water quality in wastewater treatment facilities where equipment is not sufficient or in the field.