Purification and characterization of recombinant human granulocyte colony-stimulating factor (rhG-CSF) derivatives: KW-2228 and other derivatives

Various derivatives of recombinant human granulocyte colony-stimulating factor (rhG-CSF) have been overproduced in Escherichia coli with the strong, inducible trp promoter. A derivative designated as KW-2228 in which the amino acids were replaced at five positions showed more potent granulopoietic activity and stability than those of wild-type both in vitro and in vivo. The purification involved a sequential renaturation process and three-step chromatography. Refolding succeeded in very high yield using a urea system. The purity of KW-2228 was greater than 99% as measured by SDS-PAGE and HPLC analysis. According to circular dichroism and nuclear magnetic resonance spectroscopy, rhG-CSF and KW-2228 have very similar conformations. This suggests that the substitution of five amino acids does not appreciably change the conformation of hG-CSF. KW-2228 ([Ala1, Thr3, Tyr4, Arg5, and Ser17]-hG-CSF) and derivative A ([Ala1, Thr3, Tyr4, Arg5]-hG-CSF) are easily crystallized and they show similar in vitro activity. On the other hand, neither rhG-CSF nor derivative B ([Ser17]-hG-CSF) are crystallized under the same conditions. Thus, the four amino acid substitution (Ala1, Thr3, Tyr4, Arg5) of the N-terminal sequence may facilitate crystallization. The change of Cys17 to Ser may not influence the stability and activity of hG-CSF.     

Study on local immune response in diabetic mice, in which bactericidal capacity of the neutrophils had been damaged--Escherichia coli induced experimental urinary tract infection

An experimental ascending urinary tract infection was induced by transurethral instillation of Escherichia coli in streptozotocin (STZ)-induced diabetic mice, in which bactericidal capacity of the perineal exudating neutrophils had been damaged. The local immune response in uninfected diabetic mice and the response time fluctuation of local immune responses in diabetic mice following infection were compared with those in normal mice, and the nature of induced changes in the local immune response was determined. The diabetic model was prepared by a single administration of 280 mg/kg of STZ after it had been fasted for 15 hours. The mouse was then fasted an additional 2 hours to induce the diabetic state. Compared with the normal mice, the diabetic mice prepared using this method were found to have a significant suppression of the bactericidal capacity of the perineal exudating neutrophils, 3 weeks after induction of diabetes. The study of the cellular distribution in uninfected urinary tract tissue demonstrated a reduction in CD4-positive T cell distribution in bladder submucosa of diabetic mice at 3 weeks after induction of diabetes (diabetic group), compared with that in the normal mice (control group). In the diabetic group, probably in order to compensate for the reduction in CD4-positive T cell distribution in the bladder submucosa, the distribution of macrophages was increased in the bladder epithelium, compared with the control group. This finding suggested that diabetic mice are thought to have protective mechanisms against infection different from those of normal mice in the uninfected state. The study of the response time fluctuation of local immune response at the infected sites demonstrated infiltration of macrophages was the same grade as that of neutrophils in the control and the diabetic group. However, in the diabetic group, a marked infiltration of macrophages was recognized when compared with the control group in the early phase of infection. These findings suggested that macrophages aid in protection against infection when damage to the bactericidal capacity of neutrophils results in insufficient elimination of microorganisms. On the other hand, infiltration of T and B cells was weaker in the diabetic group, than in the control group.     

Development of Millimeter Wave Fabry-Pérot Resonator for Simultaneous Electron-Spin and Nuclear Magnetic Resonance Measurement

We report a Fabry-Pérot resonator with spherical and flat mirrors to allow simultaneous electron-spin resonance (ESR) and nuclear magnetic resonance (NMR) measurements that could be used for double magnetic resonance (DoMR). In order to perform simultaneous ESR and NMR measurements, the flat mirror must reflect millimeter wavelength electromagnetic waves and the resonator must have a high Q value (Q?>?3000) for ESR frequencies, while the mirror must simultaneously let NMR frequencies pass through. This requirement can be achieved by exploiting the difference of skin depth for the two frequencies, since skin depth is inversely proportional to the square root of the frequency. In consideration of the skin depth, the optimum conditions for conducting ESR and NMR using a gold thin film are explored by examining the relation between the Q value and the film thickness. A flat mirror with a gold thin film was fabricated by sputtering gold on an epoxy plate. We also installed a Helmholtz radio frequency coil for NMR and tested the system both at room and low temperatures with an optimally thick gold film. As a result, signals were obtained at 0.18 K for ESR and at 1.3 K for NMR. A flat-mirrored resonator with a thin gold film surface is an effective way to locate NMR coils closer to the sample being examined with DoMR.     

Holographic femtosecond laser processing using optimal-rotation-angle method with compensation of spatial frequency response of liquid crystal spatial light modulator

Holographic femtosecond laser processing performs high-speed parallel processing using a computer-generated hologram (CGH) displayed on a liquid crystal spatial light modulator. A critical issue is to precisely control the intensities of the diffraction peaks of the CGH. We propose a method of compensating for the spatial frequency response in the design of CGH using the optimal-rotation-angle method. By applying the proposed method, the uniformity of the diffraction peaks was improved. We demonstrate holographic femtosecond laser processing with two-dimensional and three-dimensional parallelism.     

Ultrahigh-Q nanocavities written with a nanoprobe

High-Q nanocavities have been extensively studied recently because they are considered key elements in low-power photonic devices and integrated circuits. Here we demonstrate that ultrahigh-Q (>10(6)) nanocavities can be created by employing scanning probe lithography on a prepatterned line defect in a silicon photonic crystal. This is the first realization of ultrahigh-Q nanocavities by the postprocess modification of photonic crystals. With this method, we can form an ultrahigh-Q nanocavity with controllable cavity parameters at an arbitrary position along a line defect. Furthermore, the fabricated nanocavity achieves ultralow power all-optical bistable operation owing to its large cavity enhancement effect. This demonstration indicates the possibility of realizing photonic integrated circuits on demand, where various circuit patterns are written with a nanoprobe on a universal photonic crystal substrate.     

Material-binding peptide application--ZnO crystal structure control by means of a ZnO-binding peptide

Recently, a zinc oxide (ZnO)-binding peptide (ZnOBP) has been identified and has been used to assist the synthesis of unique crystalline ZnO particles. We analyzed the influence of ZnOBP on the crystal growth of ZnO structures formed from zinc hydroxide. The addition of ZnOBP in the hydrothermal synthesis of ZnO suppressed [0001] crystal growth in the ZnO particles, indicating that the specificity of the material-binding peptide for specific inorganic crystal faces controlled the crystal growth. Furthermore, the dipeptides with a partial sequence of ZnO-binding "hot spot" in ZnOBP were used to synthesize ZnO particles, and we found that the presence of these dipeptides more strictly suppressed (0001) growth in ZnO crystals than did the complete ZnOBP sequence. These results demonstrate the applicability of dipeptides selected from material-binding peptides to control inorganic crystal growth.     

Phase relations in the Fe-Pu-U ternary system

An isothermal section of the Fe-Pu-U ternary system at 650 °C was assessed in a previous study. In the present study, the predictions of the phase relations in the Fe-Pu-U system to higher and lower temperatures were performed by applying the interaction parameters determined at 650 °C. Differential thermal analysis (DTA) for the Fe-Pu-U alloys was also carried out to confirm the phase relations in the temperature region of 500 to 800 °C. Both results agreed well. On the basis of the predicted ternary phase diagram, the phase relations for a region surrounded by Fe₂Pu, Fe₂U, U, and Pu were described by a reaction scheme and a projection of the liquidus surface.     

The distributed constraint satisfaction problem: formalization andalgorithms

We develop a formalism called a distributed constraint satisfaction problem (distributed CSP) and algorithms for solving distributed CSPs. A distributed CSP is a constraint satisfaction problem in which variables and constraints are distributed among multiple agents. Various application problems in distributed artificial intelligence can be formalized as distributed CSPs. We present our newly developed technique called asynchronous backtracking that allows agents to act asynchronously and concurrently without any global control, while guaranteeing the completeness of the algorithm. Furthermore, we describe how the asynchronous backtracking algorithm can be modified into a more efficient algorithm called an asynchronous weak-commitment search, which can revise a bad decision without exhaustive search by changing the priority order of agents dynamically. The experimental results on various example problems show that the asynchronous weak-commitment search algorithm is, by far more, efficient than the asynchronous backtracking algorithm and can solve fairly large-scale problems     

Helical Antimicrobial Peptide Foldamers Containing Non-proteinogenic Amino Acids

Antimicrobial peptides (AMPs) are potential novel therapeutic drugs against microbial infections. Most AMPs function by disrupting microbial membranes because of their amphipathic properties and ordered secondary structures. In this minireview, we describe recent efforts to develop helical AMP foldamers containing non-proteinogenic amino acids, such as α,α-disubstituted α-amino acids, β-amino acids, γ-amino acids, side-chain stapling and N-alkyl glycines.