Detection of pathogenic bacteria by using zinc finger protein fused with firefly luciferase

We constructed a novel bacterial genome detection system using zinc finger protein (ZF) fused with firefly luciferase (ZF-luciferase). Taking advantage of the direct recognition of double-stranded DNA (dsDNA) by ZF, we previously constructed bacterial genome detection systems that did not require dehybridization processes. To detect polymerase chain reaction (PCR) products rapidly and with a high sensitivity, we constructed two kinds of ZF-luciferase, Sp1-fused luciferase (Sp1-luciferase), and Zif268-fused luciferase (Zif268-luciferase). ZF-luciferase not only maintains luciferase activity but also shows dsDNA-binding ability and specificity. Furthermore, we succeeded in the detection of 10 copies of the genome of Legionella pneumophila and Escherichia coli O157. ZF-luciferase would be a useful tool for highly sensitive detection of pathogenic bacterial genome.     

Mercury and gold concentrations of highly polluted environmental samples determined using prompt gamma-ray analysis and instrument neutron activation analysis

The authors have established a method of determining mercury and gold in severely polluted environmental samples using prompt gamma-ray analysis (PGA) and instrumental neutron activation analysis (INAA). Since large amounts of mercury are constantly being released into the environment by small-scale gold mining in many developing countries, the mercury concentration in tailings and water has to be determined to mitigate environmental pollution. Cold-vapor atomic absorption analysis, the most pervasive method of mercury analysis, is not suitable because tailings and water around mining facilities have extremely high mercury concentrations. On the other hand, PGA can determine high mercury concentrations in polluted samples as it has an appropriate level of sensitivity. Moreover, gold concentrations can be determined sequentially by using INAA after PGA. In conclusion, the analytical procedure established in this work using PGA and INAA is the best way to evaluate the degree of pollution and the tailing resource value. This method will significantly contribute to mitigating problems in the global environment.     

Serial Changes of Serum Growth Factor Levels and Liver Regeneration after Partial Hepatectomy in Healthy Humans

This study aimed to investigate the associations of the serial changes of serum levels of various growth factors with liver regeneration after hepatectomy in healthy liver donors. Sixteen healthy liver donors who underwent conventional liver resection were included. Serum levels of various growth factors before hepatectomy and on postoperative day (POD) 1, 3, 5 and 7 were measured. Liver volume data calculated by multi-detector computed tomography using workstation. The ratio of remnant liver volume on POD 0 to liver volume before the operation was 51% ± 20%. The ratio of liver volume on POD 14 to liver volume on POD 0 were inversely correlated with remnant liver volume on POD 0 (r = −0.91). The ratio of liver volume on POD 14 to liver volume on POD 0 were significantly correlated with serum hepatocyte growth factor (HGF) levels on POD 1 (r = 0.54), serum leptin levels on POD 1 (r = 0.54), and serum macrophage colony-stimulating factor (M-CSF) levels on POD 5 (r = 0.76) and POD 7 (r = 0.80). These results suggest that early-phase elevation of serum levels of HGF, leptin and M-CSF may be associated with the acceleration of liver regeneration after hepatectomy in humans.     

Synthesis of cyclic poly(methyl methacrylate) by the intramolecular cyclization of α-amino, ω-carboxyl heterodifunctional poly(methyl methacrylate)

Summary α-Amino, ω-carboxyl heterodifunctional poly(methyl methacrylate) was prepared by a living anionic polymerization of methyl methacrylate using N,N'-diphenylethylenediamine monolithium amide and succinic anhydride as an initiator and terminator, respectively. Its intramolecular cyclization was carried out to obtain a well-defined cyclic poly(methyl methacrylate). Received: 27 June 2001/Accepted: 16 July 2001     

Development of a High-Affinity Antibody-Binding Peptide for Site-Specific Modification

Immunoglobulin G (IgG)-binding peptides such as 15-IgBP are convenient tools for the site-specific modification of antibodies and the preparation of homogeneous antibody–drug conjugates. A peptide such as 15-IgBP can be selectively crosslinked to the fragment crystallizable region of human IgG in an affinity-dependent manner via the ϵ-amino group of Lys8. Previously, we found that the peptide 15-Lys8Leu has a high affinity (K_d=8.19 nM) due to the presence of the γ-dimethyl group in Leu8. The primary amino group required for the crosslinking to the antibodies has, however, been lost. Here, we report the design and synthesis of a novel unnatural amino acid, 4-(2-aminoethylcarbamoyl)leucine (Aecl), which possesses both the γ-dimethyl fragment and a primary amino group. A peptide containing Aecl8 (15-Lys8Aecl) was synthesized and showed a binding affinity ten times higher (K_d=24.3 nM) than that of 15-IgBP (K_d=267 nM). Fluorescein isothiocyanate (FITC)-labeled 15-Lys8Aecl with an N-hydroxy succinimide ester at the side chain of Aecl8 (FITC-15-Lys8Aecl(OSu)) successfully labeled an antibody (trastuzumab, Herceptin^®) with the fluorophore. This peptide scaffold has both strong binding affinity and crosslinking capability, and could be a useful tool for the selective chemical modification of antibodies with molecules of interest such as drugs.     

A novel biomarker for hyperglycemia, MRX isolated from hydrolysate of glycated proteins

Long-lived proteins can undergo non-enzymatic glycation to form highly crosslinked structures with characteristic fluorescence during aging and diabetes processes. In this paper, a typical fluorophore, named Maillard reaction product X (MRX), was isolated from the hydrolysate of glycated proteins. MRX could be formed by incubation of bovine serum albumin with glucose, followed by acid hydrolysis. The structure of MRX was determined to be 8-hydroxy-5-methyldihydrothiazolo[3,2-alpha] pyridinium-3-carboxylate. MRX was also found to be formed by the incubation of cysteine and arginine with glucose, followed by hydrolysis. We found the formation of MRX in the recently developed genetically diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats and compared them with that in the control Long-Evans Tokushima Otsuka (LETO) rats. Significantly higher levels of MRX were observed from the serum (p < 0.005) and urinary protein (p < 0.001) of OLETF rats in comparison with those of LETO rats. MRX must be a potential candidate as a biomarker for hyperglycemia.     

Low torque pulsation and high-power control of permanent magnet motor with distributed electromotive force

This paper describes low torque pulsation and high-power control of permanent magnet synchronous motor (PM motor) with distorted electromotive force (emf). The experimental model of PM motor is a 40-kW, 190 r/min, salient-pole, six-phase machine. An individual single-phase transistor inverter is connected to each phase of PM motor as a power source. By adopting full pitch and concentrated winding in the armature of the motor, the rate of flux utilization and output torque per mass can be increased. This winding causes trapezoidal distortion in the phase voltage waveform at no load. For realization of high-performance control of PM motor, the current waveform should be determined so as to minimize torque pulsation and to maximize the output torque of the motor under the rated current. This paper proposes also the method to determine the optimum current waveform. The currents and emfs are expressed as N-dimensional vectors (N: the number of phases). Due to the characteristics of the particular structure of the motor, the armature reaction is small enough to be neglected. In this case, the optimum current vector can be derived under two conditions: (1) the direction of the current vector always agrees with that of the emf vector; and (2) the scalar product of the two vectors is held constant. Supplying the optimum current vector provides no torque pulsation and the nearly maximum output torque under the rated current. This method also can derive the new optimum current corresponding to open-phase operation. The validity of the method is confirmed experimentally. The characteristics of the experimental model of PM motor also are described in this paper.     

Selective adsorption of bilirubin by macroporous poly(glycidyl methacrylate-co-divinylbenzene) beads

Macroporous glycidyl methacrylate (GMA)-divinylbenzene (DVB) copolymer beads have been synthesized and the adsorption of bilirubin, albumin and other serum components were studied. The beads composed of DVB/GMA in the weight ratio of 75/25 provide an excellent bilirubin adsorption ability. When the surface of the beads was previously coated with albumin, discriminating adsorption of the bilirubin in the serum was attained and the adsorption of albumin was depressed. In addition, when the coated albumin was crosslinked with glutaraldehyde, the excellent bilirubin adsorption ability was maintained, however, an adsorption of albumin from the serum occurred.