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8-Oxoguanosine (8-oxoG) is derived from the oxidation of guanosine (dG) and is known to induce transversion mutations (G:C to T:A) in DNA. We have already reported the fluorescent probe "8-oxoG-clamp", which shows a selective fluorescence quenching phenomenon toward 8-oxoG. In this study, attachment of an additional fluorophore to 8-oxoG-clamp was investigated as a strategy for the detection of 8-oxoG by a fluorescence color change attributed to the 8-oxoG-clamp.

8-Oxoguanosine, which is derived by the oxidation of guanosine (dG), is known to induce transversion mutations (G:C-->T:A) in DNA. The compounds with small molecular weight for recognizing 8-oxoG were designed on the basis of the structure of the G-clamp, which is reported to have selective affinity toward guanosine. The G-clamp derivatives with the additional binding units toward 8-oxoG were effectively synthesized and named "8-oxoG-clamps". Binding properties were measured by fluorescence quenching of 8-oxoG-clamps. Among the synthesized derivatives, 8-oxoG-clamp with N-benzyloxycarbonyl (Cbz) unit completely discriminated 8-oxoG from other nucleosides by fluorescence quenching.

The 5'-amino-2',4'-BNA, bearing a locked N-type sugar conformation and a P3'-->N5' phosphoramidate linkage, was found to be rapidly cleaved by acid-mediated hydrolysis when they formed a triplex with the target dsDNA. The fluorophore and quencher moieties were introduced into the 5'-amino-2',4'-BNA-modified oligonucleotide which was used as a novel and innovative DNA detection probe.

A novel bridged nucleic acid, 3'-amino-2'-deoxy-3',4'-BNA, having an N3'-->P5' phosphoramidate linkage and an S-type conformation, was successfully synthesized. It showed selective hybridization ability toward ssDNA complements, and high enzymatic stability against snake venom phosphodiesterase.

5'-Amino-2',4'-BNA, a novel analogue of BNA series compounds, was synthesized and its incorporation into oligonucleosides via P3'-->N5' phosphoramidate linkage was successfully achieved on a DNA automatic synthesizer. The 5'-amino-2',4'-BNA modified oligonucleotides were found to have a potent hybridizing affinity for the single stranded RNA and double stranded DNA complements, and also to show a good resistant ability against SVPDE.

The level of ceruloplasmin, which is a more negatively charged protein than albumin, was measured by an immunoradiometric assay in timed overnight urine and serum samples from patients with non-insulin-dependent diabetes mellitus and healthy controls. None of the plasma proteins examined showed any cross-reactivity in this assay. A linear correlation was seen between the ceruloplasmin level and the serial dilution of the sample. Western blot analysis using concentrated urine samples showed that the molecular weight of ceruloplasmin in the urine sample was the same as that of ceruloplasmin in the serum and standard samples. These findings indicated that the substance detected by this assay was truly ceruloplasmin. The urinary ceruloplasmin excretion rate (CER) and clearance of ceruloplasmin increased in parallel with the progression of albuminuria. The highest CER was found in macroalbuminuric patients, followed by micro- and normoalbuminuric patients and the healthy control subjects, the differences between the groups being significant. In view of the fact that the isoelectric point of ceruloplasmin (4.4) is more acidic than that of albumin, the present findings suggested that an enhanced CER was due either to the alteration of charge selectivity in the glomerular basement membrane with unaltered tubular function or to a defect of the non-discriminatory pores (shunt pathway) with unaltered tubular function.

We have established a specific radioimmunoassay (RIA) for C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, and have elucidated its tissue distribution and molecular form. In rats, high concentrations of CNP-like immunoreactivity (-LI) were detected in the anterior lobe (19.8 +/- 8.6 pmol/g) and neurointermediate lobe (4.64 +/- 0.74 pmol/g) of the pituitary gland. CNP-LI was present throughout the brain with its high concentrations in the hypothalamus and cerebellum. Small amounts of CNP-LI were also detected in the lower part of gastrointestinal tract and the kidney. However, no significant amount of CNP-LI was present in other organs including the heart. Considerable amounts of CNP-LI were detected throughout the human brain. High performance-gel permeation chromatography coupled with the RIA detected two peaks of CNP-LI in the rat brain; CNP and presumably its N-terminally elongated form with 53 amino-acid residues, CNP-53. These findings indicate that the tissue distribution and processing pattern of CNP are clearly different from those of atrial natriuretic peptide and brain natriuretic peptide and suggest possible roles of CNP as a neurotransmitter or neuromodulator rather than as a cardiac hormone.

Antithyroglobulin (anti-Tg) antibodies cytophilic for human monocytes were detected in the serum of 30 of 45 patients with Hashimoto's thyroiditis using the passive rosette technique. These antibodies conferred on normal monocytes the ability to form rosettes with Tg-coated erythrocyres (E-Tg) in vitro. The percentage of E-Tg rosette-forming monocytes was correlated with serum anti-Tg antibody titers measured by tanned sheep red cell hemagglutination. Most serum cytophilic activities were recovered in the immunoglobulin G fraction and were not affected by heating to 56 C for 30 min or ultracentrifugation at 105,000 X g for 60 min. Passive E-Tg rosette formation by monocytes was immunologically specific and was inhibited by the addition of small amounts of free Tg into the medium but was not inhibited by the addition of normal human serum. The anti-Tg antibody-armed monocytes became cytotoxic against Tg-coated chicken erythrocytes and lysed target erythrocytes by an extracellular mechanism. It was suggested that monocytes might be armed by cytophilic antibodies in vivo, since monocytes of patients with Hashimoto's thyroiditis showed increased E-Tg binding (rosette formation) relative to monocytes from control subjects. These findings support the possible pathogenetic involvement of monocytes in human autoimmune thyroiditis.