Halorhodopsin from natronomonas pharaonis forms a trimer even in the presence of a detergent, dodecyl-beta-D-maltoside

Halorhodopsin (HR) is a transmembrane seven-helix retinal protein, and acts as an inward light-driven Cl⁻ pump. HR from Natronomonas pharaonis (NpHR) can be expressed in Escherichia coli inner membrane in large quantities. Here, we showed that NpHR forms the trimer structure even in the presence of 0.1% (2 m m ) to 1% (20 m m ) dodecyl-β- d -maltoside (DDM), whose concentrations are much higher than the critical micelle concentration (0.17 m m ). This conclusion was drawn from the following observations. (1) NpHR in the DDM solution showed an exciton-coupling circular dichroism (CD) spectrum. (2) From the elution volume of gel filtration, the molecular mass of the NpHR–DDM complex was estimated. After evaluation of the mass of the bound DDM molecules, the mass of NpHR calculated was approximately equal to that of the trimer. (3) The cross-linked NpHR by glutaraldehyde gave the SDS-PAGE corresponding to the trimer. Mass spectra of these samples also support the notion of the trimer. Using the membrane fractions expressing NpHR ( Escherichia coli and Halobacterium salinarum ), CD spectra showed exciton-coupling, which suggests strongly the trimer structure in the cell membrane.     

Complementary structural information of positive- and negative-ion MSn spectra of glycopeptides with neutral and sialylated N-glycans

Positive- and negative-ion MSn spectra of chicken egg yolk glycopeptides binding a neutral and a sialylated N-glycan were acquired by using electrospray ionization linear ion trap time-of-flight mass spectrometry (ESI-LIT-TOFMS) and collision-induced dissociation (CID) with helium as collision gas. Several characteristic differences were observed between the positive- and negative-ion CID MSn (n = 2, 3) spectra. In the positive-ion MS2 spectra, the peptide moiety was presumably stable, but the neutral N-glycan moiety caused several B-type fragmentations and the sialylated N-glycan almost lost sialic acid(s). In contrast, in the negative-ion MS2 spectra, the peptide moiety caused several side-chain and N-glycan residue (e.g., N-acetylglucosamine (GlcNAc) residue) fragmentations in addition to backbone cleavages, but the N-glycan moieties were relatively stable. The positive-ion MS3 spectra derived from the protonated peptide ion containing a GlcNAc residue (203.1 Da) provided enough information to determine the peptide amino-acid sequence including the glycosylation site, while the negative-ion MS3 spectra derived from the deprotonated peptide containing a 0,2X1-type cross-ring cleavage (83.1 Da) complicated the peptide sequence analysis due to side-chain and 0,2X1 residue related fragmentations. However, for the structural information of the N-glycan moiety of the glycopeptides, the negative-ion CID MS3 spectra derived from the deprotonated 2,4A6-type cross-ring cleavage ion (neutral N-glycan) or the doubly deprotonated B6-type fragment ion (sialylated N-glycan) are more informative than are those of the corresponding positive-ion CID MS3 spectra. Thus, the positive-ion mode of CID is useful for the analyses of peptide amino-acid sequences including the glycosylation site. The negative-ion mode of CID is especially useful for sialylated N-glycan structural analysis. Therefore, in the structural analysis of N-glycopeptides, their roles are complementary.     

Separation of isomeric 2-aminopyridine derivatized N-glycans and N-glycopeptides of human serum immunoglobulin G by using a zwitterionic type of hydrophilic-interaction chromatography

Isomeric oligosaccharides and isomeric glycopeptides are sometimes difficult to separate on normal-phase (NP) and reversed-phase (RP) columns. A zwitterionic type of hydrophilic-interaction chromatography column with sulfobetaine groups (called ZIC-HILIC column) was first applied to the separation of 2-aminopyridine derivatized (PA) N-glycans and tryptic peptides of human serum immunoglobulin G (IgG). It is shown that the ZIC-HILIC column has high capability for structural recognition of isomeric N-glycans as well as high selectivity for glycopeptides. The former feature (i.e., structural recognition) was proven by sufficient separation of neutral PA N-glycan isomers, which are usually difficult to separate on NP and RP columns. In addition, it is noteworthy that IgG glycopeptides consisting of isomeric N-glycans and the same peptide sequences can be sufficiently separated on a ZIC-HILIC column. The latter feature (i.e., selectivity) was also demonstrated by easily separating two peptide groups with/without N-glycans. Thus, we note that the ZIC-HILIC column is highly promising for a simple analysis of N-glycans and N-glycopeptide samples.     

Coaxial holographic data storage without recording the dc components

A technique of recovering the data pages from Fourier holograms recorded without the dc components is demonstrated theoretically and experimentally by use of a coaxial holographic storage system. A reconstructed image is obtained by adding a phase-modulated dc component of the signal beam on reading. The bit error rate of the reconstructed image is comparable with that for the hologram recorded with the dc component as well. Since high intensities of the dc components are not recorded in this technique, the dynamic range of the recording media can be saved, which potentially contributes to increasing the number of multiplexed holograms.     

Microfluidic control of the internal morphology in nanofiber-based macroscopic cables

A matter of orientation: The nanofibers in cables that consist of assemblies of these nanofibers can be oriented parallel or perpendicular to the longitudinal axis by regulating the fluidic velocities of the core and sheath flows in coaxial microfluidic devices. Control of the internal morphology enables fabrication of cables with improved electrical conductivity and mechanical properties.     

Single-vesicle estimation of ATP-binding cassette transporters in microfluidic channels

We have developed a method to analyze the substrate transport of ATP-binding cassette (ABC) transporters, which are associated with drug resistance in tumor cells. Our microfluidic method is well suited to the single-vesicle estimation of substrate transport and the rapid drug screening of ABC transporters. Using this method, we have demonstrated, for the first time, the analysis of substrate transport by a single transporter and performed drug-inhibition experiments in less than 3 h.     

Microbubble formations in MEMS-fabricated rectangular channels: A high-speed observation

A microfluidic device that consists of MEMS-fabricated rectangular channels is developed for stable and sequential production of monodispersed microbubbles. The central inlet for gas phase is located between two inlets for liquid phases, where the device works as a two-fluid atomizer. The interfacial flow mechanism of microbubble formation at the junction of the inlets in the device is investigated using a high-speed visualization technique and digital image processing. The periodic formation process is successfully realized by the consideration of the wettability between the microbubble and the channel wall. The produced microbubbles are relatively uniform in size, and the size is controlled from 113 to 153 μm by changing the flow rates of the liquid and gas phases. Furthermore, a simple theoretical model to predict the equivalent diameter of microbubbles is developed by considering the mass balance of the gas phase in the formation process, where the experimental and theoretical results are in reasonable agreement.     

Thermal decomposition of propane in shock waves

The thermal decomposition of propane was studied behind reflected shock waves over the temperature range 1100–1450 K and the pressure range 1.5–2.6 atm, by both monitoring the time variations of absorption at 3.39 μm and analyzing the concentrations of the reacted gas mixtures. The rate constants of the elementary reactions were discussed from the results. The rate constant expressions, k₁ = 1.1 × 10¹⁶ exp (?84 kcal/RT) s^?1 and k₄ = 9.3 × 10¹³ exp(?8 kcal/RT) cm³ mol^?1 s^?1, of reactions C₃H₈ → CH₃ + C₂H₅ and C₃H₈ + H → n-C₃H₇ + H₂ were evaluated, respectively.     

High temperature pyrolysis of formaldehyde in shock waves

Thermal decomposition of formaldehyde diluted with Ar was studied behind reflected shock waves in the temperature range of 1200–2000 K at total pressures between 1.3 and 3.0 atm. The study was carried out for compositions from the concentrated mixture, 4% CH₂O, to the highly dilute mixture, 0.01% CH₂O by using time-resolve IR-laser absorption and IR-emission, and a single-pulse technique. From a computer-simulation study, the mechanism and the rate-constant expressions that could explain all of our data and previously reported ARAS data were discussed. This data obtained over a wide concentration range from 50 ppm CH₂O to 4% CH₂O were satisfactorily modeled by a five-reaction mechanism. © 1993 John Wiley & Sons, Inc.