A novel enzyme immunoassay based on potentiometric measurement of molecular adsorption events by an extended-gate field-effect transistor sensor

We developed a novel enzyme immunoassay based on a potentiometric measurement of molecular adsorption events by using an extended-gate field-effect transistor (FET) sensor. The adsorbing rate of a thiol compound on a gold surface was found to depend on the concentration of the compound. To construct an electrochemical enzyme immunoassay system by using the sensor, the enzyme chemistry of acetylcholinesterase (AChE) to generate a thiol compound was used and combined with the enzyme-linked immunosorbent assays (ELISA). After the AChE-catalyzed reaction, the amount of the antigen was obtained by detecting the adsorbing rate of the generated thiol compound on the gold electrode using the FET sensor. The measurement stability was also found to improve when a high frequency voltage of 10 kHz or more was superimposed to the reference electrode. The signal corresponding to a range between 1 and 250 pg/mL of Interleukin 1β was obtained by the FET sensor when a voltage of 1 MHz was superimposed onto the reference electrode. The FET sensor based ELISA used in this measurement technique can successfully detect Interleukin 1β at concentrations as low as 1 pg/mL.     

Monitoring of environmental arsenic by cultures of the photosynthetic bacterial sensor illuminated with a near-infrared light emitting diode array

Recombinant Rhodopseudomonas palustris, harboring the carotenoid-metabolizing gene crtI (CrtIBS), and whose color changes from greenish yellow to red in response to inorganic As(III), was cultured in transparent microplate wells illuminated with a light emitting diode (LED) array. The cells were seen to grow better under near-infrared light, when compared with cells illuminated with blue or green LEDs. The absorbance ratio of 525 to 425 nm after cultivation for 24 h, which reflects red carotenoid accumulation, increased with an increase in As(III) concentrations. The detection limit of cultures illuminated with near-infrared LED was 5 microgram/l, which was equivalent to that of cultures in test tubes illuminated with an incandescent lamp. A near-infrared LED array, in combination with a microplate, enabled the simultaneous handling of multiple cultures, including CrtIBS and a control strain, for normalization by the illumination of those with equal photon flux densities. Thus, the introduction of a near-infrared LED array to the assay is advantageous for the monitoring of arsenic in natural water samples that may contain a number of unknown factors and, therefore, need normalization of the reporter event.     

Unusual Accumulation of Demethylspheroidene in Anaerobic-Phototrophic Growth of crtA-Deleted Mutants of Rhodovulum sulfidophilum

Rhodovulum sulfidophilum produces carotenoids in the spheroidene pathway. Spheroidene monooxygenase, CrtA, catalyzes the conversion of spheroidene to spheroidenone. crtA-deleted mutants of R. sulfidophilum did not produce spheroidenone and demethylspheroidenone. In these mutants, the ratio of demethylspheroidene to spheroidene increased with exposure to light. One mutant exhibiting a spheroidene-predominant phenotype did not grow under anaerobic-light conditions and was devoid of bacteriochlorophyll a, even under semiaerobic-light conditions There was no difference in the growth of the mutants under aerobic-dark conditions. These data suggest that demethylspheroidene is important for photosynthesis in R. sulfidophilum.     

A novel NADP+-dependent L-1-amino-2-propanol dehydrogenase from Rhodococcus erythropolis MAK154: a promising enzyme for the production of double chiral aminoalcohols

AIM: A novel NADP(+)-dependent L-1-amino-2-propanol dehydrogenase was isolated from Rhodococcus erythropolis MAK154, and characterized. METHODS AND RESULTS: The enzyme was inducibly produced on cultivation with aminoalcohols such as 1-amino-2-propanol, 1-amino-2-butanol and 2-aminocyclohexanol. The enzyme catalyses the NADP(+)-dependent oxidation of several aminoalcohols, and also the NADPH-dependent asymmetric reduction of an aminoketone compound to a double chiral aminoalcohol, d-pseudoephedrine. Amino acid sequence analysis showed that the enzyme might belong to the short-chain dehydrogenase/reductase family. CONCLUSIONS: NADP(+)-dependent L-1-amino-2-propanol dehydrogenase isolated from R. erythropolis MAK154 reversibly catalysed dehydrogenation of aminoalcohols, and exhibited a unique sterospecifity for the reduction reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: The enzyme is a promising catalyst for the production of double chiral compound, d-pseudoephedrine, from prochiral substrate.     

Peptide nanotube composed of cyclic tetra-β-peptide having polydiacetylene

A cyclic tetra-β-peptide composed of three β-alanine residues and a L-β-homolysine residue having 10,12-pentacosadiynoic amide at the side chain was synthesized. The cyclic tetra-β-peptide formed peptide nanotubes in CDCl(3) . The diacetylene units along the peptide nanotubes were polymerized by UV irradiation to obtain peptide nanotubes with polydiacetylene along the nanotube. TEM and AFM observations of the polymerized cyclic tetra-β-peptides confirmed that the peptide nanotube structure was preserved after polymerization.     

Near-infrared fluorescent labeled peptosome for application to cancer imaging

Nonionic amphiphilic copolypeptides, which were composed of hydrophilic poly(sarcosine) and hydrophobic poly(gamma-methyl L-glutamate) blocks, were synthesized with varying chain lengths of the blocks. The polypeptides having a suitable hydrophilic and hydrophobic balance were found to form vesicular assemblies of 100 nm size in buffer, which was evidenced by the TEM observation, the DLS analysis, and the encapsulation experiment. The genuine peptide vesicles, peptosomes, were labeled with a near-infrared fluorescence (NIRF) probe. In vivo retention in blood experiment showed long circulation of the peptosome in rat blood as stable as the PEGylated liposome. NIRF imaging of a small cancer on mouse by using the peptosome as a nanocarrier was successful due to the EPR effect of the peptosome. Peptosome is shown here as a novel excellent nanocarrier for molecular imaging.     

Comparative study of hydrogen peroxide- and 4-hydroxy-2-nonenal-induced cell death in HT22 cells

Several studies have indicated that lipid peroxidation often occurs in response to oxidative stress, and that many aldehydic products including 4-hydroxy-2-nonenal (HNE) are formed when lipid hydroperoxides break down. In order to clarify the mechanism of oxidative stress-induced neuronal death in the nervous system, we investigated H(2)O(2)- and HNE-induced cell death pathways in HT22 cells, a mouse hippocampal cell line, under the same experimental conditions. Treatment with H(2)O(2) and HNE decreased the viability of these cells in a time- and concentration-dependent manner. In the cells treated with H(2)O(2), significant increases in the immunoreactivities of DJ-1 and nuclear factor-kappaB (NF-kappaB) subunits (p65 and p50) were observed in the nuclear fraction. H(2)O(2) also induced an increase in the intracellular concentration of Ca(2+), and cobalt chloride (CoCl(2)), a Ca(2+) channel inhibitor, suppressed the H(2)O(2)-induced cell death. In HNE-treated cells, none of these phenomena were observed; however, HNE adduct proteins were formed after exposure to HNE, but not to H(2)O(2). N-Acetyl-L-cysteine (NAC) suppressed both HNE-induced cell death and HNE-induced expression of HNE adduct proteins, whereas H(2)O(2)-induced cell death was not affected. These findings suggest that the mechanisms of cell death induced by H(2)O(2) different from those induced by HNE in HT22 cells, and that HNE adduct proteins play an important role in HNE-induced cell death. It is also suggested that the pathway for H(2)O(2)-induced cell death in HT22 cells does not involve HNE production.     

A physical map with yeast artificial chromosome (YAC) clones covering 63% of the 12 rice chromosomes.

A new YAC (yeast artificial chromosome) physical map of the 12 rice chromosomes was constructed utilizing the latest molecular linkage map. The 1439 DNA markers on the rice genetic map selected a total of 1892 YACs from a YAC library. A total of 675 distinct YACs were assigned to specific chromosomal locations. In all chromosomes, 297 YAC contigs and 142 YAC islands were formed. The total physical length of these contigs and islands was estimated to 270 Mb which corresponds to approximately 63% of the entire rice genome (430 Mb). Because the physical length of each YAC contig has been measured, we could then estimate the physical distance between genetic markers more precisely than previously. In the course of constructing the new physical map, the DNA markers mapped at 0.0-cM intervals were ordered accurately and the presence of potentially duplicated regions among the chromosomes was detected. The physical map combined with the genetic map will form the basis for elucidation of the rice genome structure, map-based cloning of agronomically important genes, and genome sequencing.     

Dynamics of noninvasively estimated microvascular O2 extraction during ramp exercise.

Utilization of near-infrared spectroscopy (NIRS) in clinical exercise testing to detect microvascular abnormalities requires characterization of the responses in healthy individuals and theoretical foundation for data interpretation. We examined the profile of the deoxygenated hemoglobin signal from NIRS {deoxygenated hemoglobin + myoglobin [deoxy-(Hb+Mb)] approximately O(2) extraction} during ramp exercise to test the hypothesis that the increase in estimated O(2) extraction would be close to hyperbolic, reflecting a linear relationship between muscle blood flow (Q(m)) and muscle oxygen uptake (Vo(2)(m)) with a positive Q(m) intercept. Fifteen subjects (age 24 +/- 5 yr) performed incremental ramp exercise to fatigue (15-35 W/min). The deoxy-(Hb+Mb) response, measured by NIRS, was fitted by a hyperbolic function [f(x) = ax/(b + x), where a is the asymptotic value and b is the x value that yields 50% of the total amplitude] and sigmoidal function {f(x) = f(0) + A/[1 + e(-(-c+dx))], where f(0) is baseline, A is total amplitude, and c is a constant dependent on d, the slope of the sigmoid}, and the goodness of fit was determined by F test. Only one subject demonstrated a hyperbolic increase in deoxy-(Hb+Mb) (a = 170%, b = 193 W), whereas 14 subjects displayed a sigmoidal increase in deoxy-(Hb+Mb) (f(0) = -7 +/- 7%, A = 118 +/- 16%, c = 3.25 +/- 1.14, and d = 0.03 +/- 0.01). Computer simulations revealed that sigmoidal increases in deoxy-(Hb+Mb) reflect a nonlinear relationship between microvascular Q(m) and Vo(2)(m) during incremental ramp exercise. The mechanistic implications of our findings are that, in most healthy subjects, Q(m) increased at a faster rate than Vo(2)(m) early in the exercise test and slowed progressively as maximal work rate was approached.