Microfabricated on-chip-type electrochemical flow immunoassay system for the detection of histamine released in whole blood samples

This paper describes an on-chip-type electrochemical flow immunoassay system with a multichanneled matrix column. The multichanneled matrix column was functionally coated with cation-exchange resin and used for separation of proteins. Antihistamine immunoglobulin G (IgG) antibody conjugated with ferrocenemonocarboxylic acid (Fc) was also prepared and used as a novel analytical reagent. Antibody-antigen complexes were separated from free Fc-conjugated IgG antibody (Fc-IgG) on the basis of differences in isoelectric point (pI) using the multichanneled matrix column coated with cation-exchange resin. The assay yields a good relationship between current and histamine concentration in the range of 200-2000 ng/mL. This simple technique enables the assay of histamine released in whole blood within 2 min. Furthermore, a good correlation was found between the response of the electrochemical immunoassay described in this paper and the conventional RIA (radioimmunoassay). This on-chip-type electrochemical flow immunoassay requires only minute quantities of whole blood samples and generates highly reproducible results.     

Dynamic structures of motor proteins myosin and kinesin, and switch protein troponin as detected by SDSL-ESR

We have studied biological nano-machines, motor and switch proteins operating as supramolecular complexes by electron spin resonance (ESR) and found key features of their molecular movements. In all the systems, the specific movements of elements or domains were detected and quite dynamic at nanometer scale. We have observed two broad but distinct orientations, separated by a 25 degrees axial rotation, of a spin label attached specifically to the light chain (LC) domain of myosin motor in the muscle fibers. The distribution became only narrower upon muscle activation. ESR spectrum from the spin label of the neck-linker of dimeric kinesin motor consisted of immobilized and mobilized components and did not exhibit nucleotide-dependent mobility change. The distance between two labels of kinesin dimer was also measured by spin dipole-dipole interaction, showing a broad distribution and a nucleotide-dependent change on the nanometer scale (>1.5 nm). These results suggest that two LC domains of myosin and two neck linkers of kinesin play a similar role for sliding movement using two conformations. The spin label of the skeletal (Tn)-I regulatory domain (TnIreg) showed a large mobility change by Ca2+ ion suggesting a Ca-induced switch movement of TnIreg. Spin dipole-dipole interaction showed that in reconstituted muscle fibers both skeletal and cardiac TnC undergo Ca2+-induced structural change that is thought to be essential for TnIreg movement. We also succeeded in fixing the newly-synthesized bifunctional spin label rigidly on the TnC molecule in solution, indicating that we can determine the precise coordinate of the spin principal axis of troponin on the oriented filament.     

Mechanochemistry and permeability of polyelectrolyte complex membranes composed of poly(vinyl alcohol) derivatives

Two polyanions and two polycations were prepared by the esterification of poly(vinyl alcohol). Properties of their neutral polyelectrolyte complex membranes were compared with those of polyelectrolyte membranes. The charged state on the membranes was related closely to the mechanochemical reaction, the salt rejection, and the solution permeability. Both the salt rejection and the water flux of neutral polyelectrolyte complex memberanes were lower than those of the corresponding acidic or basic complex membranes.     

Characteristics of alpha-glucosidase production from recombinant Aspergillus oryzae by membrane-surface liquid culture in comparison with various cultivation methods

alpha-Glucosidase was produced using recombinant Aspergillus oryzae by membrane-surface liquid culture (MSLC), a method previously developed by the authors and the results compared with other methods, including shaking flask culture (SFC), agar-plate culture (APC), culture on urethane sponge supports (USC), and liquid surface culture (LSC) to determine possible reasons for the advantageous features of MSLC. When yeast extract was used as a nitrogen source, the amount of enzyme produced by MSLC was 5 or more times higher than those for SFC and LSC, but similar to that using APC. Enzyme production in USC was slightly lower than in MSLC and APC. Cell growth was similar irrespective of the cultivation method used. When NaNO3, a typical inorganic nitrogen source was used, enzyme production in all the cultures was lower than that using yeast extract. However, even using NaNO3, the amount of the enzyme produced by MSLC was 8 to 20 times higher than those by SFC, APC, USC, and LSC. Although cell growth using NaNO3 was similar to that for yeast extract in MSLC, it was markedly decreased in SFC, APC, and LSC. The reason for the difference in enzyme productivity for various cultivation methods using yeast extract and NaNO3 as a nitrogen source is discussed, on the basis of the experimental findings. The role of the oxygen transfer effect and gene expression levels in enzyme production were also examined.     

Neuroimmunological Implications of AQP4 in Astrocytes

The brain has high-order functions and is composed of several kinds of cells, such as neurons and glial cells. It is becoming clear that many kinds of neurodegenerative diseases are more-or-less influenced by astrocytes, which are a type of glial cell. Aquaporin-4 (AQP4), a membrane-bound protein that regulates water permeability is a member of the aquaporin family of water channel proteins that is expressed in the endfeet of astrocytes in the central nervous system (CNS). Recently, AQP4 has been shown to function, not only as a water channel protein, but also as an adhesion molecule that is involved in cell migration and neuroexcitation, synaptic plasticity, and learning/memory through mechanisms involved in long-term potentiation or long-term depression. The most extensively examined role of AQP4 is its ability to act as a neuroimmunological inducer. Previously, we showed that AQP4 plays an important role in neuroimmunological functions in injured mouse brain in concert with the proinflammatory inducer osteopontin (OPN). The aim of this review is to summarize the functional implication of AQP4, focusing especially on its neuroimmunological roles. This review is a good opportunity to compile recent knowledge and could contribute to the therapeutic treatment of autoimmune diseases through strategies targeting AQP4. Finally, the author would like to hypothesize on AQP4’s role in interaction between reactive astrocytes and reactive microglial cells, which might occur in neurodegenerative diseases. Furthermore, a therapeutic strategy for AQP4-related neurodegenerative diseases is proposed.     

Enzymatic assay method for measuring mizoribine levels in serum

A sensitive and specific method for assaying serum mizoribine levels that can be applied to general automatic clinical analyzers was developed. Regression analysis of the enzymatic assay (y) vs. the HPLC method (x) produced the following relation: y=0.964x+0.090 (n=262, Sy, x=6.37 ng/mL).     

Resveratrol derivative-rich melinjo (Gnetum gnemon L.) seed extract suppresses multiple angiogenesis-related endothelial cell functions and tumor angiogenesis

Angiogenesis is a promising target for cancer prevention and treatment. This study aimed to determine the antiangiogenic effects of melinjo (Gnetum gnemon L.) seed extract and its resveratrol derivative components, such as gnetin C (GC), gnetin L (GL), gnemonoside A (GMA), gnemonoside C (GMC), and gnemonoside D (GMD). An ethanol extract of melinjo seeds (EEMS) and the two gnetins markedly inhibited the proliferation and tube formation of human umbilical vein endothelial cells (HUVEC) stimulated with vascular endothelial growth factor and basic fibroblast growth factor. The inhibitory effects of GC and GL were much stronger than those of resveratrol. GMC and GMD inhibited only proliferation, whereas GMA had almost no effect on the two endothelial cell functions. The EEMS and GC also reduced the cell viability of tube-forming HUVEC, with accompanying ERK1/2 inactivation, and suppressed the migration of HUVEC. Furthermore, dietary intake of EEMS significantly inhibited tumor angiogenesis in a mouse dorsal air sac assay. In conclusion, we found that the EEMS and its resveratrol derivatives, particularly GC, suppress multiple angiogenesis-related endothelial cell functions and/or tumor angiogenesis, indicating that the melinjo seeds and the natural resveratrol derivatives may be useful for cancer prevention and treatment.     

A computational model for distributed knowledge systems with learning mechanisms

This paper addresses the issues of machine learning in distributed knowledge systems, which will consist of distributed software agents with problem solving, communication and learning functions. To develop such systems, we must analyze the roles of problem-solving and communication capabilities among knowledge systems. To facilitate the analyses, we propose a computational model: LPC. The model consists of a set of agents with (a) a knowledge base for learned concepts, (b) a knowledge base for problem solving, (c) prolog-based inference mechanisms and (d) a set of beliefs on the reliability of the other agents. Each agent can improve its own problem-solving capabilities by deductive learning from the given problems, by memory-based learning from communications between the agents and by reinforcement learning from the reliability of communications between the other agents. An experimental system of the model has been implemented in Prolog language on a Window-based personal computer. Intensive experiments have been carried out to examine the feasibility of the machine learning mechanisms of agents for problem-solving and communication capabilities. The experimental results have shown that the multiagent system improves the performance of the whole system in problem solving, when each agent has a higher learning ability or when an agent with a very high ability for problem solving joins the organization to cooperate with the other agents in problem solving. These results suggest that the proposed model is useful in analyzing the learning mechanisms applicable to distributed knowledge systems.