Masanori Yoshioka

Spider toxin (JSTX) blocks glutamate synapse in hippocampal pyramidal neurons

Effects of spider toxin (JSTX)--a specific blocker of glutamate receptors--on single pyramidal neurons of the hippocampus were studied using tissue slices in vitro. JSTX blocked the synaptic response in CA1 pyramidal cells evoked by Schaffer collateral stimulation without affecting the antidromic spike potential. The toxin suppressed glutamate-induced cell firings whereas it had little effect on aspartate-induced responses. The results suggest that glutamate is a neurotransmitter of the Schaffer collateral input to CA1 pyramidal neurons.

Presence of digitalis-like factor in mammalian plasma

We attempted to purify a digitalis-like factor from volume expanded dog plasma using an inhibitory effect on the binding of [3H]ouabain to intact human erythrocytes to monitor digitalis-like activity. A highly polar [3H] ouabain displacing compound was purified to a high degree using a combination of chromatographic procedures including reverse phase and gel filtration high performance liquid chromatography. This compound, a reversible inhibitor of [3H]ouabain binding, closely resembles ouabain in its polarity and significantly increases during saline infusion. Its molecular weight was estimated to be 343Da. Moreover, similar compound was consistently detected in other mammalian plasma.

Purification and characterization of human urine-derived digitalis-like factor

We were able to purify a digitalis-like factor to apparent homogeneity from human urine based on the inhibitory effect on [3H]-ouabain binding to intact human erythrocytes. This ouabain displacing compound closely resembles ouabain in its polarity, molecular weight, non-peptidic nature and mode of action except for its UV absorbance spectrum. This compound sharing many biological activities of ouabain may be the endogenous ligand for the Na+, K+-ATPase and serve as a specific regulator of the sodium pump.

Immobilization of ultra-thin layer of monoclonal antibody on glass surface

When preparing an affinity column and a biosensor, it is desirable to immobilize a unimolecular layer of pure protein on a matrix. In this work, we tried to immobilize a monoclonal antibody on a surface of a glass test-tube as a model, to confirm the stability of this ultra-thin layer by an enzyme immunoassay, and to estimate the thickness of the layer on a slide glass by Fourier transform infrared reflection spectrometry. A new test-tube was washed and dried. The tube was filled with 5% 3-aminopropyltriethoxysilane. The 3-aminopropylsilylated surface was treated with glutaraldehyde and 5.6.10(-2) mg/ml solution of a normal mouse monoclonal antibody. The Schiff base between glutaraldehyde and the antibody was further reduced with 7.9.10(-3)% NaBH4. The tube was washed with 0.05% Tween 20 to block non-specific binding. The antibody immobilized on the surface was measured by an enzyme immunoassay based on a reaction of anti-mouse immunoglobulin G labelled with alkaline phosphatase, with which p-nitrophenol was produced from p-nitrophenylphosphate as a substrate. Meanwhile, various amounts of the antibody were immobilized on slide glasses in the same manner. The antibody on each surface was measured by Fourier transform infrared reflection spectrometry. The antibody immobilized under the final conditions was detectable by the enzyme immunoassay, and stable at 4 degrees C for ten days. The antibody on the slide glass was a unimolecular layer, as judged from the Fourier transform infrared spectra referred to -CONH- band semiquantitatively. Thus, we found the optimal conditions for immobilizing an ultra-thin layer of the monoclonal antibody on the glass surface.(ABSTRACT TRUNCATED AT 250 WORDS)

The peak height ratio of S-sulfonated transthyretin and other oxidized isoforms as a marker for molybdenum cofactor deficiency, measured by electrospray ionization mass spectrometry

Molybdenum cofactor deficiency is a fatal neurological disorder, which follows an autosomal-recessive trait and is characterized by combined deficiency of the enzyme, sulfite oxidase, xanthine dehydrogenase and aldehyde oxidase. Early detection of molybdenum cofactor-deficient patients is essential for their proper care and genetic counseling of families at risk. We demonstrate the use of S-sulfonated transthyretin (TTR) as a marker for molybdenum cofactor deficiency. Plasma or sera obtained from 4 patients with molybdenum cofactor deficiency and 57 controls were studied by electrospray ionization mass spectrometry (ESIMS) following selective enrichment of TTR by immunoprecipitation using protein G/A agarose. The data obtained from molybdenum cofactor deficiency samples indicated a strong increase in the peak height of S-sulfonated TTR. A more significant difference was revealed if the peak height ratio of S-sulfonated TTR and the sum of the other oxidized TTR were determined. By accurate determination of the ratio, the samples of molybdenum cofactor deficiency patients could clearly be distinguished from controls without molybdenum cofactor deficiency.

Direct injection of blood samples into a high-performance liquid chromatographic adenine analyser to measure adenine, adenosine and the adenine nucleotides with fluorescence detection

Adenine (Ade), adenosine (Ado) and its nucleotides such as AMP, cAMP, ADP and ATP in blood or plasma were determined by a high-performance liquid chromatographic (HPLC) adenine analyser with fluorescence detection. In order to inject samples directly into the HPLC system without pretreatment except dilution, the analyser consisted of two systems each, having three columns (pre-, mini- and analytical). A precolumn with an inlet filter of pore size 40 microns was common to both systems and packed with Butyl-Toyopearl 650-M to remove hydrophobic compounds and blood cell membranes. In the system for analysis of the nucleotides, a mini-column of Hitachi anion-exchange gel 3013-N was used for adsorbing AMP, cAMP, ADP and ATP. The adsorbed nucleotides were separated by the Hitachi gel 3013-N analytical column. In the other system for analysis of Ado and Ade, they were adsorbed on a Develosil ODS-5 mini-column and separated by an Asahipak GS-320H size-exclusion analytical column. The adenine compounds in each eluate were derivatized on-line in a 15-m reaction coil at 115 degrees C with bromoacetaldehyde as the fluorescent reagent in each mobile phase for the analytical column, and detected by spectrofluorimetry. ATP, ADP and AMP were accurately determined by the direct injection of hamster, rat and human whole blood. Authentic Ade and Ado were well separated and Ado in human plasma was determined, but it was difficult to determine it in rat plasma owing to interference from an unknown compound.

Effect of aspartame on N-methyl-D-aspartate-sensitive L-[3H]glutamate binding sites in rat brain synaptic membranes.

Aspartame (L-aspartyl-L-phenylalanine methyl ester), an artificial low-calorie sweetener, was shown to dose-dependently inhibit L-[3H]glutamate binding to its N-methyl-D-aspartate-specific receptors. L-Aspartic acid, a major endogenous metabolite of aspartame, inhibited the binding more stronger than aspartame, while the other metabolites, L-phenylalanine and methanol, had no effect at the same concentration. Aspartame caused a significant change in the affinities of L-[3H]glutamate binding without altering the Vmax values of the binding, suggesting the inhibition is competitive. These in vitro findings suggested that aspartame may act directly on the N-methyl-D-aspartate-sensitive glutamate recognition sites in the brain synaptic membranes.

Sensitive fluorimetry of adenine-containing compounds with high-performance liquid chromatography.

A definitive method to determine adenine compounds simultaneously was established by introducing a new fluorescent reagent into high-performance liquid chromatography. Bromoacetaldehyde was the best reagent among the haloacetaldehydes examined. A quantitative reaction was obtained even for unstable ADP and ATP. A high resolution of adenine nucleotides was obtained using a column of Hitachi gel No. 3012-N. The method was applied to the measurement of cyclic AMP in urine, and ADP and ATP in brain and blood. Further, the sensitivity of the method was increased by a new fluorescence spectrophotometer constructed for micro-HPLC. Femtomole amounts of the adenine nucleotides were clearly separated.

Flow injection analysis for measurement of activity of matrix metalloproteinase-7 (MMP-7)

A simple and convenient method for measuring the activity of a recombinant human matrix metalloproteinase 7 (MMP-7, matrilysin) was developed by flow injection analysis (FIA). For this method, purified recombinant MMP-7 zymogen expressed in E. coli and the substrate peptide (MOCAc-Pro-Leu-Gly-Leu-A2pr(DNP)-Ala-Arg-NH2) were used. Following the incubation of substrate peptide with activated r-proMMP-7, the resulting fluorescent product peptide (MOCAc-Pro-Leu-GLY) was monitored with a fluorescence detector (lambda ex 328 mm, lambda em 393 mm) without chromatographic separation. In this FIA system, the analysis time is 2 min and the standard curve is linear from 5 to 100 pmol of the product peptide injected. In order to use this FIA system as a method for screening inhibitors against MMP-7, the effects of CaCl2, EDTA and of the tissue inhibitor of metalloproteinase-1, and -2, were tested. A synthetic PRCGXPD-containing peptide (BS-10) was also observed to inhibit MMP-7 activity, with an IC50 value of 104 microM. Thus, it was concluded that the activity of r-MMP-7 can be reliably measured by the proposed system. Furthermore, to confirm the utility of this FIA system as a screening method, the inhibitory activity of the MMP-related substance in Joro spider (Nephilia clavata) venom was measured by this method. This inhibitory activity was observed in an extract of a venom diluted 1000-fold. Thus, the FIA method is not only simple and quick, but also sensitive enough to screen and analyze the inhibitory properties of a large number of test compounds.

PLASMA ADENOSINE CONCENTRATIONS ARE ELEVATED IN CONSCIOUS SPONTANEOUSLY HYPERTENSIVE RATS

1. Plasma levels of adenosine were measured in unrestrained, conscious spontaneously hypertensive rats (SHR) to examine the potential role of adenosine, a naturally occurring substance with profound cardiovascular actions, in cardiovascular regulation in these genetically hypertensive rats.