Specific modification at the C-terminal lysine residue of the green fluorescent protein variant, GFPuv, expressed in Escherichia coli

Green fluorescent protein (GFP) is amenable to recombinant expression in various kinds of cells and is widely used in life science research. We found that the recombinant expression of GFPuv, a commonly-used mutant of GFP, in E. coli produced two distinct molecular species as judged by in-gel fluorescence SDS-PAGE. These molecular species, namely form I and II, could be separately purified by anion-exchange chromatography without any remarkable differences in the fluorescence spectra. Mass spectrometric analyses revealed that the molecular mass of form I is almost the same as the calculated value, while that of form II is approximately 1 Da larger than that of form I. Further mass spectrometric top-down sequencing pinpointed the modification in GFPuv form II, where the epsilon-amino group of the C-terminal Lys238 residue is converted into the hydroxyl group. No equivalent modification was observed in the native GFP in jellyfish Aequorea victoria, suggesting that this modification is not physiologically relevant. Crystal structure analysis of the two species verified the structural identity of the backbone and the vicinity of the chromophore. The modification found in this study may also be generated in other GFP variants as well as in other recombinant expression systems

Theoretical Consideration on Influences of Cavity or Pillar Shape on Band Structures of Silicon-Based Photonic Crystals

This paper describes the physical manifestations of the various influences of cavity (or pillar) shape and the filling factor of dielectric material on band structures in two-dimensional photonic crystals. The influences of circular or rectangular cross-sections of cavity (or pillar) arrays on photonic band structures are considered theoretically, and significant aspects of square and triangular lattices are compared. It is shown that both the averaged dielectric constant of the photonic crystal and the distribution profile of photon energy play important roles in defining optical properties. For the triangular lattice, especially, it is shown that cavity array with a rectangular cross-section breaks the band structure symmetry. So, we go on to discuss this from the perspective of band structure, and consider the optical properties of a lattice with a circular cross-section cavity

High energy electron observation by Polar Patrol Balloon flight in Antarctica

We accomplished a balloon observation of the high-energy cosmic-ray electrons in 10-1000GeV to reveal the origin and the acceleration mechanism. The observation was carried out for 13 days at an average altitude of 35km by the Polar Patrol Balloon (PPB) around Antarctica in January 2004. The detector is an imaging calorimeter composed of scintillating-fiber belts and plastic scintillation counters sandwiched between lead plates. The geometrical factor is about 600cm^2sr, and the total thickness of lead absorber is 9 radiation lengths. The performance of the detector has been confirmed by a test flight at the Sanriku Balloon Center and by an accelerator beam test using the CERN-SPS (Super Proton Synchrotron at CERN). The new telemetry system using the Iridium satellite, the power system supplied by solar panels and the automatic flight level control operated successfully during the flight. We collected 5.7×10^3 events over 100GeV, and selected the electron candidates by a preliminary data analysis of the shower images. We report here an outline of both detector and observation, and the first result of the electron energy spectrum over 100GeV obtained by an electronic counter

Study on NOx and Soot Emissions in Diesel Engines with Multi-Stage Injection Based on a Stochastic Combustion Model

In this study, a phenomenological combustion model, which had been developed based on a stochastic combustion model for simulating combustion with multi-stage injection in diesel engines, was improved to estimate the NOx and soot emissions with modifications in the model for the interaction of sprays from sequent injection stages. Model validation was performed by comparison with experimental data from a single-cylinder diesel engine. To understand the variation of NOx and soot production with injection conditions, analysis of combustion process was conducted based on the calculation results from different injection parameters including injection timing and quantity

Both Neurons and Astrocytes Exhibited Tetrodotoxin-Resistant Metabotropic Glutamate Receptor-Dependent Spontaneous Slow Ca²⁺ Oscillations in Striatum

<div><p>The striatum plays an important role in linking cortical activity to basal ganglia outputs. Group I metabotropic glutamate receptors (mGluRs) are densely expressed in the medium spiny projection neurons and may be a therapeutic target for Parkinson's disease. The group I mGluRs are known to modulate the intracellular Ca²⁺ signaling. To characterize Ca²⁺ signaling in striatal cells, spontaneous cytoplasmic Ca²⁺ transients were examined in acute slice preparations from transgenic mice expressing green fluorescent protein (GFP) in the astrocytes. In both the GFP-negative cells (putative-neurons) and astrocytes of the striatum, spontaneous slow and long-lasting intracellular Ca²⁺ transients (referred to as slow Ca²⁺ oscillations), which lasted up to approximately 200 s, were found. Neither the inhibition of action potentials nor ionotropic glutamate receptors blocked the slow Ca²⁺ oscillation. Depletion of the intracellular Ca²⁺ store and the blockade of inositol 1,4,5-trisphosphate receptors greatly reduced the transient rate of the slow Ca²⁺ oscillation, and the application of an antagonist against mGluR5 also blocked the slow Ca²⁺ oscillation in both putative-neurons and astrocytes. Thus, the mGluR5-inositol 1,4,5-trisphosphate signal cascade is the primary contributor to the slow Ca²⁺ oscillation in both putative-neurons and astrocytes. The slow Ca²⁺ oscillation features multicellular synchrony, and both putative-neurons and astrocytes participate in the synchronous activity. Therefore, the mGluR5-dependent slow Ca²⁺ oscillation may involve in the neuron-glia interaction in the striatum.</p></div

The slow Ca²⁺ oscillations in both putative-neurons and astrocytes were mainly due to Ca²⁺ release from the intracellular Ca²⁺ store via the IP₃ receptor.

<p>A–D, Typical time courses of the slow Ca²⁺ oscillations during the administration of 10 µM CNQX and 50 µM AP5 (CNQX + AP5), 1 µM TTX, 2 µM thapsigargin (Thapsi), and 100 µM 2-APB in putative-neurons and astrocytes. Horizontal bars under the time courses indicate the application period of the agents. Scale bar, 200 s, µR  = 0.02. E, F, Transient rates of the slow Ca²⁺ oscillations during the administration of various pharmacological agents in putative-neurons (E) and astrocytes (F). The transient rates of the slow Ca²⁺ oscillations are normalized by the transient rates under control conditions. The number of cells recorded is shown above each bar graph. ****p<0.001; one-sample t-test.</p