Optical fiber biosensor for the determination of low biochemical oxygen demand

An optical fiber biosensor was developed for the evaluation of low Biochemical Oxygen Demand (BOD) values in river waters. Artificial wastewater (AWW) solution was employed as standards for the calibration of the BOD sensor. The response time of the sensor was 15 min, and the optimal BOD response was observed at 30 degrees C, pH 7.0. A linear relationship was obtained between the output voltage and BOD5 values, and the range of determination was 1-10 mg l(-1) BOD. The sensor response was almost not influenced by chloride ion up to 1000 mg l(-1), and also not affected by heavy metal ions (Fe3+, Cu2+, Mn2+, Cr3+, Zn2+). The BOD of river waters was estimated by using the optical fiber biosensor, and good correlation between the sensor and BOD5 test was obtained (r2 = 0.971).     

Determination of human serum 8-hydroxy-2′-deoxyguanosine (8-OHdG) by HPLC-ECD combined with solid phase extraction (SPE)

Human serum 8-hydroxy-2′-deoxyguanosine (8-OHdG) was measured by HPLC-ECD method combined with solid phase extraction (SPE) developed by our group: (our proprietary kit, named 8-OHdG Pre-treatment Kit (TANITA Corporation)). The major interfering substances and proteins in serum were removed by 8-OHdG Pre-treatment Kit. This measurement method was highly reproducible (CV = 2.2–7.1%) and demonstrated the lower detection limit for control serum sample of less than 10 pg/ml without the sample evaporation. The other hand 8-OHdG concentration in serum for healthy people was in the range of 0–70 pg/ml (25.5 ± 13.8 pg/ml, n = 37). Secondary a relationship between the HPLC-ECD and ELISA methods was investigated. ELISA method could not detect 8-OHdG concentration in serum for healthy people, because the detection limit of 130 pg/ml was higher than the normal range for healthy people. These results show our SPE method has high sensitivity and quantitative accuracy for 8-OHdG analysis.     

Chelating Compound,Chrysoidine, Is More Effective in Both Antiprion Activity and Brain Endothelial Permeability Than Quinacrine

1. As an extension of our previous study of quinacrine and its derivatives, chelating chemicals were screened to obtain more effective, better brain-permeable antiprion compounds using either prion-infected neuroblastoma cells or brain capillary endothelial cells. 2. Eleven chemicals were found to have antiprion activity. Most of them shared a common structure consisting of benzene or naphthalene at either end of an azo bond. Structure–activity data suggest that chelating activity is not necessary but might contribute to the antiprion action. 3. Chrysoidine, a representative compound found here, was about 27 times more effective in the antiprion activity and five times more efficiently permeable through the brain capillary endothelial cells than quinacrine was. 4. These chemicals might be useful as compounds for development of therapeutics for prion diseases.     

Application of peptide probe for evaluating affinity properties of proteins using quartz crystal microbalance

This study proved a possibility of a peptide probe for evaluating affinity properties of proteins. We have designed and synthesized three different peptide probes, H-Ala3-(Gly-Pro5)3-Gly-OH (peptide A), H-Ala3-(Gly-Pro5)-Gly-OH (peptide B) and H-Ala3-Gly-OH (peptide C) for testing their affinities to profilin. Each peptide probe was immobilized on a quartz crystal microbalance (QCM) sensor. The QCM sensor with the peptide A showed a 93 Hz decrease of resonant frequency which indicated profilin bound to the QCM sensor in a single layer. In a successive reaction with actin, the QCM analysis resulted in a 123 Hz decrease of resonant frequency which showed actin bound to the QCM sensor. A fluorescence microscope image of the sensor surface exhibited clear fluorescence after binding a rhodamine labeled actin on the sensor surface. These results supported stepwise reactions of profilin binding to the peptide A and actin binding to profilin. In the three peptide probes, the peptide A showed the highest affinity to profilin, i.e., sequence dependent affinity was confirmed.     

Purification of acidic fibroblast growth factor from bovine heart and its localization in the cardiac myocytes

We found an endogenous growth factor, referred to here as heart-derived growth factor (HDGF), that stimulates the proliferation of vascular endothelial cells. HDGF was purified from bovine myocardium using a procedure that involves denaturation of undesired proteins with methanol and chloroform. Soluble HDGF was purified essentially to homogeneity in a single step by heparin affinity chromatography. The purified HDGF was identified to be acidic fibroblast growth factor based on the following properties: molecular weight of 18,000, isoelectric point of 5.2, amino acid composition and sequence, its dissociation from a heparin affinity column at 0.9 M NaCl, potentiation of activity in the presence of heparin, and antigenicity. Our yield of HDGF was 500 micrograms/kg of tissue. Antiserum raised to HDGF localized HDGF in the cardiac myocytes in culture. These data indicate that a large amount of acidic fibroblast growth factor is present in the heart, and the cardiac myocytes are likely to be a major source of it.     

Identification of proteins encoded in Escherichia coli hydA, hydB and analysis of the hydA locus

The hydB gene of Escherichia coli, which is related with the expression of hydrogenase activity, was cloned into the plasmid (pES1). Using the maxicell protein-labeling method, the molecular weight of hydB gene product was estimated. Comparing between the gene products from the mutant strains and that of the hydB genes cloned strains, the molecular weight of the gene product was 35,000 Mr. Similarly, the molecular weight of the gene product of hydA, which had been previously cloned, was determined by maxicell analysis. The molecular weight of hydA gene product was estimated to be 80,000 Mr. Using deletion analysis and Tn1000 insertional inactivation of hydA's function, the hydA coding region was estimated between 2.2 kb and 2.8 kb in a 3.1 kb EcoRI-MluI fragment on the recombinant plasmid pEH3.